Separation, Cryopreservation By Vitrification Of The Cord Blood Stem Cells And Viability Test Of The Recovered Cells

The quantity and quality of hematoblast in umbilical cord blood (UCB) can be compared with bone marrow. Furthermore, it has other advantages: widely sourced, no contamination of pathogeny, weakly antigenicity, and no mature lymphocyte, et al.In this paper, we compared the regain-rate of two kinds of procedure for separating UCB stem cells: with Ficoll and with HES. Obviously, the procedure with HES is better than the former. Also, we found that the UCB stem cells would remain a high survival-rate if it is separated within 24h, above 93%.With the development of umbilical cord blood (UCB) bank, how to preserve the UCB stem cell effectively has been one of the key factors during UCB bank-upbuilding.Cryopreservation by vitrification is a kind of biotechnology sprung up in 1980's. It is superior in simple-equipment and facilitated-operation. Vitrification refers to the physical process by which concentrated cryoprotectant and cells treated are totally vitrificated into a ice-free and homogenous state. Vitrification is a simple and efficient technique for Cryopreservation.On the base of UCB stem cells separated with HES, we cryopreserved the cells by vitrification. Cells were disposed to a series concentration of PVS2 for different time and equilibrated in complete strength of the vitrification solution before plunging the cryotubes containing treated cells and cryopretectant into liquid nitrogen directly. The survival-rate of the recovered cells was tested by MTT and FDA-PI. Therefore, we got the preferable condition of vitrification, i.e., the concentration of cryoprotectant is 60% and the time of disposal is 15min. Under this condition, the value of FDA/PI is 46.43.In our report, we selected trehalose to substitute sucrose in the cryoprotectant. The process of vitrification is under the same preferable condition. The survival-rate of the recovered cells was tested by FDA-PI and CD34+ cell-count. As FDA/PI, trehalose is 20.25% higher than sucrose, and as CD34+ cell-count, trehalose is 25.64% higher than the former. It is evidently trehalose is superior to sucrose during Cryopreservation.This paper is the first report of Cryopreservation of UCB stem cells by vitrification. It is also the first report of introducing trehalose into Cryopreservation and got the expected results.