Abstract: BACKGROUND: The human umbilical cord mesenchymal stem cells isolation methods mainly include enzymatic digestion and tissue attachment. It is important to understand the biological characteristics of the two methods isolated cells in two-dimensional and three-dimensional culture conditions; it is of great significance to the selection of cell isolation methods and culture condition in tissue engineering.  
OBJECTIVE: To compare the biological characteristics of human umbilical cord mesenchymal stem cells from the enzymatic digestion and tissue attachment methods in two-dimensional and three-dimensional culture conditions.
METHODS:  Human umbilical cord mesenchymal stem cells were isolated by digestion method and tissue attachment method, amplified and cultured to passage 3 and divided into two-dimensional and three-dimensional culture. Scanning electron microscope was used to observe the cell spheroids. Live\dead staining was utilized to detect cell spheroid survival. Alamar Blue was applied to detect cell proliferation. Flow cytometry was used to detect the expression of immunophenotypes CD34, CD44, CD45, CD90, and CD105. Immunofluorescence and western blot assay were used to detect the expression of pluripotent transcription factors Sox2, Nanog, and Oct4.  
RESULTS AND CONCLUSION: (1) Enzymatic digestion method took shorter time to obtain cells than the attachment method. The cell proliferation ability of the attachment method was better than that of the enzyme digestion under two-dimensional culture, but the proliferation was slow and there was no difference under three-dimensional culture conditions. After 3 days of spheroid culture, the cells became compact spheroids and survived well. (2) The cells isolated by the two methods all expressed CD44, CD90, and CD105 under two-dimensional and three-dimensional culture conditions, but did not express CD34 and CD45. Under two-dimensional culture, the expression of CD105 cells isolated by enzymatic digestion was higher than that of tissue attachment method; under three-dimensional culture, the expression of CD90 and CD105 cells isolated by enzymatic digestion was also higher than that of tissue attachment method. (3) Both isolation methods under two-dimensional and three-dimensional culture expressed pluripotency transcription factors Sox2, Nanog, and Oct4. Under the two-dimensional culture, the expression of the three transcription factors of the cells extracted by the enzymatic digestion method was higher than that of the tissue attachment method. However, under the three-dimensional culture, the expression of Sox2 in the tissue attachment method was higher than that of the enzymatic digestion method, and the expression levels of Nanog and Oct4 were not different between the two groups.

Key words: stem cells, umbilical cord, mesenchymal stem cells, three-dimensional culture, cell pellets, biological characteristics, immunophenotype, transcription factors