There are two ways to split iPSCs: single cell or in clumps. Single cell splitting is usually used to count the cells and seed them for differentiations. Clumps used to regularly passage cells, because stem cells survive better when in contact with one another.

NB! For both protocols: pre-warm wash media at 37 (DMEM/F12 normally)

We usually use Accutase (link at Thermo webpage)for it. TrypLE (link at Thermo webpage) and other similar enzymes can be also used.

Logic behind: when you split single cell - cells de-attach and float in the solution. You need to collect them with centrifugation.

*pre-coat plate, after incubation with the coating agent - add culturing media to each well (1.5mL per well), anti-apoptotic agents are optional/cell line dependent. place the plate to the incubator.

1. Wash cells with PBS once (1.5-2mL per well)
2. Add 400uL of accutase per one well
3. Incubate for 4-6 minutes at 37C (depends on the cell line!)
4. Check under the microscope that all cells detached
5.Add 1mL of warm wash media (DMEM/F12)
6. Resuspend cell suspension with p1000 pipette for around 10 times (depends on the cell line!)
7. Transfer cell suspension to a falcon and add approximately 4-5mL DMEM-F12
8. Centrifuge for 5 min at 300g
9. Resuspend the cell pellet in warm culturing media with anti-apoptotic
10. Count or seed cells for differentiation

*pre-coat plate, after incubation with the coating agent - add culturing media to each well (1.5mL per well), anti-apoptotic agents are optional/cell line dependent. place the plate to the incubator.

NB! Time of incubation is always variable from cell line to cell line and sometimes even from batch to batch. Always look under the microscope how the colonies look like. The edges of colonies should “lift up” and the cells start looking different, more round. You can also wait until the smallest colonies de-attach completely and start floating - it means the incubation was enough. But be careful to not over incubate them at this point, because otherwise all the colonies will float away.

Logic behind: when you split in clumps - cells stay don't float after you aspirate Versene, ReLeSR or other splitting solution, but the colonies de-attach slightly. You collect cells after aspirating the splitting solution by adding a warm media and washing them off from the plate by the pressure of the media, released by p1000 pipette. You don't have to centrifuge them after

1. Wash cells with PBS once (1.5-2mL per well)
2. Add 500uL of Versene per one well
3. Incubate for 3-5 minutes at room temperature (depends on the cell line!)
4. Check under the microscope that all colonies rounded and became more wide. Here cells not supposed to detach! if some of the cells detached - stop the incubation
5.Aspirate Versene
6. Add 1mL of culturing media (anti-apoptotic is opntional, depends on cell line)
7. Resuspend cell suspension with p1000 pipette max 3 times (depends on the cell line and the size of clumps you want to get)
7. Add the required amount of cells in suspension to the pre-warmed plate
*example: if you want to split cells 1:10, after resuspending in 1mL of culturing media - add 100uL per well of a new plate
8. Distribute the cells in the wells
9. Place the plate to the incubator

ReLeSR splitting protocol is commercially available
I modify it by using 500uL of reagent per well. For most of the cell lines, incubation at 37C for 5 min works well.

Usually splitting with Versene (see above), but they need a bit longer to de-attach, they form tighter colonies. Add versene, wait for 3-4 min, aspirate versene and add again, wait for 2-3 min and proceed as normal Versene splitting.