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Preparation method of serum-free freezing medium of human umbilical cord mesenchymal stem cells (hUC-MSCs)

A stem cell, serum-free technology, applied in the field of cryopreservation solution, can solve problems such as pollution, increase of animal pathogens, increase of uncertain factors in cell use, etc.

Inactive Publication Date: 2019-03-26
青岛麦迪赛斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the deficiencies of the prior art, the present invention provides a preparation method of a serum-free cryopreservation solution for human umbilical cord mesenchymal stem cells, which does not contain unsafe components such as fetal bovine serum and DMSO, and can maintain the activity of UC-MSCs for a long time , significantly improve the survival rate of cells, simple preparation, safety and other advantages, solve the problem that the existing cell cryoprotectants are basically cell basal medium + fetal bovine serum (FBS) 10~20% + dimethyl sulfoxide (DMSO ) 10%, DMSO is a osmotic protective agent, it can quickly penetrate into cells and reduce the time for cells to be exposed to harmful environments, but it has a toxic effect on cells at room temperature, and the operation time at room temperature needs to be as soon as possible; FBS will increase animal pathogens The probability of contamination, which will add uncertain factors to the future use of cells

Method used

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  • Preparation method of serum-free freezing medium of human umbilical cord mesenchymal stem cells (hUC-MSCs)
  • Preparation method of serum-free freezing medium of human umbilical cord mesenchymal stem cells (hUC-MSCs)
  • Preparation method of serum-free freezing medium of human umbilical cord mesenchymal stem cells (hUC-MSCs)

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Experimental program
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Effect test

Embodiment 1

[0036] 1. A serum-free cryopreservation solution for human umbilical cord mesenchymal stem cells, comprising the following composition and volume percentage thereof:

[0037] Basal medium 58%;

[0038] Platelet lysate 25%;

[0039] Instant proteoglycan mother liquor 2%;

[0040] Trehalose mother liquor 15%.

[0041] A preparation method of human umbilical cord mesenchymal stem cell serum-free cryopreservation solution, comprising the following preparation method:

[0042] S1, preparation raw material: get appropriate F12 culture medium and DMEM culture medium, mix by volume ratio as 1:1, obtain DMEM / F12 culture medium, extract instant proteoglycan from spirulina, and mix with pure water, obtain For the instant proteoglycan mother solution with a concentration of 50 mg / ml, take an appropriate amount of trehalose and mix it with purified water to obtain a trehalose mother solution with a concentration of 1 mol / L.

[0043] S2. Sterilization by filtration: Take an appropriate am...

Embodiment 2

[0053] Example 2: Detection of cell characteristics of human umbilical cord mesenchymal stem cells frozen in serum-free freezing solution for 1 year

[0054] 1. Detection of expression levels of cell surface markers by flow cytometry

[0055] Take the same batch of cells as in Example 1, and detect the expression level of surface markers before freezing; after 1 year of freezing, the invention group and the control group were quickly recovered in a 37°C water bath, and the surface markers were expressed after 48 hours of culture level detection.

[0056]

[0057] As a result, it was found that after one year of cryopreservation in the serum-free cryopreservation solution of human umbilical cord mesenchymal stem cells and the control group, the cells after 48 hours of resuscitation highly expressed CD44, CD73, CD90, and CD105, and did not express or lowly expressed CD34, CD45, There was no significant difference between HLA-DR and the cells before cryopreservation, indicati...

Embodiment 3

[0063] A serum-free cryopreservation solution for human umbilical cord mesenchymal stem cells, comprising the following compositions and volume percentages thereof:

[0064] Basal medium 80%;

[0065] Platelet lysate 5%;

[0066] Instant proteoglycan mother liquor 10%;

[0067] Trehalose mother liquor 5%.

[0068] A preparation method for human umbilical cord mesenchymal stem cell serum-free cryopreservation liquid, comprising the following volume ratio is 1:1 mixed to obtain DMEM / F12 medium, extract instant proteoglycan from spirulina, and mix with purified water Mix to obtain a mother solution of instant proteoglycan with a concentration of 50 mg / ml, and mix an appropriate amount of trehalose with purified water to obtain a mother solution of trehalose with a concentration of 1 mol / L.

[0069] S2. Sterilization by filtration: Take an appropriate amount of mother liquor of instant proteoglycan and mother liquor of trehalose to pass through a 0.2 μm filter membrane for ster...

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Abstract

The invention relates to the technical field of freezing mediums, and discloses a preparation method of a serum-free freezing medium of human umbilical cord mesenchymal stem cells (hUC-MSCs). The serum-free freezing medium of the hUC-MSCs is prepared from the following components in percentage by volume: 20-80% of a basal culture medium, 5-30% of platelet lysate, 1-10% of instant-dissolving protein polysaccharide mother liquor, and 5-25% of trehalose mother liquor. According to the preparation method of the serum-free freezing medium of the hUC-MSCs, the platelet lysate and trehalose are usedfor replacing original animal serum and DMSO, and thus the preparation method has the advantages that the activity of the UC-MSCs can be maintained for a long time, the survival rate of the cells is significantly increased, and preparation is easy and safe; and the problems that the DMSO serves as a permeating protective agent and can penetrate into the cells quickly, the time that the cells are exposed in the harmful environment is shortened, however, the DMSO has the toxic effect on the cells at the room temperature, operation at the room temperature needs to be quick as soon as possible, FBS can increase the probability of animal pathogenic pollution, and consequently, uncertain factors are added for future using of the cells are solved.

Description

technical field [0001] The invention relates to the technical field of cryopreservation solution, in particular to a preparation method of a serum-free cryopreservation solution for human umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are adult stem cells derived from early mesoderm, which have high self-renewal ability and multi-directional differentiation potential. For example, bone, cartilage, muscle, ligament, tendon, fat and stromal cells proliferate and differentiate, and have weak immunogenicity, so they are the source of seed cells for tissue engineering projects. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are derived from Wharton's jelly in the umbilical cord of newborns. In addition to all the characteristics of MSCs, they are also rich in content, pure in cells, and less immunogenic , the advantages of primitive cells, stronger differentiation ability, and no ethical restrict...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 王玉娟张道强刘传杰王金环徐矫健葛淑娟刘燕丽
Owner 青岛麦迪赛斯生物科技有限公司
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