The HSCI iPS Core facility was created to accelerate research in the stem cell field by facilitating the derivation and distribution of iPS cell lines. Disease-specific iPS lines provide us with a unique opportunity to study the mechanisms of disease and ultimately to develop new treatments. The iPS Core serves as a repository for iPS cells produced by HSCI scientists and functions as a laboratory to produce disease-specific lines for sharing with the HSCI and broader research community.
Standard Operating Procedures (SOP) for the culture of human iPS
In general, caring for hiPS cells is identical to caring for hESCs, which you are assumed to have experience with prior to requesting these cell lines. While there are different ways of culturing these cell lines, the general concepts are the same. Provided are the general instructions we have used to culture hiPS cells (primarily adapted from the protocols from WiCell):
Standard hES Media (500 ml)
- 400 ml DMEM/F12
- 100 ml KOSR
- 5 ml L-Glutamine
- 5 ml P/S (optional)
- 5 ml MEM-NEAA
- 3.5 ul 2-Mercaptoethanol
- 5 ug bFGF (although WiCell suggests 50 ug, we found that 5 ug is sufficient)
MEF Media (500 ml)
- 450 ml DMEM
- 50 ml FBS
- 5 ml P/S
- 5 ml L-Glutamine
2X Freezing Media (10 ml)
8 ml defined FBS 2 ml DMSO
The iPS cells are typically maintained on 0.1% gelatin coated plates with MEFs. One can alternatively plate these cells on Matrigel and use mTeSR1 media for a feeder-free condition.
Commercial irradiated MEFs may be obtained from Global Stem Cell (CF-1; 6001G) containing about 4-5 million MEFs per vial. It is recommended to plate 1 million MEFs per 10 cm plate (~170,000 per well of a 6-well plate). You should optimize your MEF density as you see appropriate, judged by the level of differentiation of your iPS cells. MEFs should be given 8 hours minimum to settle after plating, although overnight is best (ideally used within 2 days).
Thawing Shipped hiPS Vial
Each vial shipped should be thawed in 1 well of a 6 well plate. The passage number and the name of the cell line can be found on the vial. All other information on the label can be ignored.
These cells should be kept in as large of clumps as possible to increase survival efficiency so one must minimize the amount of pipetting when thawing these vials.
- Set up 2x 15 ml conical tubes. In tube 1, add 1 ml of pre-warmed hES media. In tube 2, add 9 ml of pre-warmed hES media.
- Partially thaw the frozen vial of iPS cells at 37ºC, until there is a small piece of ice remaining. Spray the vial with 70% ethanol to sterilize.
- Taking 1 ml of media at a time from tube 2, slowly add the pre-warmed media dropwise to the vial and transfer the liquid content with cells into tube 1. Repeat until all 9 ml used.
- Spin at 1000 RPM for 2 min.
- Meanwhile, wash with PBS one well of a 6 well plate that was plated with MEFs atop gelatin one day prior. Add 2 ml hES media. Although not required, it is highly recommended that you add 10 μM ROCK inhibitor Y-27632 (both to 9 mL thawing media in tube 2 and to the final 3 ml of plating media) to improve survival efficiency. Do not add this ROCK inhibitor to any subsequent feeds. (The ROCK Inhibitor Y-27632 Enhances the Survival Rate of Human Embryonic Stem Cells Following Cryopreservation; Li et al.)
- Aspirate the media from the spun down tube 1, and gently resuspend the pellet with 1 ml of hES media. Pipet slowly 1 or 2X maximum, trying to avoid disrupting the chunks of cells, and transfer to one well of a 6-well plate.
- Change the medium after 36 to 48 hours.
- Feed cells daily with 2 ml medium. Colonies should emerge anywhere from 5 to 10 days.
- The first split should be mechanical (ratio depending on cell density observed).
NOTE: It is highly recommended that you perform a mycoplasma test upon successful thawing of these cells. It is also recommended that you karyotype the line about every 10 passages.
Passaging hiPS cells
Note: These instructions are for passaging cells grown on MEFs. For cells grown on Matrigel, one should use Dispase in place of Collagenase IV. Trypsinization is not recommended.
1. Before splitting, remove differentiated colonies under a microscope in sterile conditions (i.e. via slow-vacuum aspiration or pipet scraping). Be careful not to leave plate out too long and make sure cells do not dry out if using vacuum method. 2. Wash cells with either warm hES medium or PBS 3. Add 1 ml of Collagenase IV per well of a 6 well plate and incubate at 37ºC for 5-10 minutes (expect to see visible curling or thickening of colonies around the edges). 4. Aspirate off the enzyme and add 1 ml of hES medium. Using a cell lifter (i.e. Corning #3008), scrape the entire well to lift the colonies. 5. Pipet the solution into a conical tube; wash the well with an additional 1 ml hES medium and combine into tube. 6. Centrifuge 1000 RPM (200xg) for 2 min. 7. Aspirate off the media, and resuspend pellet in 1 ml media per well of a 6 well plate that you wish to plate (ratio depends on cell density just prior to splitting). Triturate to get medium-small fragments (~50-200 cells per fragment). Avoid over-triturating since that will lead to cell death, especially when colonies are broken down to single cell suspensions. 8. Plate 1 ml each into a well of a 6 well plate of MEFs that was pre-washed with PBS and containing 1 ml of hES media. 9. We recommend splitting 1:3 if the cells are close to confluency.
Note: For cells grown on Matrigel, the cells should be frozen in same manner, except using 500 ul of mFreSR per 6-well.
As with thawing, it is very important to minimize the amount of pipetting to ensure cell survival later on.
- Prepare the cells as described in steps 1-6 of "Passaging hiPS cells."
- Aspirate the media and carefully add 250 ul of hES media for every vial you intend to freeze (should freeze either 1 vial per well of a 6 well plate, or 5 vials per 10 cm dish).
- Add 250 ul of 2X freezing media for each vial you intend to freeze, and carefully resuspend the pellet in the combined media (keeping cells in as large of chunks as possible; generally pipetting 2x should be enough).
- Quickly transfer 500 ul per cryo-vial, and place inside isopropanol-containing freezing container (ie Mr. Frosty; VWR 55710-200). Store 24-48 hrs at -80C and then transfer to liquid nitrogen. (Once DMSO in contact with cells, work quickly and ideally get the cells at -80 within 3 min of contact).
For further information, please consult the following protocols:
- WiCell protocol
- Lerou et al. Nature Protocols 2008; 3:923-33
- Doug Melton protocol
- DMEM/F12: Invitrogen cat# 11330-057
- KOSR: Invitrogen cat# 10828-028
- L-Glutamine: Invitrogen cat# 25030-156
- Penicillin/streptomycin: Invitrogen cat# 15140-155
- MEM-NEAA: Invitrogen cat# 11140-050
- 2-Mercaptoethanol: Sigma cat# M-7522
- bFGF: Millipore cat# GF-003
- DMEM: Invitrogen cat# 11965-118
- FBS: Invitrogen cat# 16000-044
- Defined FBS: Hyclone cat# SH30070.01
- DMSO: Sigma cat# D-2650
- Irradiated CF1 MEFs: GlobalStem cat# 6001G
- Collagenase IV: Invitrogen cat# 17104-019
- Dispase: Invitrogen cat# 17105-041
- Rock inhibitor Y27632: Calbiochem cat# 688000
- 0.1% gelatin: Millipore cat# ES-006-B
If you have any question, please contact us at: Laurence_Daheron@harvard.edu