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Serum-free cryoprotectant, and application thereof in cryopreservation of mesenchymal stem cells

A technology of stem cells and cryopreservation solution, which is applied in the field of cell biology, can solve the problems of increasing the risk of exogenous protein rejection, expensive reagents, and limited application range, so as to maintain activity and surface antigen characteristics, Good freezing effect and simple freezing method

Active Publication Date: 2017-12-22
章毅 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing MSCs cryopreservation solution and method still have many disadvantages, for example: it contains a high proportion of fetal bovine serum (FBS), which may increase the risk of rejection caused by exogenous proteins in clinical research and application; Serum-free cryopreservation method, due to the use of serum substitutes, the reagents are expensive and the scope of application is limited

Method used

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  • Serum-free cryoprotectant, and application thereof in cryopreservation of mesenchymal stem cells
  • Serum-free cryoprotectant, and application thereof in cryopreservation of mesenchymal stem cells
  • Serum-free cryoprotectant, and application thereof in cryopreservation of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Cryopreservation of umbilical cord mesenchymal stem cells in serum-free mesenchymal stem cell cryopreservation medium

[0031] The umbilical cord of newborns was taken as the source of mesenchymal stem cells, and the preparation and primary culture of umbilical cord mesenchymal stem cells were completed under sterile conditions within 48 hours, and then subcultured. Take the P3 umbilical cord MSCs, and when the cell confluence reaches 80-90%, discard the culture medium, add 5mL PBS buffer to gently wash the cells, discard the washing liquid, and digest the cells with 0.25% trypsin preheated at 37°C , the amount of trypsin should be just enough to soak the bottom of the culture container, such as: When the cells were observed under a microscope as a transparent spherical shape and no longer adhered to the wall, immediately add DMEM complete medium, the amount of which is as follows: Next, gently blow down the cells with a sterile dropper, transfer the cell...

Embodiment 2

[0048] Example 2 Cryopreservation of umbilical cord mesenchymal stem cells in serum-free mesenchymal stem cell cryopreservation medium

[0049] The MSCs isolated from the umbilical cord were cryopreserved according to the method described in Example 1.

[0050] The serum-free freezing solution used contains the following components: in terms of final concentration,

[0051] DMSO: 9% (v / v);

[0052] DMEM basal medium: 91% (v / v);

[0053] bFGF: 25ng / mL;

[0054] Insulin: 16.5 μg / mL;

[0055] Growth hormone: 3.0ng / mL;

[0056] TF: 0.15 μg / mL;

[0057] BMP-4: 0.12ng / mL;

[0058] Glutamine: 350mg / L;

[0059] Sodium pyruvate: 65mg / L;

[0060] β-mercaptoethanol: 50 μM;

[0061] hEGF: 15ng / mL;

[0062] Sodium selenite: 42μM;

[0063] PGF: 16ng / mL;

[0064] Glutamic acid: 15.6mg / L, alanine: 10.5mg / L, glycine: 7.0mg / L, aspartic acid: 9.5mg / L, proline: 13.0mg / L, serine: 9.5mg / L ;

[0065] Vitamin B12: 0.82mg / L, Vitamin C: 0.25mg / L, Vitamin B6: 65μg / L, Vitamin B2: 0.25mg / L. ...

Embodiment 3

[0066] Example 3 Cryopreservation of umbilical cord mesenchymal stem cells in serum-free mesenchymal stem cell cryopreservation medium

[0067] Umbilical cord MSCs were cryopreserved according to the method described in Example 1.

[0068] The serum-free freezing solution used contains the following components: in terms of final concentration,

[0069] DMSO: 10% (v / v);

[0070] DMEM basal medium: 90% (v / v);

[0071] bFGF: 9ng / mL;

[0072] Insulin: 9 μg / mL;

[0073] Growth hormone: 2.7ng / mL;

[0074] TF: 0.09μg / mL;

[0075] BMP-4: 0.09ng / mL;

[0076] Glutamine: 270mg / L;

[0077] Sodium pyruvate: 50mg / L;

[0078] β-mercaptoethanol: 35 μM;

[0079] hEGF: 9ng / mL;

[0080] Sodium selenite: 15μM;

[0081] PGF: 10.6ng / mL;

[0082] Glutamic acid: 13.2mg / L, alanine: 15.3mg / L, glycine: 6.1mg / L, aspartic acid: 11.7mg / L, proline: 10mg / L, serine: 7.9mg / L;

[0083] Vitamin B12: 0.54mg / L, Vitamin C: 0.18mg / L, Vitamin B6: 50μg / L, Vitamin B2: 0.4mg / L.

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Abstract

A serum-free cryoprotectant includes: 8-15 v / v% of DMSO, 85-92 v / v% of a DMEM basic culture medium, and a nutritional additive. The nutritional additive includes: fibroblast growth factors, insulin, growth hormone, transferrin, bone morphogenetic protein 4, glutamine, sodium pyruvate, beta-mercaptoethanol, human epidermal growth factor, sodium selenite, and various amino acids and vitamins. The cryoprotectant is free of animal sourced serum and avoids pollution and risk of allergen, and has better clinical safety. The serum-free cryoprotectant is suitable for cryopreservation of human placenta sourced, umbilical cord sourced and cord blood sourced mesenchymal stem cells; compared with common serum cryoprotectants, perinatal mesenchymal stem cells preserved in the cryoprotectant have high cell survival rate after resuscitation, have excellent adherence growth status and maintain biological characters well.

Description

technical field [0001] The invention belongs to the field of cell biology, and specifically relates to a serum-free cryopreservation solution and a cryopreservation method for mesenchymal stem cells during the perinatal period. By optimizing the components and formula of the cryopreservation solution, growth factors and nutrients are used to replace animal serum, Cryopreservation of mesenchymal stem cells is carried out in a way that does not contain exogenous serum and can maintain good cell activity and biological characteristics. Background technique [0002] Mesenchymal Stem Cells (MSCs) are a type of stem cells with self-proliferating ability and multi-directional differentiation potential, which can differentiate into osteoblasts, chondrocytes, adipocytes, nerve cells, muscle cells under specific conditions Wait. People first isolated MSCs from bone marrow, and later found that a small amount of MSCs also existed in various tissues such as liver, skin, muscle, cartila...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 章毅伍婷陈侃俊陈亮李冉祁成李萍王磊
Owner 章毅
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