We plan to conduct one study for Diabetic wounds regeneration by adipose derived Mesenchymal Stem Cells from a patient's own adipose tissue. For this purpose we plan to isolate and grow MSCs from human adipose tissue. I want to know about the serum free growth of MSCs, which kind of growth supplement can be used to replace serum. For initial tissue digestion which enzyme is recommended, can we use collagenase or trypsin/ Triple E for initial tissue digestion, and disassociation solution used to detach MSCs proceeding to transplantation?
Using bone marrow MSC, I have cultured the cells with ITS, PDGF BB and bFGF. But I have 2% of FCS. Why do you use autologous human serum ?
Hi i would not use stem cells from adipose tissue, they are very slow at reproducing (no fast expansion there) and also they are much harder to have as a pure population. i normally use msc from bone marrow, p1 is not pure p2 (coupled with low sugar dmem) you have a near 95% pure msc population ready to transplant. its is a good substitute and we were doing study in the past using purified human serum from iceland (due to genetic segregation) try using that too
@ Antonio spadaccino Can you provide me some more information about media components U r using for growth of MSCs. can U give me cat # and company name for serum.
The dmem i was using is from invitrogen (21885-108 low glucose + glutamax) and i was using fbs because it was on mice and rats but in the past i used the same protocol just to test human serum (never transplanted in humans though) and cells were growing well up to only 2% of serum, just make sure you use pen strep when you isolate cells so they will be free of infections then stop using it in the expansion passages but careful you need more sterility
If you are planning for a clinical studies, then all clinical grade products should be used. Previously there was a supply of human serum, but this banned nowadays. Autologous serum is best source and no ethical issues connected to it as well. Clinical grade trypsin is advisable. If you follow these, you can be free from worries about any side effects to the patient.
You can use an autologous platelet lysate for rapid cell proliferation,
check http://www.ncbi.nlm.nih.gov/pubmed/17655588
it may replace any serum
We use human Platelet Lysate preparade by the Blood Bank from a pool of healthy donors inactivated with psoralen. You can see the method in:
" Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in good manufacturing practice conditions."
Castiglia S, Mareschi K, Labanca L, Lucania G, Leone M, Sanavio F, Castello L, Rustichelli D, Signorino E, Gunetti M, Bergallo M, Bordiga AM, Ferrero I, Fagioli F.
Cytotherapy. 2014 Feb 12. pii: S1465-3249(13)00840-2. doi: 10.1016/j.jcyt.2013.12.008. [Epub ahead of print]
Hello Irfan,
You can use collagenase for trypsinization of cells. I also agree that you can use patients serum to grow these cells as it is required for transplantation. Regarding serum free media you can use growth factors such as EGF & platelet derived growth growth factor, hormones such as Insulin, Steroid such as hydrocortisone can be added in the growth medium as a supplements. BSA also can be used. In this combination cells grow well as I have done lots of work using the said medium DMEM is a basic media with PEN STREP as an antibiotics.
All the best
DR. PRAVIN D. POTDAR
Dr. Pravin D. Potdar I appreciate it. Thank U for all these information.
what kind precaution I need to take before transplantation of Mesenchymal stem cells to the patient.
If we disassociate the cells, what will be maximum time limit to transplant these cells to patient.
is there any tests/methods available for possible immediate adverse reaction.
is there will be any effect for increasing passage numbers. what is maximum limit.
Hello Irfan,
As per my knowledge, you need to grow the cells in GMP standard laboratory recognized by your country regulatory body. As per literature, early passage cells are more effective. These cells should be free of any infectious diseases such as HBV, HCV and HIV. You need to do Mycoplasma contamination test also.
So far we have not started any therapies at our hospital thus, I have no idea about adverse effect of this type of treatment. Soon I am looking forward for this program.
You will need ethical committee approval as well as to fill up consent form sign by you and patient before this treatment.
All the best
Regards,
DR. PRAVIN D. POTDAR
For implanting in animal models DMEM+10% FBS+PENSTRP could be used successfully , as we have used, but for clinical research on human you should use the clinical grade medium. You may also use free serum medium which is safe.
In animal and in vitro stemspan could be used to increase the proliferation.
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