Umbilical cord mesenchymal stem cell protein-free non-programmed freezing medium and preparation method thereof
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A high-quality stem cell, protein-free technology, applied in the field of stem cell culture, can solve the problems of decreased cell expansion ability, time-consuming and laborious, high cost, etc., to improve the recovery rate and vitality, avoid the body's immune response, and reduce mutual extrusion Effect
Active Publication Date: 2022-05-31
大连博格林生物科技有限公司
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Problems solved by technology
These conventional cryopreservation solutions for umbilical cord mesenchymal stem cells still have some problems, such as: 1. It is often necessary to add exogenous serum at least personnel or recombinant albumin to achieve a better cryopreservation effect, but the use of exogenous serum increases exogenous The possibility of pathogen contamination, and large differences between batches, the source of human and recombinant albumin is difficult, the cost is high, and foreign proteins may cause the body's immune response, low safety, and subsequent clinical promotion of cell therapy will be affected
2. During the cryopreservation process of umbilical cord mesenchymal stem cells, the cryopreservation solution is likely to cause the cells to stick together and cause a certain amount of damage to the cells. Decreased capacity, decreased total cell number
4. Programmed cooling is required during the cryopreservation process, which is time-consuming, labor-intensive, time-consuming, and special equipment is expensive, which cannot meet the needs of large-scale cell processing
5. Studies have shown that using the same cryopreservation medium to freeze mesenchymal stem cells from different tissues has different survival rates after resuscitation, so the commercialized cryopreservation solution formula is not optimized and dedicated to umbilical cord mesenchymal stem cells of
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Embodiment 1
[0034] Preparation method of 1L of umbilical cord mesenchymal stem cell protein-free non-programmed cryopreservation solution:
[0035] 1. Preparing the base solution: Prepare the base solution according to the DMEM / F-12 formula, in which the glucose content is reduced to 1000mg / L, and the contents of other components remain unchanged.
[0036] 2. Weigh 0.4mg sodium molybdate dihydrate, 0.1mg nickel chloride, 0.15mg manganese dichloride tetrahydrate, 0.28mg cholesterol, 0.36mg linoleic acid, 6mg linolenic acid, 2.5mg spermine hydrochloride, 4g Progesterone, 10g erythritol, 15g trehalose, 25nmol Q-VD-OPh, 7.5mg sodium selenite, 0.03mg verbisoflavone were dissolved in the base solution in turn;
[0037] 3. Add 50ml of dimethyl sulfoxide, 10mg of sodium borate hydrate, 50g of hydroxyethyl starch, 40g of polyvinylpyrrolidone, 10g of dextran, and 15g of Lutrol F68 to it in turn, and fully dissolve;
[0038] 4. Adjust the pH to 6.9 with hydrochloric acid, then dilute the volume to ...
Embodiment 2
[0040] Preparation method of 1L of umbilical cord mesenchymal stem cell protein-free non-programmed cryopreservation solution:
[0041] 1. Preparing the base solution: Prepare the base solution according to the DMEM / F-12 formula, in which the glucose content is reduced to 1000mg / L, and the contents of other components remain unchanged.
[0042] 2. Weigh 0.8mg sodium molybdate dihydrate, 0.2mg nickel chloride, 0.05mg manganese dichloride tetrahydrate, 0.1mg cholesterol, 0.1mg linoleic acid, 0.5mg linolenic acid, 4mg spermine hydrochloride, 1.5mg g progesterone, 6 g erythritol, 20 g trehalose, 30 nmol Q-VD-OPh, 5 mg sodium selenite, 0.02 mg mullein isoflavones were dissolved in the base solution in turn;
[0043] 3. Add 45ml of dimethyl sulfoxide, 40mg of hydrated sodium borate, 30g of hydroxyethyl starch, 30g of polyvinylpyrrolidone, 20g of dextran, and 30g of Lutrol F68 successively to it, and fully dissolve;
[0044] 4. Adjust the pH to 6.9 with hydrochloric acid, then dilut...
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Abstract
The invention relates to the technical field of stem cell culture, in particular to a protein-free non-programmed freezing medium for umbilical cord mesenchymal stem cells as well as a preparation method and application of the protein-free non-programmed freezing medium. The cryopreservation solution comprises a basic solution, a nutritional supplement, a permeable protective agent, a non-permeable protective agent, a cell sedimentation stabilizer, a cell membrane protective agent, an apoptosis inhibitor and an antioxidant, the basic solution is DMEM / F-12 with the glucose content being smaller than or equal to 1000 mg / L. The cryopreservation liquid is suitable for directly cryopreserving umbilical cord mesenchymal stem cells at-80 DEG C after in-vitro amplification and before stem cell treatment. The cryopreservation liquid is free of serum and protein and clear in chemical component, and the cryopreserved umbilical cord mesenchymal stem cells are free of exogenous pollution risk and safer to use; after the cells are recovered, the viability is high, the adherence rate is high, and the cell expansion is fast; surface marker characteristics (phenotypes) and three-line differentiation potential of the mesenchymal stem cells can be maintained; optimization is carried out according to the cryopreservation and culture characteristics of the umbilical cord mesenchymal stem cells, the cryopreservation effect is improved, programmed cooling is not needed, and time and labor are saved.
Description
technical field [0001] The invention relates to the technical field of stem cell culture, in particular to a protein-free non-programmed cryopreservation solution of umbilical cord mesenchymal stem cells and a preparation method and application thereof. Background technique [0002] Stem cells (SCs) are primitive cells with self-replication and multi-directional differentiation potential in the human body. Under certain conditions, stem cells can be induced to differentiate into more than 220 functional cells that constitute the human body, such as nerve cells, liver cells, and cardiomyocytes. Stem cells are found in early embryos, placenta and its appendages, bone marrow, peripheral blood and adult tissues. Stem cells can be divided into totipotent stem cells, pluripotent stem cells and unipotent stem cells according to their developmental potential. Totipotent stem cells are stem cells with infinite differentiation potential that can differentiate into all tissues and org...
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