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Preserving fluid for long-term preservation of endothelial precursor cell for over-expressing VEGF

An endothelial progenitor cell, long-term preservation technology, applied in the preservation, application, animal husbandry and other directions of human or animal body, can solve the problem of the decline in the ability of cells to express VEGF, the inability to maintain the biological characteristics of cells well, and the unclear composition of the preservation solution and other problems, to achieve the effect of clear and definite ingredients, maintenance of proliferation function and biological characteristics

Active Publication Date: 2018-07-06
暨赛国际再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the composition of the serum-containing preservation solution is not clear, and contains not only cytokines, growth factors, hormones and other components, but also unknown components
In the course of the research, the inventor found that using the above-mentioned commonly used cryopreservation solution to preserve the endothelial progenitor cells overexpressing VEGF would cause the ability of the cells to express VEGF after thawing and recovery to decline, and the biological characteristics of the cells could not be well maintained, and the cell proliferation function Poor and other issues

Method used

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  • Preserving fluid for long-term preservation of endothelial precursor cell for over-expressing VEGF
  • Preserving fluid for long-term preservation of endothelial precursor cell for over-expressing VEGF
  • Preserving fluid for long-term preservation of endothelial precursor cell for over-expressing VEGF

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of vascular endothelial progenitor cells overexpressing VEGF

[0036] experimental method:

[0037] 1) Isolation, culture and identification of vascular endothelial progenitor cells: 3-4 weeks old Vistar rats were killed by neck dislocation, soaked in alcohol for disinfection, the long bones were taken, cut open, and the bone marrow was washed, and the cell suspension was added to the rat lymphocyte separation medium Surface, centrifuge, take the cloudy cells between the two layers of liquid, wash, add EGM-2MV culture medium, and culture in a CO2 cell incubator. Adherent cells on days 0, 4, 7, and 10 were taken for immunohistochemical detection of markers CD34, FLK-1, CD133, and vWF, and cells on day 14 were examined by transmission electron microscopy to show specific W-P bodies.

[0038] 2) Construction of the PcDNA3.0-hVEGF165 eukaryotic expression vector: Synthesize the cDNA of VEGF165 according to the VEGF165 sequence of GENEBANK and introdu...

Embodiment 2-3 and comparative example 1-3

[0041] After the vascular endothelial progenitor cells were transfected with VEGF165, for the obtained VEGF-overexpressing vascular endothelial progenitor cells, the 10th passage cells were taken for cryopreservation. Examples 2-3 and Comparative Examples 1-3 respectively use different preservation solutions. The components of the preservation solution used in Example 2-3 and Comparative Example 1-2 are shown in the table below, and the preservation solution used in Comparative Example 3 is 20% bovine serum medium containing 10% dimethyl sulfoxide. The albumin is human serum albumin, which was purchased from ThermoFisher Company (product number 191297010); bovine serum was purchased from ThermoFisher Company (product number 10099158).

[0042]

[0043]

[0044] Embodiment 2-3 and comparative example 1-3 all carry out the cryopreservation of cell according to the following method:

[0045] (1) Take the vascular endothelial progenitor cells overexpressing VEGF in the loga...

Embodiment 4

[0051] Example 4 Detection of the proliferation function of recovered vascular endothelial progenitor cells overexpressing VEGF

[0052] Detect the proliferation function of the cells preserved in Example 2-3 and Comparative Example 1-3 after 6 months of frozen storage:

[0053] (1) Take out the cryovial from the liquid nitrogen, put it into a 38°C water bath quickly, and shake it from time to time to completely melt it within 1 minute, and then take out the cells in a sterile environment;

[0054] (2) Centrifuge at a speed of 1000r / min for 5-10 minutes, discard the supernatant, add an appropriate amount of culture medium (DMEM+20%FBS+bFGF) and inoculate it in a culture bottle at a concentration of 1×10 9 / L, placed in a 37°C cell culture incubator for static culture.

[0055] (3) After 24 hours, the cells in the culture flask were divided into 1.0×10 4 The density per well was inoculated in a standard 24-well culture dish for culture. 1-7 days after inoculation, the cells ...

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Abstract

The invention provides preserving fluid for long-term preservation of endothelial precursor cell for over-expressing VEGF. The preserving fluid comprises the following components: albumin, glucose, glycol, dimethyl sulfoxide, epidermal growth factor, magnesium sulfate, L-glutamine and betamethasone. The invention further provides an application method of the preserving fluid. Through the application method, the endothelial precursor cell for over-expressing VEGF is preserved with the preserving fluid, the endothelial precursor cell for over-expressing VEGF can be preserved for a long time, andresuscitated cell can properly maintain a proliferation function and biological characteristics of the endothelial precursor cell.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a preservation solution for long-term preservation of vascular endothelial progenitor cells overexpressing VEGF. Background technique [0002] Various types of bone defects caused by trauma, infection, tumor and other reasons are common clinical problems. According to the latest statistics in the United States, there are more than 1 million surgical operations involving bone defect repair every year; while in my country, accidental injuries caused by traffic, work injuries and sports are increasing at a rate of 7.3% per year. In recent years, the gradual rise of bone tissue engineering technology provides an effective solution for the repair of bone defects. Early studies have proved that when tissue engineered bone is used to repair small bone defects, the effect of early osteogenesis and later bone healing is obvious. However, when using large tissue engineered bone to repair large...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/021A01N1/0226
Inventor 徐丽婉吴琨徐家科朱思品
Owner 暨赛国际再生医学科技有限公司
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