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A Microfluidic Device for Single Cell Isolation

1. DraftSight software A photomask was created at CAD Art Services Inc and returned back to us Using standard photolithographic processes the designs were transferred from the photomask to a silicon wafer some steps are shown in Figures 9 and 10 FIGURE 9 SILICON WAFER SPINCOATED WITH PHOTORESIST 39 FIGURE 11 SILICON WAFER EXPOSED TO UV LIGHT The photomask contained 3 wafers of designs each wafer containing 12 devices PDMS was poured over the silicon wafer and baked The devices were then cut from the PDMS mold and plasma bonded to a glass slide shown in Figure 11 FIGURE 10 PDMS DEVICE ON GLASS SLIDE 42 5 2 Device Setup In order to start testing the device a syringe setup needed to be attached to the device The device setup is shown in Figure 12 One vertical syringe was to hold the suspension and the horizontal one was to hold fluid in order to flush or prime the device The tubing from the syringe was then inserted into the inlet hole in the microfluidic device FIGURE 12 DEVICE SET UP 5 3 Proof of Concept Testing In order to perform proof of concept testing we used polyethylene fluorescent beads ordered from the company CosphericO Product ID UVPMS BR 1 20 with diameters ranging between 38 and 45 microns This size was chosen because they are similar in size to trypsinized PANCI cells To prevent clumping of the beads in the devices a solution of Tween surfactant was created Appendix D
2. FIGURE 1 OBJECTIVES TREE Compatible with common cell culture techniques Since cells are to be flowing through our device we thought that compatibility with cell culture techniques was most important Without the correct cell culture technique the cells will not survive therefore our device will not be useful Compatible with common microscopes Once single cells have been isolated they need to be analyzed In order for our system to be useful in a wide range of labs it has to be compatible with common laboratory equipment By bonding PDMS to a glass slide it gives the system transparency and the cells inside can be analyzed using a common light microscope Accurate The main function of our system is isolating single cells so it is important that it can do so accurately If cells clump or if more than one is isolated in the same bubble or 24 pocket the user can t analyze them as single cells and extra steps would need to be taken to further separate them Precise Ideally we want our device to be able to do a large number of tests and analyze a large number of cells at once so the device should be able to isolate a large number of single cells Inexpensive This is most applicable to completing our project while staying within our 365 budget The resulting device is going to be extremely inexpensive compared to other single cell separating technologies that are currently on the market which will be one of its ke
3. 12 As beads exited the device they flowed out of the outlet and into a small petri dish as waste 7 4 Cell Testing 1 Water was flowed through the device to reduce particulates in the device this created air bubbles in the small channels underneath the wells 55 2 Approximately 20 uL of Pluronic 127 was pipetted into the inlet in order to coat the device so cells would remain in solution and not get stuck in channels and would be less likely to clump 3 Media was flowed through the device to coat the surfaces that cells would be in contact with and to flush the Pluronic 127 4 About 1 mL of media was pulled into the left horizontal syringe 5 Asuspension of cells at a density of 20 000 cells 1 mL was added to 10 mL of media 6 5 mL ofthe cell suspension was poured into the top vertical syringe 7 The media from the left syringe was manually pushed through the device to clear any of the remaining Pluronic 127 and dust 8 The syringes were primed and again hydrostatic flow was created with a height change of 12 inches between the syringe setup and the device 9 Flow from the top device was turned on allowing cells to flow into the device 10 Again cells would flow through the main channel and out the outlet into a petri dish In our test the cells were too small for our devices and they were able to travel through the small horizontal channels We successfully created a microfluidic device that demonstrated potential to
4. 4 After 5s turn on unit power and wait for purple plasma as described in steps 3 4 above Start a time when it appears and adjust needle valve to generate brighter plasma 5 Treat PDMS surfaces for 60s 6 Turn off unit power and the vacuum pump power Slowly open the exhaust valve until the vacuum has been released As before HOLD ONTO THE DOOR or it will fall 7 Carefully remove the plasma activated PDMS and glass 8 Gently invert the glass onto the activated PDMS surface Bonding is covalent and instantaneous so there is no opportunity to realign Make sure you align before any contact and be as gentle as possible 79 9 Once the PDMS is sealed apply light pressure the remove any air bubbles that may have been trapped inside 10 Wait about 15 30s and test an edge for bonding by very gently peeling up at the corner A successfully bonded PDMS piece will not peel away from the substrate and will break internally before debonding 80 Appendix D Tween 20 Surfactant Preparing Tween Solutions Fill the container for making the solution with desired amount of de ionized water and place it on the heating surface such as the hotplate Heat until the water reaches a rolling boil and leave water boiling for 5 minutes While the water is heating using a precise scale and a pipette measure 0 10grams of Tween per each 100ml of water Example 1500ml de ionized water 0 10g x 15 1 5g of Tween Slowly add pre measured Tween
5. Appendix 1 for programming If you make any changers or additions note your changes in 64 the MFL logbook Edit Program 10 Step 001 002 Vac req Step 002 002 Vac req Time 00 10 0 Cpm 00 Time 00 30 0 Cpm 00 Rpm 00500 Loop 000 Rpm 01600 Loop 000 Acel 0100 Goto 001 Acel 0300 Goto 001 Valv Valv Sens Sens The first step is a slow ramp to 500 rpm at 100 rpm s and is designed to slowly spread the resist across the wafer The second step spins faster to determine the final resist film thickness Only the spin speed in rpm needs to be changed for different resist thicknesses all other parameters should remain unchanged 4 Remove the spin coater lid and verify the presence of a foil liner If the foil is not present line the bowl with foil to catch photoresist that is removed from the wafer during spinning Ensure that the bowl periphery is covered above the height of the chuck and wafer and also completely covering the bottom to the chuck Rotate the chuck and ensure that the foil does not touch the chuck or impede rotation 5 Select Run Mode 6 Turn on the N2 supply by opening the main tank valve Ensure an output pressure of 60 70 psi If the display reads Need CDA open the round valve attached to the pressure regulator Open the vacuum valve by aligning the black handle with the tubing 7 Make sure that the wafer is clean and dry Visible dust on the wafer can be removed by gently blowing the wafer using t
6. The Postbake procedure is required to stabilize and harden the developed photoresist prior to processing steps that the resist will mask Typical post bake temperature is 150 RIC for 30 min for SU8 or 90 120FPIC for 5 min for other thin resists This procedure uses any of the Dataplate hotplates 1 Place the developed wafer on a hotplate at no more than 65 FIC 2 Set the ramp rate to 6 IC min or 360 BlC hr SET Ramp BC hr 6 3 6 0 ENT Set temperature to 1500C Set the timer for 45 minutes Set the hotplate to automatically turn off then the timer ends by pressing Auto Off 8 Cover with a foil tent 3 The hotplate will slowly ramp up to 1508C over about 15 mins maintain temperature for 73 30mins then turn off and slowly return to room temperature This will take around 1 hr total 4 After the wafer has returned to room temperature inspect the wafer again and verify that surface cracks have disappeared Document selected microscope fields with a camera 74 Appendix C Soft Lithography SOP PROCEDURE 1 Fluorination of the Micropatterned Substrate This procedure facilitates mold release by covalent treatment of Si or glass surfaces with a fluorosilane chemical by vapor deposition The treatment renders the Si or glass hydrophobic and maintains the Micropatterned SU8 features as long as possible without delamination by reducing the forces applied during PDMS de molding 1 Set up the vacuum des
7. then transfer it to a cleanroom wipe on the work surface to cool to room temperature At this point the mask pattern should be clearly visible If not exposure and or baking times were too short Procedure 6 Development The development step dissolves away the unexposed negative photoresist or exposed positive photoresist It is performed by immersing the wafer in developer liquid and agitating until the resist is dissolved and only the insoluble pattern remains This procedure uses a glass dish and developer chemical in the fume hood Developers are located in the flammable cabinet below the fume hood left side 1 Ensure the glass dish is clean Clean and dry with a cleanroom wipe if necessary Pour developer in the dish to about 0 5 1 cm depth 2 Immerse the wafer in developer and gently slosh agitate taking care not to splash developer out of the dish Start a timer on the hotplate with the desired development time 3 Observe the wafer periodically Bare Si regions will become visible after 30s 1 min The resist at the edge is thicker than in the center and therefore tends to be the last part to dissolve away 71 4 When all resist appears dissolved remove it from the developer bath with wafer tweezers and run under a gentle stream of water in the hood sink Grasp the wafer in your hands at the edges to ensure it doesn t fall and break Note the time of development in your lab notebook 5 After both front and b
8. A monomer and Part B cross linker A typical ratio is 10 1 w w For simplicity we typically weigh out the components into a single weigh boat on a balance 1 Set up a paper tower on the balance ensuring it does not hang over the edges and a large weigh boat Remove any visible dust 2 Determine your desired PDMS volume Each wafer requires about 50 60g PDMS Ideally you should make about 80 120 g PDMS per weigh boat up to three boats at a time 3 Tare the weigh boat set weight to 0 0g Pour Part A into the weigh boat until the desired weight e g 91 2g Then divide this value by 10 for 10 1 ratio tare again to 0 0g and pour Part B to the desired weight e g 9 1g Within 0 2 0 5 is ok 4 Using a transfer pipette slowly and gently fold as in baking the low viscosity Part B into the high viscosity Part A Once Part B is no longer visible on the surface increase your folding speed Ensure that all edges have been mixed Mixing should take at least 1 min ideally gt 2 Technique is more important than time here There should be lots of bubbles 5 Place the weigh boat into the vacuum chamber If more than one is prepared invert a second weight boat on top rotated such that the PDMS in the lower boat is visible and place the second PDMS boat on top Repeat one more time for three total as needed 6 Apply a vacuum and observe bubble enlargement Release the vacuum after 1 min as necessary if bubbles appear as tho
9. A eed na xd 34 IS PEN iurium 35 Figure 8 Alternative Design 5 Microscope View miii trente tnnt tnn tttn tentent tton 36 Figure 9 Silicon Wafer Spincoated with Photoresist sentent tnn ttnnnns 39 Figure 11 PDMS Device on Glass Slide EEN 42 Figure 10 Silicon Wafer Exposed to UV Light isses tenter tentent ttn ttn tent tette tton 42 teure 12 Device Set Oderen 43 Figure 13 Bead Capture at 0 5g Density miii 45 Figure 14 Multiple Rows of Single Bead Capture sesenta tnnt tentent ttn 46 Figure 15 Bead Capture at 0 25g Density sies tentat ttt tent tenta tenta tent tot tst tton 46 Figure 16 Det Oger cato AAA oec is nde re e oce itl eunte 47 Figure 17 Poorly Bonded Feature with Bead sessi ENEE 48 Figure 18 Air Bubbles Present in Cell Testing seien tentent trennt tent tentent tnnt 49 Figure 19 sulersi didlp e 54 Figure 20 Tubing and Metal Pins Inserted into Device cnica 54 List of Tables Table 1 Pairwise Comparison Chart eise eene rte ttn tnna tenta tnnt tent tette ttn ttta a nao nis Table 2 Preliminary Data Grid of Wells Device eese tentent ttnntttn tente nnns Authorship All members of the team contributed equally to the writing and editing of this report Acknowledgements The team would like to thank Worcester Polytechnic Institute the Biomedical Engineering department our advisors Professor S
10. Post exposure bake 2 min 65 PIC 7 min 95 BIC 6 Development time 7 min The bake times directly relate to the experimental plan but the UV exposure time must be calculated from the exposure energy relative dose the illumination intensity and an empirical correction factor The illumination intensity of the UV KUB should be stable at 23 4 mW cm2 and the correction factor is 1 5 due to the narrow spectrum of UV exposure at 365 nm For example from the data above the UV exposure time should be 215 mJ cm2 x 1 multiplier x 1 5 correction factor 23 4 mW cm2 13 8 s Procedure 1 Dehydration Bake The dehydration bake removes residual water molecules from the wafer surface by heating up the wafer on a hot plate or convection oven Removing residual moisture increases the adhesion of the photoresist on the substrate 1 Turn on the blower and light on the cleanhood Let it run for a few minutes before working inside 2 Power on the PMC Dataplate hot plate in the clean hood Ensure the hotplate surface is clean 3 Set the desired temperature to 120 BC Press the following buttons in order SET Plate Temp 1 1 2 0 ENT The display cycles between the set temperature and current 63 temperature about once per second 4 Place a clean new wafer onto the hotplate surface The whole wafer should completely fit on the hotplate surface so that heat can conduct evenly to the wafer 5 Once the plate reaches the des
11. The fluorescent beads were added to the Tween and water solution and spun Depending on the desired density 0 25g or 0 5g of the beads and Tween were added to mineral oil and placed in the vertical syringe The device was flushed with oil to clear dust or PDMS particles and the syringes were primed to remove bubbles Hydrostatic pressure was created in 43 the device to initiate flow and flow from the top syringe was turned on causing beads to flow into the device Beads flowing through the main channel and were too small to flow through the small vertical rectangular channels causing some to get trapped in the wells 5 4 Cell Testing Fibroblast cells were used to test in the devices to determine ifthey were compatible with cell culture techniques and to determine ifthe devices were able to capture single cells Since cells were able to go in suspension in the water water and media were used to flush the device and prime the syringes Since a surfactant wasn t being added to the solution Pluronic 127 was pipetted into the device and let sit to coat the sides of the device in order to reduce the clumping of cells A suspension of cells at a density of 20 000 cells 1 mL was added to the top syringe Again hydrostatic pressure was created to initiate flow and cells were allowed to flow through the device 44 Chapter 6 Discussion 6 1 Proof of Concept Testing The results of this device were unique because when using beads the small chan
12. any bubbles or dust remaining 7 Surface bubbles can be removed by mouth blowing from about 10 cm away Deeper bubbles can be left until they rise to the surface Bubbles adherent to the Si or SU8 surface can be dislodged by tilting the vessel back and forth causing shear forces Be careful not to spill any PDMS It s messy sticky and hard to clean off 8 Large dust particles can be moved or aspirated with a disposable transfer pipette 9 Once you are satisfied with the casting place it onto a level shelf in the 65C oven and bake for at least 3hrs Leaving overnight is also OK PROCEDURE 4 Preparing a PDMS device This procedure completes a PDMS device including punching inlet and outlet holes for microfluidic devices 1 Demold the cured PDMS from the Si master Peel off the foil and carefully remove the Si wafer If PDMS coated the underside of the wafer you may need to cut it out with a scalpel or razor blade Store the Si master in a safe place ideally a wafer holder 2 Set up the rubber cutting pad Use a straight razor blade to identify the indentation line that separates adjacent devices if present Then align the razor vertically and apply pressure to complete the cut If 77 necessary move the razor to the next position and cut with downward pressure Do not slide the razor through the PDMS It will deform as you cut 3 Once your device has been trimmed determine the size of any inlet and outlet holes 4 Apply Sco
13. correct exposure If anything is awry immediately press the large red button to abort and retry 3 The exposure will end automatically and alert with a loud beep silence by touching the screen The drawer opens automatically 4 Gently lift the glass slide with wafer tweezers and set aside Gently lift the photomask with wafer tweezers and set aside 5 Observe the resist surface At this point no pattern should be easily visible If it is the exposure time was too long 6 Wave the drawer closed when done exposing then touch the screen and select Cancel Procedure 5 Post Exposure Bake PEB The post exposure bake completes the process of crosslinking a negative resist or solubilizing a positive resist As in the prebake a two step heating and cooling is required to minimize resist layer thermal stresses This step uses the two 7 Dataplate hotplates in the fume hood 70 1 Transfer the wafer from the UV KUB to the 65 FIC hotplate in the fume hood Be sure to place your hand underneath as you move the wafer so it doesn t drop Set the timer for the desired time at this PEB temperature 2 Observe the resist surface With ideal exposure the mask pattern will become slightly visible in 5 30 s Cover with a foil tent 3 Transfer the wafer from the 65 EIC hotplate to the 95 EIC hotplate and cover Set the timer for the desired time at this temperature 4 Return the wafer to the 65 PIC hotplate for 3 minutes
14. created Rather than performing experiments using existing cell lines which may be easy to grow but do not demonstrate the behavior found in rare cells these cell lines could be used in order to create a more realistic cell culture where specific conditions need to be met in order for the experiment to be successful Another application is cell cell interactions By isolating individual cells certain cell types can be forced to interact in order to see their behavior and the way the cells interact with each other Further experimentation can be done by co culturing the individual cells together in hopes of advancing tissue engineering By studying how the cells interact with each other it can help to determine what cells types and environmental conditions are necessary for organ growth Another limitation is that some methods depend on the size of the cell but whether the cells are in in the process of dividing or already divided the cell is always changing its size which makes it difficult to use some methods Carlo 2012 A promising application that stems from single cell analysis is personalized medicine Personalized medicine tailors drug regimens to a specific patient based on how their cells respond to certain therapies While personalized medicine is not the creation of novel drugs for individuals it is the classification of individuals to sub populations who will most benefit from preexisting and defined therapies especially cancer the
15. devices out of a hydrogel After making the PDMS mold a hydrogel is cast over it to create an entirely hydrogel device Variable hydrogel stiffness could be obtained to match the tissue of origin of the cells being studied Also it would be beneficial to conduct work in a clean room Particulate contamination via dust particles frequently clogged channels within the microfluidic device Clogging prevented flow through the device and caused device failure Decreasing the likelihood of dust entering the system would allow devices to function more successfully and over longer periods of time 57 Finally well size could also be altered to tailor the device to more specific application or to isolate more specific cells A variety of well sizes could also be used to isolate from a heterogeneous population as opposed to the homogenous population we used throughout the course of this project 58 References 2015 Single Cell Analysis Program Available https commonfund nih gov Singlecell index D Carlo Methods in Molecular Biology vol 853 New York NY Humana Press 2012 M Chabert and J L Viovy Microfluidic high throughput encapsulation and hydrodynamic self sorting of single cells Proceedings of the National Academy of Sciences vol 105 pp 3191 3196 2008 J Clausell Tormos D Lieber J C Baret A El Harrak O J Miller L Frenz et al Droplet based microfluidic platforms for the encapsulation and screenin
16. hold the key to understanding the human body at its simplest level For example in tumor biopsies there are many different types of cells present By studying the cell population as a whole the average behavior of the cells is studied rather than the behavior of the individual cells Specific cells like cancer stem cells and certain aggressive cancer cells may have very different behavior from a typical cell in the population but their behavior is being shadowed by the other cells Therefore single cell analysis is a technique to overcome the inaccuracy of the current methods Carlo 2012 10 The most common single cell separation and analysis method is flow cytometry Flow cytometry is currently the gold standard because it is incredibly high throughput some 10 000 cells per second can be analyzed but flow cytometry was not designed to perform multiple assays on the same cell Carlo 2012 Flow cytometry is able to collect data from a single cell at a single time point but after the assay is complete cells are discarded as waste This makes it difficult to identify which cells are behaving abnormally to study them further and determine the cause of their behavior Flow cytometry is also a very expensive method of single cell isolation which limits its use to labs that can afford or have access to the equipment In order to create a low cost device for single cell analysis the team was tasked with creating a microfluidic device to iso
17. in mineral oil FIGURE 2 DESIGN PROCESS After establishing each of our objectives and ranking them in order of importance we used them to assess different design ideas A thorough review of current literature on microfluidics gave our team an idea of what is currently being done in terms of single cell separation with microfluidics and what is possible and realistic We were able to use some of our original ideas in combination with concepts demonstrated in contemporary literature to develop a set of preliminary designs Figure 2 demonstrates a brief outline of our design process Each cycle begins with a basic drawing in DraftSight In order to determine the effectiveness of each design the devices need to be fabricated and tested After each design is tested it is either eliminated or improved upon and then the next iteration begins 28 Chapter 4 Alternative Designs 4 1 Needs Analysis Our device needs to isolate single cells and allow for further analysis of individual cells The single cell isolation would create a homogenous cell population so that the behavior of the cells is specific to that individual cell and the testing can take into account the different cell types present in a tumor biopsy We would like the single cells to be able to be removed from the device for further testing but we realize this may not be possible due to the time constraints of our project Our device must create a system to efficiently isolate t
18. the wells With further testing and modifications to the size and shape of the wells this device could have potential We were unable to establish a method of removing any captured cells or beads and we eliminated this design because of its initial inability to capture any single beads Figure 6 FIGURE 6 ALTERNATIVE DESIGN 4 34 4 3 5 Alternative Design 5 This brought us to our last design Figure 7 FIGURE 7 ALTERNATIVE DESIGN 5 Similar to the previous design it would have wells along the horizontal part of the channel Connecting the well and the next horizontal portion of the channel flowing underneath one row of wells would be small rectangles that are smaller than the size of the beads The wells were 60 microns in diameter and the small channels were 20 microns wide designing the pocket in which cells would ideally be captured These small rectangles provided the suction that the beads needed to be isolated in the wells This design was simple and did not require a precise flow rate so hydrostatic pressure could be used to drive fluid flow through the device A microscope image of this device can be seen in Figure 8 35 FIGURE 8 ALTERNATIVE DESIGN 5 MICROSCOPE VIEW 4 4 Feasibility Study Experiments Based on the complexity of using microfluidic devices we determined that removing the cells from the device would not be feasible for our team The process of manufacturing and testing devices included many s
19. 2 M Tibbett and K Anseth Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture vol 103 ed Biotechnol Bioeng 2009 pp 655 663 H Yin and D Marshall Microfluidics for single cell analysis Current Opinion in Biotechnology vol 23 pp 110 119 2 2012 59 Appendices Appendix A Single Cell Isolation Methods Method Advantages Limitations Process Serial Dilution Compatible with standard Manual process labor Cell suspensions are Ishii 2010 microtiter plates easy to intensive time repeatedly diluted until culture consuming low only single cells remain throughput low chance of finding rare cells Microscale Oil Simple no microfabrication Plasma treatment Glass slide is treated to Covered Cell required inexpensive array extends beyond the make it hydrophobic the Array MOCCA formation only takes 2 micropatterend filter and plasma treated with an minutes number of droplets can be changed only requires common laboratory supplies causes larger droplets to be formed Variability in droplet size Most of the process is done manually aluminum screen to make small hydrophilic circles where drops will form Cell suspension is poured over glass followed by mineral oil that forms and seals droplets Flow Cytometry gold standard High throughput up to 10 000 cells s cells can be sampled at multiple time points semi quantitative data Compatible with FACS fluorescence ac
20. Project Number SA2 1401 A Microfluidic Device for Single Cell Isolation A Major Qualifying Project Report WORCESTER POLYTECHNIC INSTITUTE By Meghan Hemond Kelsey Krupp Emily Tierney Date May 2014 Approved Professor Sakthikumar Ambady Professor Dirk Albrecht This report represents the work of WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement WPI routinely publishes these reports on its website without editorial or peer review For more information about the projects program at WPI please see http www wpi edu academics ugradstudies project learning html Abstract There exists a need for inexpensive and efficient methods to isolate single cells especially single tumor cells for single cell analysis to improve treatment methods We developed a microfluidic device that traps single beads ranging from 38 to 45 um similar to mammalian cells Our results suggest our device could trap single beads in 60 um microwells indicating this device could allow isolation of similarly sized cells Our device could be used for pharmacological testing for personalized medicine and other applications Contents ee APA O PE PO O O E Un Taa adaa aA aiia 2 LAS COLES ia 6 IR ee 7 AUTO tods 8 Acknowledgements ici iii 9 Chapter 1 Introduction teet Lee ud A sia dav eee 10 Chapter 2 Literature Review oae rtr ea 13 2 1 History of Cell Culture s 13 22 NA acier eec Dr taedet unde A B
21. Traps Offers dynamic control over cell environment Only capable of short term analysis need thermal control to prevent cell damage Ultrasonic waves create pressure gradients that isolate cells Droplets Low risk of cross contamination between droplets the small volume of droplets allows concentrations to reach detectible levels quickly droplets can be sorted and manipulated cells can be incubated within their droplets drops can be merged or split high throughput 10 7 Risk of drops coalescing stabilizing droplets to prevent this requires the use of expensive surfactants channel dimensions and microfluidic design must be extremely accurate Droplets are formed to encapsulate single cells typically using an aqueous cell suspension surrounded by a carrier oil 62 Appendix B Photolithography Process Preliminary Setup Determine photolithography parameters Before beginning any photolithography process the entire procedure must be planned The primary determinants to spin speeds and duration of baking and development steps are the photoresist material and the desired resist thickness Refer to the photoresist spec sheets for more information For example for a 80um thick process using SU8 2035 we find the following information from the data below 1 Spin speed 1600 rpm 2 Soft bake times 3 min 65 DC 9 min 95 AC 3 Exposure energy 215 mJ cm2 4 Relative dose 1x 5
22. ability The most expensive part of manufacturing the device is the cost of a clean room Assuming a company already had access to a clean room the only costs would be printing photomasks and transferring the designs onto the silicon wafer The photomask is approximately 120 including shipping and the silicon wafers are approximately 7 per wafer The photomask makes 3 wafers so each wafer costs about 47 Each wafer will make 12 devices A company could make the silicon wafers for 47 dollars and send them to labs who would only have to pour the PDMS which would 51 be inexpensive for them For us to pour the PDMS as well each device costs around 4 91 Buta company could sell the silicon wafers instead of manufacturing the PDMS molds so each device would come out to around 3 91 1 silicon wafer being 47 The device is very reproducible Once the design is made in DraftSight the steps following are very standard procedures The photomask is made from the computer image and the design is transferred to the silicon wafer PDMS is then poured over the wafer If the protocols are followed correctly the device will be very reproducible 52 Chapter 7 Final Design and Validation 7 1 Device Fabrication 1 Devices were created using the DraftSight software Features were dimensioned and the polarity was indicated to determine which features were raised and which were channels 2 1 silicon wafer was produced with 12 devices includin
23. ack sides are rinsed in H20 dry both sides with the N2 gun Bring the nozzle close to the wafer and sweep side to side especially in areas with small resist features 6 Inspect the wafer as described in Procedure 7 below and then perform a final cleaning development by holding the wafer with tweezers horizontally over the dish and squirting a small amount of fresh developer on the wafer Gently slosh side to side for about 15s Rinse with H20 and dry with a N2 gun Procedure 7 Inspection Inspection is a step to verify general process quality and the development process This section will outline the main feature distortions that are encountered in photolithography process The Zeiss Stemi 2000 stereo microscope is equipped with a fiber optic light ring and is used to visualize the wafer in reflectance mode After initial development and rinsing the wafer will appear dirty This is OK Itis due to the resist that has dissolved in the developer and will be cleaned to a shiny surface after brief wash with fresh developer Also sharp corners and large resist fields will likely display surface cracks This is also OK It is due to the thermal stresses during bakes which were minimized by gradual heating and cooling but not fully eliminated These cracks will be eliminated with the Post bake Procedure 8 1 Development time Pay attention to the smallest features in the resist pattern Lines should be sharp with no evidence of resist ma
24. akthikumar Ambady BME and Professor Dirk Albrecht BME for their guidance with the project Lisa Wall BME Lab Manager and Ross Lagoy and Laura Aurilio for their assistance with device fabrication Chapter 1 Introduction Despite all of the major technological advances over the last century basic laboratory and cell culture techniques have remained nearly the same Scientists are comfortable with the techniques they are using they are well understood and they have been standardized to make results easier to produce interpret and share with scientists around the world Despite the advantages of using these techniques the tremendous opportunities to improve upon them should not be ignored Current cell analysis techniques have two issues that must be addressed in order for more accurate cell analysis to be performed cells are cultured in heterogeneous populations and data is recorded on bulk properties of these cell populations Both bulk analysis and heterogeneous population samples add a layer of complexity to cell culture Bulk analysis only presents the average behavior of the cells and nuanced behaviors may be misrepresented or masked While a population may appear homogeneous rare cell types may exist within the population that display many interesting and unique properties and their behaviors may be masked Tibbett and Anseth 2009 If these cells cannot be studied individually we are unable to understand these behaviors which may
25. cally Remove the glass slide if present Mask alignment stage 1 Transfer the room temperature resist coated wafer to the UVKUB tray centering it in the circular pattern 2 Observe the position of any defects in the resist layer You will try to rotate your photomask such that these defects are removed during development i e they are covered with black mask regions if a negative resist or are covered with clear mask region if a positive resist 3 Cut out the photomask circle using scissors taking care not to kink the transparency film Ensure it is free of dust and gently wipe with a lint free cleanroom wipe or blow with the N2 gun if necessary 4 Place the photomask over the resist coated wafer and orient it such that any defects will be removed during development 5 Place the 4 x 5 glass slide over the wafer and mask to keep it flat and in direct contact First tilt the 5 side to the back corner supports then gently move it toward you so it rests on the bottom tray surface 69 Finally gently lower the glass plate onto the wafer ensuring it is fully covering the mask and water and that it did not move the mask while lowering Exposure stage 1 When you are satisfied with the mask orientation and glass plate placement wave the door closed Touch the screen 2 When is asks What do you want to do touch Continue on the screen The last used program will begin automatically after 1 2s Verify the
26. cause of the costs of a clean room and the photomasks but after those are acquired the cost is cheap All that is needed to produce the devices would be silicon wafers and the materials to make PDMS These devices while they have demonstrated potential have not been high throughput up until this point Optimization of suspension density and minimization of dust are factors that could greatly improve the throughput of our device Once our device yields a higher throughput we would be able to determine how well the device meets the objectives of accuracy and precision We were unable to obtain numerical data and further testing is required to determine the accuracy and precision of this device To obtain this data we would want to flow bead suspensions through the device until wells were filled We would then calculate the percentage of single beads isolated in wells compared to empty wells or wells containing multiple beads We would run these trials in triplicate and then repeat these same tests with a suspension of PANC1 cells While we are able to make very preliminary assessments about the success of our device more testing is required and more data must be gathered before any conclusive statements can be made Our trials were not reproducible and adjustments to the device protocol must be made 6 3 Design Considerations 6 3 1 Economics and Society Our device provides a low cost method for single cell isolation leading to the possibility o
27. ce because these could later be utilized to help removed the cells from the device These were formed because the channels were too small to allow any water to enter After flowing the water media was flowed through to coat the device before flowing cells When the fibroblasts entered the devices they were too small to get trapped in the wells The cells would flow into the wells and the small channels and none were getting isolated Because of time constraints we were not able to change the cell type or the size of the device For future testing of these devices the first method of testing would be using a larger cell type like the PANC1 cells BE Le FIGURE 18 AIR BUBBLES PRESENT IN CELL TESTING Our device was able to meet some of the objectives we established for this project The device is able to be used with common microscopes We have been able to use our devices successfully with both a fluorescent and a light microscope Our device is also compatible with common cell culture techniques It has the ability to be used in the hood and it is able to be sterilized by autoclaving which is a common sterilization technique that is available in most labs Microfluidic devices made of PDMS are frequently used for cell culture applications and while we 49 did not specifically test for cell viability we can assume they will be biocompatible It is also relatively inexpensive The cost to start producing these devices would be expensive be
28. cells span a diverse spectrum There are a number of mechanisms utilized and the processes vary greatly in complexity A general overview of the methods that have been used successfully in the past will highlight the main advantages and limitations of each method to exemplify how they are selected for specific applications Here we present a sampling of common single cell isolation techniques A more detailed list can be found in Appendix A 16 One of the earliest and simplest methods of single cell isolation is serial dilution This process entails repeatedly diluting a cell suspension until only single cells remain Serial dilution is not a complicated process and requires only basic lab equipment such as micropipettes microtiter well plates and a microscope Since the process results in cells contained within microwells the cells are accessible and are compatible with many assays and standard cell culture techniques Ishii 2010 Despite the simplicity of this method many of its limitations derive from its lack of automation This process is done manually and is therefore very labor intensive and time consuming The technique has very low throughput as a result which reduces the probability of finding target cells especially if they are rare cell types Ishii 2010 Another method that is relatively straightforward and fits the capabilities of most labs is micropatterning Micropatterning can utilize a variety of techniques material
29. combinations and surface treatments depending on the application Micropatterning uses different surface modification mechanisms to create cytophilic and cytophobic regions that guide cell attachment By designing cytophilic regions only large enough to permit a single cell to adhere single cell isolation is achieved An advantage of this method is that the cell containing regions can be made into any size or shape to adjust to specific cell types and the pattern can be scaled up to achieve desired throughput Another attractive feature of this methodology is that the cells are accessible and therefore easily maintained Media and other supplemental nutrients can be flowed over the immobilized cells for convenient exchange but one must consider the shear stress caused by media perfusion and ensure it will not result in cell lysis Different techniques of micropatterning vary in complexity For example this process can be simplified so that it doesn t require a microfabrication process 17 and can be done with standard laboratory materials as described by Lin et al 2009 in a Microscale Oil Covered Cell Array MOCCA In this process a glass slide is treated to make it hydrophobic It is then plasma treated while covered with a micropatterned aluminum screen that created small hydrophilic circles where cell containing drops will form on the slide A cell suspension is poured over the treated glass slide followed by mineral oil that forms and seals dro
30. cud de E ru tno de ER eda 14 ZAS APplCaONS e 15 2 4 Current single cell analysis devices ENEE 16 ZAM Gold standard e 16 2 4 2 Single Cell Isolation Methods sssini is ENEE 16 2 4 3 Limitations of Current Single Cell Analysis Techniques mociones 20 PESE atte 20 2 5 1 eet IO e 20 Chapter 3 Project rate iii iaia Rn kcu Renee ae n adn tenu Saee 23 3 TOTAL B ICSU ENTE IE I 23 3 2 ObJectiVeS ui RU RERO RERUM Eer E EE EHI 23 3 3 Revised Client Statement iua caede sese a am de Uva S ra eR Dn d ERE ode 27 JA Project ee Te 28 Chapter 4 Alternative Designs viii 29 4 1 Needs Analysis 2st a tao 29 4 2 Functions Specifications nyisiran EES 29 4 3 Conceptual Designs eet Deet Eege 30 4 3 1 Alternative Design EE 30 4 3 2 Alternative Design 2 ese ee a tierce eebe dr toni pui Dto det cep SEA eege qe 31 4 3 3 Alternative Design 3 assirinassi drania niaaa iiaeaa idadaan tame Kitana e Dae ck getan Lade 33 4 3 4 Alternative Design 4yrs iiaia p cer tin RIDERE tee dE nnt EENS 34 4 3 5 Alternative Design Dt A ee AA rod uctor 35 4 4 Feasibility Study Experiments ies tentem tentent ttn ttn tent totas ttt tta tento tse tttn stas 36 45 Modeling EE Ee 36 4 5 1 Design Calculations dace deti Dee iet ia ae eto deno dde 36 EA le EE 37 4 5 3 Optimization iia 37 4 6 Prelimi
31. d SV A hissing noise should be heard in the chamber 78 2 Close both valves on the round metal door Align it to the glass vacuum chamber and after a few seconds ensure that itis firmly held onto the chamber Support it and do not let it drop 3 Start a timer After about 15s turn on the power and set power level to High A purple glow should be visible through the vent holes after a few seconds 4 Once a purple glowing plasma is visible slowly open the needle valve a very small amount to let in room air and oxygen The plasma should brighten and become more orange If it dims too much close the needle valve slightly and observe the bright plasma return after a few seconds 5 Allow the chamber to clean for 1 2 minutes 6 When plasma treatment is completed turn off the unit power and the vacuum pump power Slowly open the exhaust valve until the vacuum has been released HOLD ONTO THE DOOR or it will fall PDMS Bonding 1 Optional Prepare a test bonding sample such as a scrap of clean PDMS and a clean glass fragment or two PDMS scraps Remove dust with tape Then follow Steps 2 10 and if successful repeat Steps 2 10 with the desired parts to be bonded 2 Seal the PDMS on the tray slide with treatment side facing up Next to it place the glass fragment or the second PDMS piece 3 Insert the tray into the chamber Ensure the door valves are closed turn on the vacuum pump and align the door until it is held in place
32. e bte a aote adt itte 63 Procedure 2 Spin co ting iecur dit sg dtu ge rr de rp d due Eed 64 Procedure 3 Prebake Soft Bake cie ddr dae t e i dg decern 67 Procedure 4 UV Enges t De dete tie de ite a A edd bred eret 68 Procedure 5 Post Exposure Bake PERL ENEE 70 Procedure 6 Developmierit eee da tetra ithe di rd pee nc Pei inca RE RE 71 Procedure 7 Inspection gege tede n dede s fade E bnt di deo d EH india 72 Procedure 8 Post bake ee recte n etdeuiDnied eege 73 Appendix C Soft Lithography SOP eene 75 PROCEDURE 1 Fluorination of the Micropatterned Substrate ees 75 PROCEDURE 2 Preparing the PDMS Mixture sessi eerte trennen tante ttt tton tette tton 76 PROCEDURE 3 Casting and Curing PDMS essere tente tentat tnnt tente tetto 76 PROCEDURE 4 Preparing a PDMS device isst tttn rentre tte tton 77 PROCEDURE 5 Plasma Bonding rite te eee tene tdt ete ee ice ode 78 PDMS BOMBA 79 Appendix D Tween 20 Surfactant esee trennen tette tttn teta tanto ttti tonto a 81 Preparing Tween Solutions eiat E cnet enue ded Rn de dE n ecce 81 Appendix E BME Educational Objectives essere tentent tent tante ttn ttta tenta 83 List of Figures Figure 1 Objectives ret ii i 24 j ASPERIS I Pr ds 28 Figure 3 Alternative Design Tennis 30 Figure 4 Alternative Design Li ha a a 32 Figure 5 Alternative Design Benin ENEE 33 Figure 6 Alternative DESTA 4 teet
33. e sufficient for a mixed cell population When the device is designed a specific well size is chosen and all the wells are the same size If there were varying cell sizes only some of the wells would be trapping single cells because some may be trapping 2 smaller cells or they would be unable to trap the larger cells Another problem with this device is that cells would not be able to be easily removed from the device A biopsy punch could be used to punch out a cell of interest but once the cells started growing the cells would become mixed populations if they were to flow out of the microfluidic device Though we liked the simplicity of this device we decided to pursue a different device that would be easier to use and would be useful for a wider variety of applications 4 3 2 Alternative Design 2 We created a device that uses a droplet generator as the mechanism of single cell isolation This design idea came from a recent publication Chabert 2008 In this device a cell suspension would flow through the center channel where it is met by two streams of mineral oil one on each side in a flow focusing channel This forces the formation of droplets ideally capturing one cell in each droplet The droplets are then sorted using 31 hydrodynamic flow so that that large cell containing droplets drift to one side of a barrier while empty droplets move to the other This leads empty droplets to flow out one outlet to be discarded while ce
34. ells is created each well being small enough that only a single cell can fit within each Micropatterning Large arrays can be made to increase throughput many Only compatible with adherent cells that will A surface is treated to make cytophilic and 60 different combinations of surface treatments have been used cells can be replenished easily by flowing media or nutrients over the array bind to the surface can t easily remove cells of interest substances used to attract and bind cells can affect their behavior flowing media over cells can cause shear stresses cytophobic regions that guide cell attachment and arranges single cells in an array Mechanical Compatible with microscopy Not designed for long Cell suspension flows over Traps high throughput time term analysis lt 24hrs traps that physically efficient cells can be flow has to be separate single cells organized into arrays of traps considered to prevent cell damage Compatible with cell culture can be transferred out of traps Magnetic Traps Specific cell types of cells of Sort term analysis Uses immunomagnetic interest can be selectively magnetic components labeling or binds a sorted out cells can be could have an effect on magnetic marker to cells sorted according to a variety cells so they can be sorted and of factors trapped when they interact with a magnetic field at designated time points Hydrody
35. et separately i multiple realistic constraints economic environmental social political ethical health and safety manufacturability page s 54 55 ii appropriate engineering standards page s 23 28 4 An ability to function on multidisciplinary teams 3d page s 8 6 Anunderstanding of professional and ethical responsibilities 3f i Professional page s 23 27 ii Ethical page s 54 7 Anability to communicate effectively 3g page 24 8 The broad education necessary to understand the impact of engineering solutions in a global economic environmental and societal context 3h both economic AND environmental need to be addressed i Economic page s 54 ii Environmental page s 54 10 A knowledge of contemporary issues 3j page s 13 20 83
36. f analyzing gene expression or clonal expansion for varied applications such as development of pure populations of cells drug and molecule testing The device size can also be increased to lead to higher throughput and increased cost effectiveness 50 6 3 2 Environmental Impact The devices and associated set up are single use only and would therefore create some plastic waste However only the research community would be using these devices and the impact should remain relatively small The protocol could be optimized to reduce waste and this would also increase the sustainability of the device 6 3 3 Political Ramifications This project has very minimal projected political ramifications This device would be used for research purposes and would therefore have limited impact on cultures of other countries even though it may affect the culture of scientific research by producing a shift in the paradigm of cell analysis and traditional culture techniques 6 3 4 Ethics Our project follows good ethical practices because it does not require any animal or human testing The only testing done in our devices uses previously established cell lines When eventually using human tumor samples privacy considerations should be upheld to protect patient confidentiality 6 3 5 Health and Safety As long as the device is used for the purposes described in the report we do not see any health and safety concerns for users 6 3 6 Manufactur
37. fficult to get flow from hydrostatic pressure in the device Oil had to be used because the beads would not stay in suspension when they were in water The beads wanted to stick to the sides of the syringe so they would not flow into the device Therefore we decided to use mineral oil but because of the change in viscosity between the mineral oil and water or media the flow rate drastically changed and it was more difficult to achieve natural flow without forcing fluid into the device This often caused the 3 way valve to get clogged with oil and beads and would prevent anything from flowing into the device Another challenge that occurred when flowing fluid through the device was dust or PDMS particles clogging the channels Because we were not in a clean room and not under sterile 46 conditions dust or remnant PDMS particles were often appearing in the device after it was flushed initially with oil Since the channels were only 80 microns wide this meant that single particles of dust or PDMS would completely clog the channels and not allow beads to get trapped in the wells Figure 16 amp FIGURE 16 DusT CLOG We also faced challenges in the proper fabrication of our device We needed to incorporate small features into our device to capture beads and cells but plasma bonding such small features posed a problem In Figure 17 we show a bead that was able to flow under small features that had not been plasma bonded to the glass s
38. g 2013 Microfluidics is an emerging field that has been show to effectively isolate single cells and culture cells in three dimensional constructs over a period of time In these systems it is easier to control the cells The suspension flows into the device and the geometry and channels arrange the cells to be cultured These microfluidic devices typically incorporate a network of small channels that range from about 10 microns to 200 microns in width The microfluidic systems can have very specific designs for certain studies making them more customizable than a simple petri dish For example there can be gradients valves channels wells or pillars incorporated into the device Microfluidics is beneficial for single cell analysis because features like wells or pillars can be used to capture the single cells and allow them to stay isolated from each other Microfluidics has the ability to control fluids at a very small scale and can create systems with laminar flow rather than turbulent flow Using different types of flow driven by either hydrostatic pressure or syringe pumps gives precise control of flow rates in the devices and allows cells to be processed without being damaged by rapid or uncontrolled flow rates Mehling and Tay 2014 Like common cell culture techniques microfluidics can allow cells to be maintained over a long period of time but the system is more automated 21 because there is limited need for pipetting fluids
39. g of Mammalian cells and multicellular organisms Chem Biol vol 15 pp 427 37 May 2008 A Folch Introduction to BioMEMS Boca Raton FL CRC Press 2013 HT Stec Ltd 2014 October Single Cell Technologies Trends 2014 Online Available http www reportsnreports com reports 29962 single cell technologies trends 2014 html X Huang W Hui C Hao W Yue M Yang Y Cui et al On site formation of emulsions by controlled air plugs Small vol 10 pp 758 65 Feb 26 2014 S Ishii K Tago and K Senoo Single cell analysis and isolation for microbiology and biotechnology methods and applications Applied Microbiology and Biotechnology vol 86 pp 1281 1292 2010 L I Lin S h Chao and D R Meldrum Practical Microfabrication Free Device for Single Cell Isolation PLoS ONE vol 4 p e6710 2009 S Lindstrom and H Andersson Svahn Overview of single cell analyses microdevices and applications Lab on a Chip vol 10 pp 3363 3372 2010 L Mazutis J Gilbert W L Ung D A Weitz A D Griffiths and J A Heyman Single cell analysis and sorting using droplet based microfluidics Nature Protocols vol 8 p 870 2013 M Mehling and S Tay Microfluidic cell culture Current Opinion in Biotechnology vol 25 pp 95 102 2 2014 A M Streets and Y Huang Chip in a lab Microfluidics for next generation life science research in Biomicrofluidics vol 7 ed United States 2013 p 1130
40. g some duplicates 3 Onthe photomask the features that would stay as solid PDMS were clear and the channels wells or inlet outlet holes were black 4 The Designs were sent to CAD Art Services Inc in order for a photomask to be produced with our devices 5 Using the standard photolithography process described in Appendix B the designs on the photomask were transferred to a silicon wafer 6 PDMS was then poured over the wafer and baked at 65 degrees C overnight after the wafer was fluorinated the full soft lithography process is described in Appendix C 7 The devices were cut out from the PDMS slab inlet and outlet holes were punched and the device was plasma bonded to a glass slide The protocol for plasma bonding is also described in Appendix C 8 Devices were then ready to be tested 7 2 Device Setup 1 Athree way valve was connected to two syringes and a luer valve The luer valve was then attached to plastic tubing The syringe setup is shown in Figure 19 53 FIGURE 19 SYRINGE SET UP 2 One syringe held about 5 mL of the suspension to flow into the device The second syringe had about 1 mL ofa solution used to flush the device to minimize dust before the solution would flow into the device 3 The plastic tubing connected to the luer valve was inserted using a metal pin into the inlet of the device shown in Figure 20 A second set of tubing and pin was inserted into the outlet and ran into a small petri dish to col
41. he array it is not possible to remove particular cells of interest or to manipulate single cells since all 18 cells are exposed to the same factors Lacking the ability to move or target specific cells restricts the possibility of further processing or expansion Microdroplets are an additional method of single cell isolation Droplets can be generated using multiple techniques and usually result in a single cell that is encapsulated in an aqueous solution surrounded by a carrier oil Microdroplet formation allows for high throughput some have been shown to generate droplets at rates exceeding 10 000 000 per second However not all of these droplets contain single cells and the percentage of successful single isolation may be lower than is desirable Lindstrom and H Andersson Svahn 2010 The volume of microdroplets usually ranges from several nanoliters to microliters Mazutis et al 2013 and allows the droplet to function as a microreactor for the encapsulated cell The small volume allows the cell s secretions to quickly change the concentration within the droplet to detectable levels and this information can in turn be used to analyze or sort cells Mazutis et al 2013 The individual droplets don t allow cross contamination between drops and do not allow cells to influence each other as long as coalescence is prevented In order to lower the risk of coalescence drops need to be stabilized usually with the use of a biocompatible fl
42. he nitrogen gun which is located on the right side of the fume hood 65 8 Position the 4 wafer alignment tool against the chuck and using wafer tweezers or your gloved hand touching only the edge place the wafer on the chuck aligning to the marks on the alignment tool 9 Before removing the alignment tool press the Vacuum button A hiss should be audible and the display should change from Need vacuum to Ready The wafer should now be held down on the chuck 10 Test your alignment by beginning the spin program Press START and observe the edge of the wafer as it turns It should wobble less than 5 mm If not press STOP then Vacuum to release the vacuum realign and return to step 8 Reset the spin program if necessary by pressing Edit Mode then Run Mode and ensuring the display reads Ready Coating Stage 1 Ensure the wafer is centered and the spin coater is programmed and ready to spin 2 For SU8 2035 photoresists and similar high viscosity materials pour the resist directly from a 50 mL conical tube It will flow very slowly Pour approximately 8 10 mL of resist onto the wafer in one continuous motion with the tube far enough to avoid contact with the wafer but close enough to prevent thin filaments of resist from forming about 1 cm Once the resist blob covers about 5cm diameter quickly move the tube toward the edge while tilting the tube upwards and twisting to prevent drips on the outside
43. he silver power button on the front panel lower right The touchscreen should light up and display UV KUB 2 Touch the screen to reach the main menu Touch Settings and Drawer to unlock the drawer Wave your hand near the door sensor at the lower left to open the drawer If there is a wafer or mask present remove them Place the 4 x 5 glass slide on the tray and wave near the door sensor to close it 3 Return to the Settings menu touch the X in the upper right of the screen Touch Illumination to calibrate the UV intensity It should display about 23 4 mW cm2 through the glass plate If not adjust your exposure time calculations in Preliminary Setup 4 Return to the main menu and select Full Surface then New cycle then Continuous 68 5 Program the desired exposure duration and intensity Enter the time using the touchscreen numbers then a unit h m s for hours minutes seconds then v to confirm Note that decimal values are not permitted so round to the nearest second Next enter the intensity in usually 100 and v to confirm 6 Test the exposure by touching Insolate The drawer will open Wave it closed The display should read Loading in Progress Touch the screen to start the exposure Verify that the countdown timer begins at the proper duration 7 The exposure will end automatically and alert with a loud beep silence by touching the screen The drawer will open automati
44. he single cells It is preferable that the method of separation is a semi automated microfluidic device based on the client statement Preferred method of separation Separation mechanism must not cause cell lysis Cells must be10 microns apart to be considered isolated 4 2 Functions Specifications The function of our device is to capture single cells in wells To accomplish this successfully the cells must remain isolated and not contact any other captured cells The device must capture single cells so that when treatments are tested using our device the researcher can see the behavior of every individual cell and not just a representative behavior of the entire population of cells A specification of our device is that minimally 5096 of the wells have to be filled with a single cell Some may have more than one cell or no cells This specification is necessary to create a high throughput system The single cells are the ones that will be studied so this specification ensures that there will be a higher number of single cells to investigate 29 4 3 Conceptual Designs 4 3 1 Alternative Design 1 The first device we tried was based on the publication Microscale Oil Covered Cell Array Lin et al 2009 We created a simple system where a cell suspension would be poured over a grid of microwells The suspension would be manually spread over the top surface of the PDMS to ensure cells have spread over the entire surface and reached all of t
45. he wells The goal was to have cells fall into wells that were just slightly bigger than the size of the cells so that no more than one cell could fit in each well After cells are allowed to settle into wells and the excess suspension is removed from the top surface a second microfluidic device would be flipped over and placed on top of the grid of wells The top device would have wells much bigger than the cells and would have channels connecting the wells that allow media to flow through them The diameter of the bottom wells ranged from 15 to 100 microns whereas the wells on the top device were 800 microns in diameter The top device would be the method of cell culture media perfusion The larger well size on the top would also allow for the cells to expand Figure 3 FIGURE 3 ALTERNATIVE DESIGN 1 30 After creating this device and conducting preliminary testing we discovered the difficulty associated with aligning the larger top device above the smaller wells We included square markers in the upper corners of each device to assist with alignment but the devices must be placed under a microscope to properly visualize these place markers This restriction limits the reproducibility and ease of use of this device because misalignment of the two devices is likely to cause malfunction We were able to capture single cells in over 50 of the wells in the bottom device but another reason we did not choose this device is because it would not b
46. hniques is that they do not prevent contamination between different cell types For accurate single cell analysis techniques this is essential During the process of some separation techniques the cells can be exposed to residues or chemicals and this adds an uncontrolled variable that could influence cell behavior which is another disadvantage Carlo 2012 2 5 Microfluidics 2 5 1 Why use microfluidics The goal of our project is to create a device that is inexpensive reproducible and marketable to scientists and researchers The device should also be able to view the cells over a biologically significant time period A method that has a high throughput is a necessity for our device The device needs to trap cells in a way that allows further experimentation to be performed Microfluidics is a technique that has been proven to successfully isolate single cells and expand them over a relevant time period beyond the scope of most single cell analysis techniques 20 Microfluidics is the study of fluids in the scale of nanometers to a few hundred microns Microfluidic applications were previously restricted to silicon based devices but have since expanded to life science applications since the development of soft lithography techniques that have allowed for polymer based applications Microfluidics are appealing to those performing research in cell biology because of their small size customization and diagnostic potential Streets and Huan
47. increase the chances of success with one of these devices 4 6 Preliminary Data TABLE 2 PRELIMINARY DATA GRID OF WELLS DEVICE Device Desired Cells Well Cell Density Empty Wells Single Cell Wells Capture Efficiency EMEN 40um 1 49 38271605 60 um 1 4400000 67 48971193 This table shows the preliminary data collected from the grid of wells device Alternative Design 1 After pouring the cell suspension over the device and letting the cells fall into the wells the wells were inspected to determine whether there was a cell present in the well Table 2 shows out of 81 wells how many wells were empty or filled Though this device had success capturing cells it could not be determined if there was one 37 cell or more than one cell in each well As described above there were other characteristics of this device that resulted in discontinuing experiments with the grid of microwells 38 Chapter 5 Design Verification This chapter verifies that we met our ranked objectives which were compatibility with common cell culture techniques compatible with common microscopes accurate precise inexpensive and high throughput Testing our devices with fluorescent beads verified compatibility with microscopes accuracy precision and the ability to be high throughput Testing of our devices with fibroblast cells verified the compatibility with common cell culture technique 5 1 Device Fabrication Devices were created using
48. ired temperature heat for 5 min To set a timer press the following buttons in order SET Timer h m 4 5 ENT Or SET Timer m s 5 5 0 0 ENT 6 Carefully remove from the hotplate with wafer tweezers and allow to cool to room temperature The wafer is now ready for the next procedure Procedure 2 Spin coating Spin coating is a step to apply photoresist onto the wafer This section will outline the steps of spin coating SU 8 a common type of negative photoresist that is used in the MicroFabrication Laboratory The procedure is similar for AZ1512 a positive photoresist except itis deposited via syringe rather than pouring due to its lower viscosity This step uses the Laurell spin coater in the fume hood Preparation stage 1 Turn on the spin coater using the left power strip switch under the fume hood If the display does not light up turn on the unit power switch at the back of the unit 2 Turn on the two 7 Dataplate hotplates Figure 5 using the right power strip switch under the fume hood and set the left one to 65 RIC and the right one to 95 PIC as in Proc 1 Step 2 above Note the 5 button sticks on one hotplate so use 96 PIC if necessary If foil is absent damaged or dirty replace with new foil 3 Press Select Process and choose the appropriate spin program according to your desired parameters If none exist yet you must enter a new spin program Refer to the User Manual or
49. isolate single beads from a solution These same principles can be applied to a cell suspension and the device could be used to isolate single cells from a tumor biopsy sample 56 Chapter 8 Conclusions and Recommendations Our device isolated single polyethylene beads If more design iterations were to be performed we believe there is demonstrated potential to isolate single cells from a cell suspension as well Large scale pharmaceutical testing could be done on these cells for applications in personalized medicine The cells would remain in their own wells to ensure that their behavior was from that one specific cell making it easier to understand how the patient s individual cells react to the specific drugs Though our device isolated single polyethylene beads from suspension the next step in development should allow for a retrieval method of these single beads or eventually cells Our device provides minimal space for the cells to grow and expand so the cells would not be viable in this device for a significant period of time If the isolated cells are retrieved from our device and transferred to a microfluidic cell culture platform more effective analysis could be conducted This device or a subsequent device could also be manufactured out of a hydrogel such as gelatin Cell culture would then occur in a three dimensional environment more closely mimicking the way they would grow in vivo There are existing protocols for creating
50. late single cells Since single cells should be trapped within micron sized devices using low flow rates that prevent cell damage and allow the cells to be cultured after isolation Microfluidics can be used as a high throughput method which is ideal for single cell analysis applications If rare cell types are of interest there is likely only a few in the cell population so the more cells that are isolated the higher the chance of seeing the individual cells of interest Our ranked objectives are that the device must be compatible with common cell culture techniques compatible with common microscopes accurate precise inexpensive and high throughput This device requires single cells to be trapped in order to study each cell individually Media must also be delivered to the cells as they are studied in the device To fabricate these devices a Computer Aided Design program called DraftSight and standard 11 photolithography techniques are used to transfer the designs to a silicon water from which PDMS polydimethylsiloxane devices can be fabricated To perform proof of concept testing CosphericO polyethylene fluorescent beads were used in a suspension of mineral oil in the device The beads were approximately the same size as PANC1 cells in suspension we believe the behavior of the bead suspension would mimic the behavior of cells within the device In the remainder of the report we will provide background into the differe
51. ld Single cell analysis is a field that has developed rapidly in the last decade but it still needs significant improvement and development before it can reach its full potential At this point people are taking many different approaches in order to determine what works best but there are still many unmet needs The importance of single cell analysis has caught the attention of the US National Institutes of Health NIH The agency launched a program to fund advances in single cell research with a budget of 90 million over five years The NIH recognizes the current shortcomings and challenges that come along with single cell analysis but they also recognize the importance of this research and the potential that it has to improve our understanding of cell responses which will aid in better detection and treatment of diseases This program funds research in a wide array of disciplines and applications and is geared toward changing the field of single cell analysis from a small highly specialized group of researchers and making it more widely used and accessible by promoting commercialization Single Cell Analysis Program 2015 14 2 3 Applications In order to further improve single cell analysis simple and reproducible techniques should be developed There also exists a need to develop a method for culturing and expanding single cells for an extended period of time If rare cells are captured and expanded cell lines of rare cell types can be
52. lect the fluid FIGURE 20 TUBING AND METAL PINS INSERTED INTO DEVICE 54 7 3 Proof of Concept Testing 1 The detailed protocol to make the Tween20 surfactant is described in Appendix D 2 About 90 uL of the Tween20 was added to 100 mL of boiling DI water and mixed for about 30 seconds This created a 0 196 Tween solution 3 About 2 0 mL of the Tween and water solution was added to 5g of the fluorescent beads 4 The solution with the beads was spun for 5 10 minutes and the clumped beads from the top ofthe conical tube were removed 5 Then 0 25g or 0 5g beads Depending on the desired density of the suspension and Tween were added to 10 mL mineral oil 6 The conical tube was inverted to mix the beads into the oil 7 Approximately 5 mL of the solution was added to the top vertical syringe 8 Approximately 1 mL of mineral oil from the left horizontal syringe was pushed through the device to clean out any dust particles 9 The syringes were primed to remove bubbles 10 The syringe setup was placed about 12 inches above the device to create hydrostatic pressure For this device the flow rate did not need to be precise so the height during each trial could vary The flow from the top syringe was turned on allowing the beads to flow into the device at the inlet hole 11 Beads flowed through the main channels but could not pass through the small horizontal channels This would cause some of the beads to get trapped in the wells
53. lide For future work the aspect ratio height width could be adjusted to increase the stability of these features We developed our silicon wafers with a height of 80 microns A shorter height may increase stability and the likelihood of features bonding appropriately to the glass slide 47 a IA A eco FIGURE 17 POORLY BONDED FEATURE WITH BEAD Optimization of suspension density and minimization of dust are factors that could greatly improve the throughput of our device Once our device yields a higher throughput we would be able to determine how well the device meets the objectives of accuracy and precision We were unable to obtain numerical data and further testing is required to determine the accuracy and precision of this device To obtain this data we would want to flow bead suspensions through the device until wells were filled We would then calculate the percentage of single beads isolated in wells compared to empty wells or wells containing multiple beads We would run these trials in triplicate and then repeat these same tests with a suspension of PANC1 cells 48 6 2 Cell Testing After getting results with the fluorescent beads we tested the device with cells To start we used human primary fibroblast cells When flowing water through the device to start there were air bubbles that were formed in the small rectangular channels underneath the wells Figure 18 The air bubbles were a positive result of this devi
54. ll containing droplets flow to the other outlet where they can be removed or put in a second device where further testing and manipulation can occur This device relies on delicate flowrates that dictate how efficiently the system works and these would have to be adjusted and finely tuned for each cell type which takes away from the device s adaptability Another drawback of this device is that it required fluorinated oils and surfactants to keep droplets from merging together and these far exceeded the budget of this project We decided this device would not be feasible for our team to use Figure 4 FIGURE 4 ALTERNATIVE DESIGN 2 32 4 3 3 Alternative Design 3 Next we designed a device based on a serpentine channel with one well on each side of the horizontal portions of the main channel The wells have a tiny channel connecting them which creates a valve system In this device a cell suspension flows from the inlet to the outlet ideally capturing a single cell in each well When cells pass the wells the downward flow through the tiny channel would pull them downwards and trap them in the well Once a cell is trapped it blocks the tiny channel and cuts off the downward flow preventing other cells from drifting into the well with it This device would allow for media perfusion so cells can be cultured within the device or they could be easily removed by reversing the flow from the outlet to the inlet which would push cells out of the
55. ls or a 5096 response in all cells and therefore averages can be misleading due to the difficulty of differentiating between the two scenarios Yin and Marshall 2012 There is mounting evidence regarding the cellular differences that are found in isogenic and clonal populations which were previously assumed to be identical throughout The population of cells present in tumors shows vast heterogeneity which makes them a particularly important application of single cell analysis Cancerous cells exhibit rapid changes in their genetic make up due to either genetic drift the rate of replication and age of that cell or the processes occurring and proteins that cell is expressing Yin and Marshall 2012 Individual cells exhibit unique behavior in regards to protein expression and metabolic activity Therefore the oversimplification of bulk analysis is problematic 13 because it neglects this cell to cell variability In order to understand the heterogeneity and inner workings of a cell population each cell has to be analyzed individually This enables researchers to study the factors that influence individual cell behavior and understand what causes the fundamental differences between cells These differences dictate cell to cell interactions and it is important to see how the behavior of one cell can influence those around it and how that affects the overall health and function of the entire population Yin and Marshall 2012 2 2 Growing fie
56. namic High throughput cells can be Short term analysis Most common method of Traps placed in an array potential harm or cell cell trapping in compatible with non adherent cells damage must be considered microfluidics uses small channels or holes next to the main channel that allow enough flow through them to trap single cells as they pass by Optical Traps Very high precision and control of cellular arrangement can be used to selectively move cells of interest has been improved to handle higher throughput Cells can be moved within enclosed chambers bc no physical contact is needed compatible w cell culture Extremely expensive laser energy can cause increased heat and photodamage that can harm cells careful complex planning and good understanding is required to prevent photodamage Optical tweezers focused laser beams cells are trapped at the focus point of the laser beam where they can then be repositioned in any direction Dielectrophoretic Traps High throughput 10 000 cells s allows heat removal that prevent cell damage sensitive enough to detect a rare event and sort cells according to it Controlling more cells increases complexity of the design Cells are moved by forces generated in a non uniform electric field that direct them If target cells can be labeled and bound to a polystyrene bead they can be sorted from a population 61 Acoustic
57. nary Dat efle rn ibit ta tuas 37 Chapter 5 Design Verification ii 39 5 1 Device Rabatt uiia uio ai 39 Ren D EE 43 5 3 Proof of Concept Testing seescessecssesessessssstesstessesssesssestecneesseeseecaeestesseeseeseesseeneesseeseeeaeeaeeneessesseesaeeateaneeseeatenseens 43 54 Cell Testibg EE 44 Chapter 6 Discussion zii tein t Fe b tenni ie e S Sg id en rt o fte eei ete tue 45 6 1 Proof of Concept Testing repe tte pep tir e tr det ae en pue i erin aede tarda 45 6 2 Cell Testing A A e DO ROI RR a ade tu Re Ede RE 49 6 3 Design Considerations eite d e acd dtd ga ca anata cca 50 6 3 1 Economies and SoClety i eis A d das hia E ARA 50 6 3 2 Environmental IMpaACt omnia 51 6 3 3 Political Ramificatioris Sisa 51 NR Mu c D 51 6 3 5 Healthrand Safety itte nde e ege gen pe tite pepe penus 51 6 3 6 Manufacturability EEN 51 Chapter 7 Final Design and Validation ENEE 53 ZiT Deyice FAN Seege 53 4 2 Device E A ER 53 7 3 Proof of Concept Testing iia 55 PA Cell Testingzsn oe a a Pr dos ide 55 Chapter 8 Conclusions and Recommendations miii tente tnnt tent te ntn tttn nns 57 RACE M 59 ees te 60 Appendix A Single Cell Isolation Methode EEN 60 Appendix B Photolithography Process setenta ten tttn tentent ttn ttt tent te stets ta nunten 63 Preliminary Setup Determine photolithography parameters enn 63 Procedure 1 Dehydration Bake use ee
58. nels underneath the wells created suction because of the oil flowing in the main channel above as well as below the small channels The suction was able to pull the beads into the wells causing them to get trapped and remain in the wells as others flowed past them in the main channel The design of this device was based on a previous publication On site formation of emulsions by controlled air plugs Huang 2014 where they used a similar device to create air bubbles within their device We modified the design and operating protocol to allow us to isolate single beads or cells and then contain them individually within droplets By varying the density of the beads in the suspension the number of beads getting trapped in the wells would change Using 0 5g of beads in the suspension was creatinga density of beads that was too high and multiple beads were getting trapped in one well usually up to three beads per well Figure 13 B 250 um FIGURE 13 BEAD CAPTURE AT 0 5G DENSITY When the amount of beads was reduced to 0 25g single beads were trapped in the 60 micron diameter wells Figure 14 45 ha L AO A FIGURE 15 BEAD CAPTURE AT 0 25G DENSITY Using one of the bigger devices resulted in beads getting trapped in multiple rows of the Ver tr Rer ERT device shown in Figure 15 79 A 2 9 s89 FIGURE 14 MULTIPLE ROWS OF SINGLE BEAD CAPTURE Working with an oil suspension for the beads made it very di
59. nt methods of single cell isolation and how they compare to each other We will also provide some background into microfluidics We will discuss our objectives constraints and functions and then explain the approach of our project We will then provide alternative designs and the reasons behind our design choices Later we will explain the experiments that we ran and discuss the results Finally we will draw conclusions from our experiments and discuss the overall functionality of our device 12 Chapter 2 Literature Review 2 1 History of Cell Culture Tissue culture was first introduced in the early 1900 s and has been widely used ever since Initially it allowed scientists to maintain cells and tissues in vitro so they could perform experiments and study them over time which was imperative to understanding basic biology and living systems Today cell culture is a universally accepted practice that influences many different industries and has enabled us to do things that wouldn t have been imaginable 100 years ago Traditionally cells are both cultured and analyzed as an entire population and the results are the average behavior of all the cells within that population This requires the assumption that the average response is representative of a typical cell in the population which is not necessarily accurate For example an average of 5096 protein expression in a cell population can represent either a 10096 response in half the cel
60. of the tube 3 Press the START button of the spinner to start spin coating The spin coating process takes about 1 minute depending on the program OPTIONAL Near the end of the second spin step use a piece of Al foil rolled into a rod to collect resist streams that fly off of the wafer Do not touch the edge but bring the rod close This will clean up the resist at the edge and somewhat 66 reduce the edge bead or thicker later at the edge due that forms due to surface tension 4 The spinner will stop automatically when spin coating is completed 5 Verify that the photoresist has been uniformly coated If striations and streaks are observed the spin coating was not successful Some causes may include dust particles on the surface clean it better bubbles in the photoresist heat the resist tube to 40 50 PIC in a water bath to remove them see resist datasheet for more information insufficient resist volume applied 6 Press Vacuum to release the chuck vacuum 7 When the last wafer has been coated close the vacuum and CDA valves at the N2 tank Procedure 3 Prebake Soft Bake The prebake Soft Bake procedure is required to densify the photoresist following spin coating and evaporate the solvent In order to reduce thermal stresses due to the substantial difference in coefficient of thermal expansion between Si and resist the temperature should be raised and lowered gradually in a 2 step process first a
61. oject would be to develop a system that would isolate and expand single cells from tissue biopsies The system created must be able to trap and expand the cells in micron sized hydrogels of varying stiffness representing different tissues Ideally the system should allow placement and or arrangement of cell laden microgels to produce precise geometries that can facilitate organ engineering tissue engineering and the study and analysis of cell cell interactions Our client expressed a desire for a microfluidic device because of the range of applications it s compatible with Microfluidics is an emerging field and is best suited to the resources available here at WPI There are opportunities to introduce a novel concept to the field of single cell analysis using microfluidic devices that are cost efficient After further research we determined it was necessary to expand the scope of our project 3 2 Objectives Using our initial client statement we established a list of objectives that would need to be met in order to successfully complete the project to the satisfaction of our client These objectives can be seen in the objective tree below in Figure 1 and are further explained after 23 System for Single Cell Isolation ooo Oe with Ce wit nexpensive Culture Microscopes Throughput Light amp Isolates 1 Consistently Adaptable Fluorescent Cell at a Isolates 356 Budget Time Single Cells ransportable Fit in Slide Holder
62. plets as it moves across the slide This process is done manually without the requirement of controlled flow rates that require significant background knowledge and planning to achieve This makes the process extremely practical and it appeals to more users Lin et al 2009 Micropatterning can also incorporate photolithography to create more intricate patterns For example McDevitt et al 2001 used laminin coatings on polystyrene tissue culture plates to encourage cardiomyocytes to assemble single file and form multinucleated myofibrils A linear pattern was designed and soft lithography was used to create a PDMS stamp of the device Laminin was applied to the stamp and the design was then transferred to a polystyrene tissue culture plate Individual cardiomyocytes were able to adhere to only the laminin patterned areas that were one cell wide The researchers then saw cell fusion and coordinated contractile activity McDevitt et al 2001 Here micropatterning was used to manipulate individual cells to form complex arrangements typically found exclusively in vivo to create a more accurate platform for studying cardiac activity at the cellular level Using photolithography and soft lithography significantly elevates the complexity of the process and creates a need for more expensive and complicated equipment A limitation of these methods is that they are only compatible with adherent cells that are capable of binding to the surface Once cells are in t
63. rapeutics Ideally a patient s tumor sample would be isolated into single cells then exposed to different drugs 15 so doctors can determine which treatment is most effective for that patient and the specific type of cancer cells present Cancers vary widely because of their rapidly changing genome Cancer cells even within the same tumor biopsy sample display distinct molecular differences which lead to the development of different assays to provide prognoses Bates 2010 Tailoring treatments to patients who are most likely to respond to them has incredible potential for improving cancer prognosis and patient outcomes 2 4 Current single cell analysis devices 2 4 1 Gold standard Currently a majority of single cell analysis is done using flow cytometry With flow cytometry hundreds of thousands of cells can be analyzed per minute The cells can be sorted by their size granularity and fluorescence properties Carlo 2012 The method of flow cytometry does not require a lot of time and is easy to perform Though many researchers scientists medical workers etc use flow cytometry for the goals of our project a combination of other methods seem to be more beneficial Flow cytometry requires expensive devices and the cells are discarded after each test so the cells cannot be studied over a more relevant period of time or beyond a single experiment 2 4 2 Single Cell Isolation Methods The different methods of isolating single
64. sicator inside a fume hood Line the bottom surface with foil if damaged missing or dirty Prepare a support ring cardboard or other material and line up Si wafers or glass slides along the inner part of the ring with the side to be treated facing inwards 2 Make aluminum foil boat big enough to hold 40 uL about 1 drop and place in the center of the platform CAUTION Tridecafluoro 1 1 2 2 tetrahydrooctyl trichlorosilane TFOCS Gelest SIT8174 0 or United Chemical Technology 6H 9283 is corrosive and toxic Avoid direct contact and always handle it in the fume hood 3 Pipette 40ul of the TFOCS chemical directly from stock bottle and place into the aluminum foil boat you just made Remove the pipette tip by hand and gently place into the vacuum chamber Do not eject it 4 Close the chamber and vacuum for 1 hour 5 After 1 hr remove the treated Si wafers or glass If any hazy film appears remove with 15 30s contact with isopropanol rinse with water and dry in an air stream 6 Fluorinated pieces are ready to use right away Verify hydrophobicity by observing contact angle of water drops on the treated surface Water drops should roll off the surface leaving it dry 7 After a few hours the chemical liquid will have evaporated Discard foil boat and pipette tip in hood waste bag 75 PROCEDURE 2 Preparing the PDMS Mixture This procedure prepares a PDMS mixture for casting We use Sylgard 184 which comes as a kit with Part
65. t Precise 1 0 1 0 5 1 1 3 5 Accurate 1 0 1 0 5 1 1 3 5 Compatible 0 5 1 1 0 0 1 3 5 with Microscopes Varity of Cell 0 0 0 0 0 0 0 Types 26 After identifying each of our objectives our team utilized a pairwise comparison chart to establish a ranking of our objectives according to their importance and relevance to our project In this chart each of the objectives are compared to the others one by one In each comparison the more important objective receives a 1 and the less important objective receives a 0 If the two objectives are of equal importance they each receive a score of 0 5 These scores are then totaled horizontally and their final score determines their ranking with the highest scoring being most important and the lowest scoring being least important 3 3 Revised Client Statement Our final client statement reads as follows The aim of this project is to develop an efficient system to isolate single cells from tissue biopsies Ideally the device should allow multiple applications such as a sorting of single cells from cell lines or tissue biopsies for clonal expansion and analysis personalized medicine b high throughput ability to screen multiple pharmacological agents on hundreds of clonally expanded cells 27 3 4 Project Approach Design Draftsight Software Assessment Fabrication Eliminate or Photolithography improve designs amp Bonding Testing 35 48 um beads
66. t 65 BC then at 95 BIC then back to 65 PIC This step uses the two 7 Dataplate hotplates in the fume hood 1 Use the removal tool to transfer the wafer from the spinner chuck to the 65 PIC hotplate Set the timer for the desired time at this temperature and cover the wafer with a foil tent 2 Transfer the wafer from the 65 BC hotplate to the 95PIC hotplate Set the timer for the desired time at this temperature and cover with a foil tent Use wafer tweezers to lift up the edge but 67 don t grab the wafer edge since the resist is still very soft Instead slide the removal tool underneath and lift 3 Return the wafer to the 65 BC hotplate for 3 minutes covered then transfer it to the clean hood to cool to room temperature Be sure to place your hand underneath as you move the wafer from the fume hood to clean hood if you drop it it ll shatter Procedure 4 UV exposure The UV exposure procedure exposes the photoresist layer to collimated 365 nm UV light via an LED source through a photomask A negative resist becomes cross linked and insoluble in developer when exposed whereas a positive photoresist becomes soluble in developer when exposed This procedure assumes that a transparency photomask will be used in direct contact with the resist layer This step uses the UV KUB exposure system in the clean hood Preparation stage 1 Turn on the UV KUB via the power switch at the back left just above the power cord Press t
67. tch Magic tape to the micropatterned side If desired mark the center of each hole for easier viewing 5 Flip over the device tape and channel side on the rubber cutting pad 6 Using a dermal punch of desired diameter punch downward and in a straight line until contact with the rubber cutting pad 7 Lift up the device leaving the punch inserted and a cored PDMS piece should protrude from the channel tape side Remove it before gently removing the punch 8 Repeat steps 6 7 until all holes are punched 9 Clean the punched holes by squirting water through each hole with a wash bottle Repeat with ethanol and water again Then dry in an air stream This process removes any PDMS particles that may have been left behind during punching PROCEDURE 5 Plasma Bonding This procedure covalently binds PDMS to glass Si or PDMS by oxygen plasma treatment of clean surfaces After plasma activation surfaces are brought into contact forming an instant and irreversible bond Oxygen plasma is also useful for cleaning substrates and vaporizing organic materials This is a relatively slow process and it will remove organic thin films not clean off dust Materials and equipment needed glass tray test slide and scrap PDMS piece tape plasma cleaner vacuum pump Plasma bonder cleaner setup Set up required only if plasma system has not been used recently 1 Turn on the vacuum pump and open the specialty vacuum valve on the fume hood labele
68. teps and took a long time therefore creating a device solely for single cell isolation would be the only process feasible in our timeframe The next step in improving our device would be creating a method to remove the cells from the wells and place them in another microfluidic device where they could be cultured for a longer period of time in order for long term studies to be performed 4 5 Modeling 4 5 1 Design Calculations We trypsinized PANC1 cells to determine what size would be required for the wells to be able to isolate just one cell The trypsinized cells were approximately 40 microns so we decided to design the wells to have a diameter of 60 microns This size would allow enough room for a single cell to settle in the well and prevent another from entering The 36 small vertical rectangular channel underneath the well had a width of 20 microns so this prevents the cell from flowing through 4 5 2 Decisions Because of our budget we decided that using the expensive surfactants needed for the droplet generator or the design iterations that required a new photomask for the other designs were not possible for our team This left the air pocket design with the wells as the primary option for our team 4 5 3 Optimization We decided to create our device out of PDMS because it would allow us to make more devices for a lower cost We also produced designs with a variety of well sizes on the same photomask to reduce cost and
69. terial in regions where it should be removed If not development is incomplete Return the wafer to the developer bath and repeat for 30s then rinse dry and re inspect Instead if the 72 resist layer that should remain looks especially cloudy or rough the wafer may be over developed Additionally overdevelopment may narrow a resist feature or widen a resist hole and underdevelopment may do the opposite 2 Bake times and temperatures The extent to which a feature deviates from its ideal size is a function of the exposure time prebake temperature prebake time development temperature and development time Any of these parameters could be the cause for overdevelopment or underdevelopment and it is therefore important that one understand some important troubleshooting techniques The key idea troubleshoot the distorted feature is to observe the effect of changing a parameter while holding the other parameters at constant The following example illustrates this idea It can be observed that by changing the exposure time while holding the other parameters at constant there is atime window where the feature size is optimal i e between 15s and 25s in this example If the changing of this parameter does not produce the desired feature size the problems are most likely to be caused by other parameters or combinations of several parameters Repeated troubleshooting with other parameters should be carried out Procedure 8 Post bake
70. tivated cell sorting single cells can be encapsulated in droplets and cultured It wasn t designed to work with single cells one cell can t be followed or identified over time expensive Flow cytometry FCM is an approach to quantitatively analyze multiple characteristics of millions of single cells and other particulate matter from a heterogeneous population Brehm Stecher and Johnson 2004 Microscopy automated microscopy high throughput microscopy ect Time dependent data can be collected and a single cell can be followed qualitative data regarding cell division and expansion Low throughput multiple cell parameters can t be analyzed a lot of time is spent collecting data cumbersome process not ideal for single cells difficult to get single cells in wells Cells are fixed or placed in a multi well plate and a microscope takes hundreds of pictures of them then they go through automated image analysis to find useful information Microwells The number of wells and their shape size depth and dimensions can be customized according to cell types and applications Different materials and fabrication methods can be used Capable of holding cells for a longer period of time Compatible with microscopy Throughput is limited to the number of wells It is difficult to remove cells of interest from the array it has to be done manually Cells are mechanically separated An array of w
71. to hot water and mix with immersion mixer for about 30 seconds Wait until water cools and any bubbles have settled before using solution When finished the solution should look clear and uniform Using Tween Solutions to Suspend Particles The specifics of this section will pertain to creating Cospheric s Density Marker Beads DMB products which are defined to have 2096 solids in 2 5ml The process is easily modified to other situations Using a 2 5ml vial add 0 5g of the desired microspheres Any container and hydrophobic particles may be used Using a pipette or syringe dispense 2 0ml of the 0 196 Tween solution on top of the spheres 81 http Weigh the microsphere Tween solution and add any additional Tween solution to each vial to ensure vial weights are equal to better balance the centrifuge while itis operation Secure the cap on the vial Centrifuge for 5 10 minutes to get the spheres wetted and into solution www cospheric com tween solutions density marker beads htm 82 Appendix E BME Educational Objectives 3 An ability to design a system component or process to meet desired needs within realistic constraints such as economic environmental social political ethical health and safety manufacturability and sustainability ABET 3c while incorporating appropriate engineering standards ABET Criterion 5 need to assess each of these separately but since or and such as not all need to be m
72. ugh they may overflow This pops many of them and reduces the likelihood of spillage 7 Degas for 1 hr Atthis point all bubbles should be gone and PDMS is ready to pour in Procedure 3 Be careful when releasing vacuum Air rushing in could knock over the PDMS boats PROCEDURE 3 Casting and Curing PDMS During this procedure mixed PDMS is poured over the Si SU8 mold master in a dish or foil vessel bubbles and or dust particles are removed and the PDMS is cured by baking at 65C for 3hrs 76 1 Prepare casting vessels by bending a foil sheet over the bottom of a 500 mL Erlenmeyer flask Flatten the edges until they are about 10 15mm high Ensure the bottom surface is flat 2 Set up the masters to be cast on the bench top covered with absorbent mats and a paper towel If dust is visible blow with the air gun Weigh the master and vessel recording the weight 3 Once the PDMS mixture has been degassed for 1hr and surface bubbles are gone bring them to the masters 4 Pour PDMS mixture across the wafer from one side to the other in a continuous movement This reduces the number of bubbles formed At this stage only the wafer needs to be covered 5 Weigh the PDMS master vessel and subtract the master and vessel weight About 60g PDMS is the target If more is needed bring the vessel back to the absorbent pad and pour more Repeat until the desired PDMS weight is achieved 6 Cover to prevent dust and observe after a few minutes
73. uorinated surfactant These surfactants can be very expensive around 1 000 mL One of the most appealing characteristics of cell containing microdroplets is that they can be sorted and manipulated in many ways while still maintaining integrity and isolation Droplets are compatible with cell culture and are shown to survive for several days without being removed from their original droplets Clausell Tormos et al 2008 All of these characteristics make this method versatile and give it great potential in current and future applications 19 2 4 3 Limitations of Current Single Cell Analysis Techniques Limitations of some previous techniques are that they are not high throughput Only a few cells can be tested at a time so a large number of tests have to be performed one after another This is much less efficient than running parallel experiments where all cells can be tested at once under the same conditions Another limitation is that cells are not able to be analyzed over a long period of time Often cells are discarded mixed and not cultured in media so they will not survive long enough to be analyzed further Also since many experiments are done on a larger scale on 96 or 364 well plates the environment is much larger than the size of the cell so it s hard to control the environment of the cell The experiments and assays that can be performed are also limited because of the single cell analysis method Another disadvantage to some tec
74. wells When testing the device we discovered that the channels connecting the wells were too wide so our suspension flowed in a vertical line from the inlet to the outlet rather than back and forth through the channels and nothing was trapped in the wells Time constraints prevented us from redesigning this device with more appropriately sized wells and channels but we do believe this device has potential as a simple method of single cell isolation Figure 5 FIGURE 5 ALTERNATIVE DESIGN 3 33 4 3 4 Alternative Design 4 The next device that was tested was a simple serpentine device From the inlet to the outlet the device had one channel shaped as a serpentine that was aligned vertically On the horizontal segments of the channel there were 3 wells that took the shape of a semicircle and were approximately 60 microns in diameter In this device cells would start in the inlet and flow through the single channel towards the outlet The goal was to have cells fall in the wells as they flowed through the device Since the wells ideally would only be large enough to hold one cell single cells would be isolated When watching the flow through the microscope as the device was being tested there were beads flowing over the tops of the wells without being trapped We determined that the flow rate was too high Once we lowered the flow rate the beads were still not getting trapped there was nothing pulling the beads from the main channel into
75. which reduces user variability and gives more predictable and reproducible results Microfluidics also offers precise control over the microenvironment of the cells due to the small volumes of reagents used and the microenvironment can be adjusted to more closely mimic the in vivo environment Folch 2013 Since the volumes of reagents required are on the nanoliter scale devices are cost efficient to run and produce very little waste Another advantage to microfluidics is the high throughput Microfluidic devices allow for parallel experiments so many cells can be tested at once to yield a large quantity of results Folch 2013 For example droplet generation is an efficient method of isolating single cells in a microfluidic device while retaining high throughput This technique preserves the viability of cells and allows for cells to be manipulated within the droplet rather than having to be removed from the system and manipulated manually with a micropipette Mazutis et al 2013 We have provided the necessary background for understanding all aspects of our project Single cell analysis by way of single cell isolation and single cell culture has been explained in depth Next we will talk about the objectives and constraints of our projects and the specific functions of our designs 22 Chapter 3 Project Strategy 3 1 Initial Client Statement After speaking with our advisor the initial client statement read The aim of the pr
76. y features High Throughput This objective isn t essential in order for our device to function however it would be a desirable feature It would make the device compatible with applications that require a large number of cells to be processed and screened and it would make it more marketable However 100 is the median number of isolated single cells used per experiment Single Cell Technologies Trends 2014 so this is a realistic number to aim for Primary Objectives e compatible with common cell culture techniques e compatible with common microscopes e accurate precise e inexpensive 25 e high throughput Secondary Objectives e compatible with 2D and 3D cell culture e compatible with a variety of cell culture techniques e compatible with light fluorescent microscopes e should be transparent e should be transportable e should fit in a typical slide holder e should capture a single cell e should be able to capture a large number of single cells at once e should not exceed 356 We then ranked our objectives using a pairwise comparison shown below in Table 1 TABLE 1 PAIRWISE COMPARISON CHART Pairwise Comparison Chart Compatible Compatible Variety with Cell High with of Cell Culture Inexpensive Throughput Precise Accurate Microscopes Types Score Compatible 1 1 0 0 0 5 1 3 5 with Cell Culture Inexpensive 0 0 1 1 0 1 3 High 0 1 0 0 0 1 2 Throughpu

A Microfluidic Device for Single Cell Isolation

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