Optimized cell transplant, and preparation method and use thereof
Disclosed in the present invention is an optimized cell transplant. The optimized cell transplant is formed by performing gene induction and modification on a mesenchymal stem cell in the form of a small molecule and protein composition. The expression levels of CD200 gene, Galectin-9 gene and VISTA gene can be increased synchronously after cell culture. Vector virus infection and plasmid transfection are not required in the cell preparation process, so that high biological safety and great clinical application value of cells are achieved. The optimized cell transplant is suitable for the technical field of mesenchymal stem cells applied to cell transplantation therapy, and the therapeutic effect of the optimized cell transplant is more excellent than that of the non-modified mesenchymal stem cell.
1 . A preparation method of an optimized cell transplant, for preparing an optimized cell transplant with high survival rate and simultaneous high expression of multiple genes; the preparation method comprising the following steps:
a cell modified culture step: mesenchymal stem cells are cultured in a first medium at a cell density of 6,000-15,000 cells/cm 2 , wherein the surface of a culture dish used has an oxygen-containing functional group and thus features a net negative surface charge due and hydrophilic; the first medium is removed after a first culture time to obtain a modified cell intermediate; and
a cell optimized culture step: the modified cell intermediate is cultured in a second medium, and the second medium is removed after a second culture time to obtain an optimized cell transplant, wherein
the mesenchymal stem cells are any one selected from adipose-derived stem cells, bone marrow stem cells, peripheral blood stem cells and umbilical cord blood stem cells;
the first medium is a serum-free DMEM/F12 medium comprising TGF-b1, FGF-2, hydrocortisone, human serum albumin, L-glutamine, leucine aminopeptidase, an HEPES buffer and lipid;
the first culture time is between 1 day and 10 days;
the second culture time is between 7 days and 28 days;
the optimized cell transplant has simultaneously and highly expressed CD200, Galectin-9 and VISTA genes; and
the expression levels of the CD200, Galectin-9 and VISTA genes of the optimized cell transplant are at least two times higher than those of the mesenchymal stem cells.
2 . The preparation method of the optimized cell transplant according to claim 1 , wherein the expression level of the CD200 gene of the optimized cell transplant is at least 8 times higher than that of the mesenchymal stem cells.
3 . The preparation method of the optimized cell transplant according to claim 1 , wherein the expression level of the Galectin-9 gene of the optimized cell transplant is at least 2 times higher than that of the mesenchymal stem cells.
4 . The preparation method of the optimized cell transplant according to claim 1 , wherein the expression level of the VISTA gene of the optimized cell transplant is at least 2 times higher than that of the mesenchymal stem cells.
5 . The preparation method of the optimized cell transplant according to claim 1 , wherein the survival rate of the optimized cell transplant is 90% or more.
6 . The preparation method of the optimized cell transplant according to claim 1 , wherein the second medium is a serum-free DMEM/F12 medium comprising nicotinamide, exendin-4, pentagastrin, a B-27 serum-free supplement and an N-2 supplement.