Serum-free cryopreservation liquid for neural stem cells
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A neural stem cell and stem cell technology, applied in the field of stem cell culture, can solve problems such as pollution, and achieve the effects of overcoming serum pollution, promoting the maintenance of stem cell characteristics, and promoting proliferation
Inactive Publication Date: 2018-03-02
XUZHOU NORMAL UNIVERSITY
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Problems solved by technology
[0006] The technical task of the present invention is to solve the deficiencies of existing cryopreservation conditions, provide a cryopreservation solution for neural stem cells, solve the problem of animal-derived components and pathogen contamination caused by fetal bovine serum in conventional cryopreservation solutions, reduce cell death and Maintain stem cell properties
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Embodiment 1
[0027] see figure 1 , technical scheme of the present invention is realized in the following manner, and the step of this experimental method is:
[0028] 1) A neural stem cell cryopreservation solution proposed by the present invention contains the following components in volume percentage: 65%-75% of pre-prepared human neural stem cell serum-free medium, 9-12% of human serum albumin, dimethylformamide A mixed solution of 9-12% sulfoxide, 3-5.5% adenosine, 2.5-5% exosome solution derived from human neural stem cells and 2.5-5% short peptide and polypeptide compound solution derived from human neural stem cells.
[0029] 2) In a specific embodiment, the cryopreservation solution contains human neural stem cell serum-free medium, and the volume percentage can be 65%, 67%, 69%, 71%, 73%, 75%; the volume percentage of human serum albumin can be 9%, 9.5%, 10%, 10.5%, 11%, 12%; the volume percentage of dimethyl sulfoxide can be 9%, 9.5%, 10%, 10.5%, 11%, 12%, the volume of adenosi...
Embodiment 2
[0038] The experimental steps for human nerve cells to be frozen in the above-mentioned freezing solution, and then to be revived, subcultured and expanded are as follows:
[0039] 1. Cell recovery steps:
[0040] a) Quickly take out 1 tube of frozen neural stem cells from the liquid nitrogen tank and place it in an ice box equipped with liquid nitrogen for use;
[0041] b) Turn on the water bath to 37°C;
[0042] c) Use a 10mL pipette to draw 5mL of serum-free medium for neural stem cells into a 15mL centrifuge tube for use;
[0043] d) Take the cells out of the ice box with tweezers, thaw them quickly in a 37°C water bath, and shake gently until there is only a small piece of ice, and the recovery time is controlled within 1 minute;
[0044] e) Use 100-1000μL Tip to suck the recovered cells into the centrifuge tube, shake gently to mix well, the tip can absorb a small amount of cell suspension in the centrifuge tube to wash the cryopreservation tube once, centrifuge at 400...
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Abstract
The invention provides cryopreservation liquid for human neural stem cells, belongs to the technical field of stem cell cultures, and is a stem cell cryopreservation liquid composition suitable for clinic application. Various ingredients are used for optimizing the cryopreservation conditions; the cryopreservation liquid is prepared from the following ingredients in percentage by volume: 65 to 75percent of serum-free culture media of human neural stem cells, 3 to 5.5 percent of traditional Chinese medicine of rhizoma gastrodiae sourced monomeric compound solution, 9 to 12 percent of dimethylsulfoxide, 3 to 5.5 percent of adenosine, 2.5 to 5 percent of human neural stem cell sourced exosome solution and 2.5 to 5 percent of human neural stem cell sourced oligopeptide and polypeptide compound solution. After the cryopreservation of the human neural stem cells in the cryopreservation liquid, the recruitment rate reaches 85 percent or higher. No fetal calf serum is contained in the cryopreservation liquid; the exogenous unclear ingredient and pathogenicity pollution is prevented; the quality stability and safety and the standardized production of the stem cell treatment product are effectively ensured; the foundation is laid for the clinic treatment.
Description
technical field [0001] The invention relates to the technical field of stem cell culture, in particular to a serum-free cryopreservation solution of neural stem cells. Background technique [0002] Neural stem cells refer to a kind of stem cells isolated and cultured from embryonic and adult brain, spinal cord and other nervous tissues. Glial cells, stem cells that provide a large number of nerve tissue cells. Therefore, the purpose of cultivating neural stem cells is to obtain a sufficient number of neural stem cells with proliferation and differentiation potential to treat central nervous system dysfunction and attack tumor growth. At present, a large number of preclinical experimental studies have shown that neural stem cells have a certain therapeutic effect on stroke and traumatic brain injury, and have made encouraging substantial progress. [0003] Nerve is a complex and highly differentiated tissue in the human body. It is difficult to cultivate neural stem cells, ...
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