Comparison of different methods for the isolation of human umbilical cord mesenchymal stem cells

Objective To explore the most appropriate method for the isolation of human umbilical cord mesenchyamal stem cells (MSCs) through a comparison of different methods. Methods Fifteen umbilical cord specimens from full-term healthy fetus with caesarean birth were completely rinsed with phosphate buffer saline (PBS) and sliced into 1 mm3 tissue blocks after removal of umbilical vessels and external membrane.These tissue blocks were averagely divided into 4 groups after washing and centrifuge.Then four methods for the isolation of human umbilical cord MSCs were compared: an explant culture and three enzymatic methods of collagenaseⅡ, collagenaseⅡ/trypsin and collagenaseⅡ/hyaluronidase.The count of living cells was evaluated by trypan blue dye exclusion test.Cell morphology was observed under inverted microscope.The expressions of cell surface markers CD105, CD90, CD73, CD31, CD44, CD45, human leukocyte antigen-Ⅰ (HLA-Ⅰ) and human leukocyte antigen classⅡ molecules (HLA-DR) were detected by immunofluorescent staining.Cell proliferation was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Results The human umbilical cord MSCs were successfully isolated by four isolated methods.However the isolation method used profoundly altered the cell number and proliferation capacity of isolated cells.Isolated cells using four methods were counted at (5.44±0.21)×105, (4.03±0.24)×105, (4.91±0.33)×105and (5.94±0.40)×105respectively.More cells were obtained with collagenaseⅡ/hyaluronidase than other three methods (all P<0.05).Cells out of tissue blocks were observed at Day 9-11 and cells were observed at Day 2 with three types of enzyme digestion.The fusion time of cells were (18.5±3.5), (8.0±1.0), (7.5±1.5) and (3.5±0.5) days respectively.The fusion time of cells obtained with collagenaseⅡ/hyaluronidase was lower than other methods (all P<0.05).Cell morphology: polygonal, irregular and of large volume for explant culture; relatively short and small for collagenaseⅡ and collagenaseⅡ/trypsin methods; thin spindle for collagenaseⅡ/hyaluronidase method.Immunofluorescent staining revealed that CD105, CD73, CD90 and CD44 were expressed in all groups while there was no expression of CD31, CD45 or HLADR.And the cells obtained with collagenaseⅡ/hyaluronidase method were in a higher cell proliferation rate and activity compared to other methods. Conclusion The collagenaseⅡ/hyaluronidase method is optimal for the isolation of human umbilical cord MSCs than other methods. Key words: Mesenchymal stem cells; Umbilical cord; Collagenases; Trypsin; Cell separation