干细胞培养
Human embryonic stem (hES) cells can be maintained on a layer of mitotically inactivated feeder cells such as irradiated mouse embryonic fibroblasts (iMEF, Catalog # PSC001). The protocol below has been used with the BG01V line of hES cells.
Please note that other hES cell lines may require modifications of this protocol. Optimal culture conditions must be determined by the investigator for each hES line. Please read the protocol in its entirety before starting.
SUPPLIES REQUIRED
REAGENTS
♦ Fetal bovine serum
♦ Knockout? serum replacer (Invitrogen, Catalog # 10828)
♦ Non-Essential Amino Acids (100x)
♦ L-Glutamine (200 mM)
♦ Penicillin/Streptomycin (100x)
♦ b-mercaptoethanol
♦ DMEM/F12
♦ High glucose DMEM
♦ Recombinant human FGF basic (R&D Systems, Catalog # 233-FB;or Tissue Culture Grade, Catalog # 4114-TC)
♦ Accutase (Innovative Cell Technologies, Catalog # AT104 or equivalent)
♦ 0.1% w/v solution of gelatin in sterile deionized H2O
MATERIALS
♦ BG01V human embryonic stem cells (ATCC # SCRC-2002)
♦ iMEF Feeder Cells (R&D Systems, Catalog # PSC001)
♦ Tissue culture plates(60 mm; the protocol can be adapted for other plate sizes)
♦ 15 mL centrifuge tubes
♦ 0.2 mm sterile filter unit
♦ Pipettes and pipette tips
EQUIPMENT
♦ 37°C, 5%CO2 incubator
♦ Centrifuge (low speed clinical or equivalent)
♦ Hemocytometer
♦ Microscope
REAGENT & MEDIA PREPARATION
Note: Sterile technique is required when handling the reagents.
♦ MEF Media - MEF media consists of high glucose DMEM, 10% fetal bovine serum, 2 mM L-glutamine, and if desired, add a 1:100 dilution of penicillin/streptomycin (100x) stock. Filter sterilize the media using 0.2 mm sterile filter unit.
♦ hES Media - hES media consists of DMEM/F12, 15% fetal bovine serum, 5% Knockout serum replacer, 1:100 dilution of non-essential amino acids stock (100x), 1:100 dilution of penicillin/streptomycin (100x) stock, and 0.1 mM b-mercaptoethanol. Filter sterilize the media using 0.2 mm sterile filter unit. Just prior to use, the media should be supplemented with recombinant human FGF basic at 4 ng/mL.
PROCEDURES
Note: When handling biohazard materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.
I. Thawing and Plating of iMEF Feeder Cells (plate feeder cells one day prior to stem cell seeding) (Figure 1)
1. Coat the appropriate sized plate(s) for the desired number of cells by covering the surface of the dish with 0.1% sterile gelatin for 15 minutes. For example, one vial of 6 x 106 iMEF can be plated on two 100 mm dishes, six 60 mm dishes, or two 6 well plates.
2. Warm MEF media to 37° C.
3. Thaw the desired number of vials of iMEF cells by quickly warming the cryotube(s) in a 37° C water bath until the cells are just thawed and then immediately transferring the contents of one vial to a 15 mL conical tube containing at least 5 mL of pre-warmed MEF media. Rinse the cryotube with an additional 1 mL of media to ensure the removal ofall the cells.
4. Spin at 200 x g in a clinical centrifuge for 5 minutes.
5. Remove the supernatant and gently flick the pellet.
6. Aspirate the 0.1% gelatin from the plate(s).
7. Resuspend the iMEF cells in MEF media and transfer to the gelatin-coated plates at a density of approximately 1 x 106 cells/60 mm plate.
8. Incubate overnight in a 37° C, 5% CO2 incubator before seeding with stem cells.
II. Thawing the BG01V hES cells
1. Warm the hES media to 37°C.
2. Place the cryovial of hES cells in a 37° C water bath until just thawed and then transfer the cells immediately to a 15 mL centrifuge tube containing at least 5 mL of pre-warmed hES media. Rinse the cryovial with an additional 1 mL of media to ensure the removal of all the cells.
3. Spin at 200 x g in a clinical centrifuge for 4 minutes.
4. Remove the supernatant and gently flick the pellet.
5. Resuspend the pellet in an appropriate amount of hES media freshly supplemented with 4 ng/mL of recombinant human FGF basic (typical volume is 5 mL per 60 mm plate).
6. Remove the MEF media from a plate containing iMEF cells and add the hES cell suspension. Typically 1 x 106 hES cells are thawed for a 60 mm plate.
7. Place the cells in a 37° C, 5% CO2 incubator. Cells should be fed daily with hES media freshly supplemented with recombinant human FGF basic. Passage the cells before the hES colonies touch at their edges.
III. Passaging of the BG01V hES cells (Figure 2)
Note: Plate iMEF feeder cells on the desired number of plates 1 day prior to passaging.
1. Warm the hES media to 37° C.
2. Remove the hES media from BG01V cells. Add 1 mL of Accutase solution to each 60 mm plate. Incubate at room temperaturefor 5 - 10 minutes or until cells begin to slough off the plate.
3. Pipette Accutase gently over the plate until all the cells have been detached.
4. Gently pipette cell suspension up and down to break up large cell clumps.
5. Transfer the cell suspension to a 15 mL centrifuge tube containing 5 mL of hES media and spin at 200 x g for 4 minutes.
6. Remove the supernatant and gently flick the pellet.
7. Resuspend the pellet in pre-warmed hES media and count the cells using a hemocytometer.
8. Plate the desired number of cells (approximately 0.5 -1.0 x 106 cells/60 mm plate) on the iMEF monolayer in hES media supplemented with 4 ng/mL of recombinant human FGF basic.
9. Feed the cells daily with hES media freshly supplemented with recombinant human FGF basic.
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