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A kind of inducing differentiation medium and method for inducing fat stem cells to differentiate into chondrocytes

A technique for inducing differentiation of adipose stem cells, which is applied in the field of inducing differentiation medium for inducing the differentiation of adipose stem cells into chondrocytes, can solve the problems of low expression efficiency of type II collagen, increase chondrocyte anabolism, and low differentiation rate, and achieve the promotion of cartilage Cell anabolism and chondrogenesis, low cost, improved expression effect

Active Publication Date: 2020-11-24
FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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  • Description
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AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a kind of inducing differentiation medium and method for inducing the differentiation of adipose stem cells to chondrocytes, in order to solve the problem of low differentiation rate, long cycle and expression efficiency of type II collagen after the differentiation of adipose stem cells to chondrocytes Low problem, through the scientific combination of TGF-β3, FGF-18, insulin, selenium, transferrin and sodium pyruvate and other components, FGF-18 can strongly promote TGF-β3 induced adipose-derived mesenchymal stem cells to Chondrocyte differentiation promotes the expression of Sox-9, a key gene for chondrocyte differentiation, increases chondrocyte anabolism, promotes chondrogenesis, components complement each other, synergize, and significantly increase cartilage extracellular matrix type II collagen and glycosaminoglycans expression, and reduces the time required for in vitro induction culture

Method used

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  • A kind of inducing differentiation medium and method for inducing fat stem cells to differentiate into chondrocytes
  • A kind of inducing differentiation medium and method for inducing fat stem cells to differentiate into chondrocytes
  • A kind of inducing differentiation medium and method for inducing fat stem cells to differentiate into chondrocytes

Examples

Experimental program
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Effect test

Embodiment 1

[0023] 1.1 Primary culture of human adipose stem cells: Collect 20 mL of adipose tissue from patients undergoing surgery, wash it three times with PBS phosphate buffer, cut up the adipose tissue and transfer it to a 15 mL centrifuge tube, add an equal volume of 0.1 wt.% type Ⅰ collagenase, and keep at 37°C Digest in a water bath for 1.5 hours, centrifuge at 2000r / min for 5min; discard the supernatant, resuspend in PBS phosphate buffer, filter and suspend into a new centrifuge tube with a 200-mesh cell sieve, centrifuge at 2000r / min for 5min again; discard the supernatant, add 10% FBS (volume fraction), 1wt.% double antibody DMEM / F12 medium resuspended, the cells were vortexed and mixed, inoculated on a 10cm culture dish, and placed at 37°C, 5% CO 2 cultured in an incubator. Change the medium for the first time after 48 hours, and then change the medium every 1-2 days. When 80% confluent, digest with trypsin-EDTA. Observe under the microscope. When the cells appear in a spheric...

Embodiment 2

[0034] In this example, the induction effect at different periods was measured, and the P3 generation adipose-derived mesenchymal stem cells were obtained by referring to the primary culture method of human adipose-derived stem cells in Example 1, and the following Pellet culture system was further respectively used to induce and cultivate chondrocytes: collect the P3-generation adipose-derived mesenchymal stem cells Stem cells, resuspend and count, add 10×10 to each 15mL centrifuge tube 5 cells, a total of 48 centrifuge tubes were set up, and they were divided into four groups: the control group, the positive control group A, the positive control group B and the experimental group. Three parallel sets were centrifuged at 300 g for 5 min, the supernatant was discarded, and each group was processed as follows.

[0035] Each of the experimental groups was added with 2 mL of the induction differentiation medium of the present invention, the induction differentiation medium was fo...

Embodiment 3

[0042] In this example, a three-dimensional cell hydrogel scaffold culture system, specifically a fibrin hydrogel scaffold three-dimensional culture system, is used to test the effects of the inducer, differentiation induction medium and induction method of the present invention. The fibrin hydrogel forming components used include liquid A and liquid B. Liquid A is a thrombin solution for resuspending cells. The preparation method is to dissolve thrombin in calcium chloride to obtain a solution with a concentration of 5 U / mL, namely It is liquid A; liquid B is fibrinogen solution, the preparation method is that fibrinogen is dissolved in sodium chloride solution to make the concentration 100 mg / mL, and contains aprotinin 10000 KIU / mL, and liquid A and liquid B are mixed to form Fibrin hydrogel.

[0043] First, refer to the primary culture method of human adipose stem cells in Example 1 to obtain P3 generation adipose-derived mesenchymal stem cells, and perform in vitro culture...

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Abstract

The invention discloses an inducer and a method for inducing differentiation of adipose-derived stem cells to cartilage cells, belonging to the technical field of cell biology. According to the technical scheme, the inducer comprises TGF-Beta 3, FGF-18, dexamethasone, insulin, selenium, transferrin, linoleic acid, BSA, sodium pyruvate solution, L-ascorbic acid-2-phosphate and proline. The inducer and the method have the advantages that the synthesis and metabolism of the cartilage cells are rapidly promoted, the generation of cartilage is promoted, the expression of cartilage extracellular matrix II type collagen and glycosaminoglycan is remarkably improved, and the time required for in-vitro induction culture is reduced; and the inducer and the method are widely suitable for various culture systems, especially a pellet system and a three-dimensional cell scaffold culture system.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a differentiation medium and a method for inducing the differentiation of fat stem cells into chondrocytes. Background technique [0002] Articular cartilage is mainly composed of chondrocytes and extracellular matrix (ECM). It is a special form of connective tissue. Its function is to reduce the pressure on the subchondral bone during joint movement and reduce the friction of the articular surface. Since cartilage tissue has no blood supply, it can only rely on joint fluid to provide nutrition. Therefore, once cartilage tissue is damaged, its ability to regenerate and repair is extremely poor, and it is difficult to heal itself. There are many reasons for cartilage damage, such as external impact, autoimmune diseases such as rheumatoid arthritis, body degeneration caused by aging, etc., resulting in a high incidence of cartilage damage and high treatment costs, which is a m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2500/10C12N2500/12C12N2500/24C12N2500/30C12N2500/32C12N2501/119C12N2501/15C12N2501/33C12N2501/998C12N2506/1384C12N2513/00
Inventor 翁土军易玲娴张春丽黄亮节何莹侯树勋
Owner FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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