Primate cells culturing protocol

Platform Degenerative Diseases - Deutsches Primatenzentrum by Ignacio Rodriguez-Polo 26.04.2018

-The following protocol has been tested for feeder-free culture of Human iPSCs, Rhesus macaque ESCs and iPSCs, Baboon iPSCs and marmoset ESC and iPSCs.
-All parts of the protocol should be carried under antibiotic-free and serum-free conditions.

PSF and PSC are the same. PSF - pro-survival factor, PSC - pro-survival compound

According to manufacturer's instructions

Plate the cells in 6cmǾ, or 6 well plates coated with Geltrex.
Geltrex coated plates, after 1h at 37°C could be stored in the fridge for one week, but before using incubate them at room temperature for at least 30min.
Change medium everyday (iPSC+), using 2ml per well. Medium should be chilled at room temperature, never 37°C.

*iPSC or ESC should be split when 60-80% confluence have been reached.
*Split ratios can vary between 1:5 and 1:15.
1. Remove the medium from the plate
2. Add 1 ml Versene Solution
3. Incubate 2 min at room temperature
4. Remove the Versene
5. Repeat steps 2-4
6. Add fresh medium and pipet up and down to detach the cells
7. Transfer the cells onto a Geltrex coated plate with fresh medium (iPSB+ and PSC) with the desired dilution
*Do not use PSC for more than one day.

1. Remove the medium from the plate
2. Add 1 ml Versene Solution
3. Incubate 2 min, at room temperature
4. Remove the Versene
5. Repeat steps 2-4
6. Add freezing medium (iPSB+, 20%DMSO and 10μM PSC)
7. Pipet up and down to detach the cells
8. Transfer the cell suspension to a CRYO tube
9. Place the CRYO tube in a Freezing Container (Mr. Frosty™)
10. Store at least 2h at -80°C
11. Transfer CRYO tube to -150°C for long term storage

1. Thaw frozen cells rapidly in a water bath, at room temperature (aprox 2-3min)
2. Pipet the content of the CRYO tube in a 15 ml falcon tube
3. Add 5ml DMEM (DMEM Media - KnockOut™ Gibco)
4. Centrifuge 200g 5min, at room temperature
5. Remove the supernatant without disturbing the pellet
6. Resuspend the pellet in 2 ml fresh medium (iPSB+ and 5μM PSC)
7. Plate the cell suspension in a Geltrex coated plate