Nov 13, 2023
iPSCs Maintenance and Banking
- 1Rutgers University
Protocol Citation: Mahmoud ElAchwah, Sol Diaz de Leon Guerrero, Zhiping Pang 2023. iPSCs Maintenance and Banking. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qd5ogr2/v1
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: September 15, 2023
Last Modified: November 13, 2023
Protocol Integer ID: 87845
Abstract
This protocols offers a thorough description of the maintenance and banking of induced pluripotent stem cells.
Materials
Matrigel:
Matrigel catalog number: 354234 Matrigel LoT number: 13823002
Matrigel stock concentration: 9-11 µg/µL Matrigel working concentration: 90-110 µg/mL
Matrigel Stock aliquot volume: 400 µL
Culture Antibiotic:
Primocin catalog number: ant-pm-1 Primocin LoT number: NA
Primocin stock concentration: 50 mg/mL Primocin working concentration: 100 µg/mL
Primocin stock aliquot volume: 1 mL
CEPT:
Chroman catalog number: HY-15392 Chroman working concentration: 50 nM
Chroman stock concentration: 0.5 mM
Emricasan catalog number: S7775 Emricasan working concentration: 5000 nM
Emricasan stock concentration: 50 mM
Polyamine supplement catalog number: P8483 Polyamine supplement working concentration: NA
Polyamine supplement stock concentration: NA
Trans-ISRIB catalog number: 5284 Trans-IRIB working concentration: 700 nm
Trans-IRIB stock concentration: 7 mM
Culture Media:
MEM catalog number: 51200-038 MEM LoT number: 2451155
Accutase catalog number: AT-104 Acutase LoT number: 2B2527A
mTesr+ basal medium catalog number: 100-0274 mTesr+ LoT number: AH29845535
mTesr+ 10x suplement: 100-0275
mTesr Plus kit: 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:
mFreSR media catalog number: 05855 mFreSR media LoT number: NA
mFreSR media stock concentration: NA mFreSR media working concentration: NA
mFreSR media Stock aliquot volume: 50 mL
Diluting Matrigel
Diluting Matrigel
Thaw Matrigel stock 9-11 µg/µL stored at -80 °C on ice
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 (Working Concentration 90-110 µg/mL)
Mix well before use. Store at 4 °C protected from the light
Matrigel Coating
Matrigel Coating
1h
Coat wells with 1 mL of Matrigel per well of 6 well plates or per 35 mm plate
Leave coated plates for 02:00:00 to 04:00:00 in the incubator at 37 °C
6h
Store diluted matrigel at 4 °C
Medium Preparation For Passaging
Medium Preparation For Passaging
Prepare mTesr+ by adding 100 mL of mTesr+ 10x supplement into 400 mL of mTESR+ media and filter it.
Add 1 mL of 50 Molarity (M) Primocin (final concentration 100ug/mL)
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 acutase 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
Washing iPSCs
Washing iPSCs
Put iPSC plate out of the incubator at Room temperature
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 Wash
Detaching Adherent Cells
Detaching Adherent Cells
4m
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
Centrifuging Cells
Centrifuging Cells
Get plate out of incubator and check that cells are detached, avoid pippeting too much.
Add 2 mL of MEM
Transfer detached cells with MEM (3 mL total volume) into 15 mL conical tube
Place conical tubes in centrifuge and spin at 1000 rpm for 00:05:00 at Room temperature
5m
Preparing Seeding Medium
Preparing Seeding Medium
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)
Mix mTESR+ with CETP
Aspirate matrigel from coated plates
Add 2 mL of mTesr+ containing CETP to each well
Collecting Cells
Collecting Cells
Aspirate supernatant from 15 mL conical tube containing cells (should have pellet)
Resuspend pellet with 1 mL of mTESR+ very gently to avoid dissociating iPSCs into single cells
Add 50 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
Incubation
Incubation
5m
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 Overnight
5m
Changing iPSC medium
Changing iPSC medium
Aspirate old medium the next day to wash the CETP
Add 2 mL of fresh mTESR+ to each well of the 6 well plate or per 35mm plate (no wash with MEM first day after splitting)
Wash and iPSC Medium Change
Wash and iPSC Medium Change
Wash CETP from each well/plate with 1 mL of MEM
Aspirate wash
Add 2 mL of fresh mTESR+ to each well of the 6 well plate or per 35 mm plate
No need to keep washing plates after the first wash, you can directly change medium
iPSC Maintainance
iPSC Maintainance
Keep feeding cells everyday until they are ready for splitting again (cells are usually ready for another splitting in 2-3 days, when their confluency reach 70%)
Check cells under microscope to estimate confluency
iPSC Freezing
iPSC Freezing
For freezing purposes, follow the same protocol shown above for cell maintenance for the following sections: Washing iPSCs, Detaching Afdherent cells, centirfuging cells (steps 10-23)
Instead of collecting cells have freezing vials set and barcoded with iPSC line number, passage number, gene mutation and date
Resuspend pellet with 1 mL of mFreSR very gently
Add 500 µL of cell solution to freezing vials
iPSC Banking
iPSC Banking
Store freezing vials at -80 °C in a cryogenic freezing container to prevent ice crystals from forming within the cells in the freezing vials
After 24 hours, move freezing containers with vials from -80 °C to a nitrogen tank