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Cryopreservation protective agent for vitrification cryopreservation of marrow mesenchymal stem cells

A bone marrow mesenchyme and vitrification cryopreservation technology, which can be used in applications, preservation of human or animal bodies, animal husbandry, etc., can solve problems such as cytotoxic damage, ice crystal mechanical damage, protective agent permeability damage, etc., to improve recovery rate, reduce the content, and reduce the effect of cytotoxic damage

Inactive Publication Date: 2016-02-17
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, thereby causing cytotoxic damage; the freezing process may also cause osmotic damage to the protective agent, and at the same time form ice crystals to cause mechanical damage, these damages will greatly reduce freezing. Recovery rate of stored cells

Method used

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  • Cryopreservation protective agent for vitrification cryopreservation of marrow mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Such as figure 1 As shown, a cryoprotectant for vitrification of bone marrow mesenchymal stem cells, including 8.2% osmotic protection solution, 0.8% monosaccharide, 20% human serum albumin, 2.1% Rho inhibitor , the remaining components are physiological saline, and the Rho inhibitor is Y-27632.

[0032] Experimental cryopreservation materials: animal, 2-year-old Beagle dog, male, weighing 22.7kg.

[0033] experimental method:

[0034] 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 cultured to the P2 generation.

[0035] Step 2: Add the protective agent of this example ...

Embodiment 2

[0045] A cryoprotectant for vitrification of bone marrow mesenchymal stem cells, including 8.6% osmotic protection solution, 1% monosaccharide, 20% human serum albumin, 2.3% Rho inhibitor, and the rest Divided into normal saline, the Rho inhibitor was fasudil.

[0046] Experimental cryopreservation materials: animal, 2-year-old Beagle dog, male, weighing 22.7kg.

[0047] experimental method:

[0048]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 cultured to the P2 generation.

[0049] Step 2: Add the protective agent of this example to the P2 generation bone marrow mesenchymal ...

Embodiment 3

[0059] A cryopreservation agent for vitrification of bone marrow mesenchymal stem cells, including 8.3% osmotic protection solution, 0.9% monosaccharide, 20% human serum albumin, 2.2% Rho inhibitor, and the rest Divided into normal saline, the Rho inhibitor is hydroxyfasudil.

[0060] Experimental cryopreservation materials: animal, 2-year-old Beagle dog, male, weighing 22.7kg.

[0061] experimental method:

[0062] 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 cultured to the P2 generation.

[0063] Step 2: Add the protective agent of this example to the P2 generation bone ma...

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Abstract

The invention discloses a cryopreservation protective agent for vitrification cryopreservation of marrow mesenchymal stem cells and belongs to the field of protective agents. The cryopreservation protective agent for vitrification cryopreservation of the marrow mesenchymal stem cells is prepared from 8.2%-8.6% of permeable protection fluid, 0.8%-1% of monosaccharide, 20% of human serum albumin, 2.1%-2.3% of Rho inhibitor and normal saline. The cryopreservation protective agent for vitrification cryopreservation of the marrow mesenchymal stem cells has the advantages of low cytotoxicity to the marrow mesenchymal stem cells, low cytotoxicity damage and low permeability damage in the vitrification cryopreservation process and increasing of the recovery rate of cryopreservation cells.

Description

technical field [0001] The invention relates to a cryopreservation agent, in particular to a cryopreservation agent for vitrification cryopreservation of bone marrow mesenchymal stem cells. 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, vitrificatio...

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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