HARVESTING AND CRYOPRESERVATION OF HUMAN EMBRYONIC STEM CELLS (hescs)

HARVESTING AND CRYOPRESERVATION OF HUMAN EMBRYONIC STEM CELLS (hescs) OBJECTIVE: can be cryopreserved in a liquid nitrogen (LN 2 ) freezer for long-term storage. This Standard Operating Procedure (SOP) details how hescs are harvested and frozen in LN 2, after samples are collected for characterization. Cells are generally frozen at one cryovial/well (approximately 1-2 x 10 6 cells/vial). SCOPE: This procedure applies to all Massachusetts laboratory personnel responsible for the harvesting and cryopreservation of hescs in LN 2 freezers. RESPONSIBILITY: It is the responsibility of the Laboratory Operations Manager and Quality Assurance Officer to ensure all laboratory personnel are properly trained in and follow this SOP. SAFETY: All laboratory personnel should be in compliance with UMASS Employee Health and Safety regulations when working in the laboratory. Protective equipment, such as a lab coat and disposable gloves must be worn when working in the lab. When placing cryovials into a liquid nitrogen freezer additional eye protective safety glasses and insulated gloves should always be worn. ABBREVIATIONS AND DEFINITIONS hescs: Human Embryonic Stem Cells MEF: Mouse Embryonic Fibroblast DMEM: Dulbecco's Modified Eagle Medium HI-FBS: Heat-Inactivated Fetal Bovine Serum FM: Freezing Medium. CM: Culture Medium LN 2 : Liquid Nitrogen SOP: Standard Operating Procedure UMASS: University of Massachusetts Medical School REFERENCES Not applicable 1. MATERIALS REQUIRED 1.1. EQUIPMENT Sterile biosafety cabinet (tissue culture hood) 37ºC incubator 37ºC water bath or a warm room Inverted microscope Pipette-aid Liquid aspiration waste disposal system Bench-top centrifuge with swinging-bucket rotor Balance 65

Timer Cryo-grade label maker -80 C freezer Liquid nitrogen freezer 1.2. SUPPLIES 5 ml sterile glass pipettes (Fisher 136667D) 10 ml sterile glass pipettes (Fisher 136667E) 5 ml sterile serological pipettes (Costar 4487) 10 ml sterile serological pipettes (Costar 4488) 15 ml sterile centrifuge tubes (BD Falcon 352097) 50 ml sterile centrifuge tubes (BD Falcon 352098) 250 ml sterile conical bottles (Corning 430776) Sterile Pasteur pipettes (Fisher brand 13-678-20D) Cryovials (Nalgene 5000-0020) Cryovial labels (Electric imaging 94511) Cryovial holder (Nalgene 5030-0510) Freezing containers (Nalgene 5100-0001) Disposable nitrile gloves (World Wide Medical Supplies 71011000-3) 1.3. REAGENTS DMEM-F12 (Invitrogen 11330-057) Heat inactivated defined FBS (HyClone SH30070-03) DMSO (Dimethyl sulfoxide) (Sigma 068K2342) Isopropanol Collagenase solution 1mg/ml (see SOP-RP-005 Preparation of Collagenase Solution). hesc Culture Medium (see SOP-RP-004 Preparation of Human Embryonic Stem Cell (hesc) Culture Medium). 6-well plates with MEFs (see SOP-CC-002 Thawing and Seeding of Frozen Inactivated Mouse Embryonic Fibroblasts (MEFs)). 2.1. DETERMINE WHEN TO HARVEST HESCS Note: Harvest cells when they are in the log phase of growth, which is normally between 3 to 5 days after plating. 1. Under a microscope, evaluate hesc growth and quality carefully. 2. All of the following observations will be used to determine when hescs are ready to be harvested. Harvest hescs when: There is less than 10% differentiation in the previous passage according to flow cytometry data. Colonies of hescs reach 70% confluence. MEF feeder cells in the culture are less than two weeks old. 66

2.2. DOCUMENTATION 1. Once it is decided to harvest cells, obtain a hesc Cryopreservation Log Sheet from the Log Sheet binder. 2. Fill out necessary information on the Log Sheet. 2.3. PREPARE CRYO-LABELS 1. On the cryo-labels, record each item below on a separate line: Stem cell bank code (MAyyXXX). For example, MA08002 is for the 2nd cell line acquired in year 2008. Barcode which is generated according to the barcode number. Barcode number which is the Stem code number followed by a three-digit lot number. The lot number is created sequentially for each line with 000 designated for original frozen vials the bank acquired. Provider s cell line name and passage number. Date (yyyymmday, corresponds to the date when cells were harvested). For example: MA08001 08001002 H1-p99 20081003 2. Print enough cryo-labels for all cryovials and at least three extra for the Log sheet and the two LN 2 log books. 3. Place a cryo-label on the hesc Cryopreservation Log Sheet 2.4. CALCULATE HOW MUCH CULTURE MEDIUM AND COLLAGENASE SOLUTION IS NEEDED FOR THIS PROCESS 1. For harvesting hescs, the following are required: Collagenase solution hesc culture medium 2. On the hesc Cryopreservation Log Sheet, calculate and record the amount of the above materials required for this process, including the lot number. 2.5. PREPARE THE BIOSAFETY CABINET (TISSUE CULTURE HOOD) 1. Make sure there are sufficient materials listed below, near the hood: 5 ml, 10 ml sterile glass pipettes Absorbent paper towels (or Kimwipes) 70% ethanol spray Appropriate-size disposable gloves Place enough freezing containers on the bench near the hood for freezing hescs. Make sure all freezing containers have a sufficient amount of fresh isopropanol in them. Each freezing container can hold 18 cryovials. 3. PROCEDURE 3.1. STERILIZATION PREPARATION BEFORE WORKING IN THE HOOD 1. Wash hands and arms thoroughly (about one minute) with soap. 2. Rinse completely with warm tap water. 3. Dry hands and arms with paper towel. 67

4. Put on appropriate-size gloves. 5. Spray 70% ethanol on the gloves and a paper towel until fully saturated. 6. Thoroughly clean the hood working surface with the ethanol-sprayed paper towel. 7. Spray the surface of everything taken into the hood with ethanol. Dry them with paper towels if needed. 8. Make sure the following items are in the hood: Sterile Pasteur pipettes 15 ml, 50 ml sterile centrifuge tubes Black extra-fine alcohol proof pen Cryovial holders 3.2. ALIQUOT MEDIUM AND COLLAGENASE SOLUTION IN THE HOOD 1. According to the volume calculated in the hesc Cryopreservation Log Sheet, label both the cap and side of: One or two sterile 50 ml tubes or an appropriate sterile bottle as ESC.M for Embryonic Stem Cell Medium One or two sterile 50 ml tubes as Collag for collagenase. 2. According to the lot number in the Log Sheet, take out the appropriate ESC medium and collagenase solution bottles from the refrigerator. After thoroughly cleaning with 70% ethanol, place them in the hood. 3. Transfer appropriate volume of ESC medium to the ESC.M tube(s) or bottle. 4. Transfer appropriate volume of collagenase solution to the Collag tube(s). 5. Return the stock ESC medium and collagenase solution bottles to the refrigerator. 6. Place the aliquot of ESC medium in a 37 C water bath or warm room for 15 minutes. 7. Leave the aliquot of collagenase solution in the hood for 15-30 minutes to reach the room temperature. 3.3. PREPARE 2 X CRYOPRESERVATION MEDIA (OR FREEZING MEDIUM, FM) IN THE HOOD Note: Prepare 2 x FM media just before use (fresh). 1. Determine the amount of cryopreservation media required: The media required = 0.5 ml x the number of cryovials + 4 ml extra for pipette and filter error, + 3 ml extra for internal sterility testing. 2. According to the volume calculated above, label one sterile 50 ml conical tube or a larger size sterile bottle as 2 x FM (on both the cap and side). 3. Add the following, proportionately, to the labeled container: 68

Ingredient Ratio in the 5 ml total 10 ml total 2 x FM HI-Defined FBS 60% 3 ml 6 ml hesc culture medium 20% 1 ml 2 ml Sterile DMSO 20% 1 ml 2 ml 4. Mix and filter through a 0.22 µm filter unit 5. Keep the above solution on ice or in a 4-8 C refrigerator and use it within the day. Note: This 2 x FM will be added to hesc suspension at a 1:1 volume ratio, resulting in 1x freezing medium with a final concentration of 10% DMSO and 30% FBS, respectively. 3.4. PLACE CRYO-LABELS ON THE CRYOVIALS THAT WERE PREVIOUSLY PLACED IN THE HOOD 3.5. HARVEST SPENT MEDIUM FOR TESTING Note: Before hescs are harvested, spent medium will be collected for sterility and Mycoplasma testing. 1. Label a sterile 50 ml tube as: Spent Medium Cell line with passage number Date Initials 2. Take out hesc plates (3-4 at a time) from the incubator and place them in the hood. 3. Collect 0.5 ml of spent medium from each well and transfer it into one labeled tube. 4. Repeat the above for all plates to be harvested. 5. Gather all tubes containing spent medium together in one container. 6. Store the container in the designated refrigerator. 3.6. INCUBATE HESCS WITH COLLAGENASE SOLUTION (PREPARE NO MORE THAN TWO PLATES AT A TIME): 1. Place the warm ESC medium aliquot in the hood after thoroughly cleaning with 70% alcohol. 2. Label a sterile container as ESC for collecting hescs at a later time. 3. Set the timer at 5 minutes and 10 minutes, respectively. 4. Take out one or two hesc plate(s) from the incubator and place it/them in the hood. 5. Aspirate the medium completely from the hesc plate(s) using a Pasteur pipette. 6. Add 1 ml collagenase/well. 69

7. Return the plate(s) to the incubator. 8. Start the timer. 9. After 5 minutes of incubation, check the plate(s) under a microscope to determine whether the cell colonies are ready to be harvested. When they are ready, the perimeter of the colony should appear folded back, separating from the MEFs. If they are not ready, return the plate back to the 37ºC incubator for another 3-5 minutes of incubation. Note: Incubation time depends on the freshness of the collagenase solution. The older it is, the longer the incubation will take. However, do not exceed 15 minutes. 10. When collagenase incubation is complete, place the hesc plate in the hood. 11. Aspirate collagenase from the wells. Be careful not to disturb the cell/ colony layer. Note: If there is a significant amount of the colonies floating, scrape the cells/colonies directly in the collagenase solution rather than aspirating the collagenase off the well. 12. Combine cell/colony solution from all wells into one 15 ml or 50 ml tube. 13. Centrifuge and resuspend the cell pellet with 5 ml ESC medium. 14. Continue with Section 3.8, to harvest the cells from the remaining plates. 3.7. HARVEST HESCS 1. Add 2 ml ESC medium to each well. 2. Using a 5 ml glass pipette, take up most of the medium from one well. Scrape the cells/colonies in the well using the pipette while slowly releasing the medium into the well to wash the cells/colonies off the surface. Gently pipette the medium up and down during the wash to minimize bubbles and to disrupt cell clumps. Note: hescs recover better from freezing and thawing when frozen in large aggregates. 3. Repeat these steps until most of the cells/colonies are detached from the surface. Note: Leave the contents in the well until all wells are scraped. 4. Detach cells/colonies in the other wells by repeating the above steps. 5. When all wells are complete, combine the cell/colony solution from all wells into the bottom of the labeled sterile ESC container. 6. Take another 3 ml ESC medium and rinse all of the wells in one plate. Transfer the cell/colony solution to the same sterile ESC container. 70

3.8. REPEAT SECTION 3.6 AND 3.7 TO HARVEST HESCS FROM REMAINING PLATES 3.9. COLLECT CELL SAMPLES FOR CHARACTERIZATION Note: When hescs are harvested, cell samples will be collected for mycoplasma testing and RT-PCR analysis. 1. Label two sterile 1.5-2 ml tubes as mycoplasma test and RT-PCR, respectively. 2. Mix hescs well in the ESC container. 3. Transfer 1 ml hescs into each of the labeled 1.5-2 ml tubes. 4. Store these 1.5-2 ml tubes in a designated refrigerator for later characterization. 3.10. FREEZE HESCS Note: Once in DMSO freezing medium, cells should be frozen as quickly as possible (within 5 minutes). Note: Cells will be frozen in two sets of freezing containers in order to be easily transferred (the next day) to two LN 2 freezers. 1. Centrifuge hescs at 200 x g (about 1000 rpm) for 5 minutes. 2. During the centrifugation period, loosen all cryovial caps in the hood without taking them off. 3. When centrifugation is complete, return ESC container to the hood after cleaning with 70% ethanol. 4. Aspirate the supernatant off the hesc pellet. 5. Resuspend the cell pellet gently in appropriate volume of ESC medium (0.5 ml /well plus 0.5 ml extra). 6. After spraying with 70% ethanol, place the cold 2 x freezing medium (FM) in the hood. Loosen the cap. Note: When freezing a large lot, different parts of the freezing process may be completed by different personnel in order to reduce the time that hescs are in the freezing medium. For example, while one person aliquots the cells to the cryovials, another person may tighten the caps of cryovials and another may transfer the cryovials to the freezing containers and place them in a -80ºC freezer. 7. Due to the capacity of the freezing container, only 18 vials can be frozen at a time. Repeat steps below if there are more than 18 cryovials to be frozen. Label a new sterile 50 ml tube as F.ESC. Gently pipette up and down twice to mix the cell suspension in the ESC container and transfer 9.2 ml cells to the F.ESC tube. From the FM tube, take the same volume of 2 x FM (9.2 ml) and drop-wise, add it to the cell suspension in the F.ESC tube while gently tapping the tube. After pipetting gently to mix the cells, transfer cells to cryovials at 1 ml/vial. Tighten caps for all cryovials and transfer them to a freezing container. 71

Place the freezing container in a -80 C freezer overnight. 8. Repeat the above steps to freeze the rest of hescs. 9. Fill out the hesc Cryopreservation Log Sheet to record the location and number of vials of cells frozen in the -80ºC freezer. 10. Transfer the tubes containing spent medium and cell samples collected in Section 3.5 and Section 3.9 to the designated molecular lab for testing and characterization. 3.11. TRANSFER CELLS TO TWO DIFFERENT LN 2 FREEZERS THE NEXT DAY Note: Cells will be cryopreserved in two LN 2 freezers for safety issues. There should be one LN 2 freezer logbook for each freezer. 1. On each LN 2 freezer, find the Log Sheet. 2. Place a cryolabel (if available) on the sheet. Date and initial. 3. Mark the locations on the Log Sheet where vials containing hescs will be stored. 4. Take the freezing containers placed in the -80ºC freezer the previous day and place them next to the LN 2 freezer. 5. Quickly transfer half of the hesc cryovials into the LN 2 freezer. 6. Transfer remaining cryovials to another LN 2 freezer. 7. When all vials have been transferred, return the freezing containers to appropriate storage location. 8. Complete the hesc Cryopreservation Log Sheet and return it to Lab Manager for review. 9. Upon completion of review, the Lab Manager will place the Log Sheet in the binder/folder for the specific cell line 72

HESC CRYOPRESERVATION LOG SHEET Day Date (day/month/year) Performed by 1. Cell line information: - Provider s cell line name Cumulative hesc passage number 2. Cells at freezing: Colony quality Colony confluence level (%) Total well# to be harvested: 6-well plate # of vials to be frozen hesc density at freezing (select one): a: vial/well b: other Label on cryovial (Attach a cryo-label here if available) 3. Medium and solution needed for harvesting and freezing hesc cells: Collagenase solution required = 1ml/well x well# + 2ml extra = 1x _wells + 2 = ml hesc medium for harvesting = 2ml x well# + 2ml x plate# = 2x wells + 2x P# = ml hesc medium for freezing = 0.5ml/well x well# = 0.5x wells = ml Total medium required = ml for harvesting + ml for freezing + 5ml extra = ml Collagenase lot #, expiration, prepared by hesc medium lot #, expiration, prepared by Cryopreservation media required: Ingredient Ratio in the 2xFM 10ml total Actual vol = ml Defined FBS 60% 6 ml hesc culture medium 20% 2 ml Sterile DMSO 20% 2 ml Defined FBS lot #, expiration, prepared by Sterile DMSO lot #, expiration 4. Storage location in the -80 o C freezer 5. Storage location in LN2 tank(s): Room# Tank ID Rack# Box# & location Room# Tank ID Rack# Box# & location 6. Update LN2 Freezer Log Sheets: Initials Date 7. Update electronically LN2 Freezer Database: Initials Date Comments: Cryopreserved by Date Submit this form to Lab manager for review Reviewed by Date 73