Jan 07, 2025
Version 2
iPSCs Maintenance and Banking V.2
- Mahmoud ElAchwah1,
- Sol Diaz de Leon Guerrero1,
- Zhiping Pang1
- 1Rutgers University
Protocol Citation: Mahmoud ElAchwah, Sol Diaz de Leon Guerrero, Zhiping Pang 2025. iPSCs Maintenance and Banking. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qd5ogr2/v2Version created by Sol Diaz de Leon Guerrero
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: January 07, 2025
Last Modified: January 07, 2025
Protocol Integer ID: 117835
Keywords: hiPSC, cell culture, pluripotent stem cell, induced pluripotent stem cell, ipscs maintenance, cell, thorough description of the thawing
Abstract
This protocols offers a thorough description of the thawing, maintenance and banking of human induced pluripotent stem cells.
Materials
Matrigel:
Matrigel catalog number: Cornig 354234
Matrigel stock concentration: 9-11
Matrigel working concentration: 90-110 µg/mL (1:100 dilution)
Matrigel Stock aliquot volume: 400 µL
Culture Antibiotic:
Primocin catalog number: InvivoGen ant-pm-1
Primocin stock concentration: 50 mg/mL
Primocin working concentration: 100 µg/mL
Primocin stock aliquot volume: 1 mL
CEPT:
Chroman 1 catalog number: MedChemExpress HY-15392
Chroman 1 stock concentration: 50 μM
Chroman 1 working concentration: 50nM
Emricasan catalog number: SelleckChem S7775
Emricasan stock concentration: 5 mM
Emricasan working concentration: 5 μM
Polyamine supplement catalog number: SigmaAldrich P8483
Polyamine supplement working concentration: 1000x
Polyamine supplement stock concentration: 1x
Trans-ISRIB catalog number: R&D systems 5284
Trans-ISRIB stock concentration: 700 μM
Trans-ISRIB working concentration: 700 nm
Culture Media:
DMEM/F-12 catalog number: cytiva SH30023.01
MEM catalog number: Gibco 51200-038
Accutase catalog number: Innovative cell technologies AT-104
mTESR+ basal medium catalog number: Stem Cell Technology 100-0274
mTESR+ 10x suplement: Stem Cell Technology 100-0275
mTESR Plus kit: Stem Cell Technology 100-0276
mTESR+ supplemented media
- Add 100ml of mTESR 10x supplement into 400ml of mTESR+ media
- Filter and aliquot 45ml into conical tubes, keep at -20°C.
mFreSR media catalog number: Stem Cell Technology 05855
Troubleshooting
Matrigel preparation
Thaw Matrigel stock stored at-80 °C by placing on ice and leaving at 4 °C Overnight
Place a 10ml pipette, 1 ml pipete tips and 1.5ml eppendorf tubes to cool before preparing Matrigel aliquots
Place Matrigel stock on ice and mix with the previously cooled 10 ml pipette. Make 400 µL aliquots of Matrigel to avoid multiple freeze-thaw cycles using previously cooled tips and tubes.
Store 400 µL Matrigel aliquots at -20 °C until use
To prepare matrigel working solution thaw 400 µL Matrigel aliquots On ice or at 4 °C
Dilute 400 µL Matrigel aliquot in 40 mL of MEM (1:100 dilution)
Mix well before use. Store at 4 °C protected from the light
Medium Preparation
Prepare mTESR+ media by adding 100 mL of mTESR+ 5x supplement into 400 mL of mTESR+ media and filter it.
Add 1 mL of Primocin (50mg/ml) to the mTESR+ media (final concentration 100ug/mL)
Matrigel Coating
5h
Before thawing or passaging cells, coat wells with 1 mL of Matrigel per well of 6 well plates or per 35 mm plate
Leave coated plates for 01:00:00 to 04:00:00 in the incubator at 37 °C
5h
Store diluted matrigel at 4 °C
iPSC thawing
5m
Take out 2 mL of mTESR+ per cell line and leave at room temperature to warm up
Take out 5 mL of DMEM/F-12 per cell line and leave at room temperature to warm up
Label 15 mL conical tubes per line to thaw
Take out iPSC vials in dry ice
Thaw iPSC vials using a 37 °C water bath until a small piece of ice is left in the vial
Using a 1 ml pipette mix the cells gently and transfer to labeled 15 mL conical tubes.
Add 5ml of DMEM/F-12 dropwise to the conical tube with the cells
Place conical tubes in centrifuge and spin at 300 x g for 00:05:00 at Room temperature
5m
Supplement mTESR media with CETP
Add 1 µL of CET per 1 mL of mTESR+ (1:1000 ratio)
Add 1 µL of P per 1 mL of mTESR+ (1:1000 ratio)
Aspirate matrigel from coated plates
Add 1 mL of mTESR+ containing CETP to each well
Take out cells from the centrifuge. Aspirate supernatant from 15 mL conical tube containing cells, avoid disturbing the cell pellet
Resuspend pellet with 1 mL of mTESR+ very gently to avoid dissociating iPSCs into single cells. Transfer all the volume into the well of a 6 well plates or per 35 mm plate, label with the line.
Gently mix the plate to evenly spread the cells
Ensure the presence of cells in each well by viewing the plate under the microscope (should see floating cells)
Incubate plate in the incubator at 37 °C
iPSC passaging
10m
Take out 3 mL of MEM per well of 6 well plates or per 35 mm plate in a conical tube
(1 mL /well to wash old medium and 2 mL /well to wash out acutase) and leave at room temperature to warm up
Take out 1 mL of accutase per well of 6 well plate or per 35mm plate in 15 mL conical tube to warm up at RT
Take out 3 mL of mTESR+ per well of 6 well plate or 35 mm plate (2 mL /well for plating and 1 mL /well for resuspension) to warm up at RT
Aspirate old medium
Add 1 mL of MEM per well of 6 well plate or per 35 mm plate, rock the plate to wash
Aspirate MEM
Add 1 mL of acutase per well of 6 well plate or per 35 mm plate
Place plate in the incubator at 37 °C for 00:05:00
5m
Get plate out of incubator and tap plate gently to check that cells are detached. Pippete cells once before removing from plate, avoid pippeting too much.
Transfer detached cells into a labeled 15 mL conical tube with 2 mL of MEM
Place conical tubes in centrifuge and spin at 300 x g for 00:05:00 at Room temperature
5m
Supplement mTESR plating media with CETP
Add 1 µL of CET per 1 mL of mTESR+ (1:1000 ratio)
Add 1 µL of P per 1 mL of mTesr+ (1:1000 ratio)
Aspirate matrigel from coated plates
Add 2 mL of mTESR+ containing CETP to each well
Take out cells from the centrifuge. Aspirate supernatant from 15 mL conical tube containing cells, avoid disturbing the cell pellet
Resuspend pellet with 1 mL of mTESR+ very gently to avoid dissociating iPSCs into single cells
Add 70-100 µL of cell solution per well in 6 well plate or per 35 mm plate (on top of the mTESR+ with CETP)
Gently mix the plate to evenly spread the cells
Ensure the presence of cells in each well by viewing the plate under the microscope (should see floating cells)
Incubate plate in the incubator at 37 °C
iPSC Maintainance
After thawing or passaging cells aspirate old medium the next day to wash away the CETP
Add 2 mL of fresh mTESR+ to each well of the 6 well plate or per 35mm plate
Cell can be fed every other day (or daily depending on cell confluency and growth) until they are ready for splitting again when cells are 70-80% confluent. Cells are usually ready for splitting every 4 days.
Check cells under microscope to estimate confluency
iPSC Freezing
Have freezing vials set and labeled with the correct info (iPSC line number, passage number, gene mutation, date, etc).
Take out 3 mL of MEM per well of 6 well plates or per 35 mm plate in a conical tube
(1 mL /well to wash old medium and 2 mL /well to wash out acutase) and leave at room temperature to warm up
Take out 1 mL of accutase per well of 6 well plate or per 35mm plate in 15 mL conical tube to warm up at RT
Follow the same protocol shown above for cell passaging for the steps 32-39.
Take out cells from the centrifuge. Aspirate supernatant from 15 mL conical tube containing cells, avoid disturbing the cell pellet
Resuspend pellet with 1 mL of mFreSR very gently
Add 500 µL of cell solution per freezing vial.
Place freezing vials in cryogenic freezing container to prevent ice crystals from forming within the cells and store at -80 °C Overnight
Transfer vials to a liquid nitrogen tank for storage
Protocol references
Chen, Y., Tristan, C.A., Chen, L., Jovanovic, V.M., Malley, C., Chu, P.H., Ryu, S., Deng, T., Ormanoglu, P., Tao, D., et al. (2021). A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells (Springer US).