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Cryopreservation protection solution and cryopreservation method for placenta amniotic mesenchymal stem cells

An amniotic membrane mesenchymal stem cell technology, 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 placental amniotic membrane mesenchymal stem cells, etc., and achieve the effect of excellent cell proliferation ability.

Active Publication Date: 2015-12-23
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 for cryopreservation of placental amniotic mesenchymal stem cells are not ideal, and the cell recovery rate and cell viability after recovery are low.

Method used

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  • Cryopreservation protection solution and cryopreservation method for placenta amniotic mesenchymal stem cells
  • Cryopreservation protection solution and cryopreservation method for placenta amniotic mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Preparation of conditioned medium for placental amniotic mesenchymal stem cells: take P1 generation placental amniotic mesenchymal stem cells with a confluence of 90%, wash them once with PBS, and add 0.015 mL / 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.

[0049] Before cell cryopreservation, prepare placental amniotic mesenchymal stem cell cryopreservation protection solution: mix 80% placental amniotic mesenchymal stem cell conditioned medium and 10% fetal bovine serum, then add 10% DMSO, place in an ice water bath until the placenta ...

Embodiment 2

[0053] Preparation of conditioned medium for placental amniotic mesenchymal stem cells: Take P5 generation placental amniotic mesenchymal stem cells with a confluence of 80%, wash them with PBS three times, 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 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.

[0054] Before cryopreservation of cells, prepare placental amniotic mesenchymal stem cell cryopreservation protection solution: mix 30% placental amniotic mesenchymal stem cell conditioned medium and 50% fetal bovine serum, then add 20% DMSO, place in an ice water bat...

Embodiment 3

[0058] Preparation of conditioned medium for placental amniotic mesenchymal stem cells: take P3 placental amniotic mesenchymal stem cells 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.

[0059] Before cell cryopreservation, prepare placental amniotic mesenchymal stem cell cryopreservation protection solution: mix 55% placental amniotic mesenchymal stem cell conditioned medium and 30% fetal bovine serum, then add 15% DMSO, place in an ice water bath until frozen The storage...

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Abstract

The invention relates to the field of stem cells and discloses a cryopreservation protection solution and a cryopreservation method for placenta amniotic mesenchymal stem cells. The cryopreservation protection solution for the placenta amniotic mesenchymal stem cells is prepared from 10-20 v / v% of DMSO, 30-80 v / v% of placenta amniotic mesenchymal stem cell conditioned media and 10-50 v / v% of fetal calf serums. After the cryopreservation protection solution is adopted for cryopreservation before resuscitation, the cell recovery rate is obviously higher than that of a conventional cryopreservation solution, and the cell reproductive capacity is superior to that of a conventional cell cryopreservation solution. According to the cryopreservation method, the placenta amniotic mesenchymal stem cells are digested and centrifuged, the placenta amniotic mesenchymal stem cell conditioned media are added for adjusting cell density, and the cells are cryopreserved after the cryopreservation protection solution with the same volume as the media is added. After the cells cryopreserved through the cryopreservation method resuscitate, the cell recovery rate is obviously higher than that of a conventional method, and the cell reproductive capacity is superior to that of a conventional 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 placental amniotic 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. ...

Claims

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Application Information

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IPC IPC(8): A01N1/02
Inventor 葛啸虎陈海佳王一飞麦锦连李平
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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