====== Primate cells culturing protocol ======
===== Original protocol from Göttingen =====
Platform Degenerative Diseases - Deutsches Primatenzentrum
by Ignacio Rodriguez-Polo
26.04.2018
==== General specifications ====

-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.\\

{{:wetlab:cell_culture:cell_lines_list:screenshot_2024-05-14_at_12.59.38.png?400 |}}

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{{:wetlab:cell_culture:cell_lines_list:screenshot_2024-05-14_at_13.10.34.png?400 |}}



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

==== Plate coating using Geltrex ====
According to manufacturer's [[https://www.thermofisher.com/order/catalog/product/A1413301|instructions]]
==== Culture ====
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.\\
==== Splitting ====
*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.
==== Freezing ====
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\\
==== Thawing Frozen Cells ====
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\\

==== Morphology of different non-human primate iPSCs ====


{{:wetlab:cell_culture:cell_lines_list:screenshot_2024-05-14_at_13.12.52.png?400 |}}