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A cryopreservation solution and cryopreservation method for mesenchymal stem cells

A technology of stem cells and cryopreservation, which is applied in the preservation, application, and animal husbandry of human or animal bodies. It can solve the problems of inability to precisely control the cooling rate and cell damage, and achieve good stem cell characteristics, high expression rate, and reduced effect of concentration

Active Publication Date: 2017-03-15
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cooling rate cannot be precisely controlled during the gradual cooling process of the cryopreservation box, which will cause more ice crystals to form in the cryopreservation solution, resulting in greater damage to the cells

Method used

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  • A cryopreservation solution and cryopreservation method for mesenchymal stem cells
  • A cryopreservation solution and cryopreservation method for mesenchymal stem cells
  • A cryopreservation solution and cryopreservation method for mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1, Isolation and Culture of Umbilical Cord Mesenchymal Stem Cells

[0046] 1. Take the bovine umbilical cord tissue block, cut it into pieces, add the complete medium to the tissue block, pipette evenly and transfer it to the cell culture dish; the composition of the complete medium is: Lonza universal UltraCULTURE serum-free medium + PALL Serum replacement + 2mM / L-glutamine + 100mg / L NEAA;

[0047] 2. Shake the culture dish to make the tissue pieces evenly distributed in the culture dish, transfer to 5% CO 2 , Cultured in a 37°C cell incubator, and regularly added to observe the growth of the cells;

[0048] 3. When the cells crawl out, discard the old medium and tissue pieces, wash the culture dish with PBS, add complete medium and continue to culture in the cell culture incubator;

[0049] 4. When the cell confluency reaches 80%-90%, discard the old medium, wash the culture dish with PBS, add EDTA-containing trypsin mixture to digest for 1-2min, add complet...

Embodiment 2

[0051] Embodiment 2, cryopreservation of umbilical cord mesenchymal stem cells

[0052] 1. Preparation of cryopreservation solution: prepare a cryopreservation solution of mesenchymal stem cells in advance according to the ratio of each component of the cryopreservation solution, which contains 5% DMSO (V / V), 1% dextran-40 (mass volume ratio , g / mL), and the remaining components are Ultra CULTURE serum-free medium. Pre-cool the prepared cryopreservation solution in a 4°C refrigerator.

[0053] 2. Get the P2 generation cells obtained in Example 1. When the cell confluency reaches 80% to 90%, discard the old culture medium, wash the culture dish with PBS, and add EDTA-containing trypsin mixture to digest for 1-90%. After 2 minutes, complete medium was added to terminate the digestion, and the cells were pipetted into a single cell suspension to obtain P2 generation umbilical cord mesenchymal stem cells.

[0054] 3. Take a small amount of cell suspension and use a cell counter ...

Embodiment 3

[0065] Example 3, Evaluation of the effect of the mesenchymal stem cell cryopreservation solution of the present invention

[0066] Take the cell suspension of umbilical cord mesenchymal stem cells obtained in steps 1 to 2 in Example 2, and use a conventional freezing solution containing 10% DMSO, 20% FBS, and 70% DMEM / F12. The freezing method is the same as in the example 2 is the same. That is, except for the different components of the cryopreservation solution, other conditions were kept the same as in Example 2.

[0067] The above-mentioned cryopreserved cells were used as a control group, and the cell survival rate, proliferation and cell surface markers were evaluated respectively, and the cryopreservation effect of the cryopreservation solution of the mesenchymal stem cells described in the present invention and the conventional cryopreservation solution were compared.

[0068] 1. Comparison of cell viability of cells after recovery

[0069] The two tubes of cells in...

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Abstract

The invention relates to the field of stem cells, and discloses a cryopreservation solution and a cryopreservation method for mesenchymal stem cells. The cryopreservation solution for mesenchymal stem cells comprises a serum-free medium, DMSO and dextran-40, wherein the concentration of the DMSO is 3-7 v / v %, and in g / mL, the concentration of the dextran-40 is 1-3 w / v %. After the cryopreservation solution for mesenchymal stem cells disclosed by the invention is used, the survival rate of cryopreserved cells is significantly increased, and good stem cell characteristics are kept. In the cryopreservation method for mesenchymal stem cells disclosed by the invention, after cells are resuspended by using the cryopreservation solution disclosed by the invention and the cell density is adjusted, the cells are cryopreserved. After cells cryopreserved by using the cryopreservation method for mesenchymal stem cells disclosed by the invention recover, the survival rate of the cells is obviously increased, the cell reproductive capacity is also superior to that in conventional cryopreservation methods, and specific surface markers of mesenchymal stem cells also do not have obvious drop, and have a high expression rate.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a cryopreservation solution and a cryopreservation method for mesenchymal stem cells. Background technique [0002] Stem cells are a kind of pluripotent cells with self-renewing ability. Under certain conditions, it can differentiate into a variety of functional cells. Mesenchymal stem cells (MSCs) are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. MSCs have the common characteristics of stem cells, that is, the ability of self-renewal, multilineage differentiation and homing. In a specific in vitro differentiation environment, it can be induced to differentiate into various tissue cells such as nerve, heart, bone, cartilage, fat, epithelium, etc. It is considered to be one of the most promising source cells for cell therapy technology. research hotspot. As an important resource of regenerative medicine, MSC will...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
Inventor 王一飞陈海佳葛啸虎王小燕万桦罗二梅
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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