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 0 °C to -35 °C with a cooling rate of 0.5 °C/min (seeding at -10 °C), 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.