These cells should be recovered from cryopreservation and subcultured only on culture flasks that are sequentially pre-coated with 50 µg/mL poly-L-ornithine (Sigma, Cat. No. P-3655 or equivalent) and then with 1 µg/mL Human Fibronectin (Sigma, Cat. No. F-0895 or equivalent).
Note: Use flasks with vented caps for best results.
- Add 7.0 mL freshly diluted 50 µg/mL poly-L-ornithine to T-75 cm2 flask and allow flask to sit over night at room temperature.
- Remove and discard poly-L-ornithine solution. Rinse flask 3 times with sterile double distilled water. Discard last rinse and allow flask to air-dry uncapped and standing upright in a biological cabinet.
- Add 5.0 mL freshly diluted 1 µg/mL fibronectin and incubate 3 hours at 37°C.
- Remove and discard fibronectin solution. Rinse flask 3 times with sterile water. Discard last rinse and allow flask to air-dry uncapped and standing upright in a biological cabinet.
- Flask is ready for use when dry.
Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: Subculture before cells reach 80% confluency. Warm aliquots of wash medium (growth medium without bFGF) used in Step 4, freshly made complete growth medium and freshly diluted tryspin solution to 37°C prior to use.
Subcultivation ratio: A subcultivation ratio of 1:3 to 1:4 is recommended. Subculture approximately every 3 to 4 days.
- Remove and discard culture medium.
- Briefly rinse the cell layer with 10.0 mL Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS).
- Add 4.0 mL of freshly diluted, pre-warmed 0.025% Trypsin-0.1 g/L EDTA solution (see formula below) to flask and incubate for 3 minutes at 37°C. Knock the flask several times against the palm of your hand to dislodge cells. Observe flask under an inverted microscope to be sure cells have come off.
- Add 6.0 mL of pre-warmed wash medium (see Note above) and aspirate cells by gently pipetting.
- Transfer cell suspension to a centrifuge tube and spin at approximately 190 x g for 7 minutes. Discard supernatant and knock the tube against the palm of your hand to loosen the cell pellet. Using a 1 mL pipet, add 1.0 mL complete growth medium and pipet the pellet up and down to resuspend the cells (avoid creating foam or bubbles).
- Add an additional 2.0 mL of complete growth medium and dissociate cells further by pipetting up and down.
- Adjust cell concentration by adding the necessary volume of complete growth medium needed to seed new flasks. Pipet up and down to evenly resuspend cells.
- Add appropriate aliquots of the cell suspension to new pre-coated vented culture flasks.
- Incubate cultures in 5% CO2/95% air at 37°C.
Medium renewal: Every 2 to 3 days
2X Trypsin-EDTA Solution: 2X Trypsin-EDTA solution is 0.05% Trypsin-0.2g/L EDTA. Before use this must be diluted 1:1 in Ca++/Mg++ free Dulbucco's phosphate-buffered saline (D-PBS) to 0.025% Trypsin-0.1g/l EDTA
To make 1 liter of 2X Trypsin-EDTA solution:
- Add the following to 500 ml ddH2O:
- 8 g NaCl
- 0.4 g KCL
- 0.58 g NaHCO3
- 1 g Dextrose
- 0.2 g EDTA
- 0.5 g Trypsin (Sigma, Cat. No. T7409)
- Bring volume up to 1 liter with ddH2O, and pH to 7.4 with HCL.
- Incubate at 37°C for at least 1 hour to activate the trypsin.
- Sterile filter (0.2 µm) and make aliquots.
- Refrigerate at 4°C overnight and then store at -20°C.
- Before use, dilute trypsin 1:1 with Ca++/Mg++ free Dulbucco?s phosphate-buffered saline (D-PBS) and warm to 37°C.
Freeze medium: complete growth medium supplemented with 20% fetal bovine serum and 10% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%