Man Embryonic Stem Cell Cryopreservation In A Preliminary Study

This research is supported by the project of National Natural Science Foundation of China (Grant 50376040) and the project of Science & Technology Development of Shanghai.Embryonic stem (ES) cell lines are derived from the pluripotent cells of the inner cell mass of the blastocyst. These cell lines have been derived from human blastocysts and are expected to have far-reaching applications in the areas of regenerative medicine, pharmacology and basic scientific research. In order to exploit this remarkable potential of human ES cells, improvement of currently used technologies for handling and manipulating the cells is required. Effective freezing and thawing techniques would enable the efficient preservation of stocks of early passage cells, as well as the conservation of specific clones that are developed from the original cell lines such as genetically modified clones. Moreover, efficient cryopreservation will be essential if human ES cell banks are to be established . But human ES cells are so sensitive to the temperature that they are easily crushed when being frozen. It is difficult to cryopreserve HES cells . The methods, by which the samples are slow-rate frozen (-1 degree c/min) to -80 degree c and then stored in liquid nitrogen ,are most commonly used for the cryopreservation of cell lines. While these standard methods are efficient for the cryopreservation of mouse ES cells, it has been observed that the survival of undifferentiated human ES cells following slow-rate freezing is very poor, and most of the cells either differentiate or die.In this paper, human ES cells had been cryopreserved by programmed freezing. As the same time, different effects, including the cooling rate ,the method of seeding, and the terminative cooling temperature had been experimentally studied.lt was found that the constitution of cryoprotective agents was Me₂SO+FBS+DMEM(l:3:6,v/v/v) with cooling protocol of -0.5 degree c /min from 0 degree c to -35 degree c (seeding at -10 degree c) , and being plunged into the liquid nitrogen immediately. The high survival rate(81.8%)was obtained. The cryopreserved human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, they had a normal karyotype and showed histochemical staining for alkaline phosphatase.In addition, the osmotic characteristics of pig bone marrow stromal stem cells were experimental measured using electronic sizing technique. The cell volume measured is 5248.4 μm3, the relative diameter 21.6 μm, at isotonic osmolality. The cell volume...