PREPARATION AND METHOD OF CULTIVATION, STORAGE AND CRYOCONSERVATION OF STEM AND DIFFERENTIATED HUMAN AND ANIMAL CELLS Russian patent published in 2013 - IPC C12N5/07 C12N5/71 A61K31/02 A61K9/107 

FIELD: chemistry.

SUBSTANCE: group of inventions relates to field of biochemistry. Claimed is preparation for preservation of culture of stem and differentiated human and animal cells in cultivation, storage and cryoconservation. Preparation represents gel, which contains emulsion of perfluororganic compounds (PFOC) in their stabilisation in emulsified state with water solution of non-ionogenic surface active compounds (SAS) based on block-copolymer of polyethylene oxide (PEO) and polypropylene oxide (PPO) with ratio PEO/PPO from 9:1 to 7:3. SAS concentration in preparation constitutes from 20 to 90% with average molecular weight of SAS from 5000 Da to 21000 Da. Content of PFOC in emulsion constitutes from 5 to 70 vol.%. Size of PFOC emulsion particles lies in the interval from 15 nm to 2000 nm. Water phase of gel contains osmolytes of organic and/or inorganic nature for supporting in it osmotic pressure from 250 to 350 mOsm. As PFOC emulsion, emulsion of perfluorodecalin, perfluoro methylcyclohexyl piperidine and perfluorotributylamine mixture can be used. Also claimed is method of obtaining said preparation. Extrusion of coarse mixture of water SAS solution and liquid PFOCs or PFOC mixture is performed under pressure 400-700 atm until emulsion is obtained. After that, it is mixed in hot condition with concentrated water solution of inorganic salts or osmolytes are added into it until osmotic pressure equal from 250 to 350 mOsm is obtained. Then obtained composition is cooled. After that, partial freeze-drying or centrifugation of gel with observation of temperature gradient and acceleration from 15000 g to 35000 g is performed. Methods of preservation of culture of stem and differentiated human and animal cells in storage, cryoconservation and cultivation are claimed. In storage of cells preparation with PFOC content from 15 to 59 vol.% is added to culture medium with controlled value pH=7.0-7.4, temperature 37°C in CO₂ incubator atmosphere or with low pH<6.0, temperature 20°C in air atmosphere. In cryoconservation resuspended in fresh culture medium cells are placed into medium, which contains preparation with PFOC content 59-70 vol.%. After that, cooling is carried out in two stages: first at rate 1 degree per min to -80°C, then with maximally possibly rate to temperature -196°C. In cultivation of cells as medium component preparation with PFOC content from 15 to 70 vol.% is used.

EFFECT: cell cultures after cryoconservation, storage or cultivation in presence of PFOC-containing gels have on average 90% of viable cells, are easily separated from gel, re-inoculated in standard conditions, divided and fully preserve their functions and ability to differentiate.

16 cl, 23 dwg, 1 tbl, 2 ex