Hey,
I Have a problem regarding Human placenta mesenchymal stem cells culture. We tried to grow the cells in medium: DMDM low glucose, 10% inactive FBS, L-glutamin 2mM, Pen-Strap (1000 units/ml and 10umg/ml) and 10% CO2. I used FACS to verify the cells are actually stem cells (We bought the cells from a local hospital) we encountered two problems:
1. The cells stop growing normal in P 7, while we wanted placenta cells because they can reach higher passages (which is essential for our research) than other stem cells.
2. FACS results (Using R&D System human mesenchymal stem cell verification kit) indicated CD90 in 75%-90% of the cells and CD73 with 95%+ , which is fair. But CD105 resulted with a maximum of 20% (most of the tests were under 10%) of the cells. We also suffered from a lot of cell debris and cell aggregates.
To solve this we let the cells to settle for a few hours after detachment (we also tried different methods such as trypsin EDTA, Acutase, Versen and scraping, we chose Acutase), 70um filter, raising and lowering the incubation time and raising the antibodies concentrations, changing the cell line.
More ever, we witnessed an increase in the stem cell negative markers which is more troublesome. Positive and negative isotypes were also used. We even try it with a bone morrow stem cells cell line from ATCC with similar results. We are now trying to set the pH to 5% hopefully it will have a positive effect.
Can anyone share his experience or technique ?
We are also looking to buy human placenta stem cell line from the U.S. but haven't had any luck finding a trusted distributor that can send overseas.
Thank you for any help you can supply.
Best wishes,
Tzachy
From what part of the placenta are you obtaining the cells? We work with explant culture of umbilical cord tissue with good results in similar culture conditions. We usually don't test many passages, but the ones that I tested reach passage 10 without problems.
We use 5 min of trp 0.05% for passage and 5% co2 for culture. You could try replacing serum for platelet lysate, that also helps.
Dear Tzachy
I think this paper is good for you. try to see the video.
Pelekanos RA, Sardesai VS, Futrega K, Lott WB, Kuhn M, Doran MR. Isolation and expansion of mesenchymal stem/stromal cells derived from human placenta tissue. Journal of visualized experiments: JoVE. 2016(112).
Thanks for the answers. Should the platelet lysate be heat inactive? Sadly I dont have info about the part of the placenta, we dont have an alternative source, so far.
Regarding the video we know of him and work accordingly.