Thaw Matrigel stock 9-11µg/µL stored at -80°C on ice

Make 400µL aliquots of Matrigel to avoid multiple freeze-thaw cycles using previously cooled tips and tubes

Store 400µL Matrigel aliquots at -20°C until use

To prepare matrigel working solution: thaw 400µL Matrigel aliquots On ice or at 4°C

Dilute 400µL Matrigel aliquot in 40mL of MEM (Working Concentration 90-110 µg/mL)

Mix well before use. Store at 4°C protected from the light

Coat wells with 1mL of Matrigel per well of 6 well plates or per 35 mm plate

Leave coated plates for 2h 0m 0s to 4h 0m 0s in the incubator at 37°C

Store diluted matrigel at 4°C

Prepare mTesr+ by adding 100mL of mTesr+ 10x supplement into 400mL of mTESR+ media and filter it.

Add 1mL of 50Molarity (M) Primocin (final concentration 100ug/mL)

Take out 3mL of MEM per well of 6 well plates or per 35 mm plate in a conical tube

(1mL/well to wash old medium and 2mL/well to wash out acutase) and leave at room temperature to warm up

Take out 1mL of acutase per well of 6 well plate or per 35mm plate in 15mL conical tube to warm up at RT

Take out 3mL of mTESR+ per well of 6 well plate or 35 mm plate (2mL/well for plating and 1mL/well for resuspension) to warm up at RT

Put iPSC plate out of the incubator at Room temperature

Aspirate old medium

Add 1mL of MEM per well of 6 well plate or per 35 mm plate, rock the plate to wash

Aspirate Wash

Add 1mL of acutase per well of 6 well plate or per 35 mm plate

Place plate in the incubator at 37°C for 0h 5m 0s

Get plate out of incubator and check that cells are detached, avoid pippeting too much.

Add 2mL of MEM

Transfer detached cells with MEM (3mL total volume) into 15 mL conical tube

Place conical tubes in centrifuge and spin at 1000rpm,0h 0m 0s for 0h 5m 0s at Room temperature

Add 1µL of CET per 1mL of mTESR+ (1:1000 ratio)

Add 1µL of P per 1mL of mTesr+ (1:1000 ratio)

Mix mTESR+ with CETP

Aspirate matrigel from coated plates

Add 2mL of mTesr+ containing CETP to each well

Aspirate supernatant from 15mL conical tube containing cells (should have pellet)

Resuspend pellet with 1mL of mTESR+ very gently to avoid dissociating iPSCs into single cells

Add 50µL 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 0h 5m 0s

Aspirate old medium the next day to wash the CETP

Add 2mL of fresh mTESR+ to each well of the 6 well plate or per 35mm plate (no wash with MEM first day after splitting)

Wash CETP from each well/plate with 1mL of MEM

Aspirate wash

Add 2mL 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

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

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 1mL of mFreSR very gently

Add 500µL of cell solution to freezing vials

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