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Method for cryopreserving mesenchymal stem cells with low cryo-damage

A technology of bone marrow mesenchymal and cryopreservation methods, applied in the field of cryopreservation of bone marrow mesenchymal stem cells with low freezing damage, can solve the problems of reducing the recovery rate of cryopreserved cells, the penetration damage of protective agents, and cytotoxic damage, etc., to achieve Reduce cytotoxic damage, avoid mechanical damage, and reduce the effect of damage

Inactive Publication Date: 2016-03-16
黄林海
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] But vitrification has limitations
Vitrification cryopreservation agents in the prior art usually use DMSO. DMSO has cytotoxicity, which may cause cytotoxic damage during the freezing process, osmotic damage of the protective agent, and mechanical damage caused by the formation of ice crystals at the same time. These freezing damages will greatly reduce freezing. Recovery rate of stored cells

Method used

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  • Method for cryopreserving mesenchymal stem cells with low cryo-damage

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Embodiment 1

[0036] Such as figure 1 Shown, a kind of bone marrow mesenchymal stem cell cryopreservation method of low freezing injury comprises the following steps:

[0037] Step 1: Extract bone marrow, purify and isolate mononuclear cells, inoculate and culture the primary cells for 48 hours, and inoculate them with 10 mmol / L of dexamethasone, 2.16 g / L of β-glyceroglycerophosphate and 37.5 mg / L of 2 - Ascorbic acid phosphate culture medium, 5% CO 2 In an incubator, culture at 37°C for 8 days; use 0.25% trypsin-0.02% EDTA culture solution for the first passage; use 1.6×10 4 / cm 2 The cell density was inoculated and cultivated to P2 generation;

[0038] Step 2: Completely immerse the P2 bone marrow mesenchymal stem cell group in a mixed solution of 0.6% NaCl and 67.9% deionized water, maintain the temperature at 3.2°C, and add 3.4% DMSO, 5.2% polyethylene glycol, 0.3% glucose, 0.5% fructose, 20% human serum albumin, 2.1% Rho inhibitor, among which the Rho inhibitor is Y-27632, continue...

Embodiment 2

[0051] A method for cryopreserving bone marrow mesenchymal stem cells with low freezing damage, comprising the following steps:

[0052] Step 1: Extract bone marrow, purify and isolate mononuclear cells, inoculate and culture the primary cells for 48 hours, and inoculate them with 10 mmol / L of dexamethasone, 2.16 g / L of β-glyceroglycerophosphate and 37.5 mg / L of 2 - Ascorbic acid phosphate culture medium, 5% CO 2 In an incubator, culture at 37°C for 8 days; use 0.25% trypsin-0.02% EDTA culture solution for the first passage; use 1.6×10 4 / cm 2 The cell density was inoculated and cultivated to P2 generation;

[0053] Step 2: Completely soak the P2 generation bone marrow mesenchymal stem cell group in a mixed solution of 0.6% NaCl and 67.9% deionized water, maintain the temperature at 3.9°C, and add 3.2% DMSO, 5% polyethylene glycol, 0.5% glucose, 0.7% fructose, 20% human serum albumin, 2.3% Rho inhibitors, wherein the Rho inhibitors are Y-27632, fasudil and Hydroxyfasudil 1...

Embodiment 3

[0066] A method for cryopreserving bone marrow mesenchymal stem cells with low freezing damage, comprising the following steps:

[0067] Step 1: Extract bone marrow, purify and isolate mononuclear cells, inoculate and culture the primary cells for 48 hours, and inoculate them with 10 mmol / L of dexamethasone, 2.16 g / L of β-glyceroglycerophosphate and 37.5 mg / L of 2 - Ascorbic acid phosphate culture medium, 5% CO 2 In an incubator, culture at 37°C for 8 days; use 0.25% trypsin-0.02% EDTA culture solution for the first passage; use 1.6×10 4 / cm 2 The cell density was inoculated and cultivated to P2 generation;

[0068] Step 2: Completely immerse the P2 bone marrow mesenchymal stem cell group in a mixed solution of 0.6% NaCl and 67.9% deionized water, maintain the temperature at 3.5°C, and add 8.4% The osmotic protection solution, 0.35% glucose, 0.55% fructose, 20% human serum albumin, 2.2% Rho inhibitor, wherein the osmotic protection solution is 40% DMSO and 60% polyethylene ...

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Abstract

The invention discloses a method for cryopreserving mesenchymal stem cells with low cryo-damage, and belongs to the field of stem cell cryopreservation. The method comprises the following four steps: extracting bone marrow, and purifying and separating single karyocytes; inoculating and culturing primary cells for 48 hours, and culturing for 8 days at 37 DEG C in a culture solution comprising 10 mmol / L of dexamethasone, 2.16g / L of beta-glycerol phosphate sodium and 37.5mg / L of ascorbic acid-2-phosphate in an incubator having 5% CO2; conducting first generation with a culture solution consisting of 0.25 percent of trypsin and 0.02 percent of EDTA; inoculating with a cell density of 1.3*10<4> / cm<2>, culturing to P2 generation and the like. The method has the characteristics of low toxic injury and low protectant osmotic injury on cryopreserved cells in the freezing process, and can be used for preventing mechanical injury caused by ice crystal and improving the thawing rate of cryopreserved cells.

Description

technical field [0001] The invention relates to a cryopreservation method for stem cells, in particular to a cryopreservation method for bone marrow mesenchymal stem cells with low freezing damage. Background technique [0002] Bone marrow mesenchymal stem cells are important seed cells for constructing tissue engineered bone, and cryopreservation of bone marrow mesenchymal stem cells is of great significance for bone tissue engineering. Vitrification is a method in which cells and their protective agent solutions are supercooled to their glass transition temperature at a sufficiently fast cooling rate, solidified into a complete glass state and stored at low temperature for a long time in this glass state. In this process, crystallization is completely avoided inside and outside the cells, thereby avoiding various damages caused by freezing. The recovery rate of programmed cryopreservation is only 5%-10%, while the recovery rate of vitrification is >75%. Therefore, vitr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 黄林海
Owner 黄林海
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