Effective Cryopreservation of Human Embryonic Stem Cells By Programmed Freezing

Cryopreservation of human embryonic stem cells is an important and unsolved problem. A computer-controlled programmable cooler is used in the preservation of ES cells. Several effects have been experimentally studied, which include the cooling rate, the seeding temperature , the terminative temperature before the sample is plunged into liquid nitrogen. It is found that the constitution of cryoprotective agents is Me₂SO+FBS+DMEM(1:3:6,v/v/v) , and the sample is cooled from 0degC to -35 degC with a cooling rate of 0.5 degC /min (seeding at -10 degC) , and then being plunged into the liquid nitrogen immediately. The high survival rate (81.8%) is obtained. The cryopreserved human ES cells have been cultivated for prolonged periods and retained the properties of pluripotent cells, they have a normal karyotype and show the histochemical staining for alkaline phosphatase