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Method for cryopreservation and resuscitation of mesenchymal stem cells

A stem cell and cell technology, applied in the field of stem cell therapy, can solve problems such as insufficient stability and reduced survival percentage

Active Publication Date: 2019-10-11
天津瑞博斯生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regrettably, it has been found that the stem cells present in the existing cryopreservation solutions show insufficient stability during the period of several hours to tens of hours at the above-mentioned 25°C or 4°C, and this stability defect is reflected in the cells The survival rate, that is, the survival percentage, will decrease significantly as the storage time prolongs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Preparation of Mesenchymal Stem Cells from Dog's Umbilical Cord

[0063] (1) Disinfection and cleaning: in the biosafety cabinet, disinfect the surface of the dog's umbilical cord tissue (fresh isolated umbilical cord tissue) with disinfectant solution (75% ethanol), cut the umbilical cord, and lay it flat (on a surface with a diameter of 10cm culture dish), the umbilical cord tissue was washed by buffer (0.025M sodium dihydrogen phosphate buffer at pH 6.5) to reduce the red blood cells on the umbilical cord tissue;

[0064] (2) Digestion treatment: cut the umbilical cord tissue obtained in step (1) into tissue blocks (cubic blocks of about 0.1 cubic centimeters) in another cell culture plate, and put the tissue blocks into a digestive enzyme solution (preparation method: Add 0.1 g of type I collagenase to 100 ml of DMEM-F12, and then filter it with a 0.45 μm filter to obtain the digestion solution), digest it for 0.5-3 hours (in this example, digest it at 3...

Embodiment 2

[0068] Example 2, preparation of mesenchymal stem cells from the umbilical cord of dogs

[0069] (1) Disinfection and cleaning: in the biosafety cabinet, disinfect the surface of the dog's umbilical cord tissue (fresh isolated umbilical cord tissue) with disinfectant solution (75% ethanol), cut the umbilical cord, and lay it flat (on a surface with a diameter of 10cm culture dish), the umbilical cord tissue was washed by buffer (0.025M sodium dihydrogen phosphate buffer at pH 6.5) to reduce the red blood cells on the umbilical cord tissue;

[0070] (2) Digestion treatment: cut the umbilical cord tissue obtained in step (1) into tissue blocks (cubic blocks of about 0.1 cubic centimeters) in another cell culture plate, and put the tissue blocks into a digestive enzyme solution (preparation method: Add 0.1 g of type I collagenase to 100 ml of DMEM-F12, and then filter it with a 0.45 μm filter to obtain the digestion solution), digest it for 0.5-3 hours (in this example, digest ...

Embodiment 3

[0078] Embodiment 3: Referring to Example 1, but in step (5), use improved cell cryopreservation solution (which contains: 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine albumin, 0.25 %w / v dextran-40), the results show that the survival percentage of P1 to P15 generation cells after cryopreservation-thawing process is in the range of 92-96%, for example, the survival percentage of P3 generation canine umbilical cord mesenchymal stem cells is 93%.

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PUM

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Abstract

The invention relates to a method for cryopreservation and resuscitation of mesenchymal stem cells. A method for preparation and cryopreservation of the mesenchymal stem cells comprises the followingsteps of disinfection and cleaning, digestion treatment, cell culture, cell passage, addition of a cell cryopreservation solution to the mesenchymal stem cells and cryopreservation in liquid nitrogen.The cell cryopreservation solution contains DMEM-F12, dimethyl sulfoxide, canine blood albumin and the like. The method for cryopreservation and resuscitation of the mesenchymal stem cells comprisesthe following steps that the prepared mesenchymal stem cells and the cell cryopreservation solution are uniformly mixed, then the cell solution is placed in the liquid nitrogen for freezing, and cryopreservation is conducted until the cells are needed; when the cells need to be resuscitated, the cryopreserved cells are taken out of the liquid nitrogen and quickly put into a water bath of 37 DEG Cto make the cryopreservation solution completely thawed, and after the cells are thawed, the cells are immediately transferred to an ice bath to complete the resuscitation of the cells. The inventionfurther relates to the cell cryopreservation solution for the cryopreservation and resuscitation of the mesenchymal stem cells. The method and the cryopreservation solution have excellent technical advantages shown in the description.

Description

technical field [0001] The invention belongs to the technical field of stem cell therapy, such as stem cell therapy for canine diseases, and relates to a method for preparing mesenchymal stem cells (from, for example, the umbilical cord of dogs), and the use of such mesenchymal stem cells in the treatment of, for example, canine diseases the use of. The method for preparing mesenchymal stem cells from canine umbilical cord of the present invention has excellent technical effects. The prepared mesenchymal stem cells can beneficially treat arthritis, fracture, muscle injury, ligament injury, cartilage injury, joint injury, cognitive dysfunction, immune-mediated disease, dry eye, recurrent Uveitis, liver disease, heart disease, kidney disease, diabetes, gastrointestinal disease, thyroid disease, skin disease. Further, the present invention also relates to a method for cryopreserving the mesenchymal stem cells prepared from the canine umbilical cord, in particular, the cells con...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A01N1/02
CPCC12N5/0668C12N5/0667A01N1/021C12N2509/00
Inventor 王小武迟大明朱春颖郭春明
Owner 天津瑞博斯生物技术有限公司
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