Massachusetts Human Embryonic Stem Cell Bank. Human Embryonic Stem Cell Culture Methods

Transcription

1 Massachusetts Stem Cell Culture Methods

2 Massachusetts Cell Culture -Table of Contents CC001_Gelatin Coating of Culture Plates CC002_Thawing and Seeding of Frozen Inactivated Mouse Embryonic Fibroblasts (MEF) Cells CC003_Thawing and Seeding of Stem Cells (hesc s) onto a Mouse Embryonic Fibroblast Cell Feeder (MEF) Layer CC004_Replacement of Medium for Stem Cell Culture (hesc s) CC005_Enzymatic Passaging of Stem Cells (hesc s) on Fresh Mouse Embryonic Fibroblast Cell (MEF) Plates CC006_Manual Passage of Stem Cells (hesc s) on Fresh Mouse Embryonic Fibroblast (MEF) Cell Plates CC007_Harvest and Cryopreservation of Stem Cells (hesc s)

3 Massachusetts SOP-CC-001 Gelatin Coating of Culture Plates Objective: Stem Cells (hescs) are cultured on gelatin-coated plates. Gelatin is a translucent, colorless solid substance extracted from animal collagen. This SOP describes how 0.1% gelatin solution is used to coat plates for culture of MEF (Mouse Embryonic Fibroblast) cells. This SOP can be modified if other culture vessels require gelatin coating. Scope: This procedure applies to all Massachusetts laboratory personnel responsible for culture of hescs. 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. Definitions or abbreviations hescs: human embryonic stem cells MEF: Mouse Embryonic Fibroblast References Not applicable 1. Materials 1.1. Equipment: Sterile biosafety cabinet (tissue culture hood) 37 o C Incubator Pipet-Aid 1.2. Supplies 6-well tissue culture plates Costar ml sterile serological pipets Costar ml sterile serological pipets Costar 4488 Sterile Pasteur pipets Fisher D Disposable Nitrile gloves World Wide Medical Supplies Reagents 0.1% gelatin solution (SOP-RP-001, Preparation of 0.1% Gelatin Solution) Page 1

4 Massachusetts SOP-CC Preparation 2.1. Documentation and calculation 1. From the Gelatin Plate Request form, determine which kind and how many culture plates are requested. 2. Calculate total volume of gelatin solution required for all requests. o 4-well plates require 1ml of 0.1% gelatin solution to each well o 6-well plates require 2ml of 0.1% gelatin solution to each well Note: Make sure there is enough 0.1% gelatin solution available. If not, prepare according to SOP-RP-001, Preparation of 0.1% gelatin Solution Prepare the biosafety cabinet (tissue culture hood) 1. Place the following near the hood: o 5ml and 10ml sterile serological pipettes. o Absorbent paper towels (or Kimwipes) o 70% Ethanol spray o Appropriate-size disposable gloves o Appropriate cell culture plates or flasks 3. Procedure 3.1. Sterilization preparation before working in the hood 1. Wash your hands and arms thoroughly (about one minute) with soap. 2. Rinse completely with tap water. 3. Dry hands and arms with paper towel. 4. Put on appropriate-size gloves. 5. Spray 70% ethanol on the gloves and a paper towel until totally wet. 6. Thoroughly clean the surface in the hood with the ethanol-sprayed paper towel. 7. Ethanol spray the surface of everything you take into the hood. Dry them with paper towels if needed Aliquot 0.1% gelatin solution in the hood 1. Take the bottle of 0.1% Gelatin solution from the refrigerator and place it in the hood after cleaning it with 70% ethanol. 2. According to the volume calculated in Section 2.1, Documentation and calculation, do the following: o Label an appropriate-size sterile tube or bottle as GS for gelatin Solution. o Transfer an appropriate amount of gelatin Solution to the labeled tube or bottle. 3. Place the GS tube or bottle in a 37 o C water bath or 37 o C room for minutes Coat culture plates using 0.1 % gelatin solution 1. Place the plates which are to be coated in the hood. 2. Label the plates with: Page 2

5 Massachusetts SOP-CC-001 o G for gelatin o Date o Initials 3. For 4-well plates, add 1ml of 0.1% gelatin solution to each well; for 6-well plates, add 2ml of 0.1% gelatin solution to each well. 4. Tilt the plates in several directions so that the liquid covers the entire surface area. 5. Place the plates in a 37 o C incubator Post gelatin coating 6. Record necessary information on the Log Sheet for Gelatin Coating and Inactivated MEF Seeding. Note: The plates will be ready for use in 4 hours. They can be used for up to 7 days. Document History Date created: 10/28/08 (MJS) Date changed (xx/xx/xx) (initials) Page 3

6 Massachusetts SOP-CC-002 Thawing and Seeding of Frozen Inactivated Mouse Embryonic Fibroblasts Cells Objective: One of the methods to grow human embryonic stem cells (hescs) is to culture them on a feeder layer of inactivated MEF (Mouse Embryonic Fibroblast) cells. Inactivated MEF cells should be seeded one to three days prior to the plating of hescs. Either freshly-harvested or frozen inactivated MEF cells can be used for this purpose. This SOP describes how to thaw and seed vial(s) of frozen inactivated MEF cells into gelatin-coated plates. Scope: This procedure applies to all Massachusetts laboratory personnel responsible for culture of hescs. 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 tank additional eye protective safety glasses and insulated gloves should always be worn. Definitions or abbreviations hescs: human embryonic stem cells MEF: Mouse Embryonic Fibroblast CM: culture medium LN2: liquid nitrogen References Not applicable 1. Materials 1.1. Equipment Sterile biosafety cabinet (tissue culture hood) 37 o C Incubator 37 o C water bath Pipet-Aid Liquid waste disposal system for aspiration Bench-top centrifuge with swing rotors Safety glasses Insulated gloves Hemocytometer, or other cell counter Page 1

7 Massachusetts SOP-CC-002 Forceps for taking cryovials from a liquid nitrogen tank 1.2. Supplies 5ml sterile serological pipets Costar ml sterile serological pipets Costar ml sterile serological pipets Costar 4489 Sterile Pasteur pipets FisherBrand D 15ml sterile centrifuge tubes BD Falcon ml sterile centrifuge tubes BD Falcon Black extra-fine alcohol proof pen VWR Cryovial holder Nalgene % Ethanol spray Absorbent paper towels or Kimwipes 1.3. Reagents MEF culture medium (see SOP-RP-002, Preparation of MEF culture medium, for preparation) Gelatin-coated plates (see SOP-CC-001, Gelatin Coating of Culture Plates) Frozen inactivated MEF cells Ethanol 2. Preparation 2.1. Calculate the number of vials of inactivated MEF cells to be thawed 1. To seed 6 wells in a 6-well plate, x 10 6 MEF cells are required (assuming a 90% recovery of MEF cells from one freezing and thawing cycle). Note: Scale up or down proportionally if other cell culture vessels are used Calculate the amount of MEF culture medium (CM) is required for this process 1. For thawing: 9 ml CM is needed per vial of frozen MEF cells. 2. For seeding: cells will be x 10 6 MEF/ml. Total CM per MEF vial will be 9 ml for thawing + volume for seeding Total CM for all MEF vials= CM per MEF vial x number of MEF vials to be thawed = 5 ml extra to compensate for pipetting errors Documentation 1. Take out the binder with the Log Sheet for Gelatin Coating, Inactivated MEF Seeding and Culture (the form is shown at the end of this SOP). On the log sheet, Part 1 (Coat plates with gelatin solution) should already be recorded. Record necessary information about cells and culture medium on Part 2 of the log sheet (Thaw and seed inactivated MEF Cells). 2.In the LN2 Freezer log book, confirm that there is sufficient number of frozen inactivated MEF cells available for this process. Notify appropriate personnel if the total number of inactivated MEF cells is down to 15 vials or less. Page 2

8 Massachusetts SOP-CC Prepare the biosafety cabinet (tissue culture hood) 1. Place the following near the hood: o 5ml, 10ml, 25ml sterile serological pipettes. o Absorbent paper towels (or Kimwipes) o 70% Ethanol spray o Appropriate-size disposable gloves 3. Procedure 3.1. Sterilization preparation before working in the hood 1. Wash your hands and arms thoroughly (about one minute) with soap. 2. Rinse completely with tap water. 3. Dry hands and arms with paper towel. 4. Put on appropriate-size gloves. 5. Spray 70% ethanol on the gloves and a paper towel until fully saturated. 6. Thoroughly clean the surface in the hood with the ethanol-sprayed paper towel. 7. Ethanol spray the surface of everything you take into the hood. Dry them with paper towels if needed. 8. Make sure the following items are in the hood. o Sterile Pasteur pipets o 15ml, 50ml sterile centrifuge tubes o 10ml sterile serological pipets Fisherbrand F o Black extra-fine alcohol proof pen o A cryovial holder 3.2. Aliquot MEF CM in the hood 1. Take the MEF culture medium bottle from the refrigerator. 2. Place the bottle in the hood after cleaning it with 70% ethanol. 3. According to Section 2.2, calculate how much MEF culture medium (CM) is required for this process, label an appropriate-size sterile tube or bottle as MEF.M. 4. Transfer an appropriate amount of MEF CM to the labeled MEF.M tube or bottle. 5. Warm the MEF.M tube or bottle in a 37 o C water bath or 37 o C room for minutes Prepare gelatin plate(s) in the hood 1. Take out the gelatin plate(s) from the incubator and place it/them inside the hood. 2. Remove the gelatin solution from the plate(s) completely with a Pasteur pipette (be sure to remove all gelatin solution completely). 3. Return the plate(s) to the incubator for later use. Page 3

9 Massachusetts SOP-CC Thaw inactivated MEFCs Note: If thawing more than one vial of cells, thaw no more than two vials at one time. 1. Place the warmed MEF CM in the hood after thoroughly cleaning it with 70% ethanol. 2. Label a sterile 50ml tube (both on the cap & side) in the hood as MEF. (Note: label one 50ml tube for each vial of irradiated MEF cells to be thawed) 3. Transfer 9ml MEF CM into the MEF tube. 4. Put on eye protective safety glasses and insulated gloves. 5. In the LN2 freezer log book, locate the inactivated MEF cells in the log sheet. There is one sheet for each storage box in the freezer. Cross-out the vial(s) to be thawed using one line. Date & initial in the slot(s). 6. Open the freezer and take out the correct vial(s) of inactivated MEF cells. 7. Warm the vial(s) slightly with the palms of your hands as you walk to the water bath. 8. In the 37 o C water bath, immerse the vial in the water without submerging the cap. Swirl the vial gently. After about 30 sec, check frequently to see if the frozen solution is beginning to melt. This process may take up to 1 min. 9. When there is only a small piece of ice floating in the vial, remove the cryovials from the water bath. 10. Spray the vial(s) with 70% ethanol until saturated. 11. After drying with a Kimwipe, place the vial(s) in the hood. 12. In the hood, pipette the cells up and down gently a couple times, and then transfer the cell suspension drop-wise to the tube labeled MEF while gently swirling the tube. This will help to reduce osmotic shock to the cells. 13. Centrifuge for 5 minutes at 200 x g. 14. Return the 50 ml centrifuge tube to the hood after spraying it with 70% ethanol. 15. Remove the supernatant from the tube. Be careful not to touch the cell pellet. 16. Gently flick the bottom of the tube to loosen the cell pellet. 17. Transfer 10 ml of MEF CM to the 50ml cell tube. Resuspend and mix the cells by pipetting gently up and down a few times If necessary, count MEF cells. 19. Add extra MEF CM to the MEF cell tube to make the final cell concentration of cells at x 10 6 MEF/ml Seed MEF cells in 6-well or 100mm plates Seed MEF cells in 6-well plates 1. Take the gelatin plate(s) (no more than 3 plates at a time) from the incubator and place it/them in the hood. 2. Label the plates with MEF, date and initials. 3. Mix the MEF cells completely by pipetting up and down a few times. Then take 6 ml MEF cells and add 2ml/well to 3 wells. 4.Seed the remaining MEF cells to other gelatin-coated plates by repeating step 3. Page 4

10 Massachusetts SOP-CC When all plates in the hood are seeded, slide the plates in a cross motion inside the hood. 6. Transfer the plates to the incubator and make another 3-5x cross-motions with the plates. 7. Close the incubator door and repeat steps 1-6 steps if there are more MEF cells to be seeded Seed MEF cells in 100mm plates 1. Take out the gelatin plate(s) (no more than 5 plates at a time) from the incubator and place it/them in the hood. 2. Label the plates with MEF, date and initial. 3. Mix the MEF cells completely by pipetting up and down a few times. Then, take 12 ml MEF cells and drop-wise add evenly to the plate. 4. Seed MEF cells to other gelatin-coated plates by repeating the above step. 5. When all plates in the hood are seeded, slide the plates in a cross motion along inside the hood. 6. Transfer the plates to the incubator and make another 3-5x cross-motions with the plates. 7. Close the incubator door and continue and repeat steps 1-6 steps if there are more MEF cells to be seeded Post seeding 1. When all cells are plated, clean the hood thoroughly with 70% ethanol. 2. Do not disturb the freshly seeded plates for at least the next 12 hours Monitor MEF cells in culture 1. Check each plate of cells under the microscope the following day. 2. Record observations on the Log Sheet for Gelatin Coating, Inactivated MEF Seeding and Culture. 3. Repeat the above each day until the plates are used for hesc culture. 4. Notify the lab manager immediately if the culture is contaminated. 5. Complete the Log Sheet for Gelatin Coating Inactivated MEF Seeding and Culture (a template is shown on the next page). Submit the completed log sheet to the Lab Manager for review. Document History Date created: 10/28/08 (MJS) Date changed (xx/xx/xx) (initials) Page 5

11 Massachusetts SOP-CC-002 Log Sheet for Gelatin Coating, Inactivated MEF Seeding and Culture 1. Coat plates with Gelatin solution: Date (MM/day/year) - 0.1% Gelatin solution batch # Prepared by - Number of plates coated: 4-well plate 6-well plate - Number of wells/plate seeded Prepared by (Initials) Date 2. Thaw & seed inactivated MEF cells: Date (month/day/year) 2.1. Inactivated MEF cells: - MEF cell lot number - Vendor - Number of vials thawed - MEF cell inactivation method: o Irradiation Mitomycin C treatment 2.2. MEF culture medium: - MEF medium Batch# - Prepared by 2.3. MEF seeded plates: - Number of plates seeded: 4-well plate 6-well plate - Number of wells/plate seeded Prepared by (Initials) Date 3. Monitor MEF in culture: Days in culture 1 st day 2 nd day 3 rd day 4 th day Date (mm/day/year) confluence % Cell quality Initials Comments Reviewed by (Initials) Date Page 6

12 Massachusetts SOP-CC-003 Thawing and Seeding of hescs onto a MEF Feeder Layer Objective: Successful recovery after cryopreservation is important for efficient propagation of human embryonic stem cells (hescs) in culture. This SOP describes how a vial (or vials) of cryopreserved hescs is/are thawed and seeded on a feeder layer of inactivated MEF (Mouse Embryonic Fibroblast) cells in a one well or a 6-well plate. Scope: This procedure applies to all Massachusetts laboratory personnel responsible for thawing of hescs for culture. 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 removing cryovials from a liquid nitrogen freezer additional eye protective safety glasses and insulated gloves should always be worn. Definitions or abbreviations QA: quality assurance hescs: human embryonic stem cells MEF: Mouse Embryonic Fibroblast CM: culture medium LN2: liquid nitrogen References Not applicable 1. Materials Required 1.1. Equipment Sterile biosafety cabinet (tissue culture hood) 37 o C Incubator 37 o C water bath Pipet-Aid Liquid aspiration waste disposal system Bench-top centrifuge with swing rotors Safety glasses Insulated gloves Forceps for taking cryovials from a liquid nitrogen freezer Inverted microscope Page 1

13 Massachusetts SOP-CC Supplies 5ml sterile serological pipettes Costar ml sterile serological pipettes Costar ml sterile serological pipettes Costar 4489 Sterile Pasteur pipettes FisherBrand D 15ml sterile centrifuge tubes BD Falcon ml sterile centrifuge tubes BD Falcon Black extra-fine alcohol proof pen VWR Cryovial holder Nalgene % Ethanol spray Absorbent paper towels or Kimwipes MEF plate containing irradiated MEF cells 1.3. Reagents DMEM-F12 Invitrogen hes Cell Culture Medium (see SOP-RP-004 for preparation) 2. Preparation 2.1. Monitor MEF cells under microscope 1. Take out the MEF plates assigned for the process from the incubator one at a time and evaluate them under a microscope. 2. Return the plates to the incubator if there is no contamination. Note: If there is contamination in any culture plate, do the following: o Move the contaminated plate(s) to the designated area in the molecular lab (room# 232D). o Disinfect all wells in the plate(s) (whether contaminated or not) with bleach at 200 µl/well. o Record the contamination on the Log Sheet for Gelatin Coating, Inactivated MEF Seeding and Culture and report the situation to the lab manager. o Request replacement of MEF plates from the Lab manager Documentation 1. Obtain a hesc Thawing and Seeding Log Sheet from the Log Sheet binder 2. On the Log Sheet, record necessary information (e.g., the hesc line information, LN2 freezer storage location, hesc culture medium, etc.) 2.3. Prepare the biosafety cabinet (tissue culture hood) 1. Make sure there is a sufficient amount of the following materials near the hood: o 5ml, 10ml, 25ml sterile serological pipettes. o Absorbent paper towels (or kimwipes) o 70% Ethanol spray o Appropriate-size disposable gloves Page 2

14 Massachusetts SOP-CC Procedure 3.1. Sterilization preparation before working in the hood 1. Wash your hands and arms thoroughly (about one minute) with soap. 2. Rinse completely with tap water. 3. Dry hands and arms with paper towel. 4. Put on appropriate-size gloves. 5. Spray 70% ethanol on the gloves and a paper towel until fully saturated. 6. Thoroughly clean the surface in the hood with the ethanol-sprayed paper towel. 7. Ethanol spray the surface of everything you take into the hood. Dry them with paper towels if needed. 8. Make sure the following items are in the hood. o Sterile Pasteur pipettes o 15ml, 50ml sterile centrifuge tubes o Black extra-fine alcohol proof pen o A cryovial holder 3.2. Aliquot an appropriate amount of DMEM-F12 and hesc culture medium (CM) in the hood 1. Take a bottle of DMEM-F12 medium and a bottle of the appropriate hesc CM from the refrigerator. 2. Place them in the hood after cleaning with 70% ethanol 3. According to the volume calculated in the hesc Thawing and Seeding Log Sheet: o Label an appropriate-size sterile tube as DMEM. o Transfer an appropriate amount of DMEM-F12 to the tube. o Label an appropriate-size sterile tube or bottle as ESC.M. o Transfer an appropriate amount of hesc CM to the labeled ESC.M tube or bottle. 4. Warm the aliquots of medium in a 37 o C water bath or 37 o C room for 15-30min. 5. Return the bottles of stock medium to the refrigerator. Note: When the medium is warm, perform the following thaw procedure Thaw hescs Note 1: Thawing procedure will be executed by two employees; one performs the thawing, while the other (preferably the Lab manager or QA personnel) verifies the entire procedure and records necessary information on the hesc Thawing and Seeding Form. Note 2: Wear eye protective safety glasses and insulated gloves when removing cryovials from the liquid nitrogen freezer. Note 3: Thaw no more than two vials simultaneously. Page 3

15 Massachusetts SOP-CC Place the warmed DMEM-F12 and CM in the hood after thoroughly cleaning with 70% ethanol. 2. Label a sterile 50ml tube (both on the cap & side) in the hood as cell. 3. Transfer 9 ml CM per cryovial of hesc into the cell tube. 4. Put on eye protective safety glasses and insulated gloves. 5. According to the hesc Thawing and Seeding Log Sheet, locate in the LN2 freezer log book the desired vial(s) of hescs on the right log sheet and cross-out the vial(s) with one line. Date & initial next to the line. 6. Remove the hes cell vial(s) from the LN2 freezer. 7. Verify the label on the cryovial(s). 8. Warm the vial(s) slightly between your gloved hands on the way to the water bath. 9. In the 37 o C water bath, immerse the vial(s) in the water without submerging the cap. Swirl the vial gently. 10. After 30 seconds, check frequently to see whether frozen medium is beginning to melt. This process may take up to 1 minute. 11. When there is only a small piece of ice floating in the cryovial(s), remove the vials(s) from the water bath. 12. Spray the vial(s) with 70% ethanol until saturated. 13. After drying slightly with a Kimwipe, place the vial(s) in the cryovial holder that is already in the hood. 14. Pipette the cells gently up and down a few times and transfer the cell suspension dropwise to the CM in the labeled cell tube while gently swirling the medium to reduce osmotic shock to the cells. 15. Centrifuge for 5 minutes at 200 x g. 16. During the centrifugation period, label MEF plates as shown below: o o Take the approved MEF plates from the incubator and place them in the hood. Label the plate(s) in the hood with: The date (MM/day/yy) The words thaw and the cell line ID The hesc passage number from the cryovial plus one Your initials o For example: 09/10/08.thaw H1.p55.MJ. o Return the plates to the incubator. 17. When centrifugation is complete, return the centrifuge tube to the hood after cleaning with 70% ethanol. 18. Remove the medium from the tube (be careful not to touch the cell pellet). 19. According to the volume calculated for seeding in the hesc Thawing and Seeding Log Sheet, add the appropriate amount of CM to the tube and pipette cells gently a several times to mix. Page 4

16 Massachusetts SOP-CC Seed hesc cells in 6-well plates 1. Take the labeled MEF plates from the incubator and place them in the hood. 2. Aspirate the MEF medium completely from the wells with a Pasteur pipette. 3. Rinse the wells with DMEM-F12 at 2ml/well. 3. Mix the hesc cells in the tube completely by pipetting gently up & down a few times, and drop-wise add cells at 2.5ml/well to 6-well or 1ml/well to 4-well plates. 4. When all plates are seeded, slide the plates in a cross-motion inside the hood. 5. Transfer the plates to the incubator. 6. With the plates in the incubator, make another 3-5 cross-motions. 7. Close the incubator door. 8. Clean the hood thoroughly with 70% ethanol Post seeding 1. Do not move the freshly seeded plates for at least the next 12 hours. 2. Complete recording on the hesc Thawing and Seeding Log Sheet and place this sheet in the binder specific for this cell line. 3. Update the form Workflow Chart for Expansion & Cryopreservation of Human Embryonic Stem Cells and place it in the binder or folder specific for this cell line. 4. Update the electronic hesc database by eliminating the vials used. Note: From here on, monitor cell growth and change medium daily (see SOP-CC-004, Replacement of Medium for hesc culture). Colonies may not be visible for a few days. Document History Date created: 10/28/08 (MJS) Date changed (xx/xx/xx) (initials) Page 5

17 Massachusetts SOP-CC-004 Replacement of Medium for hesc Culture Objective: Successful growth of hescs requires daily replacement of culture medium. This is true regardless of what culture medium is used in the presence or absence of MEF feeder layer in the culture. This SOP describes how spent hesc medium is removed from one 6-well plate and fresh medium is added to the plate. Adjust accordingly if other culture vessels are used for hesc culture. Scope: This procedure applies to all Massachusetts laboratory personnel responsible for changing of hesc culture medium. 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. Definitions or abbreviations hescs: human embryonic stem cells MEF: Mouse Embryonic Fibroblast CM: culture medium References Not applicable 1. Materials Required 1.1. Equipment Sterile biosafety cabinet (tissue culture hood) 37 o C Incubator 37 o C water bath or a warm room. Inverted microscope Pipet-Aid Liquid aspiration waste disposal system Alcohol proof pen 1.2. Supplies 5ml sterile serological pipets Costar ml sterile serological pipets Costar 4488 Sterile Pasteur pipets FisherBrand D Disposable gloves World Wide Medical Supplies Page 1

18 Massachusetts SOP-CC Reagents hesc culture medium (see SOP-RP-004 for preparation) 2. Preparation 2.1. Documentation and calculation 1. Take the hesc Culture Log Sheet from the appropriate binder. 2. Record the hesc medium batch number to be used for this process on the sheet. 3. Calculate the amount of CM required according to the following formula and record it in the hesc Culture Log Sheet: o 6-well plate = Well# x 2.5ml/well + 2ml extra = ml 2.2. Prepare the biosafety cabinet (tissue culture hood) 1. Make sure there are sufficient materials listed below near the hood: o 5ml and 10ml sterile serological pipets. o Absorbent paper towels (or kimwipes) o 70% Ethanol spray o Appropriate-size disposable gloves 3. Procedure 3.1. Sterilization preparation before working in the hood 1. Wash your hands and arms thoroughly (about one minute) with soap. 2. Rinse completely with warm tap water. 3. Dry hands and arms with paper towel. 4. Put on appropriate-size gloves. 5. Spray 70% ethanol on the gloves and a paper towel until fully saturated. 6. Thoroughly clean the surface in the hood with the ethanol-sprayed paper towel. 7. Ethanol spray the surface of everything you take into the hood. Dry them with paper towels if needed. 8. Make sure the following items are in the hood. o Sterile Pasteur pipets o 15ml, 50ml sterile centrifuge tubes o Black extra-fine alcohol proof pen 3.2. Aliquot the appropriate amount of hesc CM required for this procedure in the hood 1. According to the batch lot number in the hesc Log Sheet, take the appropriate medium bottle from the refrigerator. 2. Place the bottle in the hood after cleaning it with 70% ethanol. 3. According to the volume calculated in Section 2.1, Documentation and Calculation, do the following: o Label an appropriate-size sterile tube or bottle as M for medium. o Transfer an appropriate amount of hesc CM to the labeled M tube or bottle. 4. Place the M tube or bottle in a 37 o C water bath or 37 o C room for minutes. Page 2

19 Massachusetts SOP-CC Monitor hesc growth 1.Take the hesc plates from the incubator one by one and evaluate cell growth under the microscope. 2.Record observations in the hesc Culture Log Sheet. 3.Return the hesc culture plates to the incubator. 4.If there is contamination in any culture plate, do the following: o Place the contaminated plate in a specifically designated tissue culture hood and report the situation to the Lab Manager or designee. o Based on the decision made, either Move the plate to the designated area in the molecular lab (room# 232D) and disinfect all wells in the plate with bleach at 200 µl/well or Disinfect only the contaminated well and place the plate in a specifically designated incubator for observation during the next a few days. o Change medium for this plate at least 30 minutes after disinfection in the specifically designated biosafety hood. o Record the contamination on the Culture Log Sheet. Note: When the medium is warm, perform the following procedure. 3.4 Change medium for hescs culture Note: If there are multiple identical plates of the same line requiring feeding, it is acceptable to change medium for up to three plates simultaneously in the hood. However, it is not acceptable to have different cell lines in the hood at the same time. 1. Place the warm M tube or bottle in the hood after thoroughly cleaning with 70% ethanol. 2. Take the hesc plates from the incubator and place them in the hood. 3. Remove the spent medium completely using a pasteur pipet. 4. Add 2.5ml fresh ESC medium to each well in 6-well plate plate. 5. Never insert a used pipet into sterile media. After pipets are used once, they must be discarded outside the hood in order to avoid any contamination. 6. Return the hesc plates back to the incubator. 3.5 Post medium change 1. Sign and date the hesc Culture Log Sheet. 2. Return the Log Sheet to the appropriate binder. Note: Repeat the above procedure daily until the cells are ready for passage or splitting to new MEF plates. Page 3

20 Massachusetts SOP-CC-004 Document History Date created: 10/28/08 (MJS) Date changed (xx/xx/xx) (initials) Page 4

21 Massachusetts SOP-CC-005 Enzymatic Passage of hescs on Fresh MEF Plates Objective: Successful growth of hescs requires regular passage of hescs. This SOP describes how hescs will be enzymatically harvested from old 6-well MEF plates and seeded on new 6- well MEF plates. This SOP can be modified by adjusting reagents proportionally if hescs are cultured in other culture vessels. If hesc differentiation level is close to or greater than 20% follow SOP-CC-006 for manual passage of hescs. Scope: This procedure applies to all Massachusetts laboratory personnel responsible for passaging hescs to new MEF plates. 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. Definitions or abbreviations hescs: human embryonic stem cells MEF: Mouse Embryonic Fibroblast CM: culture medium References SOP-CC-004, Replacement of Medium for hesc cultures Materials Required 1.2. Equipment Sterile biosafety cabinet (tissue culture hood) 37 o C Incubator 37 o C water bath or a warm room. Inverted microscope Pipet-Aid Liquid aspiration waste disposal system Bench-top centrifuge with swing rotors Balance Timer Page 1

22 Massachusetts SOP-CC Supplies 5ml sterile glass pipets Fisher D 5ml sterile serological pipets Costar ml sterile glass pipets Fisher E 10ml sterile serological pipets Costar ml centrifuge tubes BD Falcon ml centrifuge tubes BD Falcon ml sterile conical centrifuge tubes Corning Alcohol proof pen Sterile Pasteur pipets Fisherbrand D 6-well plates with MEFs 1.3. Reagents DMEM-F12 Invitrogen Collagenase Solution (see SOP-RP-005 for preparation) hes Cell Culture Medium (see SOP-RP-004 for preparation) 2. Preparation 2.1. Determine when and how to passage (or split) hescs Note: Different cell lines have different growth kinetics. Therefore, different splitting schedules may be used for different cell lines. However, it is generally true that hescs grow slowly during the first couple of weeks after being thawed, then faster until the growth rate reaches a plateau. The cell growth rate will then stay in that plateau for many passages if cultured properly. It is essential to observe cell growth daily to become familiar with the growth kinetics of each cell line. 1. Under a microscope, carefully evaluate hesc growth and quality. Observations will be used to determine when and how often the hescs are passaged. 2. Passage hescs (normally every 4-10 days) when o MEF feeder cells are two weeks old in the culture, or o hesc colonies are becoming too dense or too large, or o Increased hesc differentiation occurs (see SOP-CC-006 for passaging of hescs, if differentiation level is close or higher than 20%). Note: Passage hescs when any of the above occur. 3. How often to passage of hescs depends on the cell growth rate: o Look at the hesc Culture Log Sheets for the previous passages (up to three passages). Compare the cell splitting-ratios and the colony confluence levels at the harvest days between sequential passages. Page 2

23 Massachusetts SOP-CC-005 If cells grew significantly faster than the previous passage, increase the splitting ratio. If the grow rate is similar to the previous passage, use the same cell splittingratio. If cells grew significantly slower than the previous passage, split cells at a lower ratio than the previous one. Note: During the first few passages after thawing, hescs grow slowly and they may only be passaged to new MEF plates at a 1:1 ratio (i.e., one well to one well). Later, cells may be split using a1:3 to 1:6 ratio Documentation 1. Once it is decided to passage cells, obtain a new hesc Culture Log Sheet from the binder. 2. Check the Log Sheet to confirm that new MEF plates are prepared for this passage Calculate how much medium and solution are needed for this process 1. For harvesting and plating hescs on MEF cells, the following are required: o DMEM-F12 o Collagenase solution o hesc medium 2. On the hesc Culture Log Sheet calculate and record the amount of the above materials required for this process, including the lot number. 3. Record other necessary information on the Culture Log Sheet Prepare the biosafety cabinet (hood) 1. Make sure there are sufficient materials listed below near the hood: o 5ml, 10ml, sterile serological pipets. o Absorbent paper towels (or kimwipes) o 70% Ethanol spray o Appropriate-size disposable gloves 3. Procedure 3.1. Sterilization preparation before starting work in the hood 1. Wash your hands and arms thoroughly (about one minute) with soap. 2. Rinse completely with warm tap water. 3. Dry hands and arms with paper towel. 4. Put on appropriate-size gloves. 5. Spray 70% ethanol on the gloves and a paper towel until fully saturated. 6. Thoroughly clean the surface in the hood with the ethanol-sprayed paper towel. 7. Ethanol spray the surface of everything you take into the hood. Dry them with paper towels if needed. Page 3

24 Massachusetts SOP-CC Make sure the following items are in the hood. o Sterile Pasteur pipets o 15ml, 50ml sterile centrifuge tubes o Black extra-fine alcohol proof pen 3.2. Aliquot hesc medium and Collagenase solution in the hood 1. According to the volume calculated in the hesc Culture Log Sheet, label: o One or two sterile 50 ml tubes or an appropriate sterile bottle as ESC.M (on both the cap & side). o One or two sterile 50 ml tubes as Collagenase. o One or two sterile 50 ml tubes as DMEM-F According to the batch number in the hesc Log Sheet, take out the appropriate ESC medium and collagenase solution bottles, and a bottle of DMEM-F12 medium from the refrigerator. Place them in the hood after thoroughly cleaning them with 70% ethanol. 3. Transfer appropriate volume of ESC medium to the ESC.M tube(s) or bottle. 4. Transfer appropriate volume of collagenase solution to the Collagenase tube(s). 5. Transfer appropriate volume of DMEM-F12 medium to the DMEM-F12 tube(s). 6. Return the hesc, DMEM-F12 medium and collagenase solution stock bottles to the refrigerator. 7. Place the aliquot DMEM-F12 and ESC medium in a 37 o C water bath or warm room for minutes. 8. Leave the aliquot of collagenase solution in the hood for minutes to reach room temperature Monitor MEF cells under a microscope 1. Take the freshly-seeded MEF cell plates from the incubator one at a time and evaluate cell culture under microscope. 2. Return the plates to the incubator if there is no visible contamination. If there is contamination in any culture plate, do the following: o Move the contaminated plate(s) to the designated area in the molecular lab (room# 232D). o Disinfect all wells (whether contaminated or not) in the plate(s) with bleach at ul/well. o Record the contamination in the Culture Log Sheet and report the situation to the lab manager. o Request replacement of MEF plates from the Lab manager. Note: When the medium is warm, perform the following Prepare MEF plate(s) (up to 3 plates may be processed at a time) 1. Place the warmed DMEM-F12 and hesc medium aliquots in the hood after thoroughly cleaning the containers with 70% ethanol. 2. Take the approved MEF plates and place them in the hood. Page 4

25 Massachusetts SOP-CC Label the plates with the hesc line abbreviation, new passage number, the date and your initials. For example: H1.p55, 10/25/08, MJ. 4. Completely remove the medium from the MEF plate(s) with a Pasteur pipet. 5. Add 2 ml/well of fresh DMEM-F12 medium. 6. Gently swirl the plate to rinse the well walls. 7. Remove the DMEM-F12 medium completely from the plate(s) with a Pasteur pipet. 8. Add 1.5ml of fresh hesc medium/well. 9. Return the plate(s) to the incubator Harvest hescs (no more than two plates at a time) 1. Set the timer at 5 minutes and 10 minutes, respectively. 2. Label a sterile 15ml or 50ml tube as ESC for collecting hescs later. 3. Take the hesc plate(s) from the incubator and place them in the hood. 4. Remove the medium from the plate(s) completely with a Pasteur pipet. 5. Add 1ml collagenase/well. 6. Return the plate(s) to the incubator. 7. Start the timer. 8. After 5 minutes of incubation, check the plate(s) under a microscope to determine whether the cell colonies are ready to be harvested. If they are ready, the perimeter of the colony should appear folded back, separating from the MEF cells. If they are not ready, return the plate back to the 37 o C incubator for another 3-5 minutes of incubation. Note: Incubation length depends on the freshness of the collagenase solution. The older it is, the longer the incubation time will be. However, do not exceed 15 minutes of incubation time. 9. When collagenase incubation is complete, place the hesc plate(s) in the hood. 10. Remove 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 into the collagenase solution rather than aspirating the collagenase out of the well. Combine cell/colony solution from all wells into one 15ml/50ml tube. Centrifuge, then carefully remove the supernatant and resuspend the cell pellet with 5 ml ESC medium. Continue with Section 3.6 if there are more plates to be harvested, otherwise continue with Section Add 2 ml ESC medium to each well in the plate(s). 12. Using a sterile 5 ml glass pipet, take up most of the medium from one well. Use the pipet to scrape the cells/colonies in the well while slowly releasing the medium into the well to wash the cells/colonies off the surface. Pipette up and down gently in the medium during the wash to minimize bubbles. Repeat these steps until most of the cells/colonies are Page 5

26 Massachusetts SOP-CC-005 detached from the surface. Leave the contents in the well until cells in all of the wells are detached. 13.When cells in all wells of one plate are detached, transfer the cell/colony solution into the labeled sterile ESC tube. 14.Take another 3ml ESC medium and rinse all the wells in the plate. Transfer the cell/colony solution to the same ESC tube. 15.Harvest the cells from the other hesc plates Repeat Section 3.5, Harvest hescs, if there are more plates 3.7. Spin and disrupt hesc colonies 1. Pellet the cells in the ESC tube by centrifugation at 200 x g for 5 minutes. 2. Aspirate the supernatant from the tube (do not touch the cell pellet). 3. Resuspend the cell pellet in appropriate volume of ESC medium (1ml/well to be seeded, plus 0.5ml extra for pipetting error). 4. Using a 5 or 10ml glass pipet, disrupt the cell clusters by pipetting cells up and down gently a few times. Pipette carefully to avoid bubbles in the cell suspension. Note: In the above step, pipette the cell suspension up and down relatively fast initially to disturb big cell clusters. Check frequently to see if the big cell clusters are disrupted. When most of the colonies are small enough to float in the solution, the hescs are ready to be plated. When mixing, pipette slowly as not to break down clusters any more than needed Add hescs to MEF plates (no more than three plates at a time): 1. Return the MEF plates (containing 1.5 ml/well ESC medium) to the hood. 2. Using a 5ml glass pipet, gently pipet cells up and down a few times. Then take 3 ml of the cell suspension and drop-wise (in a circular motion) add 1ml/well to each of the three wells. 3. Repeat the above two steps until cells are distributed to all wells in the MEF plate. 4. When complete, slide the plates in a cross-motion along the inside surface of the hood. 5. Transfer the plate to the incubator. 6. Slide the plates in a cross-motion along the inside surface of the incubator. 7. Close the incubator door. 8. Continue with steps 2-7 if there are more cells to plate. 9. When all cells are plated, clean hood thoroughly with 70% ethanol. Note: Do not move the freshly seeded plates for at least the next 12 hours. Note: check cells under microscope the next day. Page 6

27 Massachusetts SOP-CC-005 Document History Date created: 10/28/08 (MJS) Date changed (xx/xx/xx) (initials) Page 7

28 Massachusetts SOP-CC-006 Manual Passage of hescs on Fresh MEF Plates Objective: Successful growth of hescs requires great attention to morphological changes of colonies in culture. It is important to keep differentiation of the stem cells to a minimum in culture. If a high level of differentiation (close to or higher than 15%) occurs, the differentiated areas should be removed. This Standard Operating Procedure (SOP) describes how differentiated colonies can be removed or high quality hescs can be selected for further culture. Scope: This procedure applies to all MA SCB laboratory personnel responsible for passaging hescs to new MEF plates. Responsibility: It is the responsibility of the Laboratory Operations Manager and Quality Assurance Officer to ensure all required personnel are properly trained for performing this SOP. Safety: All laboratory personnel should be in compliance with UMASS health and safety regulations when working in the laboratory. Specifically, wear protective equipment (lab coat, disposable gloves) while working in the lab. Definitions or abbreviations QA: Quality Assurance hescs: Stem Cells MEF: Mouse Embryonic Fibroblast CM: Culture Medium References SOP-XX-XXXX, Change Medium for hesc Culture 1. Materials Required 1.1. Equipment Sterile biosafety cabinet (cell culture hood) Sterile biosafety cabinet equipped with a stereomicroscope 37 O C Incubator 37 O C water bath or a warm room. Inverted microscope with a colony marker Pipet-Aid Liquid aspiration waste disposal system Bench-top centrifuge with swing rotors Mettler Balance Timer Alcohol burner Page 1

29 Massachusetts SOP-CC Supplies 5ml sterile glass pipets Fisher D 5ml sterile serological pipets Costar ml sterile serological pipets Costar ml centrifuge tubes Corning ml centrifuge tubes Corning inch Pasteur pipets Fisherbrand D 6-well plates with MEFs 1.3. Reagents DMEM-F12 Invitrogen, Collagenase Solution (1mg/ml, see SOP-RP-005 for preparation) hesc Culture Medium (see SOP-RP-004 for preparation) 2. Preparation 2.1. Identifying Differentiated Cells 1. Healthy, undifferentiated hesc colonies generally have well-defined uniform borders and the individual cells within the colony appear to be similar. The exact colony morphology will differ with different culture conditions (i.e., whether MEFs, mtesr1, or conditioned medium with Matrigel are used for hesc culture). 2. Spontaneously differentiated cells may resemble cobblestones or trophoblast-like cells. The differentiated cells may reside at the colony perimeter or in the center of the colony. There can also be differentiated colonies containing large clumps of swirled tissue, or little balls of cells, like embryoid bodies. Different culture conditions yield different types of differentiated cells Determine when to passage (or split) hescs 1. Examine the hesc culture daily under a microscope to assess colony size and differentiation. 2. In general, passage cells when the either of the following occurs: Mouse Embryonic Fibroblasts (MEF) feeder layer is two weeks old in culture. hesc colonies are too dense or too large. Increased rate of differentiation occurs Determine how to passage (or split) hescs Note: Regardless of the method used, always set aside one well of unpassaged hescs in case of passaging errors. 1. Two methods can be used to passage healthy hescs: Remove the differentiated areas first, then passage undifferentiated hescs to new MEF plates. Page 2

30 Massachusetts SOP-CC-006 Pick the undifferentiated healthy hescs, and transfer them to new MEF plates. 2. Which method to use depends on the differentiation level in the cultures. % Differentiation Method to use Split ratio 15-70% Remove differentiated areas before harvesting hescs 1:1 to 1:3 >70% Pick only undifferentiated areas of cells (i.e. *manual transfer) 1:1 *Manual transfer is also recommended for very sparse cultures (fewer than 20 colonies/well) such as during the first passage after thawing Prepare sterile bent Pasteur glass pipettes Note: Whatever method is chosen, modified (bent) Pasteur pipettes are required to either remove the differentiated cells or pick undifferentiated hescs to new MEF plates. 1. Turn on an alcohol burner. 2. Take a 9-inch Pasteur glass pipette and warm briefly (about 3-5 seconds) the area about two inches from the tip over the flame. 3. Once the area is pliable, remove the pipette from the flame and gently pull the tip at a 45o angle until it separates from the base of the pipette. Ideally, the pipette should now have a small 0.5 inch hook that ends with a small ball. 4. Place the bent Pasteur pipette gently in a Pasteur pipette container. 5. Repeat the above to make more bent Pasteur pipettes and place them in the same container. 6. Autoclave the bent Pasteur pipettes according to the SOP-SP-001 for sterilization Documentation Once it is decided to passage cells, obtain the appropriate hesc Culture Log Sheet from the binder. Record the selected passage method on the log sheet. Check the Log Sheet to confirm that new MEF plates are prepared for this passage Calculate how much medium and solution are needed for this process 1.The following are required for passaging hescs on new MEF cells: o DMEM-F12 o Collagenase solution o hesc medium 2.On the hesc Culture Log Sheet calculate and record the amount of the above materials required for this process, including the lot number. 3.Record other necessary information in the Culture Log Sheet Prepare the regular biosafety cabinet (cell culture hood) Page 3

31 Massachusetts SOP-CC Make sure there are sufficient materials listed below near the hood: o 5ml, 10ml, 25ml sterile serological pipets. o Absorbent paper towels (or kimwipes) o 70% Ethanol spray o Appropriate-size disposable gloves 3. Procedure 3.1. Sterilization preparation before starting work in the two hoods 1. Wash your hands and arms thoroughly (about one minute) with soap. 2. Rinse completely with warm tap water. 3. Dry hands and arms with paper towel. 4. Put on appropriate-size gloves. 5. Spray 70% ethanol on the gloves and a paper towel until fully saturated. 6. Thoroughly clean the surface in the hood with the ethanol-sprayed paper towel. 7. Ethanol spray the surface of everything you take into the hood. Dry them with paper towels if needed. 8. Make sure the following items are in the hood. o Sterile Pasteur pipets o 15ml, 50ml sterile centrifuge tubes o Black extra-fine alcohol proof pen 9.Make sure the following is in the cabinet equipped with a stereomicroscope: Sterile bent Pasteur pipettes Note: Do not pass your hands over an open culture plate or an open bottle of medium in the hood Prepare fresh MEF plates for passaging of hescs as shown in SOP-CC-005. Add 1m/well hesc culture medium instead 1.5ml/well after rinsing the wells with DMEM- F12 medium. Return the MFE plates to the incubator Remove differentiated colonies or areas by manual scraping 1. Remove a hesc plate from incubator and place it under an inverted microscope. 2. Mark the differentiated area in the hesc plate bottom using the colony marker attached to the microscope. 3. Transfer the hesc plate to a biosafety cabinet equipped with a stereomicroscope. 4. Place the plate under the stereomicroscope and use a bent Pasteur pipette to scrape all marked (differentiated) cells in the plate until they are floating in the medium. 5. Transfer the plate to a regular biosafety cabinet equipped with a vacuum aspiration system. 6. From the plate, aspirate the spent medium along with the floating differentiated cells or cell debris using a straight Pasteur pipette. Page 4