Cryopreservation, including freezing and thawing, becomes very important for cell culture technology. Cryopreservation of primate ES and iPS cells is very severe and difficult compared to murine or other cells. At present, vitrification method is considered adequate for primate ES/iPS cell, although slow-freezing method using DMSO has been popular for a wide variety of cell lines. Vitrification method needs special skills and has to avoid dry ice transportation. To address these problems, we developed a new freezing medium (BambankerTM HRM) containing Human Serum Albumin and DMSO for primate ES/iPS cells.
The experimental data shown below describe primate ES cells, cryopreserved in BambankerTM HRM or 10 %. DMSO/culture medium by slow-freezing or in a conventional vitrification medium by quick-freezing, and then subsequent storage in liquid nitrogen. After 3 days, the cells were thawed by each adequate protocol, and then plated. The different cryopreservation media were analysed by the number of alkaline phosphatase-positive colonies as recovery points. The recovery points of BambankerTM HRM directly from liquid nitrogen storage were twice higher than that of vitrification medium and four times than that of 10 % DMSO/culture medium. Assuming dry ice transportation, the cryopreserved primate ES cells by above each freezing method were put on dry ice 24 h after three days in liquid nitrogen, and then thawed and plated. The recovery points of BambankerTM HRM still remained high, but those of vitrification were considerably low.
These results indicate that BambankerTM HRM provides efficient cryopreservation and dry ice transportation for primate ES/iPS cells. Moreover, as BambankerTM HRM is xeno-free and chemically defined, it may be useful for a lot of different applications where hES/iPS cells are involved.
Cell lines succesfully stabilized by BambankerTM HRM: