Resuscitation fluid applied to vitrified cryopreserved mesenchymal stem cells

A bone marrow mesenchymal, vitrification cryopreservation technology, applied in the field of cryopreservation and resuscitation fluid, can solve the problems of reducing the recovery rate of cryopreserved cells, penetrant damage of protective agents, cytotoxic damage, etc., to promote the differentiation and growth of stem cells, reduce Cytotoxic damage, the effect of reducing the content

Inactive Publication Date: 2016-03-09
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; during the resuscitation process, it may also cause osmotic damage to the protective agent, and at the same time form ice crystals to cause mechanical damage, which will greatly reduce freezing. Recovery rate of stored cells

Method used

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  • Resuscitation fluid applied to vitrified cryopreserved mesenchymal stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Such as figure 1 As shown, a resuscitation solution for vitrification of bone marrow mesenchymal stem cells, including 20% ​​human serum albumin, 0.8% monosaccharide, 0.21% cell growth factor, 1.5% hydroxyethylpiperazine Sulfuric acid, 0.8% non-essential amino acids, 0.08% L-glutamine, 0.03% β-mercaptoethanol, and the rest are normal saline.

[0030] Experimental cryopreservation materials: animal, 2-year-old Beagle dog, male, weighing 22.7kg.

[0031] experimental method:

[0032] 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.

[0033] St...

Embodiment 2

[0043] A resuscitation solution for vitrification of bone marrow mesenchymal stem cells, including 20% ​​human serum albumin, 1% monosaccharide, 0.23% cell growth factor, 1.5% hydroxyethylpiperazineethanesulfonic acid, 1.2% non-essential amino acids, 0.12% L-glutamine, 0.04% β-mercaptoethanol, and the rest are normal saline.

[0044] Experimental cryopreservation materials: animal, 2-year-old Beagle dog, male, weighing 22.7kg.

[0045] experimental method:

[0046]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.

[0047] Step 2: Completely immerse t...

Embodiment 3

[0057] A resuscitation solution for vitrification of bone marrow mesenchymal stem cells, including 20% ​​human serum albumin, 0.9% monosaccharide, 0.22% cell growth factor, 1.5% hydroxyethylpiperazine ethanesulfonic acid, 1.1% non-essential amino acids, 0.1% L-glutamine, 0.032% β-mercaptoethanol, and the rest are normal saline.

[0058] Experimental cryopreservation materials: animal, 2-year-old Beagle dog, male, weighing 22.7kg.

[0059] experimental method:

[0060] 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.

[0061] Step 2: Completely immer...

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Abstract

The invention discloses resuscitation fluid applied to vitrified cryopreserved mesenchymal stem cells and belongs to the field of resuscitation fluid. The resuscitation fluid applied to the vitrified cryopreserved mesenchymal stem cells comprises 20% of human serum albumin, 0.8-1% of monosaccharide, 0.21-0.23% of a cell growth factor, 1.5% of N-2-hydroxyethylpiperazine-N'-2-ethane-sulphonic acid, 0.8-1.2% of non-essential amino acid, 0.08-0.12% of L-glutamine, 0.03-0.04% of beta-mercaptoethanol and normal saline. The resuscitation fluid applied to the vitrified cryopreserved mesenchymal stem cells has the characteristics of low cell toxicity on the mesenchymal stem cells, low cell toxicity damage and low permeability damage in a vitrified cryopreserved resuscitation process and increased cryopreserved cell resuscitation rate.

Description

technical field [0001] The invention relates to a cryopreservation resuscitation solution, in particular to a resuscitation solution for vitrification and storage 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, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 黄林海
Owner 黄林海
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