YERYEMYEYEV, ARTYEM VALYER'YEVICH,SVYETLAKOV, ANATOLIJ VASIL'YEVICH,BOL'SHAKOV, IGOR' NIKOLAYEVICH,SHYEINA, JULIJA IGORYEVNA,POLSTJANOJ, ALYEKSYEJ MIKHAJLOVICH,YERYEMYEYEV, ARTYEM VALYERYEVICH,SVYETLAKOV, ANATOLIJ VASILYEVICH,BOLSHAKOV, IGOR NIKOLAYEVICH
申请号：
EP11780875
公开号：
EP2557153A4
申请日:
2011.04.01
申请国别(地区):
EP
年份:
2013
代理人:
摘要：
The invention relates to biology and medicine and, more specifically, to neurology and neurosurgery, cellular nanobiotechnology and bioengineering, and transplantology, and can be used for culturing, proliferating and differentiating stem cells into cells of a neural nature for the purpose of transplantation. The problem addressed by the invention is to enhance culturing quality and stability and increase the proliferative activity of pluripotent stem cells with the differentiation thereof into neural cells, to prevent immunogenicity of the microenvironment of the cells and to produce a neural matrix which is suitable for direct transplantation. The problem is solved in that a biomass of human embryonic stem cells of a feeder-free line hESKM-05 is first of all grown in flasks covered by a 0.1% solution of gelatine, with the use and daily replacement of KO-DMEM basal medium comprising 10% serum replacement (SR), 100 mkg/ml kanamycin sulfate, 1mM L-glutamine solution, 4 ng/ml basic fibroblast growth factor (bFGF), and 1 mM solution of essential amino acids, and then the biomass is transferred with the aid of a 0.5% solution of collagenase into flasks containing a prepared collagen-chitosan matrix in a conditioned medium produced from embryonic neural cells of mice, or in a complete culture medium with the addition of neural factor N2 which is changed every three days, wherein the matrix is used in the form of a sponge, film, microspheres, gel or fibres. The proposed method for producing a neural matrix makes it possible, on the 5-7th day of culturing, to produce a sufficient quantity of cells of a neural nature, and the collagen-chitosan matrix ensures the viability and proliferative activity of said cells and conditions for direct transplantation.
