The repetitive freeze-thaw-in vivo and in vitro subculture method (RFTIVIVSCM) to indefinitely preserve human bone marrow and adipose tissue derived mesenchymat stromal cells (MSCs) for regenerative medicine.

Human vein and perivascular adipose tissue will be a precious source of MSCs for regenerative therapy, however problems in tissue culture remain as for foreign proteins, recombinant stimulants, and the resources for MSCs. Perivenous live “adipose” tissue explants obtained by surgery (82 varices) were cultured in a media including human sera, platelet lysate, limulus lysate et al in a 5 % CO2 at 37 °C and obtained confluent monolayer of MSCs in 35 mm dish. On the other hand, the adipose and vein explants, which were frozen in a 10 % dimethylsulfoxide (Me2SO) in Iscove’s modified Dulbecco’s Medium (IMDM) or in 15 % glycerol in TC 199 in cryovials at −85 °C or in a plastic bag at −196 °C for days~33 years, induced confluent MSCs too after thawing and culture, that is adipocyte-mesenchymal transition (AMT). Those confluent MSCs were gently scraped by trypsin-IMDM or PBS to be proliferated to form passaged MSCs by RFTIVISCM, which was originated from repetitive frozen autologous bone marrow transplantation (FABMT) used to rescue ablated marrow function. RFTIVIVSCM will be an essential technique in regenerative therapy to cryopreserve the MSCs induced from every internal tissue for a long term.