Jan 28, 2020
iPSC Cell Culture – Maintenance and Expansion
- 1Washington University in Saint Louis - WUSTL (MO);
- 2Washington University in St Louis
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Jacob Marsh, Celeste M M. Karch 2020. iPSC Cell Culture – Maintenance and Expansion. protocols.io https://dx.doi.org/10.17504/protocols.io.9pwh5pe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 22, 2019
Last Modified: July 19, 2024
Protocol Integer ID: 30166
Keywords: iPSC, Cell Culture, Maintenance, Expansion
Abstract
This protocol is about Maintenance and Expansion of induced pluripotent stem cells.
Attachments
Materials
STEP MATERIALS
StemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
- DMEM/F12 medium
- Matrigel
- mTesR1 media
- Rock Inhibitor
- 1x PBS
- Accutase (Gibco A11105-01)
StemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
- 2X Freezing Medium (20 % DMXO, FBS)
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
Note
Warm all media before use. Media should be Room temperature . Do not use a water bath to warm media.
Matrigel Coating
Matrigel Coating
To resuspend, thaw aliquot On ice .
Add 12.5 mL cold DMEM/F12.
Pipette up and down twice.
Add 1 mL of Matrigel per well of 6 well plate.
Store diluted Matrigel at 4 °C .
Thawing iPSC
Thawing iPSC
Prior to thawing cells, coat plate with Matrigel for 01:00:00 .
Note
1 vial of iPSC should be thawed into 1 well of a 6 well plate.
Add 9 mL DMEM/F12 to a 15 ml conical tube labeled with the iPSC line name and passage number.
Remove cells from liquid nitrogen storage.
Quickly thaw cells in 37 °C water bath and/or in hands.
Just prior to complete thaw, remove vial from water bath.
Transfer the contents of the cryo-vial (~ 1 mL ) into the 15 ml conical tube.
Spin at 750 rpm for 00:03:00 at Room temperature .
Aspirate media.
Resuspend cells in 2 mL mTesR1 (supplemented with 5 micromolar (µM) — 10 micromolar (µM) Rock Inhibitor) by pipetting two times.
Transfer the cell solution to one well of a 6-well plate.
Incubate at 37 °C Overnight in 6 % CO2.
Replace the media daily until cells are ready to split or analyze.
Cell Growth/Maintenance
Cell Growth/Maintenance
Note
Media should be changed daily. It is okay to skip a media change one time each week if double feeding is performed; however, this is largely dependent on the density of the cells and volume of media (do not double feed if cells are more than 70% confluent).
Aspirate media.
Gently add fresh mTesR1 to cells (volume depends on cell density and well size).
- 0.5 mL per well to 24 well plate
- 2 mL — 4 mL per well to 6 well plate
- 5 mL — 10 mL to 10 cm2 plate
Incubate at 37 °C in 6 % CO2.
Cleaning
Cleaning
Note
When differentiating cells appear in the culture, it is important to remove all the cells promptly.
Repeated cleaning may be necessary over the course of several days to remove all the material. If differentiation is excessive and line is precious, perform subcloning.
Under microscope, remove differentiated cells with p20 or p200 tip (depending on the amount of differentiation). Transfer the cells/media to a biohazard bag.
Gently wash cells with 1x PBS.
Add fresh mTesR1.
- 0.5 mL per well to 24 well plate
- 2 mL — 4 mL per well to 6 well plate
- 5 mL — 10 mL to 10 cm2 plate
Incubate at 37 °C in 6 % CO2 until cells are 60 — 80 % confluent. Change mTesR1 media daily until
cells are needed. Repeat cleaning as necessary.
Splitting/Expanding
Splitting/Expanding
iPSCs grow on Matrigel. Plates should be coated with Matrigel at least 1 hour prior to plating and no
longer than 24 hours prior to plating cells:
- 0.5 mL in 12 well plate
- 1 mL in 6 well plate
- 4 mL in 10 cm2 plate
Note
It is critical to keep Matrigel on ice while coating. Prior to plating cells, ensure Matrigel has not
evaporated from well.
Aspirate media.
Gently wash cells with 1x PBS (2 — 3 ml/well).
Add Accutase (Gibco A11105-01) directly to the cells and incubate at 37 °C for 00:03:00 — 00:04:00 .
- 6 well plate, add 0.75 mL — 1 mL per well
- 24 well plate, add 0.5 mL
- 10 cm2 dish, add 3 mL
Tap dish to aid in dislocation of cells.
Add DMEM/F12 directly to cells and scrape gently to remove all cells (use p1000 for 24 well plate, and cell scraper for 6 well plate and 10cm2 dish).
- 6 well plate, add 2 mL — 4 mL per well
- 24 well plate, add 1 mL
- 10 cm2 dish, add 9 mL
Collect cells in conical tube (15 ml/50 ml depending on volume).
If necessary, add 2 mL — 5 mL DMEM/F12 to dish to remove all cells from the dish and add to conical tube.
Centrifuge cells at 750 rpm for 00:03:00 at Room temperature .
Carefully aspirate supernatant.
Note
To avoid aspirating cell pellet, it is OK to leave a small amount of media (0.5 mL — 1 mL ).
Resuspend cell pellet with mTesR1 (Rock Inhibitor addition varies, see below).
- 2 mL mTesR1 per well of a 6 well plate
- Our goal is to maintain iPSC lines without using Rock Inhibitor; however, this must be done through careful weaning off Rock Inhibitor
- All cells should be thawed in Rock Inhibitor:
- 10 micromolar (µM) concentration for new iPSC lines, lines thawed from 96 well after editing.
- 5 micromolar (µM) concentration if thawing from a line without knowledge of its Rock sensitivity.
- 1 micromolar (µM) concentration for all other lines (for lines still exposed to Rock Inhibitor, use
1 micromolar (µM) . Otherwise, do not use Rock Inhibitor.)
Pipet cells 2 times only to preserve clumps.
Transfer cell suspension to appropriate plate (pre-coated with Matrigel for at least 01:00:00 ).
- For maintenance, dilute cells 1:3 in mTesR1
- For expansion, plate all cells
Incubate at 37 °C in 6 % CO2 until cells are 60 — 80% confluent. Change mTesR1 media daily until cells are needed.
iPSC Freezing
iPSC Freezing
Aspirate media.
Gently wash cells with 1x PBS (Use 2 mL — 3 mL per well in 6 well plate).
Add Accutase (Gibbco A11105-01) directly to the cells and incubate at 37 °C for 00:03:00 — 00:04:00 .
- 6 well plate, add 0.75 mL — 1 mL per well
- 10cm2 dish, add 3 mL
Tap dish to aid in dislocation of cells.
Add DMEM/F12 directly to cells.
- 6 well plate, add2 mL — 4 mL per well
- 10cm2 dish, add 9 mL
- If cells remain attached, use a cell scraper to gently dislodge cells (apply gentle pressure and use 1 — 2 passes to remove cells)
Collect cells in conical tube (15 ml/50 ml depending on volume).
Add 2 mL — 5 mL DMEM/F12 to dish to remove all cells from the dish and add to conical tube.
Centrifuge cells at 750 rpm for 00:03:00 at Room temperature .
Carefully aspirate supernatant.
Note
To avoid aspirating cell pellet, it is OK to leave a small amount of media (0.5 mL — 1 mL ).
Resuspend cell pellet with mTesR1 (No Rock Inhibitor).
- Use volume appropriate for freezing
- Assume 1 mL per cryovial total and add ½ total volume of mTesR1
- Pipet cells 1 — 2 times only to preserve cell clumps
Note
Example: to freeze 10 tubes, you will need 10 mL total and will add 5 mL mTesR1 to cell pellet (and 5 mL of 2x Freezing Media below)
Add an equal volume of cold 2x Freezing Media (20 % DMSO, FBS). Pipet cells 1 time only to preserve cell clumps.
Transfer cell suspension to pre-labeled cryovials (1 mL per cryovial).
Ensure that cryovials are labeled with the following:
- Cell Type
- Line Name
- Passage #
- Date
- Your Name
Freeze vials at -80 °C in foam racks for 48:00:00 — 72:00:00 .
Transfer vials to liquid nitrogen for long-term storage.