Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells

A technology of stem cells and protective solution, applied in the field of stem cells, can solve the problems of low cell recovery rate and cell viability rate, unsatisfactory effect of cryopreservation of umbilical cord mesenchymal stem cells, etc., and achieve the effect of excellent cell proliferation ability.

Active Publication Date: 2015-12-16
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned formulas are not effective in cryopreserving umbilical cord mesenchymal stem cells, and the cell recovery rate and cell viability after resuscitation are low.

Method used

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  • Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells
  • Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells
  • Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Preparation of umbilical cord mesenchymal stem cell conditioned medium: Take umbilical cord mesenchymal stem cells of P1 generation with a confluence of 90%, wash them once with PBS, and add 0.015mL / cm2 to the cells 2 Digest with 0.05% trypsin + 0.01% EDTA for 1min, stop the enzymolysis with complete medium 5 times the size of the digestion solution, centrifuge at 200g for 5min, reselect with complete medium, inoculate in a petri dish, the passage density is 8000 cells per cm 2 . The cells continued to grow for 72 hours, and the culture supernatant was collected, centrifuged at 200g for 5 minutes, and the supernatant was collected, aliquoted and stored at -80°C for later use.

[0051] Before cell cryopreservation, prepare umbilical cord mesenchymal stem cell cryopreservation protection solution: mix 80% umbilical cord mesenchymal stem cell conditioned medium and 10% fetal bovine serum, then add 10% DMSO, place in ice water bath until umbilical cord mesenchymal stem cel...

Embodiment 2

[0055] Preparation of umbilical cord mesenchymal stem cell conditioned medium: Take umbilical cord mesenchymal stem cells of P5 generation with a confluence of 80%, wash them with PBS three times, and add 0.04mL / cm2 to the cells 2 Digest with 0.05% trypsin + 0.04% EDTA for 1min, stop the enzymolysis with complete medium 10 times the size of the digestion solution, centrifuge at 400g for 5min, reselect with complete medium, inoculate in a culture bottle, and the passage density is 15,000 cells per cm 2 . The cells continued to grow for 24 hours, and the culture supernatant was collected, centrifuged at 400g for 5 minutes, and the supernatant was collected, aliquoted and stored at -80°C for later use.

[0056] Before cell cryopreservation, prepare umbilical cord mesenchymal stem cell cryopreservation protection solution: mix 30% umbilical cord mesenchymal stem cell conditioned medium and 50% fetal bovine serum, then add 20% DMSO, place in an ice water bath until cryopreservatio...

Embodiment 3

[0060] Preparation of umbilical cord mesenchymal stem cell conditioned medium: take umbilical cord mesenchymal stem cells of P3 generation with a confluence of 80%, wash them twice with PBS, and add 0.04 mL / cm2 to the cells 2 Digest with 0.05% trypsin + 0.04% EDTA for 1min, stop the enzymolysis with complete medium 10 times of the digestion solution, centrifuge at 400g for 5min, reselect with complete medium, inoculate in culture dish or culture bottle, passage density 10,000 cells per cm 2 . The cells continued to grow for 48 hours, and the culture supernatant was collected, centrifuged at 400g for 5 minutes, and the supernatant was collected, aliquoted and stored at -80°C for later use.

[0061] Before cryopreservation of cells, prepare umbilical cord mesenchymal stem cell cryopreservation protection solution: mix 55% umbilical cord mesenchymal stem cell conditioned medium and 30% fetal bovine serum, then add 15% DMSO, place in an ice water bath until cryopreservation solut...

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Abstract

The invention relates to the field of stem cells, and discloses a cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells. The cryopreservation protective fluid for the umbilical cord mesenchymal stem cells comprises 10-20 v / v% of DMSO, 30-80 v / v% of umbilical cord mesenchymal stem cell conditional media, and 10-50 v / v% of fetal calf serum. The umbilical cord mesenchymal stem cells revive after being cryopreserved through the cryopreservation protective fluid, and compared with common cell cryopreservation fluid, the cell recovery rate is obviously higher, and the cell reproductive capacity is better. The cryopreservation method for the umbilical cord mesenchymal stem cells comprises the steps that the umbilical cord mesenchymal stem cells are digested and centrifuged, the umbilical cord mesenchymal stem cell conditional media are added for adjusting the cell density, and cryopreservation protective fluid with the same volume is added for cryopreservation of the cells. After the cells cryopreserved through the cryopreservation method revive, the cell recovery rate is obviously higher and the cell reproductive capacity is better compared with a common cryopreservation method.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a cryopreservation protection solution and a cryopreservation method for umbilical cord mesenchymal stem cells. Background technique [0002] Stem cells are a kind of pluripotent cells with self-renewing ability. Under certain conditions, it can differentiate into a variety of functional cells. Mesenchymal stem cells (Mesenchymal stem cells, MSC) is an important member of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. MSC has the characteristics of self-replication, self-renewal, multi-directional differentiation potential, hematopoietic support and immune regulation. In a specific in vitro differentiation environment, it can be induced to differentiate into various tissue cells such as nerve, heart, bone, cartilage, fat, epithelium, etc., and is considered to be one of the most promising source cells for cell therapy technology. In a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 葛啸虎陈海佳王一飞麦锦连张维敏
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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