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Preparation method of novel human adipose-derived stem cell preparation

An adipose stem cell and human fat technology, applied in the field of biomedicine, can solve the problems of low separation and purification rate, low cell adhesion rate, and large cell damage, and achieve the effects of promoting cell proliferation, increasing expansion efficiency, and promoting angiogenesis.

Pending Publication Date: 2020-09-04
成熙(上海)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a preparation method of a novel human-derived adipose stem cell preparation, which can solve the problem of large cell damage, low separation and purification rate in the fat digestion process in the prior art, and residual heterologous proteins or antibiotics in the culture process. Allergic reaction of vaccinators, low cell adhesion rate and poor proliferation rate

Method used

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  • Preparation method of novel human adipose-derived stem cell preparation
  • Preparation method of novel human adipose-derived stem cell preparation
  • Preparation method of novel human adipose-derived stem cell preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] This example provides a method for preparing a novel human-derived adipose stem cell preparation, which includes the following steps:

[0023] S1: Collection of human adipose tissue: HIV, HBV, HCV physical examinations are performed on the donor, and then under local anesthesia, fat is obtained by tumescent liposuction with a maximum negative pressure of 430mmHg, and the collection site is the thigh. The fat is filtered out of oil and water through a 500μm filter hole to obtain light yellow fat;

[0024] S2: Preparation of primary adipose stem cells: pretreat the fat obtained in step S1, and wash it with PBS solution for several times until the washing solution is clear and translucent, and the fat is clean and light yellow; after the above-mentioned multiple washings, the fat Put it in a mixture of 0.2%-0.4% collagenase type I and collagenase VI which is equal to the volume of fat, and digest evenly at a temperature of 37°C for 30-40 minutes, and then add one-third of ...

Embodiment 2

[0030] Selection of Adipose Tissue Collection Sites

[0031]In this example, on the basis of Example 1, during the collection of human adipose tissue, the fat was obtained by tumescent liposuction on the thigh, back and abdomen of the donor respectively, and the collected fat tissue The appearance and the number of SVF cells were tested, and the experimental results are shown in Table 1.

[0032] Table 1 shows the effect of adipose tissue collection site on SVF cell recovery

[0033] experiment number collection site Adipose Tissue Profile SVF cell number / 10mL 1 thigh Golden color, uniform size of fat lumps 1.12E+06 2 lower back Golden color, uniform size of fat lumps 9.32E+05 3 abdomen Golden color, uniform size of fat lumps 8.42E+05

[0034] The experimental results showed that although the fats at the three sites were similar in shape, the recovery rate of SVF cells was significantly different. Among them, the thigh is the be...

Embodiment 3

[0036] Effect of Fat Pretreatment Method on SVF Cell Recovery

[0037] Different fat processing techniques will directly affect the content of stem cells in the raw material fat and the success rate of separation and purification. On the basis of Example 1, in this embodiment, in the process of collecting human adipose tissue in step S1, the obtained fat is separately processed through only Sedimentation separation, 300 μm filter hole filtration, 500 μm filter hole filtration and 800 μm filter hole filtration were used to filter, and the number of SVF cells and miscellaneous cells recovered by these four methods were tested. The experimental results are shown in Table 2.

[0038] Table 2 Effects of different fat pretreatment methods on SVF cell recovery

[0039]

[0040]

[0041] The experimental results prove that the number of SVF cells recovered by the four methods of sedimentation separation and filtration of 300 μm, 500 μm and 800 μm is different, and the number of ...

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Abstract

The invention discloses a preparation method of a novel human adipose-derived stem cell preparation. The preparation method comprises the following steps of: S1, performing medical examination on a donor, and then under the state of local anaesthesia, acquiring adipose by swelling method liposuction; S2, respectively performing digestion treatment on the obtained adipose in 0.2%-0.4% of mixed collagenase and 0.25% of Trypsin-EDTA digestive fluid, adding a trypsin inhibitor, performing uniform mixing, ending digestion, then performing centrifuging, cleaning bottom cells with PBS, and performingfiltration so as to obtain primary substitute adipose stem cells; S3, culturing the primary substitute adipose stem cells to obtain massive adipose stem cells; and S4, performing detection and frozenstorage. The adipose stem cells finally prepared by the preparation method are high in survival rate, uniform in cellular morphology size and safe and reliable to use, and the possibility of allergyand infection with exogenous pathogeny in the use process is eliminated.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method of a novel human-derived adipose stem cell preparation. Background technique [0002] Stem cells are cells with self-replication ability and multi-directional differentiation potential, which can differentiate into various human cells such as fat, cartilage, osteoblast, and myoblast. The research on autologous stem cell therapy has always focused on bone marrow stem cells, but bone marrow stem cells are traumatized, the cell proliferation is weak, the number of cells in the preparation is small, and the number of treatments is limited. Adipose-derived stem cells (ADSCs) have the advantages of abundant raw materials, convenient material acquisition, good cell proliferation, autologous transplantation, and low immunogenicity. The scarcity of bone marrow stem cells was discovered in 2001, and it was widely used in the medical beauty industry soon after it w...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12Q1/02A01N1/02
CPCC12N5/0667G01N33/5005A01N1/02C12N2509/00
Inventor 韩瑛璐唐健朱旭石
Owner 成熙(上海)生物科技有限公司
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