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Development of a Perfusion Based Decellularization System for

1. Figure 24 Decellularized Heart Double Decellularization Experiment Results Before Top After Bottom 61 VI Discussion 1 Economics This decellularization bioreactor was designed as a small scale project with our advisor being the primary client the device was designed for the Gaudette Lab s purposes and was customized to fit its needs Consequently we do not envision our device could produce a large scale economic impact However the development of a low cost decellularization bioreactor with similarly cost efficient monitoring capability presents a method for researchers to circumvent the need for high cost decellularization systems Effectively it could open up opportunity for lower cost devices to make their way onto the market in the future should the design be developed further in the future 2 Environmental Impact The decellularization system was designed to utilize readily available materials that could be found in any research laboratory This was done predominantly to cut down on costs and the need to purchase and wait for the shipment of additional materials during the course of this project Specifically the device uses a Nalgene container laboratory 50mL plastic conical tubes a 3mL syringe silicon based tubing along with general wiring and electronic components powered by a AA battery pack Because this device uses conventional materials and due to the nature of the research work done with it r
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4. 2 Preliminary Designs The conceptual ideas for bioreactor materials led us to develop preliminary designs We modeled these designs using the 3D CAD software SolidWorks so that we could visually evaluate the positive and negative aspects of each design Nalgene Based Bioreactor The first main preliminary design is based off of using a 1000 mL Nalgene Straight Sided Jar as the chamber and 50 mL conical tubes as holding cells for the individual heart samples Figure 14 below is the main design featuring this concept 45 Figure 14 CAD Model of Nalgene Bioreactor The main concept of this design is that a sample heart after cannulation will be attached to a luer lock that has been fitted and sealed to a hole in one of the conical tube caps The conical tube cap can then be screwed onto the conical tube which is fitted and sealed into a hole in the Nalgene container cap Solutions for decellularization will run from separate holding containers into polyethylene tubing that will then split into two lines for each individual heart This tubing is then connected to the luer locks in the conical tube caps so that fluid can then flow through the hearts The heart attachment and assembly will have to be conducted in a biological flow hood to ensure the inside of the chamber remains sterile Once assembled the system is allowed to leave the flow hood to be attached to the solution tubes A peristaltic pump will pump the solutions from the
5. 3 Perfusion with 1 SDS for 48 hours to chemically decellularize cardiac tissue followed by a wash with dH20 for 12 hours to remove SDS and residual intracellular materials 4 Perfusion with Triton X 100 for 12 hours which further decellularized the heart and aids in the removal of SDS by solubilizing the detergent 5 Perfusion with antibiotic infused PBS for 48 hours to remove any pathogens that may have accumulated during the process and flush the decellularized tissue Following this protocol through initial decellularization trials on the prototype system have shown promising results showing extensive removal of intracellular proteins and DNA when evaluated histologically However the system and process needs improvement some residual cellular material remains in the tissue This residual intracellular tissue is shown in Figure la Figure 1b is presented as a comparison done by the Ott lab which presents tissue 9 absent of cellular nuclear material the primary goal in organ decellularization Cadaveric heart Decellularized heart Cadaveric heart Decellularized heart Figure 1 A Hoescht stain of decellularized rat cardiac tissue processed with the current Gaudette lab system and protocol Scale bar 0 1 mm B Representative images of decellularized cardiac tissue versus cadaveric cardiac tissue from the Ott laboratory 13 The system itself is prone to flooding within the bioreac
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7. These scores are displayed in Table 2 Table 2 Weighted Objectives Table Objective Score Decellularizes amp Perfuses Small Organs 10 User Friendly 8 Safe 8 Versatile 6 Marketable 3 Decellularization and perfusion of small organs was established as the most vital objective as the bioreactor would ultimately have to meet this objective with full success in order for the device goals to be met completely Although we had previously established safety as our most important objective it was tied with a score of 8 10 with the user friendliness objective It was considered that a successful user friendly design would implicitly integrate safe construction and interface and that consideration of these two objectives would be done concurrently as the device went through the design and construction process Versatility was given a score of 6 the ability to adapt the decellularization device to different research objectives although not necessarily vital to the success of the device was a desirable feature worth consideration during design Marketability was ranked at 3 the device was being custom built for the Gaudette lab and while aesthetic design and adaptability to other 25 users was desirable the primary goal of the project was to produce a device specific for use in the Gaudette Lab and not a device to enter the market This 10 point scale enabled us to consider the overall importance of each object
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9. et al 2013 Effect of a Novel ORCA Bioreactor on Intact Porcine Heart Decellularization Circulation Harvard Apparatus Regenerative Technology 2013 Harvard Apparatus ORCA Cost Breakdown Annual Data Report 113 137 Retrieved from http srtr transplant hrsa gov annual reports 2012 Default aspx Badylak S F Taylor D amp Uygun K 2011 Whole Organ Tissue Engineering Decellularization and Recellularization of Three Dimensional Matrix Scaffolds Annual Review of Biomedical Engineering 13 26 Guyette J Gilpin S Charest J Tapias L Ren X amp Ott H 2014 Perfusion decellularization of whole organs Nature Protocols 9 6 17 National Heart Lung and Blood Institute 2014 March 27 How can Heart Failure be Prevented retreived from http www nhlbi nih gov health health topics topics hf prevention html 72 10 11 12 13 14 15 16 17 US Department of Health and Human Services Timeline of Historical Event Significant Milestones in Organ Donation and Transplantation retrieved from http www organdonor gov legislation timeline html Rahlf S H The Use of Skin Grafting for the Treatment of Burn Wounds in Denmark 1870 1960 37 2009 99 116 Pubmed gov Web 24 Sept 2014 US Department of Health and Human Services The Need is Real Data retrieved from http www organdonor gov about data html Abouna G M Organ Shortage Crisis Problems and Possible Solutions Transplantatio
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12. automated controls with monitoring and user friendliness The device was intended to be low cost The total cost of the whole system was 140 The system increases the time efficiency of research due to the fact that it has the ability to decellularize two hearts simultaneously in the bioreactor chamber The individual heart ports 65 give ease of use by being able to be screwed on and off with the hearts attached Also with further design this bioreactor has the potential to decellularize greater than two hearts at once The chamber is fairly simple to switch out for a larger chamber in order to house larger organs This bioreactor was able to decellularize hearts successfully shown through our histology as previously discussed The system achieved automation through our MATLAB program The MATLAB program in conjunction with the embedded light sensor monitored the decellularization process with a remote warning notification via text or e mail The MATLAB program also automatically stores the data of light translucency into a Microsoft Excel file The user interface of the MATLAB program makes it simple for the user to adapt the run time for another protocol that may differ from the Gaudette Lab protocol VII Final Design and Validation 1 Final Design Description The final design of the perfusion based cardiac decellularization system described in this report contains two major components 1 a two organ decellularization bioreactor for sm
13. cardiac tissue was obtained every 6 hours for an 80 hour period The data regarding the ambient light in the room was a negative control as the light did not change significantly over time At the conclusion of the experiments the light intensity data based on the voltage across the sensor was graphed The graphs from both trials are shown below in Figures 8 and 9 36 Light Sensor Test V1 LED Heart Ambient Light Voltage Measured 40 50 Time Hours Figure 8 Light Sensor Validation test first trial Graph shows voltage output of sensor system due to direct LED exposure exposure through decellularizing cardiac tissue and ambient light over an 80 hour period Light Sensor Test V2 LED Heart m a c 9 n L4 b v LJ m 9 Ambient 90 100 Time hours Figure 9 Light Sensor Validation test first trial Graph shows voltage output of sensor system due to direct LED exposure exposure through decellularizing cardiac tissue and ambient light over an 80 hour period As seen in Figure 8 ambient light stays relatively consistent over time as expected while the LED exposure similarly shows consistent values with the exception of a drop in 37 voltage at 10 hours which may have been due to experimental error Figure 9 shows more erratic data with both LED and ambient light sensor readings showing inconsistent values over time This may be attributable to changi
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16. suggesting that this design would best meet the needs of the overall system The customized hardware and software coupled with the low cost but effective monitoring methods showed significant progress However further consideration of the plate reader based detection system merited its re evaluation The DNA content assay offered a secondary mechanism to monitor the decellularization process in a low cost way that could be easily implemented in any biomedical laboratory Thus it was decided to incorporate this testing method into future design iterations 4 Preliminary Software Designs The establishment of a ranked conceptual design warranted the evaluation of preliminary designs incorporating these features into an integrated software system for monitoring and 32 automation In addition to system design the software that would be employed to construct the system was taken into consideration LabVIEW Based Operating System Initially LabVIEW a graphical programming software development environment by National Instruments was evaluated as a stand alone operating system to both automate the bioreactor and monitor sensor input The considerations for using this program were that it was licensed by the Worcester Polytechnic Institute campus and could be employed at no cost is integrated into the WPI curriculum and would be easy to use for any incoming volunteers to the Gaudette lab and employed a highly customizable inte
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20. AER CERTE PART ERATES 15 MEUM IM H 17 T User MOMMY sca E 18 241 FSU aciem mener eM e MEE 18 3 Decellularize and Perfuse Small Organs cccccccccccsssssessscecececessessceeeceescesseseaeseeeessesseseeaeeeeess 19 MO Jr ce 19 5 JMarketable iie un tii t e nhe titer on dd eodera EQ PR Re ANA 19 3a CONSTA EM anie aa a a aa Ea a Eiaa Ee ea Eaa TE EE O aaiae 21 As Project Approach E 22 Technical Approaching E E E ra o Dn E E exce aedes 22 2 Management Approach nite teet eri eer Her aa eva ae FERE CIR A Fan E ERR x aee Fees DER CEA ERE 24 3 Financial Approach sesaria saisan eias irain ines esed eot dpa dua a due na Danae aana ada sanaaa ieies 24 IV Alternative Designs esis nsien iee ae aaa Ee E Ee RER EEA EE EE Eaa EEES 25 I Needs Analysi8 ertt a E a E EEA EEEE EE ENESE T ATER 25 2 Monitoring and Automation Software System esseeseeseeeeeeeeeeneenee nne meer 26 Functions and Specifications Sensors amp Process Automation eee 26 2 Conceptual and Preliminary Designs of Monitoring and Automation System 28 3 Software Design Evaluation eeeeeeeeeeeeeee eene nennen enren rennen nennen 32 4 Preliminary Sottware Designs eee e eU I MERE eh ee e La ero ERE REA HERE RE PA Lead 32 5 Pre
21. KM References KM KM Appendix A LRP LRP Appendix B LRP LRP Appendix C LRP LRP Table of Contents LASHOE LADIES m viii Noc A E 0 Acknowledgements 5i rr ree EA Eie HR iat wn TRE ERXD AEG RS CEF IEEE FL XE E E 1 L JntroduCtOnics uincere t tte eR rre er rt Face ted d ETHER RE ETE MER eV EA EU xe QE dea Fe EPA tav R Ere e LEEREN ad 2 M Literat re au PD 6 le Heart Laitl re terio tern nile pae er geared ae teh EE De Yee e Ua xo cesse era de eere eu ipn 6 2 Organ Transplant Timeline ueniret dene rur ti FeH FREI RA SHREK dg le voneuaceestesslcostesiasteahdnstegeeesae 7 Se Organ Transplants ET 7 4 Organ REJOCuOn m 8 5 Regenerative and Tissue Engineering sese nennen nennen 8 6 Solution to Rejection and the Lack of Organs eeeeeeeeeeeeeeee nennen eene 9 7 Decellularization Protocol and Usage eene rennen 10 8 he Gold Standard zai niter peine ter eo e eg e aix eet va ap E an pae deed adeat ee hens 11 9 Gaudette laboratory at WEI eiie ttr eite erra ne eiii Fi Hia Ran F e RA PER SER Ra ad 12 10 Customized System for Gaudette Lab sesseeeeeeeeeeenenneeeee eee 14 III Inc zuNI DIA 15 Te Client Statement 25 oriri etr rie air E De a Leer EUR FE XRME DRE ERRARE PAET Fe LEE EY SAFER
22. Prof Dirk Albrecht include toneAC h define ANALOG IN 0 void setup Serial begin 19200 toneAC 2 10 void loop int val analogRead ANALOG IN Serial write Oxff Serial write val 8 amp Oxff Serial write val amp Oxff WPI BME2210 Arduino Oscilloscope Gives a visual rendering of analog pin 0 in realtime Allows saving of data to a text file c 2013 Dirk Albrecht dalbrecht wpi edu Modified from c 2008 Sofian Audry info sofianaudry com 0X 0X F F 0X 0X F F ox import processing serial Serial port Create object from Serial class int val Data received from the serial port int values t float zoom boolean halt String savefile int bg 0 void setup size 640 480 Open the port that the board is connected to and use the same speed 19200 bps port new Serial this dev tty usbmodem411 19200 values new int width t new int width zoom 1 0f halt false smooth int getY int val return int height val 1023 0f height 1 int getValue int value 1 while port available gt 3 if port read Oxff value port read lt lt 8 port read return value void pushValue int value for int i 0 i width 1 i values i values i 1 t i t it l values width 1 value t width 1 millis void drawLines stroke
23. all elements of the system into one integrated software environment warranted the decision to begin the prototyping process using the Arduino Uno interfaced with MATLAB Mathworks Natick MA Similarly while the Hoescht dye assay provided an accurate assessment of the progress of the decellularization process over time it did not provide real time data To effectively monitor the decellularization process real time data that a system program could respond to immediately providing the user with instantaneous process data whenever applicable was essential With this consideration in mind the decision to implement the light sensor as a process monitor was selected over the Hoescht assay Additionally because the system would be in a laboratory environment that is climate controlled a temperature sensor was eliminated as an unnecessary expense The system was comprised of an Arduino Uno microcontroller modified with an Adafruit Motor Shield V2 Adafruit Industries New York This shield connects to an 8 AA battery 12V 40 power pack and permits the control of two small peristaltic pumps Adafruit Industries New York The Arduino additionally controls the photosensors comprised of two standard CdS photoresistors built into separate voltage divider circuits with a lOkOhm resistor these detect the light from an LED positioned outside the bioreactor as the light penetrates through heart tissue As the heart decellularizes more light c
24. cardiovascular research being conducted in the Gaudette Lab The research done at WPI could potentially aid the research for decellularizing human hearts which would benefit many patients around the world who are on the waitlist for a donor ll Literature Review 1 Heart Failure Heart failure is a condition that affects almost 5 1 million people in the United States with a 50 mortality rate after four years Heart failure is not a condition that means that the heart has stopped or is about to stop working It develops over time as the heart pumping action decreases in force Heart failure can affect the heart in two ways either in the right side only or in both sides The majority of heart failure cases are affected in both sides of the heart This failure means that the heart is unable to pump the correct amount of blood to meet an individual body s needs There are two cases of heart failure The first is where the heart cannot fill with enough blood due to weakened pumping force The second case of heart failure is where the heart cannot pump blood to the extremities and other parts of the body with a strong enough force People with heart failure can have one or both of these cases There are many diseases that cause heart failure to develop The main being coronary heart disease high blood pressure and diabetes Although there is no cure for the heart failure that these diseases cause medicine regimens and lifestyle changes can i
25. easily reproduced and customizable to experimental needs The degradation rate and compatibility with the cells can be a challenge to control Natural polymers on the other hand have cell compatibility but tend to have weak mechanical properties To overcome these challenges research into decellularization a process that takes the cells from a tissue or organ and leaves behind the ECM began Decellularized organs can serve as a natural polymer template that has the innate strength as well as biocompatibility of the original It also maintains the original mechanical integrity and structure of the organ necessary to maintain normal function 6 Solution to Rejection and the Lack of Organs In 1995 Badylak was the first to decellularize a small intestine mucosa with a chemical detergent This paved the way for later on the Ott Lab at Harvard Medical School to be the first to successfully decellularize a rat heart and still retain its original functioning structure 5 Decellularized organs could become the solution to the lack of whole organs and reduce risk of donor rejections This technique would allow previously unusable organs to be potentially transplanted into patients in need When an organ is decellularized a template of ECM is left behind that has the potential to be re seeded with a patient s cells This re population could be with stem cells There are many different methods to decellularize which include chemical enzymatic physica
26. established by our client by defining the needs for the decellularization system and identifying key aspects of the design that must be implemented or met to produce a successful decellularization bioreactor Our constraints as defined by our client were e Portable e Fitted for rat hearts small mammalian hearts e Bioreactor must be large enough to manipulate organs manually e System design must allow for visual confirmation e Must be compatible with on the market fluid pumps e Must not exceed 500 The first of our constraints was for the bioreactor and system to be portable The system must be portable so that the user could have the option to take the system from a laboratory bench top to a laminar flow hood This meant that the dimensions of the bioreactor must be compatible with the maximum height that the hood hatch can be opened which is around 10 inches Our next constraint that we defined with our client was that the system must be fitted for rat hearts or similarly sized mammalian organs The main purpose of our project was to decellularize rat hearts and so the system we design must be tailored to meet the space requirements of rat hearts Additionally the bioreactor had to be large enough so that the user can adjust and manipulate the organ with their hands This meant that although the bioreactor has 21 to be compact to fit inside a laminar flow hood it must also allow for the user to easily use their hands to adjust the h
27. organs leaving the native extracellular matrix of connective tissue and proteins to create tissue and whole organ scaffolds to be reseeded with host cells Ultimately the end goal of this research is to use these decellularized organ scaffolds to generate patient specific organs or tissue including the heart for transplantation Ideally a perfusion based decellularizing system will assist in supplementing the shortage of available transplants Currently decellularization technology is beginning to be implemented in the WPI Myocardial Regeneration Laboratory led by Principal Investigator Prof Glenn Gaudette PhD An important part of the project scope is that it will focus primarily on use in early research rather than pre clinical studies The goal of this project was to create a perfusion based low cost sterilizable decellularization bioreactor that can decellularize small mammalian hearts specifically the rat for use in the Gaudette Lab Ideally this system would be user friendly and simple to assemble with an easy learning curve permitting laboratory students and volunteers to use the system quickly and efficiently The system should have the ability to monitor and acquire data on the decellularization process an advantageous feature as it presents another option to quantify the amount of decellularization instead of relying upon visual observations of the technician moderating the process Automation and monitoring would also reduce
28. output println t i t 1 1000 0f nf values i Write the coordinate to the file output flush Writes the remaining data to the file output close println Data saved to savefile The Arduino code include lt toneAC h gt define ANALOG_IN 0 void setup Serial begin 19200 toneAC 2 10 void loop int val analogRead ANALOG IN Serial write Oxff Serial write val 8 amp Oxff Serial write val amp Oxff xy 1023 0f 5 Of 1 3 78 Gaudette Lab Decellularization Senseor System For MQP Group GRG 1503 Worcester Polytechnic Institute Program edits amp modifications by Luke R Perreault Original code derived from Adafruit and Virtuabotix code This sketch was generated using open source code and code from both Adafruit and Virtuabotix sample sketches copy the sketch below and paste it into the Arduino IDE compile and run the program this sketch was created using code from both the adafruit and the virtuabotix sample sketches You can use any 4 or 5 pins define sclk 4 define mosi 5 define cs 6 define dc 7 define rst 0 you can also connect this to the Arduino reset define ANALOG IN 1 0 for cds light sensor define ANALOG IN 2 1 for cds light sensor define ANALOG IN 3 2 for resistive sensor include Adafruit GFX h Core graphics library include Adafruit ST7735 h Har
29. the decellularization protocol established by the Gaudette Lab A recommendation for the future would be to perfect the decellularization protocol so that the solutions can successfully decellularize tissue completely every time 70 The histology results also showed us that in a double decellularization experiment one heart will more likely be decellularized faster and more than the other This is due to the specific resistance in each individual heart Because of this the decellularization solutions will be more likely to flow through the heart with least resistance In order to overcome this difference in decellularization we would recommend inserting pressure catheters inside the individual hearts so that the user can know if the resistance is affecting the decellularization in each heart With the information from the catheters the user can then make adjustments as needed to ensure that both hearts will be fully decellularized by the end of the process 71 IX T References Lopez Sendo J 2011 The Heart Failure Epidemic Mediographia Vol 33 No 4 363 369 Center for Disease Control and Prevention 2013 December 3 Heart Failure Fact Sheet Retrieved from http www cdc gov dhdsp data statistics fact sheets fs heart failure htm Scientific Registry of Transplant Recipients amp Organ Procurement and Transplantation Network 2013 OPTN SRTR 2012 Harvard Apparatus Regenerative Technology n d ORCA Bioreactor Qu N
30. the hours associated with working the decellularization protocol the organ being decellularized would be able to be left unattended The technician if anything went wrong could be notified electronically by the machine or the machine could self abort the process Ultimately this project aimed to draw from and improve upon the previously developed decellularization system This system gave a basis for the initial design and from there the team made adjustments Our design is meant to be utilized in the Gaudette Laboratory to further their involvement in decellularization research The current market gold standard for decellularization technology is the ORCA Organ Regenerative Control Acquisition bioreactor and system developed and produced by Harvard Apparatus The ORCA system features a built in controller which controls all critical elements involved in 3D organ bioreactors and records and displays real time data collected from the bioreactor chamber effectively automating and monitoring many aspects of the decellularization process Preliminary studies by independent researchers have shown the ORCA system to effectively reduce the length of time needed to decellularize cardiac tissue It also monitors the pressure temperature and pH by means of pumps and sensors Despite the ORCA system s reliability and wide range of versatile customizable features including bioreactors for both small and large mammalian organs the ORCA is a co
31. the start of a general project strategy This strategy built off of the client s initial project statement and establishes and ranks objectives and constraints for the project based off of the client s requirements for the system 14 III Project Strategy Client Statement Our client the Gaudette Lab focuses primarily in research involving methods for regeneration of infarcted mammalian heart tissue and investigating new methodologies for improving cardiovascular medicine In its collaborations with other laboratories including the group led by Harald Ott MD which pioneered methodologies for decellularization as a tool to develop scaffolds for regenerative engineering of whole organs the lab has begun working extensively with decellularization technology to evaluate its efficacy for myocardial regeneration The Gaudette Lab has already conducted pilot studies into decellularization of rat hearts using a prototype system built in lab which follows previously developed perfusion based decellularization protocols by the Ott Lab and other groups Initial trials have proved promising but our client desires a customized system designed for the type of research done by the Gaudette Lab with relevant features and applications The desire for a customized system was the initial motivator for this project and our client Prof Gaudette provided us with this initial client statement Develop a closed system to decellularize mammal
32. 0 036 0 037 Week2 Avg 0 364 0 161 0 092 0 076 0 055 0 055 0 036 0 038 Figure 10 Hoescht fluorescence in decellularization fluid over 42 hours Fluorescence was stimulated at 490nm Shown are the averages for trials 1 and 2 compared against the pure SDS control As shown in Table 4 above both trials showed a decrease in Hoescht absorbancy over time and approached the absorbancy values of the control SDS samples This correlated with the visual observation of the decellularizing fluid The rapid decrease in fluorescence seen between 39 0 10 hours in both trials Figure 10 correlated with an observation that the decellularizing tissue was rapidly losing color and cellular material during the same time span This method may provide a viable counter assessment against which to validate the decellularization system effectiveness in future tests and also provide a test against which to compare the validity of decellularization monitoring methods such as the light sensor design as the design goes through further testing and iterations 6 Final Automation Monitoring System Design Selections Several decisions and developments were made to determine the final automation and monitoring system design for the decellularization bioreactor While a LabVIEW based operating system was initially envisioned for the project a desire to reduce costs and have full control over how the user would interface with the system and combine
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35. Arduino board Select Port MUST be synced Run Protocol monitoring data will save as an excel file automatically Please ensure you are in a subfolder of Projects where you want the data to be saved This program is not protected by the chkdir m file developed by John Favreau for the Gaudette Lab MATLAB data analysis environment Tech Support Sends a text message to Luke Perreault undergraduate volunteer responsible for developing this program for the Decellularization MQP o Please don t actually use this unless he s involved with the project Summon Jordan Sends a text message to Jordan Jones undergraduate volunteer currently as of Feb 2015 responsible for decellularization projects in the lab o Please don t actually use this unless he s involved with the project 100
36. Development of a Perfusion Based Decellularization System for Cardiovascular Research A Major Qualifying Project Report Submitted to the Faculty of the WORCESTER POLYTECHNIC INSTITUTE In partial fulfillment of the requirements for the Degree of Bachelor of Science by Mei Lee Amend This report represents the work of WPI Kaitlyn Marengo undergraduate students submitted to the faculty as evidence of completion of a degree requirement WPI C routinely publishes these reports on its website Luke Perreault without editorial or peer review For more information about the projects program at WPI potes please see http www wpi edu academics ugradstudies project Date 30 April 2015 learning html Approved Prof Glenn R Gaudette AUTHORSHIP Section Main Author Editing Introduction Group Group Literature Review Group MLA KM Client Statement Group MLA KM Objectives Group Group Constraints Group Group Project Approach LRP AA KM Alternative Designs Needs Analysis Group Monitoring amp Automation Software LRP AA KM Bioreactor Design System KM AA KM Final Design LRP AA KM Custom Built Semi Automated Sys for C D KM AA KM Design Verification amp Testing Photosensor based Monitoring Auto Cap LRP KM AA Histological Evidence KM KM Visual Confirmation KM KM Discussion AA MLA KM Final Design amp Validation LRP KM Conclusion amp Recommendations AA MLA KM
37. Nalgene container without the divider to DNA sample detection The final design CAD model can be seen below in Figure 18 We as a group began compiling a list of materials that we would need to build this prototype and approaches we could use to manufacture the different sized holes in the chamber We decided that we would use the drill press and milling machine to create the holes for all the components since a laser cutter is not idea for cutting polycarbonate as it produces toxic gas The Nalgene container was purchased from ThermoScientific and the conical tubes syringes and luer locks were obtained from the Gaudette laboratory A consultation with the employees of the machine shop in Higgins Laboratories on the WPI campus helped us learn that we could not precisely cut holes for the run off on the sides of the Nalgene container Because of this finding we made a modification to have the runoff samples be taken from a hole that is cut in the bottom of the chamber This hole will be sized so that a 3 mL syringe can be inserted into it Waste fluid runoff will exit the chamber through this syringes As stated before gravity will be the mechanism of waste removal The syringe will then flow the waste into a container where waste will be disposed of properly 50 4 Custom Built Semi Automated System for Cardiac Decellularization Once the design process for both the monitoring and automation portion of the project as well as the bioreacto
38. Photosensor circuit schematic e This circuit is powered by a 5V input from the Arduino Uno which also collects analog data from the sensors The sensor data and peristaltic pump control is processed by the Arduino which is programmed via MATLAB functions programmed into a MATLAB GUI Appendix B These functions are built with the Arduino Support from MATLAB add on which allows MATLAB to interface with the Arduino send it commands and receive data In this program the user can control the pumps activate the monitoring and automation system and input an email address or cellular number to receive process notifications on 4 Cardiac Decellularization Individual rat hearts were provided by the Gaudette Lab for testing of the device and cannulated for attachment into the system Rat hearts were frozen in 20 degree Celsius heparinized PBS until needed Full decellularization protocols can be monitored via the MATLAB decellularization system software per the user manual instructions detailed in Appendix C Protocols were based off a previously described protocol by Guyette et al 8 After decellularization protocols are completed histological analysis on the hearts should be performed as described previously in the Design Validation section 69 VIII Conclusions and Recommendations The group has designed and created a functional semi automated perfusion based decellularization bioreactor suitable for cardiac research This low cos
39. X color 81 Appendix B Decellularization System MATLAB Software Code 82 decell ranprogram Page 1 of 16 Contents e Decellwarization System Contro Panel Structures s Neswd Functons Program Pop Up Menu a Purp 1 Slider Values Pump 2 Slider values s Runtime Value wm RunTine Generation B Set Jserrame a Acquire user text number or email s Generate Custom V essage to User a Summon tre inimtable Jordan Jones Contact Primary System Usern Editor w PushButton Senser System Protoco Rus Activston Decellularization System Control Panel Provides graphical set up interface to run decellularization program All functionality also capable in MATLAB command window with pump testum program amp Hr sz SC WIS HEVI MATLAR SOFRO FOR ARIT NO TRS ALLE ORUM POOGRXN Develicvecizalsva MgP J014 Wer3lcr L I i dereesior B n gybeckbadbe rshitube radek ak aln Galcwny Pur t Draated sy Lure 1 Percvaarit 8 Feorrary 2675 W Seviececs rf aar Gavdazte Zauctioa cecell rarpr3ccaxi 2 2 figure vV 2zIble on Po3iticr 350 31U0J tU 2 zt0 xelis f nzuge sSecellalarzzalzcu Sy L u Conlecl sarel zu xbeclille off Y E ech tn of ote t E 80194 X hreoloxi aicvto14 8 ule hast RurEPg Piplila Lah Feccl n rb zz2508 Ryslem Pool ior 12 253 230 22U FortSzre 23 Ferntteigh bela 3t Ligh Geust 1 RvalTins Grap Go sect gl axes Units
40. Yor nor F 12 10 PM 1X5 gaudettedecell2015 qg Gaudette Lab Decell System Problem Detectec on Heart 1 Decell Aborted Check System Details gt as Fyw 2 e o Hie tat Vew imet ico Uescop Window Help OdmMaslk 09834 2080uu Heart 1 5 45 rj 535 3 5 25 PB oer ooooototooteto eto 4 gt m 5 e lt ns 0 0 5 1 1 2 2 5 3 35 4 6 Time seconds Figure 20 Problem Detected notification and accompanying MATLAB graph of light sensor readings that initiated the system response Decellularization Monitoring Validation After validating that the system automation could work as specified it was necessary to assess whether the photosensor monitoring method could effectively track decellularization and give a reasonable indication of process status To test this the monitoring system was run while decellularization protocols 3 total two with one heart perfused and 1 with two hearts perfused used to test the full decellularization bioreactor were being performed Data from the first six hours of SDS perfusion and a recording of monitoring data from 20 hours of SDS perfusion are presented below in Figures 21 and 22 As seen in the initial 6 hour trial light intensity increases gradually over time as a heart decellularizes as expected However the rate of light intensity increase appears to gradually decrease over time This is more indicative in the 20 hour monitoring test per
41. all mammalian hearts build from a modified Nalgene container and two 50mL conical tubes connected via conventional serializable tubing to a constant volume solution reservoir fed by a larger reservoir This constant volume control permits constant pressure for perfusion of the hearts being decellularized which can be modified by raising or lowering the reservoir 2 A decellularization system automation and monitoring program that controls two peristaltic pumps for circulation of solution within the system and two photosensors that monitor the light from separate LEDs that penetrates through decellularizing tissue thereby monitoring the decellularization process 66 This system is built off an Arduino Uno microcontroller and is programmed and controlled in MATLAB Using this automation and monitoring system in tandem with the bioreactor decellularization device cuts down on time required to monitor decellularization protocols and provides status updates via text or email to the specified user 2 Custom Decellularization Bioreactor The bioreactor for the perfusion system is comprised of a 1000 mL Nalgene straight sided container from Thermoscientific modified with two 50mL conical tubes cut horizontally to create separate holders for the decellularizing hearts The caps each have a hole cut in the center in which a Luer tubing connector is glued in place to allow cannulated hearts to be locked into place and facilitate easy removal o
42. amples with Masson s Trichrome Blue so that we could tell the difference between muscle fibers cytoplasm and collagen This stain marks cell nuclei with a black color cytoplasm with pink muscle fiber with red and collagen blue Column B in the figure shows the representative images of the control and experimental heart samples stained with Trichrome The control heart for this stain is represented by the second image in row 1 This image shows that the muscle tissue has been clearly stained red ad cell nuclei were black There is also indication of collagen that has been stained blue This is what is expected of native tissues We see collagen in the native tissue as it is one of the main proteins in the ECM Once tissue has been decellularized the native ECM proteins are left behind including collagen In the first experimental heart the tissue has been stained completely blue showing that only collagen remains This indicated that the genetic material and cellular fibers have been washed away during the protocol The second experimental heart was clearly stained blue indicating that lots of collagen remains however there was genetic debris that had been stained black For the third experiment the heart that was on the right side of the bioreactor shows clear indications of collagen as well as muscle fiber and genetic debris The blue stained tissue indicated that the ECM protein collagen has been partially cleared of genetic and cellular debr
43. an penetrate through the gradually more translucent tissue This automation and monitoring hardware interfaces with MATLAB Mathworks Natick MA via the MATLAB add on Arduino Support from MATLAB which offers a suite of functions to generate programs that both relay commands to the Adafruit Motor Shield and peristaltic pumps and can collect and analyze data collected from the Arduino based photosensors In order to operate the entire system as a functional whole a program was developed in MATLAB to control the pump system and photosensor monitoring apparatus This system is programmed to accept specific inputs from the user to specify how the decellularization will function and for how long Specifically the user can input the amount of time the pumps and monitors will run the speed and direction of flow for both pumps which can be activated or deactivated via this system as well specify the USB port the Arduino is connected to begin communication with the system and specify a phone number or email address for the system to send system notifications to These notifications represent a major component of the monitoring system The program is designed such that if the photosensors detect a light level above a specified threshold denoted by a value of 4 5V on the photoresistor circuit the pumps and monitoring system deactivate automatically and a message is sent to the specified user The program sends messages by remotely interfacin
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45. at is full of dark purple nuclei and pink ECM fibers and cytoplasm This image is a good representation of what is expected in native heart tissue The images that follow after the control heart in column A are of the different experimental hearts The first experimental heart shows a good representation of tissue that has been decellularized The image show no formed nuclei or genetic debris that was left behind in the tissue following the decellularization protocol The image of the second experimental heart does not show indications of any nuclei but there is a slight tinge of purple indicating that not all the genetic debris was washed away The third experiment that was conducted was a double decellularized heart experiment where two hears were decellularized at the same time The heart that was on the right side of the bioreactor appears to have no complete nuclei left in the tissue but there is an indication of genetic debris The heart that was on the left side of the bioreactor shows no formed nuclei or genetic debris and is thus a good representation of tissue that has been decellularized From this stain we could see that genetic debris was washed away in the hearts 58 that had undergone the decellularization protocol Unfortunately this stain did not show us the specific proteins and fibers that were left after decellularization as it stained ECM proteins and other fibers the same color Following the H amp E stain we stained tissue s
46. ated 27 Published with MATH ADT R29 Mer file research wpi edu 2audettelab Projects GCE builds 2013 03 0001 shtmidecell mnp 426 2015 98 Appendix C Decellularization System User Manual Decellularization MQP Gaudette Lab 2015 Protocol for Operation of Decellularization System Linked Google Account for system notifications Username WPIMQP GaudetteLab Email gaudettedecell2015 gmail com Password case sensitive Gaudette 2015 Required Software e MATLAB 2014a or later Arduino Uno with Adafruit Motor Shield v2 attachment e MATLAB Support for Arduino add on o Initialize supportPackagelnstaller in the MATLAB command prompt and follow directions as process initializes o Select Arduino from menu and install necessary add on e Requires MQP MATLAB software folder to be active in the MATLAB command space Procedure ny rS EE tie Cot View met Teele Denbttnp nde Ilep Daaidajs 5 9v 4 O DE aD GaudetteLab 2 Decellularization k 1 System v 1 0 o 07 Duriszed Set E Hun lime lasht rond Set Ure sete Pat Run Protocol Fig 1 Decell System Graphical User Interface GUI Windows 7 99 9o To initialize system activate Gaudette Data Analysis system input command startup into command prompt while in the research drive Projects folder Call the m file for the GUI decell runprogram Plug Arduino Uno into a USB port on the computer On
47. ce system is initialized select the port the Arduino is connected to Select Port in Figure 1 a Once selected MATLAB will connect to the Arduino and call the board and motor shield Input your email or phone number to Set Text Email to enable remote notifications d This is not required to initialize the system b Phone numbers should have email address format check online with your provider example 7741231234 Q vtext com for Verizon wireless users Specify the run time that the system will run independently When RunTime terminates peristaltic pumps will shut off automatically and you will receive a notification to check your experiment P1 automates Pump 1 P2 automates Pump 2 Speed ranges from 1 specifying full reverse to 1 full forward 1 100mL min a Press the P1 P2 buttons to start stop the pumps b PUMPS MUST BE RUNNING WHEN RUN PROTOCOL BUTTON IS ACTIVATED TO KEEP PUMPS RUNNING DURING PROTOCOL c Speed cannot be controlled while Run Protocol is active Once all preferences are set connect the Arduino to the sensor system on the bioreactor Activate the Run Protocol button a Two additional Figures tracking light sensor data over time along with a progress bar will be generated in MATLAB Monitoring is now activated and you will receive a notification upon completion or process error Additional Notes Pumps can be run independently of one another or the automation Run Protocol system but an
48. cells which will die without a substrate to proliferate and differentiate onto In the future this research has the potential to allow researchers to better understand the architecture of heart and for the future be able to completely create a synthetic heart made of natural materials At the moment decellularization hopes to utilize previously wasted organs to be implanted into and this project hopes to contribute to this endeavor 7 Decellularization Protocol and Usage The process of decellularization is to disrupt the homeostasis of cells by means of lysing After all the cell material has been removed from the tissue the end product of decellularization is the extracellular matrix also called the ECM There is research being conducted in many laboratories which experiment with the ECM and using it as a scaffold to seeding stem cells onto in the hopes of regenerating tissue This can be used clinically for patients who are in need of an organ instead of using donor organs in junction with the patient taking immunosuppressant drugs the patient can use their own cells to seed decellularized organs from allogeneic or xenogeneic sources and create a genetically identical organ There are many ways to decellularize tissue which are organized into two categories physical agents and chemical agents Physical agents include temperature freeze force and pressure and non thermal irreversible electroporation For chemical agents scientists p
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51. del 47 Figure 16 Four Heart Bioreactor Model Advantages to this design is that it allows for a larger space for decellularization This allows for the system to not become flooded with fluid as well as allows the user to reach into the chamber and fix problems if they arise The container also has the ability to be easily removed from the system and transported into the hood if needed A disadvantage to this system is that it requires drilling of many different hole sizes as well as additional components that need to be attached and sealed in order to maintain internal sterility of the bioreactor T 75 Flask Based Bioreactor The second main preliminary design we produced was based upon using T 75 flasks that are stacked upon each other The flask idea integrates multi heart decellularization but in separate compartments The design is flexible based on the user s needs These flasks are intended for cell culture work and are used in most laboratories around the world this system could be constructed in any lab as long as they have access to a drill press They have an air vent incorporated into the cap to keep the inside and outside pressure equal The flask design can be seen in Figure 17 below 48 Figure 17 Tissue Culture Flask Design Model Taking these flasks and standing them upright they can become a bioreactor for rat hearts Drilling a hole at the top and the bottom it will allow the solutions to flow into the cannu
52. dette Lab meeting minutes 13 August 2014 Contributed by Jordan Jones Kim YJ et al 1988 Fluorometric assay of DNA in cartilage explants using hoescht 33285 Analytical Biochemistry 174 168 176 National Instruments 2014 NI LabVIEW Retrieved on 18 December 2014 from http www ni com labview Arduino 2014 Arduino Uno Retrieved on 18 December 2014 from http arduino cc en Main arduinoBoardUno Adafruit 2014 Adafruit Motor Stepper Servo Shield for Arduino v2 Kit Retrieved 18 December 2014 from http www adafruit com products 1438 ThermoScientific 2014 Instructions Hoescht 33342 Retrieved on 18 December 2014 from https www piercenet com instructions 2 162245 pdf Nalgene 2116 1000 Straight Sided Wide Mouth Sample Jar Clear Polycarbonate with White PP Closure 1000mL Thermo Scientific http www capitolscientific com Nalgene 2116 1000 Straight Sided Wide Mouth Sample Jar Clear Polycarbonate with White PP Closure Corning 75cm Rectangular Canted Neck Cell Culture Flask with Vent Cap Product 430641 http catalog2 corning com LifeSciences en 74 US Shopping ProductDetails aspx productid 430641 Lifesciences amp categoryname Flas ks 2C Culture 2C Plastic Lifesciences 7C75cm C2 B2 Flasks Lifesciences 75 X Appendix Appendix A Preliminary Prototyping Arduino Uno Code Photosensor Testing Code MQP Group GRG 1503 Derived from Oscope Testing Code BME 2210
53. dware specific library include lt SPI h gt include dhtll h dht temp humidity sensor library dht11 DHT11 Adafruit ST7735 tft Adafruit ST7735 cs dc mosi sclk rst void setup void DHT1l attach 2 set digital port 2 to sense dht input Serial begin 9600 Serial print hello tft initR INITR BLACKTAB initialize a ST7735S chip black tab Serial println init tft setRotation tft getRotation 1 uncomment to rotate display get time to display sensor up time uintl16 t time millis tft fillScreen ST7735 BLACK time millis time Serial println time DEC delay 500 Serial println done delay 1000 tftPrintTest 79 delay 500 tft fillScreen ST7735 Bl Splash screen optimized lines testfastlines S delay 500 delay 500 tft fillScreen ST7735 Bl LACK for esthetic purposes only I 7735 RED testdrawrects ST7735 GRE void loop tft invertDisplay true delay 500 Cf tf in tf setCursor 0 0 Cursor 50 20 TextSize 2 se se se pr se se se pr se dr nt millis TextSize 1 Cursor 40 40 ntln minutes Cursor 0 60 wLine 0 50 ct Be cb OE TE bec TE oct w tft width ST7735 BLUE tft invertDisplay false SetTextColor ST7735 WHITE println De Cell has been println running for
54. each bioreactor design and decellularization protocol Some features our bioreactor and system have are a sensor to determine when the heart is decellularized and a monitoring program that analyzes the raw data from the sensor into a real time correlation graph The system is relatively easy to use as well as easy to attach the heart to the designated ports for decellularization fluid to perfuse through them The features and functionality were based off of the golden standard as well as the needs of the client By studying and drawing inspiration from relevant design aspects of these protocols and apparatus this project produced a streamlined easy to use and easy to train system that incorporates features relevant to cardiac decellularization and study while still having the versatility for use in the decellularization of similarly sized mammalian organs Our perfusion based decellularizing system design was easily useable and replicable enabling the Gaudette Lab and its collaborators to use the system design and conduct their own research to further the development of cardiac decellularization and myocardial regeneration having consistency in the results The hope was that advancements in perfusion based decellularization will eventually lead to the process being conducted on human hearts for future implantation What this project ultimately sought to achieve was to create a low cost and user friendly bioreactor specifically designed towards the
55. eart and any components in the system In addition to complex sensors that we would incorporate into our design to make it unique and specific to the needs of the Gaudette Lab the bioreactor must have components that allow for visual confirmation of the decellularization process Having a transparent material for the bioreactor would allow any user or spectator to visually monitor the heart during the decellularization process and also see if complications occurred in the bioreactor The next constraint that the bioreactor and system needed to follow was that it must be compatible with any fluid pumps that are on today s market This was so that the Gaudette Lab and any other lab looking to clone the system can use a pump that they already have on hand The final constraint of our project was that the cost of the system and the components designed must not exceed the set budget for our team Our client wanted to see the expenses of designing our prototype to not exceed the 500 that had been established If in the future our client decided to add components to the prototype the expenses are not required to remain under the 500 budget 4 Project Approach 1 Technical Approach Having established a revised client statement and analyzed and ranked specific objectives and constraints for the project a technical approach to design and construction was implemented The team spent time in the Gaudette Laboratory learning techniques used to carry out g
56. elopment of alternative designs commenced Software to run sensor systems and a pumping apparatus was researched and designed separately of the decellularization bioreactor itself to permit a more open design space and assist in the generation of a wide variety of ideas and solutions After group deliberation and a presentation of design specifications to our client we selected a preliminary design jointly with the client and merged software selections with the hardware and bioreactor to generate a preliminary design This was consequently drafted in SolidWorks while a working software prototype was developed separately for later integration into a full prototype used for testing and validation 23 2 Management Approach Management was broken down into a hierarchy with our advisor and sponsor Prof Glenn Gaudette making final decisions on purchases and project direction and our graduate advisor Joshua Gershlak guiding our work To maintain dialogue between the project team and the client weekly meetings were established to update Prof Gaudette on progress and review work from the previous week The group established eight different milestones for project management including understanding of client requirements analysis of functional requirements generation of alterative designs design evaluation selection of best alternative prototype development documentation and testing and validation To work more efficiently as a team the
57. eneral cardiac decellularization research to develop a better understanding of both the challenges associated with the current approach used in the laboratory and the skills necessary to carry out design development and testing of the project device 22 Initially we observed the preparation of a rat heart specimen for decellularization by the Gaudette Lab a process that involves the removal of a rat heart post mortem tying off of major veins and arteries except the aorta to occlude fluid flow through the heart and the cannulation of the aorta to permit retrograde perfusion of decellularization fluids through the heart Additional observation and independent research into appropriate protocols provided us with the necessary training to carry out this preparation independently Additional training involved the observation of the decellularization cycle itself including the perfusion of the heart with SDS and other detergent based solutions to purge the tissue of cellular material and the general set up this included proper tubing set up understanding of the path of fluid flow through the device and heart pump control and failure conditions that could arise such as the detachment of the heart from the cannula or inhibition of fluid flow due to clotting in the vessels of the heart With a sufficient understanding of the decellularization process and the advantages and disadvantages of the system currently used in the Gaudette Lab the dev
58. enoting a value between 1 100 reverse and 1 100 forward with 100 speed being approximately 100 mL min The Set Text Email allows input of a user s contact information to receive notifications Select Port allows the user to specify the port the Arduino is connected to and Run Protocol activates the system monitoring and pump automation for the specified runtime input in the Protocol Run Time input above the port selection dropdown box The two yellow buttons in the center left of the interface send text message notifications to laboratory volunteers primarily responsible for running the system if immediate help is needed The full MATLAB code for this decellularization system and GUI can be found in the Appendix 3 Bioreactor Design System 1 Conceptual Design Ideas When we began our bioreactor design process we referenced our original objectives in order to draft conceptual ideas Our design as stated previously needed to remain low cost and SO as a team we chose to create a bioreactor utilizing common laboratory materials These materials also needed to be sterilizable whether it be by means of an autoclave or EtOH process Because of this common plastics and materials such as acrylic would not be able to be used as the materials deform and degrade over time from such processes An important characteristic that we wanted our bioreactor to have was to be able to decellularize more than one heart at a time This aspect would allow us t
59. equiring containment within a laboratory setting at all times the environmental impact presented by the design is minimal The fact that the pumps on the device require conventional batteries may be the most significant environmental consideration future design iterations may take this fact into account and opt to use a more sustainable power source 62 3 Societal Influence The decellularization system presented in this report presents unique implications for society at large but specifically for education and research within the scientific community An affordable design for a decellularization system offers opportunities for low budget projects involving decellularization and tissue engineering By drastically reducing the cost to do this type of research it is feasible that researchers interested in applying decellularization technology on a trial basis to start new projects could do so expanding the field significantly Additionally a low cost system could be utilized in teaching laboratories enabling students to engage in projects exploring decellularization research and the properties of natural ECM scaffolds 4 Political Ramifications The political and legal implications of this device largely focus on the impact a device such as this may have on healthcare in society at large and the global medical and research device market A low cost regenerative engineering research device and indeed any medical device that plays a r
60. erent organs Automatically switches fluids Stable Minimizes Sharp Edges Settings for different organs Easy to connect organs to system Ability to be scales for different sizes Decellularizes more than 1 organ Replicable Inexpensive Aesthetically Pleasing Figure 2 Primary secondary and tertiary objectives tree 17 The primary objectives for the system were specifically for it to be user friendly be safe decellularize and perfuse small organs be versatile and be marketable 1 User Friendly The first objective user friendliness breaks down into two secondary objectives and further some tertiary objectives Firstly the system components should have the ability to monitor the process of decellularization This objective includes being able to have a remote warning system in the event of failure remote real time monitoring of the process to allow researchers to view progress when outside of lab and a visual monitoring system to track the color change associated with the decellularization These will help the system be more user friendly by reducing the time and effort associated with running a decellularization protocol and showing how far along in the decellularization process that the heart has gone Secondly the system and the monitoring components should be automated to permit an easy learning curve so users will be able to assimilate into the use of the system quickly Specifically the system could accept
61. et dat e ete e ede coutnder siete dere oe teen da 64 Te I ECeniclolim T m 64 8 jSustamability ie Rte dite RE e ud se tee osea eti rae Da cepa ea Eee va ak Te PE EE OF rtu cette 64 9 CONCIUSION tee ie etie td oe t de deside ae ere te o Paene oaa dra ceo REA EHI 65 VII Final Design and Validation eseeeeeeeeeeeeeenen aaau en rennen 66 Le inabDesien Description nh e ee the eeu er er Pene ad IRE oae Rea eek ea po nexa buco 66 2 Custom Decellularization Bioreactor eeeeeseeeeeeneeneeneeee een een emen rennen 67 3 Automation amp Monitoring System Components essere ene nren nne 68 4 Cardiac Decellularization eH RAS Uer eU a ER Tet aei eer SERE IUe ER RET Pa ea aho 69 VII Conclusions and Recommendations eese nennen een rennen rennen 70 IX References sicot ftr todetaten don tetto itio tost Pls dod tater etre a desi eod 72 Xs Appendix oec eter metre ee teet eet bt rete tere ee ate 76 Appendix A Preliminary Prototyping Arduino Uno Code esee 76 Appendix B Decellularization System MATLAB Software Code eee Appendix C Decellularization System User Manual vi List of Figures Figure 1 A Hoescht stain of decellularized rat cardiac tissue processed with the current Gaudette lab system and protocol Scale bar 0 1 mm B Representative images of decellularized cardiac tissue vers
62. f hearts A hole is bored in the center of the Nalgene container into which a 1 4 in OD T Valve from Home Depot is affixed to provide housing for the two Radioshack photoresistors that comprise the light sensors Wiring for the light sensors extends out through the top and the T valve is glued in place and sealed with Plasti Dip spray a rubber coating spring that seals the container to maintain sterility The bottom of the Nalgene container is modified with a ImL syringe that acts as a drainage port for the system This can be modified with tubing and directly connected to a sealed container to maintain sterility in the device All machining for this device was completed in the WPI Higgins Laboratories Machine Shop Our final prototype can be seen in Figure 25 below 67 Figure 25 Final Decellularization System Prototype 3 Automation amp Monitoring System Components The automation and monitoring system is built entirely around an Arduino Uno microcontroller and an Adafruit Motor Shield V2 powered by 8 AA batteries in a 12V power pack This motor shield controls two Adafruit peristaltic pumps that run off the battery power and can circulate fluid at speeds up to 100mL min in forward and reverse directions The monitoring hardware is built off of two Radioshack brand photoresistors built into separate 2 2kOhm resistor voltage divider circuits as shown in the sketch in Figure 26 below 68 L O 7 TT u Figure 26
63. formed on the same heart after the initial 6 hour test The 6 hour test was 54 performed at night and in a dark room and is free of sources of ambient light interference But the 20 hour test was done overnight As seen in the graph the time when the sun sets and the lights turn off in the laboratory about 3 hours into the test and the sun rises and lights turn on 16 hours in approximately interferes with the sensors and obscures readings Trial 1 Sensor on 6 hrs 2 6 4 2 4 v 1 8 1 6 Heart1 Sensor Voltage 1 4 4 4 6 8 Time hrs cd N Figure 21 Photosensor Test 6 hours no ambient light interference Trial 1 Sensor on 20 Hrs 3 5 2 5 1 5 Heart1 Sensor Votage V 0 5 eo Ui 10 15 20 25 Time Hrs Figure 22 Photosensor Test 20 hours ambient light interference due to day night cycle and laboratory automatic lights 55 2 Histological Evidence of Cardiac Decellularization Histological staining is a technique that can be used to visualize different biological materials structures and fibers present in a sample of tissue To ensure that our system was successfully decellularizing the hearts taken from rats we used specific stains to visualize muscle fibers cytoplasm nuclei and collagen To accomplish this we used three different histological staining protocols Figure 23 shows microscopic images of all the stained samples taken using a Leica Upright Fl
64. g with a Google Gmail account and sending a preprogrammed message via 41 the account In this way early completion of a decellularization process or process errors i e a heart falling off a cannula and no longer blocking the light source detected by the photosensor are detected in real time and an immediate notification is sent to the user The data from the light sensor is plotted by MATLAB in real time and saved in an excel file for later analysis if required This system operates off of a MATLAB graphical user interface GUI via which the user can readily input desired parameters with ease and specify the runtime and conditions the decellularization system will run in for a particular cycle The GUI itself is shown below in Figure 11 4 Figure1 e Je JL gs File Edit View Insert Tools Desktop Window Help x nge hh AS0Swudssao0gB mm Gaudette Lab m Decellularization E Rs System v 1 0 E 0 wa men sa i l Protocol mm Set Run Time S jJ Text Email 0 0 0 Set User Seconds Minutes Hours Select Port Run Protocol com1 a Figure 11 MATLAB GUI for Decellularization System Operation The GUI as shown above presents a streamlined interface to allow input of process parameters and system settings Clockwise from the top of the figure the two P1 and P2 buttons act as on off switches for the two peristaltic pumps Pump speed and direction is 42 controlled by d
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66. grated interface This software design would center around a single user interface that would allow control of pump flow time rate and allow easy visualization of sensor data as it was generated The drawback to this system was the necessity of purchasing sensors and pumps and data acquisition devices to permit communication of the software with sensor and pump hardware While a versatile and reliable system the functionality of it would consequently be dependent on several different components yielding a complex system that would require extensive training to employ and use Arduino Microcontroller Based Decellularization System The desire to reduce the overall cost of the design and enable more customization options motivated the development of a stand alone system that would operate independently of external software and both automate the bioreactor pumps while processing and outputting sensor data to enable real time monitoring of the decellularization process Research into pre existing methodologies to do so led to the Arduino Uno Figure 6 a microcontroller built by the Arduino company based on the ATmega328 The Arduino has 14 digital input output pins 6 analog inputs a USB connection for direct computer interface a power jack for operation while not 33 connected to a computer and is licensed under the Creative Commons and can be customized and used freely The Arduino is programmed in an easy to use software environmen
67. he bioreactor to be versatile To achieve this objective it was broken down into secondary objectives The first was the ability to decellularize more than one heart The current system on the market has only one chamber that can be used to decellularize the heart Creating a modular design where multiple decellularization chambers could be added as needed would set the team s bioreactor apart from current market bioreactors Secondly the team hoped to design the bioreactor so it could be scaled to different sized organs This design characteristic would be helpful in the future if the system proved to work well This would allow for the system to be adapted for other larger sized organs 5 Marketable To be marketable the bioreactor should be replicable inexpensive and aesthetically pleasing Systems that already exist in laboratories around the world have complicated and expensive bioreactors and components The goal was to make our bioreactor inexpensive so that it is more appealing to research laboratories Simple construction of the bioreactor was more desirable instead of having a complicated system Including aesthetically pleasing design components into the design helped motivate researchers in the Gaudette Lab to use our bioreactor system These design features could also encourage outside researchers to consider 19 our system for use and study These components could have included transparent surfaces to watch the process and us
68. ht intensity over time permitting real time monitoring of the decellularization process This sensor would be controlled with custom built hardware and software that would simultaneously operate pressure and temperature sensors built into the bioreactor and control automate the pumping system The use of custom built hardware and software to operate the system s automation and monitoring components allows for more freedom of design and would allow all components of the project to be run off of a single interface for a lower cost than the employment of external hardware would offer Additionally the use of a light sensor to track the decellularization process offers a low cost but effective way to monitor the decellularization status in real time and output data on its progress 31 3 Software Design Evaluation After evaluating conceptual designs for the software and hardware devices the group and client evaluated each software design by employing a design evaluation matrix that assessed the relevant omitting decellularizes and perfuses small organs objectives each design met on a 1 10 scale This table is shown in Table 4 Table 4 Numerical Design Evaluation Matrix Objective Camera Monitoring Plate Reader Photosensor User Friendly 7 4 8 Safe 8 7 8 Versatile 3 3 7 Marketable 4 4 6 Total 22 18 29 As determined in the design evaluation matrix above the photosensor design ranked highest
69. ian hearts which will monitor the decellularization process maintain sterility of the heart and incorporate user friendly construction and interface Additional communication with Prof Gaudette and research yielded more information about what features the lab desired in a customized decellularization system e Minimization of tubes and clutter to open up bench space and increase ease of use 15 The option to perfuse multiple rat hearts or other small organs simultaneously One or more sensors that monitor the decellularization process and collect data the system and organ undergoing perfusion These sensors could detect failure situations within the system and issue a remote warning to notify the user of a problem A program developed in conjunction with the device that would take video digital footage of the decellularizing organ in real time to track the color change as intracellular materials are purged from the extracellular matrix of the organ and potentially permitting remote monitoring of the decellularization process Automation of the decellularization process to facilitate easy training and use potentially using a customizable application like LabView to generate the operating program The bioreactor should facilitate easy viewing of the perfusing organ to permit visual confirmation of the decellularization process The system must be easily sterilizable with equipment available in the Gaudette laboratory and
70. image shows consistent results with the samples that were stained with H amp E and Trichrome All three stains indicate that the tissue of the first experimental heart was fully decellularized The second experimental heart shows visible nuclei and genetic debris This finding is consistent with the previous stained samples of this heart that indicate that this heart was not fully decellularized The right side heart from the third experiment shows that complete nuclei are not present but there is visible genetic debris that has been stained Along with the findings from the samples stained with H amp E and Trichrome this image indicated that this heart was not fully decellularized The image of the left side heart from this experiment shows no indications of any nucleic or genetic debris in the tissue This finding is consistent with 60 the previous statements that the left sided heart in the double decellularization experiment was fully decellularized 3 Visual Confirmation of Decellularization The final way we ensured that our system was successfully decellularizing rat hearts was by observing the change in visual properties of the organs as they were undergoing the decellularization process Figure 24 below shows two images that were taken during the double heart decellularization experiment The images are taken approximately 48 hours apart This figure allows us to confirm that the decellularization process was successfully taking place
71. iner Seen in the schematic as well is the incorporation of the photosensor in the cap of the bioreactor The photosensor holding device is situated between the two places for heart decellularization Each heart is provided with a designated photosensor Each photosensor if connected to the Arduino where data is then transferred to the MATLAB program for analysis V Design Verification amp Testing Our team conducted a series of tests to determine whether or not the decellularization system functioned as specified and could be used reliably and efficiently Tests yielded information about effective elements of our design and led to suggestions about improvements that could be made in future iterations 1 Photosensor based Monitoring amp Automation Capability Determining the functionality of the monitoring and automation system required two separate tests testing the system software and automation capability and testing of the monitoring system during a decellularization cycle 52 System Automation Validation System automation capability was evaluated by activating the peristaltic pumps and monitoring system and determining if the system would deactivate and relay a notification to the specified user if a system error was detected specifically an increase in light intensity over the specified threshold This functionality was necessary to respond to process errors properly but also to reduce the time necessary for users to ac
72. ing sterile colors and materials such as white or stainless steel After evaluating objectives for the project we presented a pairwise comparison chart PCC to our client and discussed the importance of our selected secondary and tertiary objectives The ranked primary secondary and tertiary objectives from our client s PCC are listed in Table 1 Table 1 Ranked primary secondary and tertiary objectives from client s PCC and discussion Safe Stable Minimize Sharp Edges 1 2 3 Decellularize amp Perfuses Small Organs Easy to connect organ to system Settings for different organs r Friendly Monitors Process Track color change Remote warning system Remote monitoring of process Automates Process 1 Programmable protocols for different organs 2 Automatically Switches Fluid satile Can decellularize more than one organ at a time Able to be scaled for different sized organs Marketable Inexpensive Replicable Aesthetically Pleasing Going forward this ranking was used to assist in the development of conceptual designs and evaluation of a working prototype Assuring that the decellularization system met these 20 objectives was a significant goal of the project the level to which these objectives were met was a key metric of the success of the system 3 Constraints The constraints of our project were
73. ir individual containers to the system and through the heart Once the solutions have passed through the heart they will drip down the chamber and exit through two holes cut in the side of the chamber The waste holes 46 will then lead into polyethylene tubing that flows the waste into a closed container This will allow for flow out of the chamber to avoid flooding while maintaining sterility inside the system The mechanism for waste removal will be dependent on gravity The photosensor can be incorporated into this design by creating a small hole in the cap of the container The sensor can be housed in a closed holding device that can be put into the hole created The holding device can be sealed so that the inside of the container remains sterile This design not only can be used as is in the picture but also has the ability to be changed to accommodate different criteria For example the main Nalgene chamber can be modified to have a separation between the two heart samples when running This is to accommodate run off sample collection in case the user chooses to detect for DNA to correlate how far along in the decellularization process the heart has gone Another modification that can be made is adding two more ports for heart decelllarization so that up to four hearts can be decellularized at the same time In Figures 15 and 16 the design modifications can be seen e l i k CHEN ni Figure 15 Divided Section Bioreactor Mo
74. is Red muscle fibers are apparent in the sample indicating that the decellularization process did not completely clean the ECM of these fibers Also there is a shade of gray that covers most of the sample indicating that cell 59 nuclei were broken down but not all of the genetic material was removed from the tissue The heart from this experiment that was on the left side of the bioreactor shows a sample of tissue that is comprised of collagen This image shows no nuclei or other cellular debris This image shows that this heart was fully washed of cellular structures and debris during the decellularization process The final stain that was used for histological evaluation was the Hoescht fluorescent stain We used the Hoescht 33342 dye and its accompanying protocol Hoescht is able to stain only cell nuclei and the fluorescence can be excited at a wavelength of 350 nm With a fluorescent lens we were able to visualize the nuclei in the tissue samples With Hoescht nuclei appear to be a bright blue dot on a tissue sample In Figure 23 column C shows the representative images of the control and experimental tissue samples stained with Hoescht The control heart sample can be seen in Figure 23 in the first row and third column This image shows an abundance of cell nuclei that have not been disrupted by any sort of the decellularization process The image of the first experimental heart sample shows no visible nuclei or genetic debris This
75. ity of life of patients who receive a heart transplant because of the adverse effects when a person is taking immunosuppresant drugs When a patient is on these drugs they are susceptible to infection due to the fact that their immune system cannot fight the infection An organ that has been engineered to be genetically identical to the patient will not create an immune response 7 Manufacturability Manufacturing of the bioreactor took place at Higgins Laboratory at Worcester Polytechnic Institute Two of the team members received training on the machines and also received help and supervision when using the machines As mentioned before we opted to design the device with common laboratory materials and used electronic components such as an Arduino Uno that are commonly used in educational and research settings This assists in ease of manufacturability and ease of use in a laboratory setting The streamlined design also allows easy scalability for use with larger organs if the need arise or for production of similar devices in the future 8 Sustainability Our device was designed with sustainability in significant consideration The Nalgene container bioreactor and additional components can all be cleaned sterilized and reused 64 repeatedly Tubing can be reused several times but may begin to wear out after repeated cycles of cyclic stress due to the peristaltic pumps and may require replacement 9 Conclusion Harvard Ap
76. ive to generate conceptual designs that adequately reflected the needs of the client and users of the bioreactor device 2 Monitoring and Automation Software System 1 Functions and Specifications Sensors amp Process Automation Through discussions with the client and via group discussion of project constraints and objectives we identified the following design functions and specifications for the automation and monitoring system that would be connected to the decellularization bioreactor corresponding to the project functions shown in Table 3 Table 3 Monitoring and Automation System Functions and Specifications Functions Specifications Monitor the decellularization process including providing remote warnings of failure conditions Software must transmit warnings via a readily available communication mode internet or email Track and record decellularization status and data Record at minimum every 10 minutes Monitor process parameters including bioreactor temperature flow pressure flow rate Enable remote viewing of data outside of the lab Software must output data to data format readily usable for analysis in the lab ie csv xls Sensors must be sterile and fit into system Ideally fit into a 3 16 outer diameter tubing Permit user programming input and automation of pump function on bioreactor Pumps must be independently controlled accept manual fl
77. l and a combination of the three that are used The main idea behind all three methods is to create a disruption in the cell membrane so the cellular components will become detached and rinsed away Through decellularizing all cellular components and antigens are removed which essentially wipes away the donor s DNA This reduces the chances of donor rejection by reducing foreign body reaction inflammation and immune rejection In 2008 the world s first tissue engineered whole organ transplant occurred in Spain The woman needed a left bronchus after being damaged by tuberculosis The research team of Paolo Macchiarini received a donor trachea and decellularized the organ with a mixture of chemical and enzymes Then re seeded the decellularized organ with epithelial cells and mesenchymal stem cells derived from the patient The recellularized trachea was transplanted into the patient and after four months the biopsy showed that the transplanted organ was fully integrated with its own blood supply as well as being mechanically sound Most importantly the patient did not show symptoms of transplant rejection although she was not taking immunosuppressant drugs This a prime example of the ECM being used as a template At the moment it is critical for organ regeneration to have a good scaffold Scaffolds control the growth of cells and prevents the seeded cells from migrating away from targeted area This is important for the anchorage dependent cardiac
78. l plate and read via a plate reader Using specific wavelengths to analyze the samples the plate reader data would provide a method to track the progress of the decellularization Less DNA bound would theoretically indicate the process is close to completion Pumping automation would be handled by external software either on board the pump or through controller software such as LABVIEW While a potentially very accurate method to monitor the decellularization process as it provides a quantifiable way to track the DNA content of the decellularizing organ this method is hindered by its inability to monitor and output decellularization data remotely Additionally it relies on the functionality of external equipment such as a plate reader which reduces its ease of use The necessity of extracting a sample manually to acquire data is also a hindrance Photosensor Monitored Decellularization System The photosensor decellularization monitoring system design operates under the hypothesis based on observations made in the Gaudette lab that a decellularizing organ becomes more translucent as it decellularizes and loses cellular material This design would employ a small light aimed directly at the organ as seen in Figure 5 30 Figure 5 Schematic of Photosensor As the organ decellularizes more light would be able to pass through the tissue and be detected by a photosensor on the opposite side of the heart which would output changes in lig
79. lace 10 tissue in a bioreactor a sterile chamber which holds the tissue in place while detergents are pumped through to remove cell residues 8 Each have their advantages and disadvantages Temperature freeze involves freezing the tissue which kills the cells in the process It does minimal damage to the ECM however it does not remove the remnants of the cells Force and pressure uses chemicals and mechanical abrasion to lyse cells although it does remove the cells from the ECM the ECM is not like its original state and not kept intact Non thermal irreversible electroporation uses small electrical pulses to create micropores to cause cell lysis however this method has some limitations such as it not being able to decellularize large tissue sizes Although the physical agents are valid candidates for decellularization this project will mainly focus on the chemical agents such as Triton X 100 and sodium dodecyl sulfate to decellularize tissue By perfusing and immersing the tissue in the chemical solutions the cells can be lysed and removed from the scaffold A common way to chemically decellularize tissue is using sodium dodecyl sulfate followed by Triton X 100 and finally a PBS rinse to remove all chemicals from the ECM scaffold The Gaudette Lab from Worcester Polytechnic Institute WPD follows a similar protocol 8 The Gold Standard Currently the gold standard is the Harvard Apparatus This bioreactor is controlled by a speciali
80. lated heart and have the wastes leave the bottom The flasks have ridges on them allowing the user to easily stack the flasks The number of flasks correlates with the number of tissue samples that need to be decellularized In this case it would be 1 heart per flask Fluid flow into the heart will be driven by a syringe pump Gravity will drain the waste out The flasks will be set on a stand so that the pump would be on the shelf on top of it making more counter space in the lab Another hole would be drilled near the top of the flasks for the sensors to be inserted in These sensors include temperature pressure and humidity A photosensor and a light source would be placed on each side of the flask so that the photosensor can analyze the light going through the tissue Some advantages to this design is that the hearts are in different chambers and they can easily be manipulated if there were a malfunction the 49 flask would simple be removed from the pump and be brought back into the hood One disadvantage is that the waste has to be manually changed unless it is plugged into a vacuum 3 Final Design After drawing up all of the preliminary designs and their choices for modifications in Solidworks we presented the concept to our advisor Professor Gaudette and asked him which was most pleasing to him After discussion about how each could be used in the lab the final design chosen was the bioreactor based upon using the 1000 mL
81. leading causes of death worldwide Currently heart transplantation is the treatment of choice for end stage heart failure but lack of donor hearts and risk of rejection remain a challenge Research has shown that decellularized hearts in which cells are removed from the cardiac extracellular matrix ECM can provide a scaffold for use in engineering patient specific heart transplants This project presents a low cost semi automated small organ ie rat heart decellularization bioreactor designed for cardiac research Testing has shown that this device can successfully decellularize rat hearts and offers functionality similar to commercial devices Acknowledgements All authors would like to thank the personnel of the Gaudette Laboratory to Dr John Favreau Emily Abbate Spencer Coffin and Katrina Hansen for their advice and feedback on this project and to Jordan Jones for his continuous assistance and support over the past year The authors off their sincere thanks to graduate student Joshua Gershlak and our advisor Prof Glenn Gaudette for their advice and support during the past year Without their help the successes achieved in completing this project and developing this device would not have been possible I Introduction Heart failure one of the leading causes of death affects approximately 26 million people worldwide with a 5096 mortality rate after four years In the United States alone heart failure affects approximatel
82. liminary Tesung Datacenter He ter I EN e Ve ERR Ree VERE a ena Dd 35 6 Final Automation Monitoring System Design Selections eee 40 3 JBioreactor Design System sat i ER e Le a a A Re ERU ER RR RENDER eus 43 1 Conceptual Design Ideas 4 4 eet e eh ert ee iter fae ibat deba iaaii 43 2 Preliminary Designs eerte en i gie rede tra dero ita eater sets Eai 45 3 Final Desin tics S 50 4 Custom Built Semi Automated System for Cardiac Decellularization sss 51 Ns Design Verification amp Teste tiir etit etre ee aree tees Fo det den d aea eaa ee ceceuee ERA Ee In 52 1 Photosensor based Monitoring amp Automation Capability eene 52 2 Histological Evidence of Cardiac Decellularization eene 56 3 Visual Confirmation of Decellularization eeeeeeeeeeeeeeeeneeeeenenee eene nemen enne 61 MEUS P aaa 62 Ls Economics ah e e e ve e pee e ad a e ER tie een ba etae tenes Eee ro nep pa eta 62 2 Environmental Impact cre edet e tene cea er ven vie CR e a T 62 3 Societal NUEN E ctt to teret roe ett n e arte toe lt ota a a deere te 63 4 Poltical Ramimticauons 2 nni Reim et reu e RE HOC o ER Rae E ETEA 63 3 Ethical Concerns uiii ence heti ri aariaa ka a osi eae eiae rera eei ledge 63 6 Health and Safety acne et ede e
83. lularizing heart as it becomes more transparent and loses color over time This could be quantified via the software and used to track the process until completion The pump system would be controlled via separate controller software ie LABVIEW or a related program Additional sensors recording pressure and temperature within 28 the bioreactor would consequently be output to the data analysis software for monitoring and recording While intuitive and able to track and record the decellularization process in real time the system is hindered by the various software modes it must employ to fully monitor and automate the system rather than having one integrated program Additionally camera equipment may present a higher cost consideration if a higher quality camera is to be used Plate Reader Based Monitoring System The plate reader based monitoring system design shown conceptually in Figure 4 makes use of established research into detecting the DNA content of fluids with a fluorescent dye assay Conventional temperature and pressure sensors would monitor the decellularization process and output this data to a computer Samples of the decellularization waste fluid would be manually extracted from the system via syringe Figure 4 Plate Reader Based Monitoring System Schematic 29 These samples could then be treated with a fluorescent dye such as Hoescht 33342 that binds to DNA The samples could then be placed into a 96 wel
84. maintain the sterility of the perfusing organ Revisiting the initial client statement and incorporating these specific needs and wants the project goals were laid out in further detail and the following revised client statement was established Develop a low cost perfusion based bioreactor customized for use in cardiac regeneration research to decellularize small mammalian hearts and similarly sized organs The design must maintain sterility of the organ and system facilitate easy usage through a user 16 friendly setup and interface and monitor and acquire data on decellularization process parameters and the status of the decellularizing organ This client statement highlights what was identified as the client s key specifications for the system from evaluation of their statement of wants and needs The client statement was used as a tool to define the design space for the project and determine project objectives and constraints 2 Objectives Using the revised client statement as a starting point objectives for the project were identified and defined These objectives were organized into primary secondary and tertiary objectives as seen below in Figure 2 Objectives User Friendly Safe Decellularizes and Perfuses Small Organs Versatile Marketable Monitors Process Remote warning system Remote monitoring of process Track color change Automates Process Programmable protocols for diff
85. me data output but there is no indication that the machine has fully completed the decellularization system 9 Gaudette Laboratory at WPI The Myocardial Regeneration Laboratory run by Prof Glenn R Gaudette PhD focuses primarily on research into methodologies for regenerating mammalian hearts after a myocardial infarction In particular this group evaluates methods to improve mechanical function of an infarcted region of the myocardium through regenerative engineering In pursuit of this goal the Gaudette Lab collaborates extensively with other groups including the Ott Lab in the hope of advancing research and cardiovascular medicine Consequently the lab has recently begun working with decellularization technology to evaluate its efficacy for research in cardiac regeneration and potential uses in regenerative therapies The Gaudette Lab has already conducted initial studies into the decellularization of rat hearts using a prototype system built on site with at hand materials including sterilizable bottles for use as a small bioreactor and fluid reservoir available tubing and a peristaltic pump The protocol used by the Gaudette Laboratory to decellularize rat hearts is summarized as follows 12 Cannulation of aorta and connection of heart to bioreactor 2 Perfusion with heparinized PBS for 1 hour to flush residual blood and loose tissue Heparin is added to combat potential coagulation and facilitate removal of blood
86. mprove the condition of a patient to enable them to live longer and more actively In cases that cannot be treated with medicine and diet and exercise changes doctors may implant a cardiac resynchronization therapy CRT devices such as a pacemaker These devices help correct heart rhythms when there is an abnormality When all medical implants and treatments are not advanced enough to help correct heart failure a transplant becomes necessary Heart transplant procedures for heart failure are performed only as a life saving measure for the most severe end stage heart failure cases 2 Organ Transplant Timeline The history of organ transplant dates back to 1869 when the first every organ transplant was performed This transplant was of skin and as performed by Jacques Louis Reverdin In the 150 years following 1869 many milestones have been made regarding the medical practices of organ transplantation In 1963 the first ever organ was transplanted from a brain dead donor Practices today are based off of this brain dead donor as most all organ donations come from donors who have been medically declared brain dead This does not include live organ donations where living donors can donate a partial lung liver intestine or one of their two kidneys One of the biggest post operation regimens that every patient must follow is taking an immunosuppressant drug every day for the rest of their life In 1983 the very first immunosuppressant dr
87. n Proceedings 40 1 2008 34 38 Patnaik S Wang B Weed B Wertheim J amp Liao J 2013 Decellularized Scaffolds Concepts Methodologies and Applications in Cardiac Tissue Engineering and Whole Organ Regeneration In Tissue Generation Where nano structure Meets biology Khan A Vishwakarma S Bardia A amp Venkateshwarulu J 2014 Repopulation of decellularized whole organ scaffold using stem cells An emerging technology for the development of neo organ Journal of Artificial Organs Retrieved September 23 2014 from http link springer com article 10 1007 s10047 014 0780 2 fulltext html Macchiarini MD P Jungebluth MD P Go MD T amp Asnaghi A 2008 Clinical transplantation of a tissue engineered airway The Lancet 372 9655 2023 2030 Retrieved October 15 2014 from http www sciencedirect com science article pii S0140673608615986 Khang G Lee S J Kim M S and Lee H B 2006 Biomaterials Tissue Engineering and Scaffolds Encyclopedia of Medical Devices and Instrumentation 73 18 19 20 21 22 23 24 25 26 21 28 Crapo P M Gilbert T W amp Badylak S F 2011 An overview of tissue and whole organ decellularization processes Biomaterials 32 12 10 Personal communication Glenn Gaudette Ott HC et al 2008 Perfusion decellularized matrix using nature s platform to engineer a bioartificial heart Nature Medicine 24 2 Gau
88. nd moisture build up Ideally these sensors will be small enough to fit within the system without disrupting the decellularization process The device should permit easy programming and automation of the pumping system employed by the bioreactor allowing users to pre program pump rates and durations These pumps must be independently controlled to facilitate adjustments if multiple hearts are decellularized at once and include failsafe modes to automatically shut off the pumping system in the event of a failure in the system 27 2 Conceptual and Preliminary Designs of Monitoring and Automation System We established several conceptual designs for system software and monitoring systems that could effectively track the decellularization process and automate the pumping system employed by the decellularization bioreactor Each design incorporates different monitoring methodologies and software types Sketches where applicable descriptions advantages and limitations of each design is provided in the following sections Camera Monitoring Decellularization System The camera based monitoring design uses an optical camera fixated to the outside of the decellularization bioreactor to monitor the decellularization process and record its progress drawn in Figure 3 Figure 3 Camera Monitoring Decellularization System This system would employ data analysis software available in the lab to process and analyze the color change of a decel
89. ng light levels in the laboratory over the 80 hour period but is more likely due to experimental error while collecting results and interfering light sources while collecting data In both trials light intensity through the heart shows a noticeable decrease over time supporting the initial hypothesis However the readings are erratic and suggest that future design iterations should seek to reduce exposure of the light sensor to ambient light and background noise to increase accuracy of readings Hoescht Dye Decellularization Waste Fluid Assay The objective of this study was to assess the validity of the secondary decellularization monitoring method for use in future device tests In this study decellularization fluid collected via syringe injected directly into the fluid line of the decellularization waste fluid was treated with a Hoescht dye solution and run through a plate reader assay to determine the fluorescence at 490 nm and consequently the DNA content of the waste fluid over time Based on previous research into the use of Hoescht solutions to quantify DNA content in solution it was hypothesized that sample absorbency would decrease over time in correlation with the increasing decellularization of the tissue Samples of ImL were collected from the SDS decellularization residue fluid every 6 hours simultaneously with the light sensor trials detailed on pages 35 37 of this report Samples were labeled with time extracted and fro
90. nsiderable expense costing more than 30 000 to purchase the control system which by itself the bioreactor costs 13 000 and all associated parts Additionally the system including the controller the pump bioreactor chambers and medium reservoir is essentially a table top device and is not easily portable Ideally an improved easy to use system would reduce the time involved in decellularization research at the laboratory graduate students and undergraduate volunteers typically must commit to a training period of about 2 3 days to learning the decellularization process and then devote well over 8 hours a week in the care and monitoring of the decellularization process as it currently stands Because the design was specific to the needs of the Gaudette Laboratory the size and cost of this decellularization bioreactor could be reduced significantly The goal of the project was to keep the cost within the Worcester Polytechnic Institute MQP budget of approximately 125 dollars per student member for this project approximately 500 Still decellularization is a relatively new technology and many research laboratories specially build and employ different apparatus designs and different decellularization protocols to meet the specific needs of their research These systems are customized to meet the needs of their laboratories and have both benefits and drawbacks depending on the features and functionality incorporated into
91. o make our design stand out from other bioreactors on the market as well as allow for double the amount of research to be conducted in the normal time for one decellularization This characteristic also meant that the materials and components that we decided to use must have the ability to undergo machining techniques in order to create holes and other modifications to build our custom system and create ports for multiple organ decellularization 43 These conceptual ideas then led us to search for laboratory materials that allowed us to accomplish the set goals The first material that we found to be a candidate for the final bioreactor design was a 1000 mL Nalgene Straight Sided Container This material seen in Figure 12 allowed us to remain low cost be versatile in creating holes in the cap for multiple organ decellularization and maintaining a sterilizable container The Nalgene container it made from polycarbonate which can be sterilized by the process of EtOH Figure 12 Nalgene Container used in Bioreactor Design The second material that we chose as a candidate for the final bioreactor design was a T 75 tissue culture flask Like the Nalgene container the flask allowed the bioreactor to remain low cost sterilizable and machinable In order to perform multiple organ decellularization hearts would be placed in separate flask containers This material can be seen in Figure 13 below 44 Figure 13 T 75 Tissue Culture Flask
92. ole in healthcare and therapeutics can serve to improve healthcare significantly and open up new markets for medical devices and therapies While the global political implications for a research device like this would likely be minimal bringing a new medical research device to market raises questions of how to appropriately allocate funding for medical research and may help raise political support and society questions about the ramifications of decellularization technology in healthcare 5 Ethical Concerns Decellularization research requires the usage of whole organs and tissue samples from both human donors and mammalian models Our research utilized full hearts excised from rats euthanized for both laboratory research and undergraduate teaching classes The use of animal models in research represents a long standing ethical concern we were well aware of during our 63 work and did our best to conduct the research with the respect and effort owed to sacrifice of a life However this group feels that the promise of decellularization research and the opportunity to advance healthcare and potentially save lives in the future are worth the sacrifices of animal models and human donors 6 Health and Safety Decellularization of tissue can have health benefits and well as safety If FDA approved it can decrease rejection of the organ therefore decreasing the number of people that die from heart transplants It can also improve the qual
93. ow adjustments pre programmed protocols and automatic shut offs 26 Some of the initial requirements of the software component to the project were to monitor the decellularization process including remote warnings of failure conditions tracking and recording of decellularization status and data and the monitoring of system parameters including flow pressure and rate and bioreactor temperature Our device should record and output this data in a tabular format easily usable by researchers that does not require a significant amount of data processing such as csv or xls To provide consistent and continuous data of the decellularization process the data should be recorded at a minimum of once every ten minutes and the software should automatically upload this data to a storage apparatus or cloud service Additionally the system should be able to detect failure modes within the system and transmit warnings via a readily usable communication mode such as internet or email to maximize ease of use and reduce the amount of time necessary for researchers to spend tending to the system That being said data should ideally be viewable outside of the lab via remote upload of data collected by the system to a cloud server or other method Any sensors used within the bioreactor must be sterile or contained in such a way to maintain system sterility The sensors should also be able to withstand conditions inside the bioreactor such as humidity a
94. paratus mentioned previously is the gold standard which funded research laboratories use to decellularize tissue It has an advanced system which allows for small and large organ decellularization and has multiple protocols stored within their software It can acquire data during the decellularization process too Our bioreactor which has been designed for the Gaudette Lab can replicate key functions of an ORCA bioreactor at a fraction of the cost The materials which make up the bioreactor are made from common laboratory apparatuses which are usually bought in bulk such as a conical tube cannulas and Nalgene containers It also allows for real time tracking of decellularization of the tissue Using an LED light and a photo sensor this records light intensity data in MATLAB and correlates it with the stage of decellularization As the cells lyse the light intensity will increase due to the lack of tissue This is definitely an innovative aspect of the bioreactor Also the bioreactor can be adaptable for different organs Currently the bioreactor is built to decellularize two small mammalian hearts However it can be designed to decellularize four small mammalian hearts as was previously shown Not only can it house small mammalian hearts it can do any type of tissue as long as it can fit inside the container Through this project we achieved all our major project objectives affordability time efficiency successful decellularization
95. programmed custom protocols for different organs and run those protocols automatically with limited outside interference Additionally it would be beneficial if the bioreactor system automatically switches decellularization fluids For example after perfusing the heart with Triton X 100 the bioreactor would be programmed to switch to SDS after a preprogrammed amount of time 2 Safety Safety was one of the key objectives for this device The design should enable as much stability as possible limiting the risk of breakage or failure in any component of the bioreactor which will reduce the risk of the decellularization fluids and organs used in testing to be exposed to outside elements or a failed decellularization protocol Simple design choices for the safety of 18 the user were also put into consideration specifically reducing the number of sharp edges or uncovered moving parts in the apparatus 3 Decellularize and Perfuse Small Organs The third primary objective of our project was to create a bioreactor system which has the ability to decellularize organs by means of perfusion Chemical solutions will be pumped through a native heart and will lyse the cells of the organ This will result in an extracellular matrix scaffold made mostly of collagen In the future this scaffold will hopefully be used for recellularization research to eliminate the need for exact tissue matching donor hearts 4 Versatile The team also wanted t
96. project was broken up into two sub projects 1 the design and development of the bioreactor with software design 2 sensor development and testing and validation This work breakdown and management structure permitted easy communication and optimized team efficiency to successfully complete the project 3 Financial Approach Material costs were the main source of spending In particular the pumps and required hardware were the largest investments made in the project In order to develop an easily reproducible and low cost design the team made use of readily available materials in the Gaudette Lab including solution containers conical tubes and syringes to construct various components necessary for the bioreactor container Rat hearts were provided by the Gaudette Lab for prototype testing free of charge There was no cost associated for labor in production as production assistance was provided by the WPI Machine Shop as a school service A primary goal of the project was to keep costs as low as possible to permit revisions and design improvements for a final design prototype at the conclusion of the project 24 IV Alternative Designs Needs Analysis Given the list of established and ranked objectives laid out in Chapter 3 Table 1 we established the relative importance of each objective by ranking them based on 1 10 scores 1 indicating an objective of little importance and 10 indicating an objective vital to project success
97. r portion were complete we brought all the components together to build a working system for semi automated cardiac decellularization Our final prototype includes the photosensor monitoring system the MATLAB automation and user interface and the final bioreactor design Below in Figure 18 is 3D schematic of our complete system design WASTE SPOUT Li WASTE RESEVOIR C Figure 18 Full Scale Final CAD Design Model This schematic shows every main component that allows our system to work and successfully decellularize rat heart by means of perfusion The process of decellularization starts 51 with the solutions in the top two reservoirs in the schematic Between these two reservoirs there is tubing that is transferring fluid in and out of the reservoirs at a constant rate This fluid transfer is controlled by two pumps that are controlled by the Arduino and MATLAB user interface The fluid transfer creates a constant volume in the second reservoir This reservoir is closed off from non sterile air by a filter The constant volume in this reservoir creates a constant pressure of fluid that enters the hearts during decellularization This allows for the decellularization process to be consistent The decellularization solution flow from the second reservoir to the hearts by means of gravity Once the fluid has flown through the hearts it drips down into the bottom of the bioreactor and out the waste spout into a waste conta
98. rejection of the foreign organ This rejection can vary from either acute or severe depending on the individual According to the OPTN Annual Report from 2012 acute rejection after one year ranged from 14 of patients to 39 The amount of rejection depends on the type of organ For the heart in particular the amount of acute rejection was 23 in the first year Over time the risk of rejection increases for patients At year five rejection of transplanted organs ranged from 17 of patients to 53 of patients Patients with a heart transplant have a 45 chance of acute rejection after five years 5 Regenerative and Tissue Engineering In an attempt to keep up with the high demand for organ donations The field of tissue engineering and regenerative medicine have been trying to fill those gaps with an alternative to donated organs The goal of tissue engineering is to repair damaged tissue and organs through creating tissue scaffolds or implants that can be integrated into the host organ system The three main methods currently used are 1 The implantation of cultured cells directly into the body 2 The generation of cells in situ 3 The congregation of cells and scaffolds in vitro This section will focus more on the last method since it is the most relevant to this project To create a scaffold in tissue engineering it can be synthetic polymers or naturally derived polymers Polymers most often used are PGA PLA and PCL These products are
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100. t design has salient features comparable to commercial decellularization bioreactors This will allow universities around the world to afford a decellularization bioreactor to further expand the research in this area It is also more efficient than the current laboratory custom bioreactor in the Gaudette Lab and cheaper and easier to use than the ORCA bioreactor from Harvard Apparatus Overall this system has met the need of the Gaudette Lab During the development process several improvements for future iterations of this device were documented The photosensors currently used in the bioreactor are sensitive to ambient light which generates noise in decellularization monitoring data A directed light source such as a laser would increase the efficacy and reduce interference Alternatively calibrating with an external sensor to offset ambient light values could prevent this problem This system is currently tested exclusively for decellularization purposes Modifying it to enable use as a recellularization bioreactor may be an appropriate next step in expanding this device Our results from the histology staining suggest that our bioreactor and system can work as a successful decellularization process One aspect to the system hindering the accuracy of the decellularization process is the protocol by which specific solutions are used to decellularize the tissue The overall scope of our project did not include making adjustments and changes to
101. t free to download from the Arduino company website Figure 6 Arduino UNO 4 This microcontroller based software design would employ a custom user interface programmed within the Arduino which would run photosensors temperature and pressure sensors necessary to fully monitor decellularization parameters The Arduino Uno can be adapted with a motor shield a secondary board that sits on top of the microcontroller and enables the board to drive DC motors or stepper motors permitting it to automate the pump system used by the bioreactor A preliminary prototype of this system employing a DHT11 temperature and humidity sensor from Virtuabotix and standard CdS photoresistors used as photosensors from Radioshack built into a voltage divider circuit with a 10kOhm resistor was developed to assess the validity of the idea These sensors are processed by the Arduino and output to a Sainsmart 34 1 8 ST7735R TFT LCD screen which displays the current decellularization system parameters The design effectively conveyed all the parameters that were desirous to monitor in the system but did not offer a way to interface with the system Figure 7 De Cell has been running for minutes re Temperature C Humidity re oH empet re 253 Light Sensor 2 268 Figure 7 Decellularization System Visual Interface 5 Preliminary Testing Data Before selection and development of a final design could be undertaken it
102. tively monitor experiments themselves To test this functionality the peristaltic pumps were activated and set to an arbitrary speed value about 50 speed for tests Both user cellular phone numbers and email addresses were added as notification addresses so that it could be validated that both would work as contact addresses After system parameters were set and the pumps were activated the monitoring and automation program was activated and the system was allowed to run for a pre set runtime 10 seconds After this time had elapsed the pumps and monitoring system deactivated appropriately and a notification was successfully received by the user Figure 19 below Mm Yeon X 1207 vM 1906 NP 5 4 Fyuc2 e amp llltgs file ott Wee imat Tonk Destop Window Hep lt gaudettedecell2015 g Details Osea ss Sopa 208 C Heart 1 Gaudette Lab Decell System Decellulanzation Cycle Complete Check System 35 E B5 T 1 os B Timm saconds Figure 19 Process Complete Notification and accompanying MATLAB graph of light sensor data Additionally tests were performed in which the system was stopped by exposing the photosensors to a light level above the 4 5V threshold In this case the pumps again stopped 53 once this light level was detected the monitoring system deactivated and the system sent an appropriate notification to the specified user notifying that the system needed to be checked Figure 20 ee
103. tor and accumulation of foam when the peristaltic flow agitates the SDS detergent This can hinder the decellularization process and reduce consistency and reliability between runs a challenge our client desires to overcome 10 Customized System for Gaudette Lab In an effort to optimize the protocol already in use in the Gaudette Lab and advance their progress in developing cardiac decellularization methods for their purposes the Gaudette Lab has commissioned this project to develop a custom decellularization system optimized for their use with features and applications geared toward their research This system must address the current challenges faced in their decellularization process Specifically reducing the amount of time involved in running the decellularization process by building in a monitoring system to detect failure conditions such as fluid overflow or disconnection of the decellularized organ from the bioreactor Additionally the lab desires some level of automation to increase consistency of decellularization runs and further reduce time involved in the process Supplementary features such as sensors to monitor and record the decellularization procedure inspired from systems used in other labs and current marketed decellularization bioreactors will assist in data collection and study of the cardiac decellularization process The overall goals for an improved decellularization system provided by the client helped establish
104. ug cyclosporine was approved by the FDA Cyclosporine is still widely used today to treat transplant patients Moving along in the timeline in 1984 the Organ Procurement and Transplantation Network was founded This network ensures that organs are allocated fairly and equally in the United States This network today has an online database that has up to the minute data on the number of patients on the transplant waiting list for any organ Finally one of the most recent milestones happened in 2006 In this year the organization Donate Life America was founded This organization has raised the number of registered organ donors in the country to almost 120 10 12 million people 3 Organ Transplants Out of the 120 million registered donors in the United States only a few thousand donate for transplants each year Organs are turned away from transplants for a multitude of reasons the main being disease or infection If an organ donor has any severe diseases or infections HIV or metastasized brain cancer their organs are no longer eligible for donation Organs from donors are also turned away from transplantation because of any human error Medical institutions have started education programs for their staff to learn how to harvest organs for transplantation more accurately These programs will allow for the number of possible organ transplants to increase over time 4 Organ Rejection For any type of transplant there is a risk of
105. uorescent Microscope in junction with the Leica Application Suite Version 3 7 at a 40X magnification 56 NU MU MON LUI mn EE 1 1l z y o v T e E a z z 2 2 z a MA N i a Lus 3 IE z a Y Lu 3 2 n Z i ud i 7 j a say a e ff z v i z ete 4 r n a amp iy 5 EXPERIMENT 3 L Figure 23 Results of H amp E A Trichrome Blue B and Hoescht C histological stain assays on decellularized and control heart tissues from hearts decellularized in the prototype system 57 The first stain we used is a Hematoxylin and Eosin stain This stain focuses on showing cell nuclei cytoplasm proteins extracellular ECM fibers and intracellular membranes The nuclei are stained a dark purple color and the cytoplasm ECM fibers and intracellular membranes are stained a color resembling pink or red Column A in the figure shows representative images taken of the Hematoxylin and Eosin H amp E stain for the control and experimental tissue samples To confirm if the decellularized heart tissue has been completely washed of genetic debris and other cellular structures we stained a control heart as well as our experimental hearts The control heart was a native rat heart that did not undergo any portion of the decellularization protocol In the first row of column A in Figure 23 the control heart that had been stained is represented The image shows healthy heart tissue th
106. us cadaveric cardiac tissue from the Ott laboratory ssssseseseeeeeneeeennen nnns 13 Figure 2 Primary secondary and tertiary objectives tree sssssssssseeeeeeeeenneenen nn 17 Figure 3 Camera Monitoring Decellularization System ccceccccesssneecesseeeecesseeeecesseeaecesseeaeesseeaaees 28 Figure 4 Plate Reader Based Monitoring System Schematic esee 29 Figure 5 Schematic of Photosensor cccccccesssecessececesseeeececeeaaececeeaaeeeseeaaececseaaeceeseaaeeeeseaaesecseaeeeseeaas 31 Figure 6 Arduino UNO 5 cse ot tet iot dtu tuu tom i dae LE ue UE ti M eM OO 34 Figure 7 Decellularization System Visual Interface sesssssssseseeeeeeeennnnen enne enne 35 Figure 8 Light Sensor Validation test first trial Graph shows voltage output of sensor system due to direct LED exposure exposure through decellularizing cardiac tissue and ambient light over an 80 hour period cete ee etes eee seen rest Senses ede ete ome ee YN Du uas ee en ate ca de ras 37 Figure 9 Light Sensor Validation test first trial Graph shows voltage output of sensor system due to direct LED exposure exposure through decellularizing cardiac tissue and ambient light over an 80 haur peno EET 37 Figure 10 Hoescht fluorescence in decellularization fluid over 42 hours Fluorescence
107. was necessary to validate the usage of the methodologies developed in preliminary designing to monitor the decellularization process Two separate experiments were conducted to assess the designs of the light sensor decellularization progress tracking and the fluorescent DNA assay of decellularization waste fluid Light Sensor Validation It was initially hypothesized based on general observations of the increasing transparency of the decellularizing heart as it underwent the perfusion process that tracking the light that penetrated through decellularizing tissue with a photosensor would indicate that light intensity detected by the sensor increased over time In order to test this hypothesis a 35 rudimentary CdS photoresistor light sensor wired into a voltage divider circuit with a 10kOhm resistor was programed into an Arduino Uno microcontroller which output voltage across the light sensor to a personal computer The code used to program this design is shown in Appendix A Increased light exposure yielded increased resistance and consequently less voltage was read across the graph Decreased light exposure increased the amount of voltage going across the circuit Two trials were performed during two decellularization runs Data on the photosensor output of the ambient light in the room the photosensor output when the testing LED was shined directly across the photoresistor and the light intensity of the LED through the bioreactor and
108. was stimulated at 490nm Shown are the averages for trials 1 and 2 compared against the pure SDS control 39 Figure 11 MATLAB GUI for Decellularization System Operation sse 42 Figure 12 Nalgene Container used in Bioreactor Design sessssssseeeeeneentennnes 44 Figure 13 T 75 Tissue Culture Flask sesesssseeeeneneeeeren rennen rennes treten trennen 45 Figure 14 CAD Model of Nalgene Bioreactor ccccccessececesnecesseeaececseauececseaaececeeaaeeecseaaeeeeseaeeeeeeaaes 46 Figure 15 Divided Section Bioreactor Model sse nnne nnns 47 Figure 16 Four Heart Bioreactor Model eese eee enne ener nnnns 48 Figure 17 Tissue Culture Flask Design Model essere enne enne ennt 49 Figure 18 Full Scale Final CAD Design Model sese 51 Figure 19 Process Complete Notification and accompanying MATLAB graph of light sensor data 53 Figure 20 Problem Detected notification and accompanying MATLAB graph of light sensor readings that initiated the system respOnse ccccccccccesssececsssnececsesnececesneeecseaeeeceeueeecsesaeeeceeaeeeceeaeeeeseneeeesees 54 Figure 21 Photosensor Test 6 hours no ambient light interference ccccccccessececeeseeeeeeesteeeeeeaaes 55 Figure 22 Photosensor Test 20 hours ambient light interference due to day night c
109. y 5 1 million people There are many ways in order to treat the symptoms of heart failure but a complete heart transplantation is the only cure for the most severe cases of heart failure However transplantation inherently has many drawbacks The odds of a heart being available for transplant is 3 5 in every 1000 deaths or approximately 2000 hearts available each year This poses a problem as the number of patients on the heart transplant waitlist is above or equal to 3 000 on any given day The list changes every day with people constantly being added and removed from the list In 2012 3 007 patients were added to the heart transplant waiting list and 2 784 were taken off Of the 2 784 patients taken off the list 2 008 received transplants 117 became too ill to be considered for transplant 142 became healthier and 372 passed away while waiting Even for those lucky enough to receive a transplant they run a major risk as with any type of organ transplant is rejection While the first year survival rate of heart transplants is 8896 the main cause of death is infection due to the rejection of the donor heart The percentage of patients developing acute rejection increases from 23 in the first year to 45 in the fifth year The challenge in transplant procedures is identifying tissue matches between a recipient and a donor in order to avoid rejection To overcome this limitation researchers have developed methods to decellularize tissue and whole
110. ycle and laboratory automatic lights iere yer eret dress enero ee forie ee na dena Fere e e ERE T eR URS 55 Figure 23 Results of H amp E A Trichrome Blue B and Hoescht C histological stain assays on decellularized and control heart tissues from hearts decellularized in the prototype system 57 Figure 24 Decellularized Heart Double Decellularization Experiment Results Before Top After Dope DEEP 61 Figure 25 Final Decellularization System Prototype ssesesseeeeeeeeee enne enne enne 68 Figure 26 Photosensor circuit SCHEMAtIC cccccessccessensececeeenececseaaececseaaeceeseaaececeeaaececseaaeeecseaeeeeseaaes 69 vii List of Tables Table 1 Ranked primary secondary and tertiary objectives from client s PCC and discussion 20 Table 2 Weighted Objectives Table sesesssesseseeeeeeee eene ene nnnn en ennnnn sn ennnns eene nn nnns 25 Table 3 Monitoring and Automation System Functions and Specifications susessss 26 Table 4 Numerical Design Evaluation Matrix ener ennt 32 Table 5 Fluorescence output of Hoescht dye decellularization fluid assay Duplicates of the weeks 1 and 2 studies along with averages are shown below and compared against a control of pure SDS treated with Hoescht dye Data was sample every 6 hours for 42 hours sees 39 viii Abstract Heart failure is one of the
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112. zed system called the ORCA controller Organ Regeneration Control and Acquisition This system is the most advanced system offered in the market for organ decellularization and recellularization Advanced analytics and sensor technology is combined with a controller designed specifically for controlling all the critical elements involved in 3D organ bioreactors This system includes small and large chambers for different organs It has a built in software to monitor acidity flow rate and pressure of the decellularizing tissue and the status of the bioreactor itself 11 Some advantages of this bioreactor is that it has multiple chambers it can collect different protocols that the scientists use it can acquire data from the decellularization process in forms of pictures and video and it controls the flow and pressure of the gases such as oxygen and carbon dioxide The ORCA controller uses various sensors that can take temperature and pressure readings throughout key points in the system Also there are microscope cameras that will monitor and document the decellularization process by taking photographs and video These cameras have the capability to monitor visual UV and IR spectrum One main limitation of this bioreactor is the cost The system cost 13 000 however the chamber and all the components together cost over 36 000 Another limitation is that the system does not notify the user when the organ is decellularized It has real ti
113. zen for later evaluation Hoescht solution was prepared per a ThermoScientific protocol 6 A working solution was added to samples in 200uL solution 200uL sample ratio in a 96 well plate Samples from 0 hours to 42 hours from both decellularization trials were added in duplicate to the 96 well plate from both light sensor trials The absorbancy of these are compared against 8 pure SDS samples treated with Hoescht 38 solution as a control Samples were evaluated for absorbancy at 490nm which is indicative of DNA concentration in the residue The plate reader fluorescence results are shown in Table 5 and graphically represented in Figure 10 below Table 5 Fluorescence output of Hoescht dye decellularization fluid assay Duplicates of the weeks 1 and 2 studies along with averages are shown below and compared against a control of pure SDS treated with Hoescht dye Data was sample every 6 Time Hours 0 6 12 18 24 30 36 42 E T ea t a I c T I A a u hours for 42 hours CTRL Week1 Week1 Week2 Week2 0 032 0 165 0 200 0 344 0 384 0 033 0 133 0 177 0 164 0 158 0 033 0 128 0 153 0 094 0 090 0 032 0 042 0 046 0 076 0 076 0 031 0 035 0 036 0 055 0 056 0 032 0 035 0 038 0 052 0 059 0 031 0 038 0 035 0 037 0 035 0 037 0 037 0 037 0 039 0 037 Hoescht Fluorescence in DeCell Fluid over 0 42 hours Week1 Avg 0 182 0 155 0 141 0 044 0 036 0 037

Development of a Perfusion Based Decellularization System for

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