Cryopreservation method and resuscitation method of umbilical cord mesenchymal stem cells

A technology of mesenchymal stem cells and cryopreservation method, which is applied in the field of preparation of umbilical cord mesenchymal stem cell cryopreservation solution, which can solve problems such as allergic reactions and failure of cell cryopreservation solution, and achieve the effects of stable physical properties, guaranteed survival rate, and low toxicity

Inactive Publication Date: 2020-12-04
SHANDONG QILU STEM CELL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently used fetal bovine serum cryopreservation solution can maintain cell viability for a long time, but the cell cryopreservation so

Method used

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  • Cryopreservation method and resuscitation method of umbilical cord mesenchymal stem cells
  • Cryopreservation method and resuscitation method of umbilical cord mesenchymal stem cells
  • Cryopreservation method and resuscitation method of umbilical cord mesenchymal stem cells

Examples

Experimental program
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Embodiment 1

[0032] A cryopreservation method for umbilical cord mesenchymal stem cells, comprising the following steps:

[0033] (1) Take mesenchymal stem cells, re-suspend the culture medium, re-spread it into a culture bottle, and harvest it when the cells in the culture bottle are more than 90% fused. The culture medium contains 2% fetal bovine serum, 40% MCDB201, 18 μg / L platelet-derived growth factor, 10 μg / L basic fibroblast growth factor, 10 μg / L epidermal growth factor;

[0034] (2) The culture medium in the culture bottle is poured out, after washing with 0.9% aqueous sodium chloride solution, add 2g / ml trypsin to digest for 25 seconds, add mixed physiological saline to stop digestion after the digestion is completed, and the mixed physiological saline is prepared by Made from the following components by weight: 88 parts of 0.9% sodium chloride aqueous solution, 1 part of α-arbutin, and 0.8 parts of L-glutamine;

[0035] (3) Transfer the digested cells into a centrifuge tube, ce...

Embodiment 2

[0039] A cryopreservation method for umbilical cord mesenchymal stem cells, comprising the following steps:

[0040] (1) Take mesenchymal stem cells, re-suspend the culture medium, re-spread it into a culture bottle, and harvest it when the cells in the culture bottle are more than 90% fused. The culture medium contains 2% fetal bovine serum, 40% MCDB201, 20 μg / L platelet-derived growth factor, 15 μg / L basic fibroblast growth factor, 15 μg / L epidermal growth factor;

[0041] (2) The culture medium in the culture bottle is poured out, after washing with 0.9% aqueous sodium chloride solution, add 4g / ml trypsin to digest for 35 seconds, add mixed physiological saline to stop digestion after the digestion is completed, and the mixed physiological saline is prepared by Made from the following components by weight: 100 parts of 0.9% sodium chloride aqueous solution, 5 parts of α-arbutin, and 1.6 parts of L-glutamine;

[0042] (3) Transfer the digested cells into a centrifuge tube, ...

Embodiment 3

[0046] A recovery method for umbilical cord mesenchymal stem cells, comprising the following steps:

[0047] (1) Take out the cells in the deep-low temperature refrigerator, and dissolve them in a 37°C water bath to obtain a cell suspension;

[0048] (2) Add the cell suspension to the prepared mixture of normal saline and human serum albumin, mix well, take out 1ml of the cell suspension and put it into the EP tube for trypan blue staining and counting, record the cell viability, normal saline and human serum albumin The volume ratio of human serum albumin is 15-20:1;

[0049] (3) Take 1×10 6 Add one cell into 15ml resuscitation medium, add it to culture in the culture bottle, and the resuscitation is completed when the fusion of the cultured cells reaches more than 90%, and the resuscitation medium is made of the following components by weight: 87-92 parts of low-sugar DMEM medium, 0.01-0.03 parts of isopropyl-β-D-thiogalactoside, 0.1-0.5 parts of sodium ascorbate, 4-6 parts ...

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Abstract

The invention discloses a cryopreservation method of umbilical cord mesenchymal stem cells, which comprises the following steps: (1) taking mesenchymal stem cells, resuspending the mesenchymal stem cells with a culture medium, repaving the mesenchymal stem cells into a culture bottle, and harvesting the mesenchymal stem cells when the fusion of the cells in the culture bottle reaches 90% or above;(2) pouring out the culture medium in the culture bottle, conducting washing, adding trypsin for digestion, and adding mixed normal saline to terminate digestion after the digestion is finished; (3)transferring the digested cells into a centrifuge tube, carrying out centrifugal treatment, discarding the supernatant, re-suspending the precipitate, carrying out centrifugation again, and discardingthe supernatant to obtain mesenchymal stem cells; and (4) adding the mesenchymal stem cells into a cryopreservation liquid, conducting resuspending, conducting sub-packaging in cryopreservation tubes, carrying out gradient cooling, conducting transferring to a cryogenic refrigerator, and completing cryopreservation. According to the cryopreservation method of the umbilical cord mesenchymal stem cells, the used cryopreservation liquid has a good cryopreservation effect on the umbilical cord mesenchymal stem cells, is efficient, does not contain any animal-derived component and can be directlyused as MSCs cryopreservation liquid for clinical infusion.

Description

technical field [0001] The invention belongs to the technical field of preparation of umbilical cord mesenchymal stem cell cryopreservation liquid for clinical infusion, and in particular relates to a cryopreservation method of umbilical cord mesenchymal stem cell. Background technique [0002] Stem cells (SC) are a kind of pluripotent cells with self-renewing ability, which can differentiate into various functional cells under certain conditions. Stem cells are not fully differentiated and immature cells, which have the potential to regenerate various tissues and organs and the human body. They are called "universal cells" in the medical field. Mesenchymal stem cells (MSCs) are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. Originally found in bone marrow, MSCs have attracted increasing attention because of their multi-lineage differentiation potential, hematopoietic support and promotion of stem cell i...

Claims

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Application Information

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IPC IPC(8): A01N1/02C12N5/0775
CPCA01N1/0221C12N5/0665C12N2500/38C12N2501/999
Inventor 王帅吕玲张秀涛庄肃静杨未未王玉霞李小博雒猛
Owner SHANDONG QILU STEM CELL ENG
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