CN112680408A

CN112680408A - Culture medium for inducing human mesenchymal stem cells to form cartilage differentiation and preparation method thereof

Info

Publication number: CN112680408A
Application number: CN201911010808.7A
Authority: CN
Inventors: 李艳群; 李治寰; 刘冬羽
Original assignee: Dongguan Enlian Stem Cell Biotechnology Research Institute
Current assignee: Dongguan Xuanguan stem cell regenerative medicine Co.,Ltd.
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2021-04-20

Abstract

The invention provides a culture medium capable of inducing human mesenchymal stem cells to be chondrogenic differentiation, and belongs to the technical field of stem cell culture. The chondrogenic induction culture medium comprises the following components: low-sugar DMEM basal medium, fetal bovine serum, sodium pyruvate, ascorbic acid, proline, dexamethasone, transferrin, insulin, sodium selenite and transforming growth factor beta 3 (TGF-beta 3). The culture medium is used for inducing the mesenchymal stem cells to have higher chondrogenic differentiation success rate under a two-dimensional culture environment, and the differentiation time only needs 19 days.

Description

Culture medium for inducing human mesenchymal stem cells to form cartilage differentiation and preparation method thereof

Technical Field

The invention relates to an induction culture medium for directional differentiation of human mesenchymal stem cells into cartilage.

Background

Mesenchymal stem cells are a class of cells that are self-replicating and have multi-differentiation potential. In the human body, mesenchymal stem cells are present in various tissues such as umbilical cord, fat, bone marrow, dental pulp, placenta, and the like, and can be isolated and expanded in vitro for many generations. Under the culture of a differentiation culture medium, the mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, nerve, skin and the like. Therefore, mesenchymal stem cells have been widely used in clinical studies on functional damage repair of tissues and organs. For example, in a plurality of early clinical researches, researchers use adipose-derived mesenchymal stem cells to repair cartilage tissues, so that good curative effects are obtained, and the mesenchymal stem cells are shown to have great clinical transformation potential.

At present, the mesenchymal stem cell chondrogenesis induction culture medium usually adopts DMEM/F12 high-sugar culture medium as a basic culture medium. The chondroblasts obtained by the technology have limited differentiation efficiency, the differentiation time is as long as 28 days, the number of induced chondroblasts is small, the survival rate is low, and the requirements of research and clinical cartilage repair cannot be well met. In order to obtain sufficient chondrocytes differentiated from the mesenchymal stem cells to better meet clinical requirements, mesenchymal stem cell differentiation culture conditions need to be optimized, sufficient chondrocytes are obtained in a shorter time, and favorable conditions are provided for clinical application of the mesenchymal stem cells for cartilage injury treatment, cosmetology or biological activity identification of the mesenchymal stem cells.

Disclosure of Invention

In order to solve the problems of the prior art that the chondrogenesis induction efficiency and specificity are not ideal enough, the induction time is long and the like, the invention provides a culture medium capable of inducing mesenchymal stem cells to chondrogenesis differentiation within 19 days and a preparation method thereof.

A culture medium for inducing chondrogenic differentiation of human mesenchymal stem cells, comprising: the composition comprises low-sugar DMEM medium (LG-DMEM), Fetal Bovine Serum (FBS) 5-50% by volume, sodium pyruvate 50-100 μ g/ml, ascorbic acid 20-200 μ g/ml, proline 10-120ng/ml, dexamethasone 39-500ng/ml, insulin-transferrin-solarization additive (ITS) 0.5-1% by volume, and transforming growth factor-beta 3 (TGF-beta 3)0.1-20 ng/ml.

Preferably, the composition is prepared from the following components: LG-DMEM medium, FBS 10% volume percentage, sodium pyruvate 100 mug/ml concentration, ascorbic acid 50 mug/ml concentration, proline 40 mug/ml concentration, dexamethasone 39ng/ml, ITS 1% volume percentage, TGF-beta 310 ng/ml concentration.

A preferred ITS comprises 1.0mg/ml human recombinant insulin, 0.55mg/ml human transferrin and 0.5. mu.g/ml 100 Xconcentration of sodium selenite.

Preferred basal media contain one or more inorganic salts, vitamins, amino acids, antibiotics, and the like.

Preferably, the human mesenchymal stem cells are umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells from the second generation to the fifth generation.

A preparation method of a culture medium for inducing differentiation of human mesenchymal stem cells into cartilage comprises the following steps:

1) sterilizing a culture bottle: washing the culture bottle, washing with deionized water, air drying, wrapping with paper or cloth, or placing in a metal box, sterilizing at 121 deg.C for 30min, and drying;

2) DMEM medium addition: adding LG-DMEM medium into a culture bottle under aseptic conditions;

3) mixing the components: adding the FBS, the sodium pyruvate mother liquor, the ascorbic acid mother liquor, the proline mother liquor, the dexamethasone mother liquor, the ITS mother liquor and the TGF-beta 3 mother liquor with the concentration, and uniformly mixing;

4) and (3) filtering and sterilizing: the mixed medium was sterilized by filtration through a 0.22 μm filter.

The human mesenchymal stem cell chondrogenic induction differentiation is carried out by the following method:

1) obtaining the stem cells: culturing and amplifying the human mesenchymal stem cells by using LG-DMEM medium containing 10% FBS;

2) pretreatment: digesting the cells with pancreatin, resuspending the cells in LG-DMEM medium containing 10% FBS;

3) cell inoculation: 5 μ L of the extract containing 5X 10⁵Dropping the cell suspension of each cell on a twenty-four pore plate, wherein one pore is one drop;

4) differentiation culture: the cells were placed in an incubator for 2 hours, and 1ml of the human mesenchymal stem cells were added to each well, and cultured in a chondrogenic differentiation induction medium for 19 days.

Drawings

FIG. 1 is a staining chart of Alisin blue of a chondrocyte pellet section formed 19 days after chondrogenic induction of human umbilical cord mesenchymal stem cells

FIG. 2 is a staining diagram of Alisin blue of cartilage globules formed 19 days after the human adipose-derived mesenchymal stem cells are induced into cartilage

Detailed Description

The following will further describe the present invention with reference to specific embodiments.

Example 1: chondrogenic differentiation of human umbilical cord mesenchymal stem cells

The patent provides a preparation of chondrogenic induction culture medium:

1) the human umbilical cord mesenchymal stem cells of the generation P2 are arranged according to the proportion of 1 x 10⁵The density of the cells seeded was 5. mu.l in the center of a 24-well plate and cultured for 2 hours.

2) Cartilage induction medium was prepared according to the composition and amounts shown in table 1:

TABLE 1 chondrogenic differentiation-inducing medium

Component (A)	Dosage of
		LG-DMEM	50ml
FBS	10％
		TGF-β3	10ng/ml
ITS	1％
		Dexamethasone	39ng/ml
Ascorbic acid	50μg/ml
		Pyruvic acid sodium salt	100μg/ml
Proline	40μg/ml

3) After 2 hours, 1ml of chondrogenic differentiation induction medium prepared according to the method in Table 1 was added, and then the fresh chondrogenic differentiation induction medium was replaced every two days, and chondrocytes were obtained after induction for 19 days.

4) The differentiation degree of chondrogenic tissue was identified by using aliskiren staining solution, and the results are shown in fig. 1.

Example 2: chondrogenic differentiation of human adipose-derived mesenchymal stem cells

1) The human adipose-derived mesenchymal stem cells of generation P2 are arranged according to the ratio of 1 × 10⁵The density of the cells seeded was 5. mu.l in the center of a 24-well plate and cultured for 2 hours.

2) Cartilage induction medium was prepared according to the composition and amounts shown in table 2:

TABLE 2 chondrogenic differentiation induction medium

3) After 2 hours, 1ml of chondrogenic differentiation induction medium prepared according to the method in table 2 was added, and then the fresh chondrogenic differentiation induction medium was replaced every two days, and chondrocytes were obtained after induction for 19 days.

4) The differentiation degree of chondrogenic tissue was identified by using aliskiren staining solution, and the results are shown in fig. 2.

Claims

1. A medium for inducing chondrogenic differentiation of mesenchymal stem cells, comprising: is prepared from the following components, including low sugar DMEM culture medium, Fetal Bovine Serum (FBS) 5-50% volume percentage, sodium pyruvate 50-100 mug/ml concentration, ascorbic acid 20-200 mug/ml concentration, proline 10-120ng/ml concentration, dexamethasone 39-500ng/ml concentration, insulin-transferrin-solarization additive (ITS) 0.5-1% volume percentage, TGF-beta 30.1-20 ng/ml concentration.

2. The human mesenchymal stem cell chondrogenic induction differentiation medium according to claim 1, characterized by being prepared from a low sugar DMEM medium, FBS 10% by volume, sodium pyruvate at a concentration of 100 μ g/ml, ascorbic acid at a concentration of 50 μ g/ml, proline at a concentration of 40ng/ml, dexamethasone at 39ng/ml, ITS 1% by volume, TGF- β 310 ng/ml.

3. The method for preparing a culture medium for inducing differentiation of human mesenchymal stem cells into cartilage according to claim 1, comprising the steps of,

1) sterilizing the culture bottle, washing the culture bottle with deionized water, air drying, wrapping with paper or cloth, or placing in a metal box, sterilizing at 121 deg.C for 30min, and oven drying;

2) adding a DMEM medium, and adding the low-sugar DMEM medium into a culture bottle under an aseptic condition;

3) mixing the components, adding the FBS, the sodium pyruvate mother liquor, the ascorbic acid mother liquor, the proline mother liquor, the dexamethasone mother liquor, the ITS mother liquor and the TGF-beta 3 mother liquor with the concentration, and uniformly mixing;

4) the mixed culture medium was sterilized by filtration through a 0.22 μm filter.

4. The method for preparing the culture medium for inducing differentiation of human mesenchymal stem cells into cartilage according to claim 1, wherein the differentiation of human mesenchymal stem cells into cartilage is induced by the following method,

1) obtaining the stem cells: culturing and amplifying the human mesenchymal stem cells by using a low-sugar DMEM medium containing 10% FBS;

2) pretreatment: digesting the cells with pancreatin, centrifuging at 1200rpm to obtain cell pellets, and suspending the cells in low-sugar DMEM medium containing 10% FBS;

3) cell inoculation: 5 μ L of the extract containing 5X 10⁵Dropping the cell suspension of each cell on a twenty-four pore plate, one drop and one pore;

4) differentiation culture: the cells are placed in an incubator for 2 hours, 1ml of the human mesenchymal stem cell culture medium is added into each hole, and chondrogenic differentiation induction culture medium culture is carried out for 19 days.

5. The medium for inducing the differentiation of mesenchymal stem cells into cartilage according to claim 1, wherein the human mesenchymal stem cells are derived from umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells.

6. The method of claim 5, wherein the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells for second to fifth generations.

7. The medium for inducing differentiation of mesenchymal stem cells into cartilage according to claim 1, wherein the ITS is a mixture of human recombinant insulin, human transferrin and sodium selenite, and comprises 1.0mg/ml human recombinant insulin, 0.55mg/ml human transferrin and 0.5 μ g/ml 100 Xconcentration of sodium selenite.

8. The method of chondrogenic induced differentiation of human mesenchymal stem cells according to claim 4, wherein the mesenchymal stem cells are cultured in an environment having a carbon dioxide content of 5% and 37 ℃.