System and methods for sampling materials
Contents
1. a rotary mechanism through the dispenser The freezer transports the containers to a derived location for example by use of a carousel to one of the stationary addresses For illustrative purposes only the carousel and stationary nests may have a combined capacity of 1 000 standard microplates It is con templated that various capacities may be designed An advantage of the present invention operation in a stand alone mode or can be integrated into a completely automated laboratory The systems are scalable to meet the needs of small laboratories as well as large institutions that require long term storage of large numbers of samples Another advantage of the present invention is that the apparatus can be designed as a slide in unit for existing ultra cold freezers which will keep the majority of the hardware in the door so as to be insulated from the freezer compartment minimizing both the number of low temperature hardware components and the actual alteration to the freezer itself However the freezer as described herein may be altered to accommodate additional features of the systems A reduced number of mov ing components continuously exposed to the design tempera ture of about 80 C which reduces the cost of production Further advantages of the present invention are attributed to the improved sample quality lowered operating costs accuracy of clone picking and reduced maintenance of the automated storage and retrieval apparat2. container The container is ejected from the freezer through the dispenser It should be noted that the motors for the freezer transfer device the addressable sampler and can be a variety of types of motors known to those skilled in the art including but not limited thereto servo motors and stepper motors or any direct current DC motor with suitable position or velocity control lers In the various preferred embodiments disclosed herein the motors are mounted outside of the freezer to extend the life of the component and improve the overall serviceability ofthe apparatus In one embodiment the servomotors may be of a SMART MOTOR by Animatics Corp These types of servo motors are microprocessor controlled ensuring accu rate placement and monitoring of the robotics operating within the critical environment however any position or velocity controlled motors may be used Motors are mounted near the particular component they are controlling The general features of the present invention control sys tem will be discussed as shown in the block diagram of FIG 1 The control system interfaces with a computer system that may be integral with the housing or remote via a wire or wireless communication or any combination thereof More over the control system may be in communication with and integrated with a laboratory information management system LIMS The control system is operatively connected with the various motors actuators positi
3. hollow solid or slotted In one embodiment the method further comprises moni toring the temperature of the tip In another embodiment the tip comprises a volume control mechanism In another embodiment sampling comprises replicating a sample plate Other embodiments are disclosed infra BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 is a flow chart of an exemplary system FIG 2 is a schematic of an exemplary system showing an automated freezer with dispenser a transfer device plate handler the addressable sampler picking robot tip tip heater temperature sensor the controller destination plates and reader device FIG 3 depicts a side view of a component of one embodi ment of a transfer device FIG 4 depicts a top view of a component of one embodi ment of a transfer device FIG 5 depicts a side view of a component of one embodi ment of an addressable sampler FIG 6 depicts a top view of a component of one embodi ment of an addressable sampler FIG 7 depicts a side view of a component of one embodi ment of an addressable sampler component e g a rotating tip station FIG 8 depicts a top view of a component of one embodi ment of an addressable sampler component e g a rotating tip station FIG 9A depicts a side view of a component of one embodi ment of a climate controlled receiving chamber FIG 9B depicts a top view of a component of the embodi ment of the climate controlled receiving chamber illustr
4. sampler the tip 505 and related assembly In this embodiment the tip is passively heated by a hot air gun and no heating assembly is included In some embodiments a heating assembly is included in the tip assembly The tip assembly as shown comprises a magnetic tip holder 503 a spring compensator 502 and an adapter 501 FIG 6 depicts a top view of a component of one embodiment of an addressable sampler adapter 601 with four spring com pensators 602 FIG 7 depicts a side view of a component of one embodiment of an addressable sampler e g a rotating tip rack 704 and the heater 702 The tip rack 704 is rotated by a motor 701 so that each tip is exposed to hot air from the hot air nozzle 702 The tips are rotated every 1 s to 2 minutes depending on the temperature desired for sampling FIG 8 depicts anther top view of a component of one embodiment of an addressable sampler e g a rotating tip station the tip rack 801 with four positions for tips 803 Tt is envisioned that a control system and computer system can be accessed directly by using the touch screen interface and or remotely by a stand alone personal computer or with a local area network LAN Next details of the climate controlled receiving chamber will be provided as best shown in FIGS 9A and 9B A particularly desirable feature is that the climate controlled chamber 902 prevents ambient humid air from entering the interior of freezer compartment 901 during storage con
5. samples and the samples frozen Inone embodiment a plate handler replaces used plate lids to avoid ice buildup on source plates which is problematic In one embodiment the lids are coded to be read by the reading device In other embodiments the sample plates are coded In one embodiment the system further comprises a desti nation plate handler The destination plate handler may delid and relid destination plates and transfer them to a plate hotel refrigerator freezer or other incubation station In one embodiment low profile 96 well Genetix X 6011 or 384 well Genetix X7001 plates with or without samples are stored in the freezer with the plastic lid on The system in one embodiment stores samples e g plas mid libraries in bacterial cells yeast knockout collections etc in a 80 C automatic freezer BSU BioPhile loads and unloads plates to and from the freezer and picks individual isolates from frozen source plates to destination plates The system is adapted to transfer samples from for example individual sample containers 96 well to 384 well or 384 well to 96 well format with 96 pin Genetix X5054 the system is also adapted to replicate plates e g replicate from 384 well to 384 well with 200 nl pin tool In certain embodiments the system includes for example the following components 1 Biophile BSU automatic freezer 2 Adept Cobra s600 robot component of the plate transfer device 3 Zymark Twis
6. to a piece of blotting paper Liquid transfer was visible as a spot of liquid for 51 sequential picks After this point no further liquid could be transferred Therefore the average volume transferred per pick from a 384 well source plate is approximately 1 uL Reproducibility of Viable Cell Transfers Lack of Cross Con tamination and Estimate of Number of Viable Cells Trans ferred on Picking Tip Starting materials loaded into BioPhile BSU Saturated cultures of Escherichia coli DH10B T1 strain containing an Ultimate ORF clone from Invitrogen Corporation in LB 50 micGm ml kanamycin 10 w v glycerol and Sac charomyces cerevisiae strain 4741 ho kanMX in YPD medium 200 micGm ml G418 10 w v glycerol These cultures were grown in 96 well plate wells low profile Genetix plates in 150 uL to saturation at 30 C The cultures were each diluted 10 fold 100 fold and 1000 fold into the appropriate medium as defined above These source plates were covered with sterile foil seals as well as the plastic lid and then frozen at 80 C by placing them in the BioPhile The concentration of viable cells in the undiluted starting cultures was approximately as follows S cerevisiae 108 cells per mL or 10 cells per uL E coli 10 cells per mL or 10 cells per uL The destination plates contained the appropriate growth medium They were left open to the air during the entire picking operation No special procedures were used to min
7. 10B and 10C at least 10 cells uL were viably transferred even at the highest source well dilution using this procedure What is claimed is 1 A system for retrieving a subset of a stored sample comprising an automated freezer comprising a dispenser a climate controlled receiving chamber adjacent to the dispenser a transfer device within the receiving chamber a robot operatively connected to the transfer device and positioned to access the subset of a stored sample wherein the robot comprises a tip said tip being selected from the group consisting of a metal tip with high ther mal heat capacity metal pin a ceramic pin a hollow pin and a core sampler for retrieving a portion of a sample and wherein the tip comprises a volume control mechanism 2 The system of claim 1 wherein the dispenser commu nicates with the climate controlled receiving chamber through a port 3 The system of claim 1 wherein the robot comprises a sample locator or a rotating tip station for maintaining sam pling tips at a sampling temperature 4 The system of claim 3 wherein the sample locator com prises a barcode reader 5 The system of claim 1 further comprising a tip sterilizer positioned in functional proximity to the robot 6 The system of claim 1 further comprising one or more destination plates for receiving retrieved samples positioned in functional proximity to the robot 7 The system of claim 1 wherein the automated freezer
8. A oe ak a AN NN I LL BIER RE A RN RARE ae 7 7 E VOT Sls U S Patent Dec 31 2013 Sheet 7 of 7 US 8 616 072 B2 FIG 11B FIG 11A US 8 616 072 B2 1 SYSTEM AND METHODS FOR SAMPLING MATERIALS RELATED APPLICATIONS This application claims the benefit of U S Provisional Application No 60 772 767 filed on Feb 13 2006 which is incorporated herein by reference in its entirety BACKGROUND Large biomaterial and chemical library collections require precise and selective access to a subset of material under sterile conditions The materials are often stored in multiwell plates at low temperature where each individual sample is stored in one of the compartments or wells The format of multiwell plates include for example 96 and 384 wells as well as other formats Sample plates have an ensemble of wells with a registered position for each well Such ensembles of wells may be physically linked or in a separable rack The ability to access large subsets of these materials with preci sion and without cross contamination and to avoid thawing is important to maintain the integrity of samples and material collections which may be of limited supply In addition there is also a need in the art to have a system that can without human intervention access a subset of the wells in a collec tion of source plates take a portion or all of a sample and re array the sample into a destination plate s according to need SUMM
9. ARY The present invention provides methods to store retrieve and sample a subset of frozen biological and other material e g drug and candidate drug substances while minimizing contamination and or damage to surrounding samples It fur ther describes an integrated system that allows efficient man agement of large complex libraries of biomaterials or other substances In one aspect provided herein are systems for retrieving a subset of a stored sample comprising an automated freezer comprising a dispenser a climate controlled receiving cham ber adjacent to the dispenser a transfer device plate handler within the receiving chamber and an addressable sampler picking robot In one embodiment the dispenser communicates with the climate controlled receiving chamber through a port In one embodiment the addressable sampler comprises a tip In another embodiment the tip comprises a metal tip with high thermal heat capacity metal pin a ceramic pin a hollow pin a core sampler for retrieving a portion of a sample In another embodiment the tip comprises a tip head In another related embodiment the tip is disposable In another related embodiment the tip comprises a volume control mechanism In one embodiment the system further comprises a heat sensor to monitor the temperature in the vicinity of the tip In one embodiment the addressable sampler comprises one or more of a sample locator a rotating tip station for main
10. US008616072B2 az United States Patent 10 Patent No US 8 616 072 B2 Boeke et al 45 Date of Patent Dec 31 2013 54 SYSTEM AND METHODS FOR SAMPLING 51 Int Cl MATERIALS GOIN 1 00 2006 01 75 Inventors Jef D Boeke Baltimore MD US Min 62 ee s 73 863 11 Li Lutherville MD US Heng Zhu SPC sce seriei nisi Towson MD US Shunyou Long 58 Field of Classification Search Lutherville MD US None See application file for complete search history 73 Assignee The Johns Hopkins University Baltimore MD US 56 References Cited Notice Subject to any disclaimer the term of this U S PATENT DOCUMENTS patent is extended or adjusted under 35 x USC 154 5 by 297 days 5267400 A 12 1993 Howells 22 73 86332 5 361 528 A 11 1994 Peacock s 43 6 5 21 Appl No 12 223 885 6 444 171 B1 9 2002 Sakazume et al 422 65 7 022 528 B2 4 2006 Avdeefetal 436 172 22 PCT Filed Feb 13 2007 2003 0026732 Al 2 2003 Gordon et al 2004 0014097 A1 1 2004 McGlennenetal 435 6 2005 0188705 A1 9 2005 Jones etal occ 62 86 86 PCT No PCT US2007 004139 2007 0014692 A1 1 2007 Erbetal wee 422 82 11 371 OQ 2007 0105214 Al 5 2007 Micklash et al 435 306 1 2 4 Date Aug 9 2010 cited by examiner 87 PCT Pub No WO2007 095366 Primary Examiner Robert R Raevis PCT Pub Date Aug 23 2007 74 Attorney Agent or Firm John
11. a container from a freezer transferring the sample to an addressable sampler and sampling the sample and transferring the sample to the freezer In one embodiment the method further comprises delid ding a sample plate in a climate controlled chamber In one embodiment the method further comprises deter mining the location of the sample in the container prior to sampling In one embodiment retrieving the sample comprises eject ing the container to an exterior of the freezer for transport to an addressable sampler In one embodiment sampling comprises transiently thaw ing at least a portion of the sample In another embodiment the sample rapidly refreezes upon return to the freezer In one embodiment the container com prises a multiwell plate In another embodiment adjacent samples to the sample being sampled are not thawed In one embodiment the method further comprises sealing the container with a seal prior to freezing in the freezer a first time In another embodiment the seal comprises a foil seal a plastic seal a polymer seal or a septum In one embodiment the method further comprises placing a lid on the sealed container prior to freezing the sample before and or after sampling In one embodiment the method further comprises trans ferring the portion of the sample to a destination plate In one embodiment the method further comprises sam pling one or more further samples from the container In one embodimen
12. a portion of a sample 13 The system of claim 12 wherein the tip comprises a tip head 14 The system of claim 12 wherein the tip is disposable 15 A method for sampling a frozen sample comprising retrieving a sample in a container from a freezer transferring the container to a climate controlled receiving chamber and sampling the sample with a robot and transferring the con tainer to the freezer 16 The method of claim 15 further comprising delidding the container in the climate controlled chamber 17 The method of claim 15 wherein retrieving the sample comprises ejecting the container to an exterior of the freezer for transport to the robot 18 The method of claim 15 wherein sampling comprises transiently thawing at least a portion of the sample 19 The method of claim 15 wherein the sample rapidly refreezes upon return to the freezer 20 The method of claim 15 wherein the container com prises a multiwell plate 21 The method of claim 20 wherein adjacent samples to the sample being sampled are not thawed 22 The method of claim 15 further comprising sealing the container with a seal prior to freezing in the freezer a first time 20 25 30 35 40 i 23 The method of claim 22 wherein the seal comprises a foil seal a plastic seal a polymer seal or a septum
13. arbon dioxide or the like that rapidly reduces ambient humidity to any desired level e g less than about 25 relative humidity RH and as low as about 1 RH In fact any compressed gas from which moisture has been removed will reduce the humidity in the airlock and will cool the airlock by adiabatic expansion to about 10 C to about 0 C or as desired Preferred embodiments of the present invention an auto mated storage and retrieval apparatus and related method thereof operate at an ultra low temperature from about 50 C to about 90 C It should be understood that the apparatus may operate in a range of 50 C up to ambient temperature or greater The normal design operating temperature of the freezer compartment ofthe present invention is about 80 C It should be noted that the present invention is contemplated to operate at conditions colder than ultra low temperatures in the range of about 140 C to about 90 C Conveniently if the freezer fails for whatever reason maintenance or sched uled outage then liquid carbon dioxide can be pumped into the system and keep it at approximately 78 C Thus the ultra low freezer set point of the apparatus can be backed up by installing a cylinder of liquid carbon dioxide One skilled in the art would appreciate that various types and substitutes for interior and exterior chamber doors can be used Moreover a single door can be utilized which can rotated between interio
14. ated in FIG 9A FIG 10A shows yeast S cerevisiae plates FIG 10B shows bacterial E coli plates FIG 10C shows heat map repre sentation of bacterial growth as detected by a 96 well plate reader measuring A600 Blue represents background read ings and shades of red represent different extents of growth FIG 11A depicts yeast S cerevisiae plates and FIG 11B shows bacterial E coli plates DETAILED DESCRIPTION The presented herein is an automated storage and retrieval apparatus and related method thereof providing a sample process management system to the store and retrieve samples Samples in containers are stored and retrieved automatically through a dispenser e g an airlock climate control chamber access means that is automatically dehumidified by a dry gas purge e g carbon dioxide or nitrogen purge or the like This purge rapidly reduces ambient humidity to a desirable relative humidity RH e g less than about 15 RH to minimize or eliminate the accumulation of frost on the sample plates Microplates or storage containers or the like are identified for example using barcode technology or radio US 8 616 072 B2 5 frequency technology The containers containing the samples are transferred by the transfer device to the addressable sam pler where they are sampled using pins or other described means The sample containers or plates are then transferred back to the freezer via the transport device e g
15. equently can be retrieved from the carousel 20 the plate handler 3 and by the transport device 4 and available to be taken to the sampler components 5 6 8 9 16 FIG 3 shows a side view of a component of one embodi ment of a transfer device A sample container plate is dis pensed from the freezer through the dispenser shown as 21 in FIG 2 and in one embodiment taken by a robotic arm to the transport mechanism 4 The transport mechanism comprises a slide 301 a reading device 305 a guide wheel 306 to prop erly locate the plate a cover 308 with a tip access slot 307 and a motor to drive the operations The transport device is con trolled by the controller In certain embodiments the robotic transport arm may be a conveyer belt operably coupled to the dispenser and the transport device may be integral The trans port device may be located within or outside of the climate controlled receiving chamber The receiving chamber sur rounds the dispenser of the freezer In regard to FIG 4 depicted is a top view of a component of one embodiment of a transfer device as shown in FIG 3 The sample plate 404 is shown on the slide 401 and is transported to the tip access slot 407 by the controller so that the proper sample in the plate may be accessed by the addres sable sampler The addressable sampler has access to the sample through the tip access slot 407 FIG 5 depicts a side view of a component of one embodi ment of an addressable
16. er mits contact with material in the mentioned compartment with biomaterials pins can be hollow solid slotted or have other types of recesses designed to adhere to droplets of liquid The tips may be heated passively hot air heat block oractively by conduction The temperature ofthe tip may be monitored by a real time temperature monitoring system by incorporating a thermostat allowing more precise tempera ture control thermistor may also be imbedded in the tip to provide real time monitoring on intra tip temperature In cer tain embodiments sterilized disposable arrayed tips or indi vidual tips are used The tips may also have liquid volume control mechanisms e g pipetting For example constant volumes of frozen samples could be picked up and delivered by the system for example by employing a hollow picking pin connected to a liquid volume control e g pipetting module The tip may also be a core sampling device to core a frozen samples e g without thawing the sample For 20 25 30 35 40 45 50 55 60 65 8 example a rotating coring tip could cut drill sample from a tissue slice or other frozen material without thawing it In one embodiment the system allows object sampling followed by freezing of the samples by adding visual devices to identify randomly positioned objects For example this would allow microbial colonies grown on an agar plate and randomly or non randomly placed to be
17. essary sterilize tips first 5 Turn on the N valve Step 4 Start Adept robot 1 Turn the ROBOT POWER on if necessary 2 Run AdeptWindowsPC from Start 3 IP address 172 16 180 108 4 Note if the Adept is POWER OFF the AdeptWindow sPC software has to be started 5 seconds later right after POWER ON It is not necessary to turn off ROBOT POWER for daily operation 5 DDT prompt Load from local disk D or network disk X default is D Select default 6 Release any emergency button on the control pendant 7 Press COMP PWR and white button to power on robot 8 Type Calibrate and confirm with Y 9 Load program for Adept from networking disk in PC load nfs gt xc ice mainpicking v2 10 If program exists either keep it or clean it with zero Syn 11 Tum the tip heater ON 12 Run ex mainpicking 13 Check that the proper number and type of destination plates filled with appropriate media formulation are in place Step 5 Start PC Software 1 Click SystemNet on desktop 2 Reset System Reset devices which includes Bio phile Twister II 3 Load List Load the text file generated in step 2 4 Start Picking 5 Tip Temperature gt 45 C This is the average temperature in the picking tip area above the heat gun The tip temperature should be gt 150 C The heat gun should be set to 250 C for yeast Use 300 C for bacteria Program will pause seq
18. example rests in a thermostatically controlled heating block or a rotating tip station that can be warmed precisely to the desired tempera US 8 616 072 B2 7 ture heat travels through the pin and into the sample by conduction Exemplary pin tip temperatures are for example between about 30 and about 70 C Once partial or full thaw ing occurs the pin is removed from the source well and dipped into the destination well releasing a sample of cells which can subsequently be grown and used for many pur poses e g preparation of nucleic acid PCR bioassays etc After sampling the source compartment rapidly refreezes due to the low temperature of biomaterials in adjoining wells and also by returning the sample plate to the freezer Thus sample integrity is maintained After delivering the inoculum the pin may be cleaned and sterilized before reuse In one embodiment the pin is dispos able For reusable pins there are many well known methods for sterilizing the pin e g scrubbing the pins in a brush bath sonicating dipping the pin into a series of wash baths and or heating the pin to an extremely high temperature In certain aspects the system comprises an automated freezer e g robotic freezer e g Biophile a transfer device e g plate transfer robot e g Twister 2 an optional delidding device e g robotic arm to remove the lid e g modified from motorized slide a climate controlled receiv ing chamber c
19. f the user s sample pro cess management system Furthermore the present invention apparatus enables the user to generate a variety of reports in support ofthe user s quality assurance needs Finally the user will benefit from the present invention s information technol ogy by receiving excellent sample security optimal sample visibility optimal quality assurance sample migration con trol and flexible data management The controls and processing of present invention may be implemented using hardware software or a combination 20 25 30 35 40 45 50 55 60 65 12 thereof and may be implemented in one or more computer systems or other processing systems such as personal digit assistants PDAs In an example embodiment the invention was implemented in software running on a general purpose computer Computer system includes one or more processors in a communication infrastructure e g a communications bus cross over bar or network Computer system includes a display interface that forwards graphics text and other data from the communication infrastructure or from a frame buffer not shown for display on the display unit The computer system may also include a main memory preferably random access memory RAM and may also include a secondary memory The secondary memory may include for example a hard disk drive and or a removable storage drive representing a floppy disk drive a magnetic tape drive a
20. i mize airborne contamination For example the room air was not HEPA filtered and the IcePick area was not sterilized with a germicidal lamps between picking runs These simple pro cedures are predicted to further minimize airborne contami nation FIGS 10A 10B and 10C illustrate the results of cross contamination tests done with yeast and bacterial cells Source plates with undiluted wells only and prepared as described were used Destination plate wells were inoculated ina pattern spelling the word YES As can be readily seen only these wells grew FIG 1A and 11B illustrate the results ofthe transfer experi ment Experiments are organized as follows Wells A1 A4 are picked from four independent undiluted source wells A9 A12 were picked from 10 fold diluted source wells and C5 8 were picked from 100 fold diluted source wells The remainder are blank wells to seek evidence of cross contami nation A5 A8 were touched by pins that were not dipped into source wells and the others were simply exposed to air during the entire picking operation which lasted approximately 20 minutes FIGS 10A 10B 10C 11A and 11B demonstrate that cross contamination is not detected using this procedure importantly this was true even though these experiments were performed without any special care taken to minimize contamination such as lid closure on the destination plate during the picking time US 8 616 072 B2 17 In reference to FIGS 10A
21. ion The robot dips the used tip in the well s priorto heat sterilization This was found to give more reliable sterilization than heat alone 12 N Control Valve lid dryer It controls dry N injection to the Environmental Transfer chamber to minimize con sumption of protective dry sterile N gas The N gas is directed at the underside of the lid holder that holds the current source plate lid reducing ice buildup on the lid 13 N Environmental Transfer Chamber It encloses the plate handler delid slide and barcode reader It interfaces with the port ofthe robotic freezer It can also be extended to interface with multiple freezers 14 Frozen Sample Plate This is the multi well source plate It is typically sealed with foil and covered by a loose fitting plate lid and is labeled with a unique barcode for identification tracking The sample information is stored in a database 15 Destination Plate There are one or more plates to accept the sample s delivered by picking robot 16 Hot Air Gun It heats up the tip to sterilize and or dry the tip Currently temperatures of 200 350 C are used 17 Network Router It links the major devices in the system for reliable and quick operation 18 Barcode Scanner It reads the barcode for each plate This information is used for ID validation when retrieving the plates 19 Carousel It sits inside the freezer and serves as the hotel for plates 20 Dispensing Port It is a
22. is a vertical or horizontal storage freezer 8 The system of claim 1 wherein the climate controlled receiving chamber comprises one or more of a humidity controller an atmospheric gas control mechanism a tempera ture controlling mechanism or a reading device to identify the containers as they are inserted into and retrieved from the chamber 9 A system for retrieving a subset of a stored sample comprising an automated freezer comprising a dispenser a climate controlled receiving chamber adjacent to the dispenser a transfer device within the receiving chamber a robot operatively connected to the transfer device and positioned to access the subset of a stored sample wherein the robot comprises a tip and 18 a heat sensor to monitor the temperature in the vicinity of the tip 10 A system for retrieving a subset of a stored sample comprising 5 an automated freezer comprising a dispenser a climate controlled receiving chamber adjacent to the dispenser a transfer device within the receiving chamber a robot operatively connected to the transfer device and positioned to access the subset of a stored sample and a delidder within the receiving chamber 11 The system of claim 10 wherein the robot comprises a tip 12 The system of claim 11 wherein the tip is selected from the group consisting of a metal tip with high thermal heat capacity metal pin a ceramic pin a hollow pin and a core sampler for retrieving
23. liable tip handling It has spring compensation for different plate types to compen sate for variable sample height in different sample wells It also provides a gentle and constant downwards force as the tip thaws the sample It has a magnetic tip holder that provides constant temperature control for the tip as well 7 Robot Controller This is the controller dedicated to the picking robot It provides digital control for other detection and valve controls 8 Rotating Tip Station It has 4 positions for tips When it rotates the tip at position 1 will go through the sterilization 20 25 30 35 40 45 50 55 60 65 14 process by being positioned above the hot air gun Positions 2 3 and 4 are ambient air cooling stations position 4 is the pickup station The motor is on a constant dwell time cur rently calibrated at 20 seconds This dwell time can be cali brated to adjust average tip temperature if necessary 9 Tips These are metal tips with high thermal capacity They are spring loaded to ensure reliable release from the tip head 10 Tip Heat Sensor This device monitors the hot air temperature in the vicinity of the pin at position 1 of the tip station This allows the system to determine if the pin has warmed to the correct temperature to automatically transition the system from standby warmup phase to picking phase 11 Ethanol Well Two wells hold 70 ethanol or other solvents for sterilizat
24. n hard disk drive and signals These computer program prod ucts are means for providing software to computer system The invention includes such computer program products Computer programs also called computer control logic are stored in main memory and or secondary memory Com puter programs may also be received via communications interface Such computer programs when executed enable computer system to perform the features ofthe present inven tion as discussed herein In particular the computer pro grams when executed enable processor to perform the func tions of the present invention Accordingly such computer programs represent controllers of computer system In an embodiment where the invention is implemented using software the software may be stored in a computer program product and loaded into computer system using removable storage drive hard drive or communications inter face The control logic software when executed by the processor causes the processor to perform the functions of the invention as described herein In another embodiment the invention is implemented pri marily in hardware using for example hardware components such as application specific integrated circuits ASICs Implementation of the hardware state machine to perform the functions described herein will be apparent to persons skilled in the relevant art s US 8 616 072 B2 13 In yet another embodiment the invention is implemented usi
25. n air tight port which receives and releases plate Operation Procedure Step 1 Check the following issues before starting System software 1 Power off TRANSPORT POWER if necessary 2 Open the chamber cover check the plate holder on the transport rail with the spacer for ZERO position 3 Power on TRANSPORT POWER 4 Cover the chamber 5 Turn Nitrogen manual valve on Check the Biophile with Biophile CommTest1 on the desktop if necessary Perform AAA H R in sequence Step 2 Prepare the hit list 1 Go to E Order and open Genlist xls with Macro enable 2 Click Load Order to input the hit list in Excel file which is in the proper template format 3 Chose the destination plate format 96 or 384 well 4 Click Generate List 5 Copy column M paste to ConTEXT and save as text file 6 Notice the format of the list IOH 10003 UH A 87 96 3 1 JH B 8000 384 1 1 3 Gene ID Source Plate Barcode Plate format 96 384 Row Column Destination plate Plate format 96 384 Plate position 1 4 Row Col umn The 1st line is the title US 8 616 072 B2 15 Step 3 Prepare working conditions 1 Clean up the working surface Move away any extrane ous materials from the work table 2 Replace the fresh 70 ethanol in the 6 well plate 3 Turn Hot Air Gun power on 4 Check that all four tips are in good condition and in place on the rotating tip station If nec
26. n optical disk drive etc The removable storage drive reads from and or writes to a removable storage unit in a well known manner Removable storage unit represents a floppy disk magnetic tape optical disk etc which is read by and written to by removable storage drive As will be appre ciated the removable storage unit includes a computer usable storage medium having stored therein computer software and or data The computer system may also include a communications interface Communications interface allows software and data to be transferred between computer system and external devices Examples of communications interface may include a modem a network interface such as an Ethernet card a communications port a PCMCIA slot and card etc Software and data transferred via communications interface are in the form of signals which may be electronic electromagnetic optical or other signals capable of being received by commu nications interface Signals are provided to communications interface via a communications path i e channel A channel or any other communication means or channel disclosed herein carries signals and may be implemented using wire or cable fiber optics a phone line a cellular phone link an RF link and other communications channels In this document the terms computer program medium and computer usable medium are used to generally refer to media such as removable storage drive a hard disk installed i
27. nanother embodiment the controller performs at least one of the following functions advising a user of sample avail ability advising of sample delivery date to the dispenser monitoring an amount of sample within the dispenser informing a supplier of sample purchased by a user and requesting the supplier to provide additional items to replace items dispensed from the dispenser In one embodiment a supplier communicates with the dispenser via the central controller In one embodiment the system further comprises a com munication network for providing communication between the controller and at least one user and at least one supplier In one embodiment the communication network is selected from the group consisting of an intranet an Ether net an Internet a telephone network a wireless network and a combination thereof In one embodiment the controller comprises a computer Inone embodiment the system further comprises a climate system for controlling the climate of the climate controlled chamber Inoneembodiment the climate system comprises a dry gas supply to dehumidify the chamber and or cool the chamber In another embodiment the dispenser is configured to interchange a sample container between the dispenser and the climate controlled receiving chamber while in a chamber exchange position and interchange a container between the dispenser and the transfer device while in a transfer exchange position In one embodiment
28. ng a combination of both hardware and software In an example software embodiment of the invention the methods described above were implemented in VISUAL BASIC con trol language but could be implemented in other programs such as but not limited to C programming language The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof The foregoing embodiments are therefore to be con sidered in all respects illustrative rather than limiting of the invention described herein Scope of the invention is this indicated by the appended claims rather than by the foregoing description and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein The patents patent applications references and other documents identified herein are incorporated in their entirety herein by reference EXAMPLES It should be appreciated that the invention should not be construed to be limited to the examples now described rather the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan In reference to FIG 2 1 Central computer This hosts the system main control software package and the database The control software 1 reads the sample list sends command to robotic freezer to get the correct plate 2 controls the plate handler
29. omprises a user station operatively connected to the system the user station comprising a data input means for inputting data to the pro cessor relative to the samples or the sample containers In one embodiment the system comprises a plate queuing system allowing the freezer to unload one or more additional plates while a plate is being sampled The queuing of the sample plates will allow for higher throughput of retrieving samples The queuing ofsample plates may be just after being dispensed from the freezer before or after being delidded for lidded sample plates or in line for the sampler The queuing system may also be in the freezer compartment or in a freezer 0 jak 5 20 30 40 45 50 55 6 compartment operably linked one or more of the freezer the climate controlled receiving chamber the transfer device or the sampler In one embodiment methods for automatically sampling a stored sample from a frozen sample plate comprises provid ing a carousel in the freezer compartment depositing a con tainer into a climate controlled chamber controlling the cli mate of the chamber while the container remains in the chamber sampling one or more samples and returning the container to the freezer carousel In certain embodiments exemplary freezers include for example the Biophile TekCel and the Biobank Thermo Systems Described herein are methods to store and efficiently retrieve a subset e g use
30. on sensors and identification sensors It is contemplated that that the information derived from the sample or work pieces carried in the containers while practicing the present invention will provide an information technology platform for the user The computer system is intended to be a user friendly utilizing Windows based plat form or any other operating system and may be integrated with a variety of laboratory information management sys tems It is envisioned that the control system and computer system can be accessed directly by using a touch screen interface or remotely by a stand alone personal computer or with a local area network LAN The present invention apparatus provides the user the capa bility among other things to set top level user definable parameters to control container sample access based on research groups research campaigns or individual laborato ries For instance sample data can be configured by the user to meet the user s particular research requirements The data base can then search the user s sample populations to find all the samples that match the user s requested research param eters Moreover time temperature profiles and sample access histories are maintained continuously The present invention allows the user to set sample migration thresholds This fea ture employing sample usage frequencies prompts the movement of low demand samples into longer term storage units maximizing the efficiency o
31. ontaining the transfer device and optionally the delidding device and an addressable sampler e g robotic arm or gantry system modified to hold heated pin device e g Adept Cobra and a controller e g computer to integrate the components of the system e g the heating temperature controlling system provide a user interface to operate the system In one embodiment the system further comprises a destination plate module e g a robotic arm and plate hotel to move destination plates to for example a heated incubator Exemplary freezers include for example a Biophile auto matic freezer which holds 931 96 or 384 well plates The Biophile can therefore organize up to 357 504 samples The freezer is adapted to retrieve or return any of the 931 plates in about 30 seconds In one embodiment the system comprises more than one source freezer As used herein biomaterial includes for example micro bial prokaryotes or eukaryotes cells viruses the components of cells components of viruses or component tissues or organisms The systems described herein are useful for managing col lections of bacteria Escherichia coli retroviruses bacte riophages or other recombinant viruses and yeast Saccharo myces cerevisiae for various purposes and of chemical compound libraries and frozen tissue micro samples homo genates As used herein a component of the sampler is the pin which includes for example a penetrating device that p
32. r and exterior sides With regards to control operations the present invention automation and robotic motions described herein are pro vided in part by the control system and processor It should be noted that the following exemplary sequences of operations may be varied partially omitted overlapped to reduce the total elapsed time of operation or reordered in an alternative sequence Operation for depositing a storage sample tray is provided as described below In a first step the dispenser ofthe freezer opens and the sample tray exits into the climate controlled chamber The lid ofthe tray is removed and the transfer device transports the tray to the addressable sampler through the exterior door of the climate controlled chamber Once sampled the transfer device transports the tray back to the dispenser for placement back in the freezer The reading device ofthe system may be located in the climate controlled receiving chamber or associated with the addressable sampler or both US 8 616 072 B2 11 In one embodiment a central controller locates relevant storage container information in the database and location of storage container in the freezer or stationary storage racks is determined Optionally if security is required then an access code is entered via data input device such a display panel integral with the apparatus housing or a remote processor and confirmed by central processor 81 to allow access to the desired storage
33. r defined of biological materials e g viruses bacteria yeast fungi sperm worms mamma lian cells plant tissue animal tissue from a freezer e g source compartment The methods and systems described herein reduce contamination of the sample and mistakes in sampling biological samples while increasing efficiency Por tions of the samples e g the inoculum dispensed biomate rial sampled material portion of sample may be placed into a destination compartment or plate for further processing or storage In certain embodiments the dispensed biomaterial to be managed or retrieved is for example placed in a sample plate e g single well 2 well 3 well 6 well 16 well 96 well 384 well plate 1536 well plates sealed with a foil e g alumi num gold tin etc or polymer plastic rubber etc seal and covered by a plastic lid prior to the storage This lid is removed to allow access by the addressable sampler e g picking robot Retrieval of a collection of materials in pre sealed plates e g source compartments is performed by a penetrating pin from the top of the 96 or 384 or 1526 well plate at a given well position The pin of the addressable sampler may contain a slot or other recess to facilitate uptake of a small sub sample of liquid from the well The position may be registered manually or for example by robotics con trolled by a computer The penetration of the pin through the foil seal permits contact be
34. s Hopkins Technology Transfer 65 Prior Publication Data US 2010 0294046 Al Nov 25 2010 GD ABSTRACT Related U S Application Data Provided herein are systems and methods for storage retrieval and sampling of materials 60 Provisional application No 60 772 767 filed on Feb 13 2006 23 Claims 7 Drawing Sheets 907 909 US 8 616 072 B2 Sheet 1 of 7 Dec 31 2013 U S Patent uonoejeq NLA joguo2 Jaydepy dij PIeq ePIIs SJosuag uonoejoJd Ajsjes ua HoN L Dld soneulauos JONUOD W S S xold92 do s foueBieur3 UND liy 10H di pJeog O I 1eu81q yOqOY ud JOJOW 9PIIS UONE S dij Bunejoy JOON 10 6 03 e npolw ejnjeJeduio 1o O3u02 1oq0M JOSUaS jeeH di pejeJju Je pueH ayeld 162994 ejudoig J9110W 3HOMJSN Japeay epooJeg eseqejeq BIEMYOS 0JjUO Od Z PIED MOMAN PIED YIOMJON JAPIO eutuo uoJees eqq JO N S q9M U S Patent Dec 31 2013 Sheet 2 of 7 US 8 616 072 B2 SS MM mn wn au n URDU LU C N N ITI U S Patent Dec 31 2013 Sheet 3 of 7 US 8 616 072 B2 U S Patent Dec 31 2013 Sheet 4 of 7 US 8 616 072 B2 601 602 603 FIG 6 703 e O O O 9 801 803 FIG 7 FIG 8 U S Patent Dec 31 2013 Sheet 5 of 7 US 8 616 072 B2 US 8 616 072 B2 Sheet 6 of 7 Dec 31 2013 U S Patent g80T Old JOT Old SA
35. t the method further comprises trans ferring each of the one or more further samples to a destina tion plate In one embodiment the freezer comprises a Biophile and or a Biobank In another embodiment sampling comprises lowering a heated pin through the seal ofthe container and into the frozen sample wherein at least a portion of the sample is thawed In another embodiment sampling comprises lowering a coring pin or coring saw through the seal of the container and removes a portion of the frozen sample In one embodiment the method further comprises steriliz ing the pin between sampling In another embodiment the pin is disposable Inanother embodiment the pin comprises a temperature of between about 30 and 70 C Inone embodiment once partial or full thawing occurs the pin is removed from the source well and dipped into the destination well releasing a sample of cells 0 a 5 40 45 65 4 In one embodiment the tip is sterilized between retrieving samples In another embodiment sterilizing comprises scrubbing the pin in a brush bath dipping the pin into a series of wash baths or heating the pin to a sterilization temperature In another embodiment sterilization temperature com prises from between about 121 C to about 500 C In one embodiment a pin comprises one or more of a penetrating device permitting contact with material in the mentioned compartment wherein the pin is one or more of
36. tainer jak 5 20 35 40 45 55 10 insertion and retrieval The receiving chamber 902 encom passes the dispenser 904 to allow communication between freezer 901 and receiving chamber 902 and an exterior door 903 to allow communication between the exterior environ ment or adjacent area and chamber 901 The chamber 901 has a climate control system 908 that provides an air purging capability to dehumidify and optionally cool the air in the chamber 902 before dispenser 904 is opened A scanning reader device 935 preferably a barcode reader is situated in the chamber 902 to identify storage containers as they are inserted into and retrieved from chamber 902 Information relative to the storage containers is transmitted from reader device to central and or remote processor Referring to FIG 9B the storage containers 909 are carried by a transfer device 907 to the addressable sampler picking robot The transfer device 907 can transport the container 909 to the exterior as the exterior door is open Alternatively the transfer device 907 can transport the container 909 to the interior of the freezer through the dispenser A climate control system 908 is in communication with the receiving chamber 902 that dehumidifies and optionally cools the chamber 902 while the container is isolated therein e g both exterior and interior doors are closed The climate control system 908 includes a dry gas or dry air purge e g nitrogen c
37. taining sampling tips at a sampling temperature an addressable sample picker In one embodiment the sample locator comprises a bar code reader In one embodiment the system further comprises a delid der delid slide 25 30 40 45 50 60 65 2 In one embodiment the system further comprises a tip sterilizer In one embodiment the system further comprises one or more destination plates for receiving retrieved samples In one embodiment the automated freezer is a vertical or horizontal storage freezer Inoneembodiment the climate controlled receiving cham ber comprises one or more of a humidity controller an atmo spheric gas control mechanism or a reading device to identify the containers as they are inserted into and retrieved from the chamber In one embodiment the transfer device comprises a mechanism to transport a sample tray between the dispenser and the addressable sampler In one embodiment the system further comprises a con troller in communication with one or more of the automated freezer the dispenser the transfer device or the addressable sampler Inone embodiment the controller monitors one or more of the freezer the transfer device or the sampler In another embodiment the controller receives and pro cesses orders from at least one user In one embodiment the controller comprises a processor for processing data relative to a sample being stored in and retrieved from the system I
38. ter II for plate handling component of the plate transfer device 4 Animatics Smart motor 1 for rotating tip station component ofthe addressable sampler 5 Yamaha slide for plate transfer and delidding additional optional component of the plate transfer device if the plate has a lid 6 Animatics Smart motor 2 for plate transfer component of the plate transfer device 7 IBM PC referred to as the central or controlling PC 8 Cisco router for networking 9 Advantech temperature test module 10 HAKKO 852 hot air gun component of the addressable sampler 11 Custom control console Software The System adopts a distribution control concept The System software package integrates a variety of commercial control software packages associated with off the shelf com ponents for example 1 system software 2 Biophile GenOne software which runs on the flat panel PC on the BioPhile freezer User can operate the freezer through a touch screen when the System software package 1s not activated There are 2 channels connecting the panel PC to the central PC One is TCP IP used for database management Another is an RS 232 interface allowing the System Software package to send commands for loading unloading plates to from the Bio Phile and or 3 Adept robot software which is executed by the SmartController It communicates with PC through TCP IP networking 4 Two pieces of Software for the Animatics Smart motors They are downloaded to
39. the motors They connect to the Adept robot through SmartController RS 232 ports There is no direct control from the System software package to the smart motors and or 5 Hit list preprocessing software This is another piece of custom software currently consisting of a Microsoft Excel macro which loads the tem plate order sheet and generates the hit list for robot Turning now to the drawings the present invention is sche matically shown in the plan view of FIG 1 and perspective US 8 616 072 B2 9 view of FIG 2 which includes an automated storage and retrieval apparatus having one or more storage carousels 20 disposed in an automated freezer 2 with one or more optional stationary racks a climate controlled receiving chamber 13 N2 environmental chamber a transfer device 4 The climate controlled receiving chamber 13 is generally disposed on a wall 11 of the freezer 2 A control system 1 is coupled to the reading device 18 e g barcode scanner the addressable sampler components 5 6 8 9 16 transport device 4 and climate controlled chamber 13 for controlling their opera tions Generally the control system 1 controls the operation of the apparatus so that the containers 14 can be loaded from the exterior or sampling position into the climate controlled chamber 13 for retrieval by the transport device 4 and the plate handler 3 to dispensing port 21 for insertion onto the carousel 20 in the freezer 2 Stored containers subs
40. the transfer device is configured to deliver the container to the addressable sampler In another embodiment the chamber further comprises a transporter to transport a sample from the dispenser to the sampler In another embodiment the chamber comprises one or more of a reading or writing device In one embodiment the reading device is configured to identify the sample In one embodiment the sample is contained in a sample plate containing other samples In one embodiment the sample plate comprises a 96 ora 364 well sample plate US 8 616 072 B2 3 Inone embodiment the system further comprises a control system wherein the control system is operatively connected with the carousel the interchange mechanism and chamber for controlling their operations In one embodiment the system further comprises a user station operatively connected to the apparatus the user sta tion comprising a data input means for inputting data to the processor relative to the containers In one embodiment the system further comprises a desti nation plate handler module adapted to transport destination plates to a destination In one embodiment the destination comprises one or more of a freezer a refrigerator an incubator a hood or a climate controlled environment In one embodiment the system further comprises a plate queuing system In one aspect provided herein are methods for sampling a frozen sample comprising retrieving a sample in
41. to deliver and delid the source plate by coordinating with the delid slide 3 sends the source and destination positions to the picking robot 4 controls dry nitrogen gas supply and 5 performs other detections and human machine interface operation 2 Automatic Freezer The BioPhile BSU stores the plates in the carousel inside which is protected by dry N2 gas It accepts retrieves and returns source plates through a robotic airtight port It also has its own database for plate number management 3 Transfer device The Twister II can retrieve a plate with or without lid on Its robotic arm can deliver the plate to various programmable positions Its gripper is programmed for various forces to perform different actions such as lid removal when the lid sticks due to ice buildup 4 Transfer device Delid Slide It moves along one axis This allows the plate to be delidded to have its barcode scanned to be repositioned if necessary and to be located at positions corresponding to the desired column of a multiwell plate so that it is exposed to the access slot allowing the robot to pick the desired sample This is a commercial slider modi fied with a custom delidding frame 5 Addressable sampler picking Robot This is an indus trial precision robot Adept Cobra It can access a wide working space rapidly It can be equipped with different func tional heads It can also work with a visual system 6 Tip Head This is designed for re
42. tween the tip of pin and the com partmented material either in solid frozen or liquid form Methods for thawing a frozen sample are described herein The retrieval of the pin and sample contained therein or thereon comprises the retrieved sample The pin may then be inserted into a bio compatible sample compartment e g into a specified or random well of a destination plate to transfer sample on or in the pin In one embodiment the methods comprise withdrawing a small sample liquid or solid from a well containing a frozen biomaterial e g stored at or around 80 C The sample is withdrawn under sterile conditions in a short period of time shorter than user manual selection of a sample from a sample plate and transfer a portion of the sample to a destination well e g containing growth medium The methods are accomplished by the systems described herein for example by an addressable sampler e g a robotic arm system that lowers a precision heated temperature con trolled pin through the seal of a sample and into the frozen sample block The pin is heated to a predetermined tempera ture warm enough to melt a portion of or the entire block of material in that compartment but not warm enough to harm the materials in certain embodiment or affect neighboring sample in the sample plate The temperature or temperature range for a particular sample is easily determined empirically by one of skill in the art The pin for
43. uence until proper temperature is reached Step 6 Finish day s operation 1 Turn N valve off to conserve gas 2 Clean the table Database Manipulation Sometime it is necessary to manipulate the database for the Biophile For example in case you have to unload all plates from the freezer manually to defrost the freezer Only authorized people can perform these functions 1 Go My Network Places 2 BIOPHILE on Biophile user name Administrator No Password 3 Genone Database bfiFreezerData type the password Maintenance Part 1 BioPhile Every time you open the door of BioPhile you have a lot of problems to solve One big headache is that you may not be able to home the robots inside the BioPhile 1 Unload all plates to backup 80 C freezer 2 Leave door open and thaw Biophile overnight 3 Unplug to power off completely including UPS 4 Rotate the lift all the way up to the limit and back off a few turns 5 Check the black registration marks for the lift and car ousel 6 Close the door 7 H to home the Biophile 20 40 45 50 55 65 16 8 If any plate location is damaged change the database to mark location as unavailable Estimation of Volume Transferred Per Picking Operation Starting material LB 10 glycerol in 384 well plate Plate type 384 well low profile Genetix Total starting vol ume per well 50 uL A picking pin was inserted by hand into the well and transferred
44. us In one embodiment the dispenser comprises at least one rotatable carousel located in the freezer the carousel com prising at least one cell defined by two side walls each side wall extending from a central location of the carousel an access hatch aligned with the or each carousel for accessing an interior of the at least one cell and a motor for rotating the carousel In one embodiment the controller performs at least one of the following functions rotation of the carousel by control ling the motor controlling dispensing of an item from the dispenser monitoring an amount of an item within the dis penser reporting to the central controller the amount of an item within the dispenser making an activity log and tem perature log of the dispenser informing a user of availability of an item receiving information from the central controller in relation to item delivery to the dispenser and temperature control setting of the dispenser and monitoring and control ling an internal environment of the dispenser In one embodiment the controller comprises a computer More than one controller may be associated with the system and may control discrete functions All or any subset of func tions may be controlled by one controller In some embodi ments the control system comprises a processor for process ing data relating to contents of the containers being stored in and retrieved from the apparatus In one embodiment the system further c
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