Substrates for human pluripotent stem cell cultures in conditioned medium of mesenchymal stem cells.

We aimed to establish a culture system of human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), free from xenogeneic proteins, Matrigel(™) and conditioned medium of mouse embryonic fibroblasts. The conditioned culture medium consisted of mesenchymal stem cells derived from human bone marrow. We examined surface properties suitable for hPSC culture by using self-assembled monolayers (SAMs) of alkanethiols with four different functional groups: CH(3), OH, COOH and NH(2). hPSCs neither adhered nor proliferated on surfaces with a water contact angle higher than 40°. Based on this finding, the contact angle of a polystyrene (PSt) culture dish was reduced to less than 40°, and COOH and OH groups were introduced to its surface by oxygen plasma treatment, making the PSt dish suitable for hPSC culture. This combination of a PSt dish treated with oxygen plasma treatment and conditioned medium of mesenchymal stem cells achieved a long-term maintenance of hPSCs without differentiation.

Highly efficient cryopreservation of human induced pluripotent stem cells using a dimethyl sulfoxide-free solution.

Human induced pluripotent stem (hiPS) cells have great potential for regenerative medicine and drug discovery. It is essential to establish highly efficient and reliable methods for hiPS cell cryopreservation. We examined cryopreservation of hiPS cells by the vitrification method using a dimethyl sulfoxide Me2SO-free and serum-free medium, VS2E, that uses Euro-Collins solution as a base with 40% (v/v) ethylene glycol and 10% (w/v) polyethylene glycol as cryoprotectants. This combination of vitrification and cryoprotectants resulted in a higher recovery rate of hiPS cells than with a commercially-available vitrification solution, DAP213, which contained Me2SO and serum components. After vitrification and warming, hiPS cells were cultured easily. Even after several subculturing steps, cells expressed undifferentiated cell markers, such as Oct-3/4 and SSEA-4, and also exhibited alkaline phosphatase activity. The pluripotency of hiPS cells was maintained, as demonstrated by teratoma formation upon hiPS cell transplantation into severe combined immunodeficient mice. Thus, we successfully preserved hiPS cells under liquid nitrogen with high efficiency using Me2SO-free vitrification solution and rapid cooling.

Cryopreservation of primate embryonic stem cells with chemically-defined solution without Me2SO.

Human embryonic stem (hES) cells are expected to be useful in the fields of regenerative medicine and tissue engineering due to their pluripotency. Therefore, it is necessary to establish highly efficient and reliable methods for the cryopreservation of hES cells. We have cryopreserved cynomolgus and human ES cells by the vitrification method, using a chemically-defined dimethyl sulfoxide (Me(2)SO)-free and serum-free medium composed of Euro-Collins solution as a base medium and 40% (v/v) ethylene glycol (EG) and 10% (w/v) polyethylene glycol (PEG) as cryoprotectants. When the vitrification and the cryoprotectants were combined, the recovery ratio of hES cells was 22.9+/-7.7%, compared to 0.4+/-0.2% when the conventional slow-freezing method was used. After the cryopreservation and thawing cycle, hES cells were easily cultured and expressed undifferentiated cell markers such as Nanog, Oct-4, SSEA-4, and alkaline phosphatase activity after several subculturing steps. We also found that the pluripotency of hES cells was maintained, as demonstrated by teratoma formation of ES cells transplanted into severe combined immunodeficient (SCID) mice. Thus, we conclude that we have successfully cryopreserved primate ES cells with high efficiency using a Me(2)SO-free, chemically-defined medium.