Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods

Abstract

Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P < 0.01) and lower yield of primary cells per centimetre of umbilical cord (P < 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P < 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P > 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P < 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P < 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37 °C for digestion of 16-20 h is an effective and simple modified enzyme digestion method.

Fig. 1

Morphological characteristics of hUCMSCs isolated and cultured with both enzyme digestion methods and the tissue explant method under an inverted phase contrast microscope (×100). a primary cells isolated with the tissue explant method at 5 days of culture. b primary cells isolated with the tissue explant method at 24 days of culture. c primary cells isolated with the conventional enzyme digestion method at 4 days of culture. d primary cells isolated with the conventional enzyme digestion method at 16 days of culture. e primary cells isolated with the modified enzyme digestion method at 2 days of culture. f primary cells isolated with the modified enzyme digestion method at 9 days of culture

Fig. 2

Morphological characteristics of the third-generation hUCMSCs under inverted phase contrast microscope (×100). a primary cells isolated with the tissue explant method at 5 days of culture. b primary cells isolated with the modified enzyme digestion method at 5 days of culture

Fig. 3

Cell cycle analysis of the third-generation of confluent hUCMSCs isolated with the modified enzyme digestion method

Fig. 4

Identification of cell antigen markers of the third-generation of hUCMSCs isolated by the modified enzyme digestion method (×100). Immunofluorescence staining showed positive expression for CD44 and CD90, negative expression for CD45 and CD31. The nucleus was stained blue with DAPI

Fig. 5

Multi-lineage differentiation potential of third-generation hUCMSCs isolated by the modified enzyme digestion method (×100). a Results of Oil-Red-O staining in cell cultures grown for 2 weeks in adipogenic medium. Part of the cells contained numerous Oil-Red-O-positive lipid droplets. b Control group cells grown in the regular medium, no cells contained Oil-Red-O-positive lipid droplets. c Result of Von Kossa staining in cell cultures grown for 2 weeks in osteogenic medium. Part of the hUCMSCs became Von Kossa positive. d Control group cells grown in the regular medium, hUCMSCs were Von Kossa negative