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Pretreatment method of human adipose-derived mesenchymal stem cells

A technology of adipose-derived stem cells, applied in animal cells, extracellular fluid diseases, vertebrate cells, etc., can solve the problems of difficult control of parameters, stem cell death, etc., and achieve the effect of increasing survival rate and migration ability

Inactive Publication Date: 2017-06-06
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One of the reasons that prevent the above method from being widely accepted is that the parameters required for pretreatment are difficult to control, and stem cells may die due to improper treatment.

Method used

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  • Pretreatment method of human adipose-derived mesenchymal stem cells
  • Pretreatment method of human adipose-derived mesenchymal stem cells
  • Pretreatment method of human adipose-derived mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Isolation and primary culture of hAD-MSC

[0027] hAD-MSCs were obtained by digesting and separating aseptic adipose tissue collected from adults after liposuction. Adult fat samples were obtained from a plastic surgery hospital. All samples were signed with informed consent.

[0028]Specifically, the fat collected by liposuction was stored and transported in D-Hanks' solution (purchased from Beijing Suolaibao Technology Co., Ltd.) containing double antibiotics (penicillin and streptomycin (purchased from North China Pharmaceutical Factory)). The adipose tissue collected by liposuction was washed twice with the above-mentioned D-Hanks' solution containing double antibiotics to remove blood cells and anesthetics, and centrifuged at 800rpm for 3min. Use a pipette to suck out the washed lower layer liquid, transfer the adipose tissue to a new 50ml centrifuge tube, add 0.2% collagenase P (purchased from Sigma) (fat:collagenase P volume ratio 3:1) to digest , inc...

Embodiment 2

[0029] Example 2: Subculture of hAD-MSCs

[0030] The primary cultured hAD-MSCs obtained in Example 1 were subcultured, specifically, 1) when the cells reached 70%-80% confluence, the medium was discarded, the cells were washed twice with D-Hanks' solution, and 0.25 % trypsin (containing 0.01% EDTA) digestion solution (Gibco), digested at room temperature, observed under a microscope, when the cells became round, FBS terminated the action of trypsin;

[0031] 2) Gently blow the cells in the culture flask with a pipette to separate the cells from the wall of the culture flask, and collect the cell suspension in a 15ml centrifuge tube, take out a small amount of culture solution and dilute to an appropriate multiple for counting with a cell counter, and the remaining cells are at 1200rpm Centrifuge for 5 minutes, discard the supernatant;

[0032] 3) Passage: The cells were resuspended in new medium, passaged at a ratio of 1:3, inoculated into a new culture bottle, and placed in...

Embodiment 3

[0033] Embodiment 3: low energy laser (LLL) irradiation treatment

[0034] The hAD-MSC subcultured in Example 2 was irradiated with a low-energy laser, and the cells between the first generation cells and the third generation were irradiated, and the adherent cells were directly irradiated. The irradiation time was about 1h ± 10min (irradiation dose 11- 16J / cm 2 ). Specifically, in this embodiment, hAD-MSCs were irradiated by using LXW660-II Quantum External Irradiation Apparatus from Shenyang Jixing Medical Instruments Co., Ltd. The working conditions of the quantum vascular external irradiation instrument are: power supply 220V±22V, 50hz±1HZ; light output wavelength 660nm±20nm, red to near infrared range (630-1000nm), output power of single point light source 3mw-4.5mw. All irradiations were carried out in a clean bench at room temperature, and the control group was treated under the same conditions without laser irradiation.

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Abstract

The invention provides a pretreatment method of human adipose-derived mesenchymal stem cells. The pretreatment method comprises the steps of (a) providing the human adipose-derived mesenchymal stem cells; (b) carrying out primary culture on the human adipose-derived mesenchymal stem cells; (c) carrying out secondary culture on the human adipose-derived mesenchymal stem cells; (d) adopting low energy laser with a wavelength of 660nm+ / -20nm to irradiate the human adipose-derived mesenchymal stem cells in a process of the secondary culture, wherein the irradiation dose is 11-16J / cm<2>; (e) obtaining the treated human adipose-derived mesenchymal stem cells. The method can maintain the cytological characteristics of the human adipose-derived mesenchymal stem cells (hAD-MSCs) and effectively improve the proliferation ability and transfer ability of the hAD-MSCs by means of radiation treatment, thus being used for a pretreatment scheme of stem cell transplantation.

Description

technical field [0001] The invention relates to a pretreatment method for mesenchymal stem cells, in particular to a pretreatment method for improving the proliferation and migration capabilities of human adipose-derived mesenchymal stem cells. Background technique [0002] Human adipose-derived mesenchymal stem cells (hAD-MSCs) are an important type of cells in the stem cell family with applications in regenerative medicine. Their surface expresses mesenchymal-specific markers CD73, CD90, and CD105, but does not express CD34, CD43, and CD45. Can differentiate into adipocytes, osteoblasts, chondrocytes [1] . Studies have shown that exposing stem cells to a hypoxic environment in vitro, simulating the microenvironment experienced by stem cells in damaged tissues, can enhance the resistance of stem cells in vivo after transplantation, and other programs such as heat shock treatment induce heat shock protein (HSP) expression , or hydrogen peroxide exposure increases resistanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N13/00A61K35/28A61P7/06A61P37/06A61P37/02
Inventor 赵春华尹侃朱榕嘉王世华
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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