Hi, I thawed hIPSCs bought from a former company two days ago, but the cells started adpating a spiderweb morphology and although they are well attached to the matrigel they are looosely pack. I understand that these are signs of differnetiation (although good quality vials should not presnet more than 20% of spontaneous differnetiation). I dont undertsand what might have happened if this is the case. They also do not form colonies but like a mesh. The cell density is quite high 500,000 cells/ well in a 6 well plate but I followed the instructions of the company for thawing and initial seeding density. I had them in mTesr1 medium + 10um of Rock inhibitor for 24h. Then on mTesr1 medium with 100u/ml of antibiotic next 24h and then as they looked stressed (as assumed by this morphology and by the high number of cells dying after each change in medium) i reintroduced the Rock inhinitor with the medium but at a lower concentration (5um). Today I changed again the medium to only mTesr1 by itself (no Rock inhibitor nor antibiotic (pen/strep)) as if been told antibiotics may alter pluripotency on the long run. I do not know if I should passage the cells (using Versene), keep them growing in mTesr1 until they form colonies (if this could happen) or what to do next. Could someone with more experience give me some hints please? All answers are welcome. Thank you!
Hi Pilar Maestre,
Rock inhibitor changes the morphology of iPSCs and makes them look more spiky. So to me, the pictures at 24h post thawing look as expected. However, the CNT are too full in my opinion. Although it is not recommended to passage a day or two after thawing it is also not good for the cells to have no room to grow.
So I would recommend to culture the cells in mTESR without Rock inhibitor and observe the morphology. When passaging, add Rock inhibitor and after 24h start culturing without again.
The cells do nicely show the nucleus and nucleoli which is a good sign. I would not day they look differentiated.
Good luck!
Selene
Dear Pilar Maestre,
PSC (iPSC, ESC) colonies can have different morphology depending on the medium, e.g., mTesr, E8 Medium, and depending on the ECM, e.g., Matrigel, Vitronectin. ROCK inhibitor always affects PSC colony morphology, but colonies return to a more compact and rounded appearance after its removal. Colonies should return to normal morphology if pluripotent stem cells are 1) plated at a lower density; 2) plated in small colonies/aggregates, and 3) after the ROCK inhibitor is remove. PSC do not like to be passaged as single cells; they are characterized by clonal growth and should be passaged in colonies if you want to maintain their pluripotent phenotype longer.
Good luck!
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