iNDI Maintenance protocol of iPSCs Version 1 

Aliquot concentrated Matrige l:

• Gradually thaw a 5ml bottle of Matrigel on ice in a Styrofoam container
• Pre-chill labeled Eppendorf tubes by placing in a cool rack on ice.
• Before pipetting concentrated Matrigel into pre-chilled tubes, chill a 1 ml pipet tip by pipetting ice-cold <KnockOut™ DMEM/F-12 >up and down several times, then immediately use the tip to aliquot Matrigel.
• Prepare aliquots of 500µL of concentrated Matrigel and freeze down at-80°C

Coating plates with Matrigel solution:

• Reconstitute a 500µL aliquot of Matrigel in 50mL of cold KnockOut™ DMEM/F-12, pipet 500µL cold media into the aliquot of Matrigel tube and pipet up and down several times, then transfer what has thawed to the tube containing 50mL of cold KnockOut™ DMEM/F-12, repeat until the frozen aliquot of Matrigel has been completely transferred to the 50 ml of cold media. Mix inverting several times.

* Add half of the normal culture volume of Matrigel solution to the culture surface (i.e., 1 ml per well of a 6-well plate).

• Gently rock plate to spread the Matrigel solution evenly across the plate.
• Place in 37°C incubator for 0h 30m 0s to 1h 0m 0s before use it.

* Aspirate Matrigel and add culture medium (E8).

There is another option for coating plates besides Matrigel. Vitronectin is a recombinant human protein that provides a defined surface for feeder-free culture of iPSC. When used with E8 medium, vitronectin has been proven to maintain pluripotency and normal growth characteristics in multiple iPSC lines.

Aliquot concentrated Vitronectin 0.5 mg/ ml 0.5mg/mL

• Thaw a vial of Vitronectin on ice in a Styrofoam container.
• Prepare aliquots of 60µL of concentrated Vitronectin and freeze down at -80°C

Coating plates with Vitronectin solution 5µg/mL

• Reconstitute a 60µL aliquot of Vitronectin into a 15 ml conical tube containing 6mL of PBS, gently resuspend by pipetting up and down, do not vortex.
• Add half of the normal culture volume of Vitronectin solution to the culture surface (i.e. 1 ml per well of a 6-well plate).
• Gently rock plate to spread the Vitronectin solution evenly across the plate.
• Place in 37°C incubator for before use it.
• Aspirate Vitronectin and add culture medium (E8).

Remove iPSC stock cryovial from liquid nitrogen and thaw in 37°C bead bath. Thaw quickly by gently swirling until a small piece of frozen material remains. Spray the vial with 70% ethanol before transferring to a biological safety cabinet.

Gently add the thawed cell suspension dropwise to a conical tube containing 10mL culture medium or PBS, rinse cryovial with 1ml of medium and add the rinse to the tube, gently mix cells by swirling.

Centrifuge tube 0h 5m 0s at 200 - 300 x g at Room temperature

Aspirate the supernatant and gently resuspend cells in culture medium (E8) supplemented with 50nanomolar (nM) Chroman I and transfer to Matrigel or Vitronectin-coated plates.

Gently rock plate to evenly distribute cells.

Return plate to 37°C incubator.

Next day replace the media with fresh E8 medium (2mL /well of a 6-well plate).

When the well is 80% confluent pass to expand stock.

EDTA

Aspirate culture medium and wash with PBS 1X.

Aspirate PBS and add half of culture volume (1ml/well of a 6-well plate) of EDTA 0.5millimolar (mM) in PBS.

Incubate for 0h 3m 0s at 37°C or 0h 8m 0s at Room temperature

Aspirate EDTA solution, the cells colonies should remain attached so be careful not to disturb them.

Add 1mL of culture medium supplemented with 50nanomolar (nM) Chroman I to cells to dissociate by pipetting two or three times.

Typically splitting ratios for 6 well plates are between 1:6 and 1:12, so, add the desire volume of culture medium to the cells and discard any excess of cells or re-plate into a new Matrigel or Vitronectin-coated well.

Accutase

Aspirate culture medium and wash with PBS 1X.

Resuspend the cell pellet in culture medium E8 supplemented with 50nanomolar (nM) Chroman I.

Count cells and plate cells the desire amount into a Matrigel-coated plates.

Gently rock plate to evenly distribute cells.

Return plate to 37°C incubator.

The next day replace the medium with fresh E8 (2ml/well of a 6-well plate)

When the well is 80% confluent pass to achieve an assay or to expand stock.

Aspirate PBS and add half of culture volume of Accutase.

Transfer to 37°C incubator for 0h 8m 0s

Meanwhile aspirate Matrigel/Vitronectin from plates and add culture medium E8 supplemented with 50nanomolar (nM) Chroman I.

When Incubation is ready, tilt the plate and pipet the accutase solution two to three times up and down the culture surface to break the colonies.

Quench the Accutase adding half of the culture volume of PBS.

Transfer to a new conical tube and rinse with more PBS the culture surface, combine with the cell solution in the tube.

Centrifuge 0h 5m 0s at 200 - 300 x g at Room temperature

Aspirate supernatant.

Prepare freezing medium as combining Knockout™ Serum Replacement with 10% DMSO.

Leftovers of EDTA or Accutase dissociation procedure could be cryopreserved by centrifuging as in step 14.8 and resuspending the cell pellet with freezing medium.

When freezing cells from a well/plate, prepare the cells as for EDTA split.

Aspirate EDTA and gently dissociate cells with freezing medium.

Transfer 1mL of cell suspension to a 1 mL cryovial and freeze in a CoolCell freezing container at -80°C

Next day transfer the cryovials to liquid nitrogen for long term storage.