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A cryoprotectant for ultra-low temperature injury of mesenchymal stem cells

A cryopreservation agent and mesenchymal stem cell technology, which is applied in the field of cryopreservation agent for mesenchymal stem cells damaged by ultra-low temperature, can solve the problems of decreased cell activity, affecting the proliferation and differentiation ability of umbilical cord blood mesenchymal stem cells, etc., and achieve the purpose of providing activity Effect

Active Publication Date: 2021-11-12
山东科金生物发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, long-term cryopreservation of umbilical cord blood mesenchymal stem cells will lead to a decrease in cell viability, and will also affect the proliferation and differentiation capabilities of umbilical cord blood mesenchymal stem cells to a certain extent.

Method used

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  • A cryoprotectant for ultra-low temperature injury of mesenchymal stem cells
  • A cryoprotectant for ultra-low temperature injury of mesenchymal stem cells
  • A cryoprotectant for ultra-low temperature injury of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Detection of the effect of ranacyclin B3 polypeptide pretreatment on the cryopreservation viability of umbilical cord blood mesenchymal stem cells

[0029] 1. Cell Treatment

[0030] (1) Experimental groups: control group (use DMEM / F12 medium to treat umbilical cord blood mesenchymal stem cells for 2 hours), experimental group 1 (use DMEM / F12 medium containing 25 μmol / L Ranacyclin B3 polypeptide to treat umbilical cord blood mesenchymal stem cells 2h), experimental group 2 (umbilical cord blood mesenchymal stem cells were treated with DMEM / F12 medium containing 50 μmol / L Ranacyclin B3 polypeptide for 2 hours), experimental group 3 (treated with DMEM / F12 medium containing 100 μmol / L Ranacyclin B3 polypeptide Umbilical cord blood mesenchymal stem cells 2h);

[0031] (2) Prepare cryopreservation solution according to 10% DMSO, 50% FBS, 40% DMEM / F12 medium;

[0032] (3) Collect the cells treated in step (1), add cryopreservation solution, and divide into cryopreservation ...

Embodiment 2

[0045] Detection of the effect of ranacyclin B3 polypeptide pretreatment on the proliferation activity of umbilical cord blood mesenchymal stem cells

[0046] (1) Take out the cells of the control group and experimental group 3 that were frozen in liquid nitrogen for 12 months, put them in a 37°C water bath to quickly melt and recover, and centrifuge to remove the supernatant;

[0047] (2) Seed the cells in a 6-well plate. After the cells adhere to the wall, remove the floating dead cells, add trypsin to digest the cells and adjust the cell concentration of each group;

[0048] (3) Add 100ul 1×10 5 The cells were seeded in a 96-well plate, and after 3 days, 10ul CCK-8 was added to detect the OD value of the cells.

[0049] Experimental results such as image 3 As shown, the average OD value of the control group was 0.599, and the standard deviation was 0.032; the average OD value of the experimental group 3 was 0.658, and the standard deviation was 0.025, P<0.05. Blood mese...

Embodiment 3

[0051] Detection of the effect of ranacyclin B3 polypeptide pretreatment on the adipogenic differentiation ability of cryopreserved umbilical cord blood mesenchymal stem cells

[0052] (1) Take out the cells of the control group and experimental group 3 that were frozen in liquid nitrogen for 12 months, put them in a 37°C water bath to quickly melt and recover, and centrifuge to remove the supernatant;

[0053] (2) Cells were seeded in a 6-well culture plate. When the cell confluency reached 90%, the adipogenic induction medium I was cultured for 2 days, then replaced with adipogenic induction medium II, and the culture was continued until the 8th day. Replace the culture medium once a day;

[0054] (3) After the culture, remove the medium, wash the cells with PBS 3 times, and add 10% formaldehyde to fix the cells for 40 min;

[0055](4) After removing formaldehyde, add 500ul Oil Red O staining solution and stain at room temperature for 30min;

[0056] (5) After removing the...

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Abstract

The invention provides a cryopreservation protective agent for mesenchymal stem cells damaged by ultra-low temperature, and belongs to the technical field of mesenchymal stem cells. The cryopreservation agent for ultra-low temperature injury of mesenchymal stem cells provided by the present invention is a culture medium containing Ranacyclin B3 polypeptide, and the amino acid sequence of the polypeptide is Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser ‑Ile‑Pro‑Pro‑Lys‑Pro‑Cys‑Ser‑Gly‑Lys‑Arg. By using the polypeptide to pretreat the umbilical cord blood mesenchymal stem cells, the activity of the cells after cryopreservation, as well as the proliferation and adipogenic differentiation abilities of the cells can be effectively improved.

Description

technical field [0001] The invention belongs to the technical field of mesenchymal stem cells, and in particular relates to a cryopreservation agent for ultralow temperature injury of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells are seed cells with high self-renewal and multilineage differentiation potential. Under certain conditions, mesenchymal stem cells can be induced to differentiate into various types of tissue cells such as skeletal muscle cells, chondrocytes, adipocytes, osteoblasts or cardiomyocytes, which can be widely used in the treatment of various diseases and Tissue Engineering Research. Mesenchymal stem cells come from a wide range of sources. According to their sources, they can be divided into fat-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. Among the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 王禄
Owner 山东科金生物发展有限公司
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