Density gradient centrifugation for isolation of umbilical cord blood stem cells: Screening of separation medium

doi:10.3969/j.issn.2095-4344.2013.23.002

Abstract

Abstract:

BACKGROUND: The purpose for isolating stem cells from the cord blood is to obtain the mononuclear cell populations with the stem cells as the major group, and density gradient centrifugation is one of the simplest and most effective ways. Polysucrose diatrizoate is the most commonly used separation medium for density gradient centrifugation, but there is no in-depth research on which method can obtain more stem cells.
OBJECTIVE: To investigate the optimal density of separation medium for isolating human umbilical cord blood stem cells using density gradient centrifugation method, and to establish the isolation method of stem cells for clinical application.
METHODS: Umbilical cord blood was separated with two-step method. Firstly, hydroxyethyl starch was used to sediment cord blood erythrocyte, and the suspension was obtained, then the separation media with three kinds of concentrations (1.073 0±0.000 1), (1.075 0±0.000 1) and (1.077 0±0.000 1) g/mL was used to isolate the suspension and the mononuclear cells were obtained. The harvest rate and the survival rate of the cells were counted. Their surface markers of mononuclear cells were identified with flow cytometry; histogram or scatter plot of each subset was plotted to analyze their proportions and absolute numbers of sub-cell populations.
RESULTS AND CONCLUSION: The separation medium with the concentration of (1.073 0±0.000 1) g/mL could obtain the mesenchymal stem cell populations with the largest proportion, which was the optimal density for isolating the mesenchymal stem cells. The separation medium with the concentration of (1.075 0± 0.000 1) g/mL could obtain the hemopoietic stem cell populations with the larger proportion, which was the optimal density for isolating the hemopoietic stem cells. The separation medium with the concentration of (1.077 0±0.000 1) g/mL could obtain the largest number of stem cells, but the obtained mesenchymal stem cells and hematopoietic stem cells had the lowest proportion. Application of two-step method for isolating stem cells and the establishment of strict laboratory conditions and standards are the optimal programs for isolating the human umbilical cord blood stem cells using density gradient centrifugation method.

Key words: stem cells, umbilical cord/umbilical cord blood stem cells, human umbilical cord blood, density gradient, centrifugation, mononuclear cells, mesenchymal stem cells, hemopoietic stem cells, separation media, density, flow cytometry, standard operating procedure

CLC Number:

R394.2

Guo Ji-qiang, Liu Ai-bing, Wang Dong-ping, Wang Li-ming. Density gradient centrifugation for isolation of umbilical cord blood stem cells: Screening of separation medium[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(23): 4189-4195.

Figures/Tables 5

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