CN111979185A - Mesenchymal stem cell adipogenic differentiation medium and application thereof - Google Patents

Abstract

The invention relates to a mesenchymal stem cell adipogenic differentiation culture medium and application thereof, which is characterized by comprising a basic culture medium and additive components, wherein the culture medium comprises optimally designed components such as various inorganic salts, amino acids, vitamins and the like, and components such as recombinant human serum albumin, sodium carboxymethylcellulose, 3-isobutyl-1-methylxanthine, recombinant human insulin, dexamethasone, pioglitazone hydrochloride, palmitic acid, oleic acid, linoleic acid, cholesterol and the like. According to the invention, through optimally designing the addition and the proportion of each component, the capability of the culture medium for inducing the mesenchymal stem cells to differentiate into the adipocytes is obviously improved, and the differentiation time is further shortened.

Description

A kind of mesenchymal stem cell adipogenic differentiation medium and its application

technical field

The invention belongs to the technical field of cell culture, and in particular relates to a mesenchymal stem cell adipogenic differentiation medium and use thereof.

Background technique

Mesenchymal stem cells (MSCs), originating from the mesoderm, are a kind of pluripotent stem cells that mainly exist in connective tissues and organ interstitium. It is the ability of various cells such as fat cells, bone cells, cartilage cells, and nerve cells.

Mesenchymal stem cells are rich in sources and widely exist in bone marrow, fat, umbilical cord and other tissues. They have convenient sources and are easy to separate, culture, expand and purify. After multiple passages and expansions, they still have stem cell characteristics and have low immunogenicity. Allograft rejection is relatively mild, and the matching requirements are not strict; it has an immunoregulatory function, and inhibits the proliferation and immune response of T cells through the interaction between cells and the production of cytokines, thereby exerting the function of immune reconstruction; it is precisely because of these The characteristics of mesenchymal stem cells make mesenchymal stem cells provide an ideal biological platform for scientific research and clinical applications, such as cell therapy, regenerative medicine, drug screening, establishment of gene therapy vectors, and research on molecular regulation mechanisms of cell development and differentiation , tissue engineering seed cells, etc.

Whether in scientific research or clinical application, the identification of mesenchymal stem cells has always been a problem that needs to be faced. Due to the particularity of clinical applications, the identification of mesenchymal stem cells used for cell therapy has received more and more attention, and relevant national departments have also issued relevant documents, "Administrative Measures for Stem Cell Clinical Research (Trial)" (National Health Science and Education Development [ 2015] No. 48) and "Guidelines for Quality Control and Preclinical Research of Stem Cell Preparations (Trial)" clearly stated that the differentiation potential of stem cells, the structure and physiological function of induced differentiated cells, the ability to regulate immune cells, and the secretion of specific cytokines can be clearly identified. , expression of specific genes and or proteins, etc., to judge the biological effectiveness of stem cell preparations and treatment. For mesenchymal stem cells, regardless of the source, the differentiation ability of various types of cells (such as adipocytes, chondroblasts, osteoblasts, etc.) should be tested in vitro to determine the pluripotency of cell differentiation.

At present, there are also commercial mediums with good effects, such as the adipogenic differentiation medium product of STEMCELL (STEMCELL, 05412), which has excellent differentiation effect and can achieve adipogenic differentiation in some mesenchymal stem cells for 7-14 days. Base costs are high.

At present, there are also patent applications of the same type, which usually use substances such as fetal bovine serum, insulin, 3-isobutyl-1-methylxanthine, glucocorticoids and thiazolidinediones to synergize to induce adipogenic differentiation, although these components also It can induce adipogenic differentiation of mesenchymal stem cells, but its differentiation efficiency is low, it takes a long time, and it has the defect of insufficient efficiency.

SUMMARY OF THE INVENTION

Aiming at the deficiencies in the prior art, the present invention designs and develops a new medium for mesenchymal stem cells.

The first object of the present invention is to provide a mesenchymal stem cell adipogenic differentiation medium for the shortcomings of low differentiation efficiency and long differentiation time of the existing adipogenic differentiation medium, which can induce mesenchymal stem cells in a shorter time. Differentiation into adipocytes.

The second object of the present invention is to provide the use of a mesenchymal stem cell adipogenic differentiation medium.

In order to realize the above-mentioned first purpose, the technical scheme adopted in the present invention is:

The basal medium and supplementary components are composed of inorganic salt components, amino acid components, vitamin components, nucleotide components, trace element components and carbohydrates, and the supplementary components are composed of recombinant human serum albumin , Sodium carboxymethylcellulose, 3-isobutyl-1-methylxanthine, recombinant human insulin, dexamethasone, pioglitazone hydrochloride, palmitic acid, oleic acid, linoleic acid, cholesterol.

Preferably, the added components are calculated by volume, and the content of each component is: recombinant human serum albumin 100g/L, sodium carboxymethyl cellulose 100g/L, 3-isobutyl-1-methylxanthine 50mM, recombinant Human insulin 500uM, dexamethasone 50uM, pioglitazone hydrochloride 1mM, palmitic acid 3mM, oleic acid 1mM, linoleic acid 3mM, cholesterol 1mM;

The volume ratio of the basal medium to the added components is 100:0.1 to 100:3.

Preferably, the volume ratio of basal medium to supplementary components is 100:1.

Preferably, the basal medium is composed of the following components:

In order to realize the above-mentioned second purpose, the technical scheme adopted in the present invention is:

The adipogenic differentiation medium of mesenchymal stem cells is used for inducing adipogenic differentiation of mesenchymal stem cells derived from fat, bone marrow, umbilical cord, skeletal muscle and the like in vitro.

Due to the application of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

The invention is more suitable for the culture of mesenchymal stem cells through the unique design of the basic medium formula;

The medium of the present invention is added with components such as recombinant human albumin and sodium carboxymethyl cellulose, does not contain human-derived extract material components, does not contain animal-derived components, has clear components, and does not require HIV-1/2 ) antibody, hepatitis B surface antigen (HBsAg) antibody and hepatitis C virus (HCV) detection, at the same time, it can exclude the risk of zoonotic disease, it is safer and more convenient to use, reduces the need for quality control, and the application of downstream cell induction and differentiation is safer.

The culture medium of the present invention is supplemented with various combinations of fatty acids and cooperated with various cytokines 3-isobutyl-1-methylxanthine, recombinant human insulin growth factor, dexamethasone, and pioglitazone hydrochloride to synergize, and can Rapidly induce the differentiation of mesenchymal stem cells into mature adipocytes in vitro, the differentiation efficiency is higher, and the formation of differentiated cells can be detected in a shorter time.

Description of drawings

Figure 1: The results of oil red 0 staining on the 12th day of in vitro induction of adipogenic differentiation of human adipose-derived mesenchymal stem cells (P6) by mesenchymal stem cell adipogenic differentiation medium in Example 2.

Figure 2: The results of oil red 0 staining on the 12th day of in vitro induction of adipogenic differentiation of human adipose-derived mesenchymal stem cells (P6) by the mesenchymal stem cell adipogenic differentiation medium described in M1-M5 in Example 3.

Detailed ways

As introduced in the background art, adipogenic differentiation ability is the basic ability of mesenchymal stem cells, and how to quickly and efficiently induce mesenchymal stem cells to differentiate into mature adipocytes in vitro is an important part of the research and application of mesenchymal stem cells. ring. In order to improve the speed and efficiency of mesenchymal stem cell adipogenic differentiation, it is necessary to develop a mesenchymal stem cell adipogenic differentiation-free medium. The invention achieves rapid and efficient in vitro induction of mesenchymal stem cells to differentiate into mature adipocytes by adding adipogenic differentiation inducing factors, serum, fatty acids and other components into the basal medium.

Example 1

The mesenchymal stem cell adipogenic differentiation medium provided in this example consists of the following components: basal medium and supplementary components.

(1) The basal medium is marked as Basal, and is prepared according to the following scheme:

The above-mentioned various components can be purchased from Sigma, Aladdin and other reagent companies. Each component is dissolved in 1 L of water for injection, and filtered with a 0.22-micron membrane to obtain the basic medium Basal.

(2) The added components are recorded as SUP1 components, and the composition of SUP1 components is as follows:

In this example, 3-isobutyl-1-methylxanthine, dexamethasone, and pioglitazone hydrochloride are soluble in DMSO, while palmitic acid, oleic acid, linoleic acid, and cholesterol are soluble in absolute ethanol , recombinant human serum albumin, sodium carboxymethyl cellulose can be dissolved in water for injection, and recombinant insulin can be dissolved in 0.3M hydrochloric acid solution. That is, the SUP1 solution is obtained.

In this example, the mesenchymal stem cell adipogenic differentiation medium was prepared with Basal and SUP1 in a volume ratio of 1000mL:10mL.

Comparative Example 1

It is a commercial adipogenic differentiation medium (STEMCELL, 05412).

Comparative Example 2

As a negative control, it is a conventional culture group, and the culture medium used is DMEM/F12 medium (Sigma, D2906) supplemented with 10% FBS (ExCell Bio, FND500).

Example 2

This example provides verification of the effect of the mesenchymal stem cell adipogenic differentiation medium of Example 1. This example sets up one experimental group (Example 1) and two control groups (Comparative Example 1, Comparative Example 2). Human adipose-derived mesenchymal stem cells (AD-MSCs) were used in the experiments in this example, and the cells were derived from the ATCC standard cell bank (PCS-500-011, Lot 80622175, ATCC), and the cells were used in all experiments described below.

1. Experimental method

1. Recovery of AD-MSCs

Take 1 AD-MSCs at passage P6 from the cell working bank, and dissolve them quickly in a 37°C water bath. The cells were slowly added to 10 mL of mesenchymal stem cell medium (ExCell Bio, ME000-N023), pipetted evenly, counted, and centrifuged at 300 g for 5 minutes. Resuspend the cells with an appropriate amount of mesenchymal stem cell medium (ExCell Bio, ME000-N023), and inoculate the cells in a 100mm culture dish at a density of 8000/cm ² , shake well and place at 37°C, 5% CO ₂ , saturated humidity cultivated in the environment.

2. Subculture of AD-MSCs

When the AD-MSCs reached 80-90% confluence in the petri dish, use a pipette to remove the old medium in the petri dish. 5 mL of DPBS was added to wash the cells, the DPBS was discarded, and 0.5% trypsin-0.4% EDTA solution was added to digest the cells. When the cells contracted and became round, the 0.5% trypsin-0.4% EDTA solution was immediately discarded, and 5 mL of mesenchymal stem cell medium was added. Resuspend, collect cells, count, and centrifuge at 300g for 5 minutes. The supernatant was discarded, 5 mL of mesenchymal stem cell medium was added, and the cells were re-seeded to a 12-well plate at 20,000/cm ² , and the cells were cultured for 24 hours to induce differentiation.

3. Adipogenic differentiation of AD-MSCs

1) Culture medium

Induction and differentiation of mesenchymal stem cells into adipocytes were performed using the medium described in Example 1, Comparative Example 1, and Comparative Example 2.

2) Adipogenic induction and differentiation method

20000/cm ² of passaged cells were re-seeded into 12-well plates, and cells were cultured for 24 h to induce differentiation. The culture medium described in Example 1, Comparative Example 1, and Comparative Example 2 was added to each well to induce differentiation, and fresh cells were replaced every 2 days. Medium, oil red O staining was performed 5-12 days after induction of differentiation.

3) Oil red O staining

After the differentiation, aspirate the differentiation medium from the 12-well cell culture plate, add 1 mL of DPBS, gently rinse the bottom of the well plate, and aspirate the DPBS; add 1 mL of 4% formaldehyde solution in a fume hood, and shake the culture plate gently , make the bottom surface evenly infiltrated, and let stand at room temperature for 30 minutes; discard the fixative, add 1 mL of DPBS, rinse the cells twice, add 500uL of 0.3% Oil Red O staining solution (Sigma, O1391) for 15 minutes at room temperature for staining; Add 1 mL of DPBS to rinse 3 times, observe and collect pictures under an inverted optical microscope with a 20x objective lens, and use CSI equipment to calculate the abundance of fat droplets.

The results show that: the time for the appearance of fat droplets in the cells of each culture group is shown in Table 1 below, the time for the appearance of fat droplets in the differentiation medium of Example 1 is earlier, and the differentiation effect of cells in the medium of Example 1 is better for 12 days of differentiation (as shown in the figure). 1), the fat droplet abundance values were higher (as shown in Table 1).

Table 1Example 2 Mesenchymal Stem Cell Differentiation Detection

Example 3

This example provides a test of the effect of different ratios. The basal medium (Basal) and the supplementary components (SUP1) are prepared according to the scheme of Example 1 of this example, and the basal medium and the supplementary components are prepared according to different ratios of mesenchymal stem cells. Adipogenic differentiation medium was prepared, and the effect of adipogenic induction and differentiation of each proportioning medium was tested. Oil red O staining was performed 12 days after induction of differentiation. The human adipose-derived mesenchymal stem cells and the culture method used in this example are the same as those in Example 2.

1) Culture medium

2) Adipogenic induction and differentiation method

20000/cm ² of passaged cells were re-seeded into 12-well plates, and cells were cultured for 24 h to induce differentiation. The culture medium described in Example 1, Comparative Example 1, and Comparative Example 2 was added to each well to induce differentiation, and fresh cells were replaced every 2 days. Medium, oil red O staining was performed 5-12 days after induction of differentiation.

3) Oil red O staining

Aspirate the differentiation medium from the 12-well cell culture plate, add 1 mL of DPBS, gently rinse the bottom of the well plate, and aspirate the DPBS; in a fume hood, add 1 mL of 4% formaldehyde solution, shake the culture plate gently to infiltrate the bottom surface Evenly, let stand at room temperature for 30 minutes; discard the fixative, add 1 mL of DPBS, rinse the cells twice, add 500uL of 0.3% Oil Red O staining solution (Sigma, O1391) for 15 minutes at room temperature for staining; remove the staining solution, add 1 mL Rinse with DPBS three times, observe and collect pictures under an inverted optical microscope with a 10x objective lens, and use CSI equipment to calculate the abundance of fat droplets.

As shown in Table 2 and Figure 2 below, the results show that: the ratio of M3 medium Basal: SUP1, the differentiation medium prepared in the ratio of 100:1 has the best differentiation effect; M1, M2, M4 medium Basal: SUP1 mix The ratio of medium can also induce mesenchymal stem cells to differentiate into adipocytes, and the differentiation effect of M2 is better than M3, and M3 is better than M1; M5 medium, the ratio of Basal:SUP1 is 100:5, which has cytotoxicity. Cells are lethal and cell differentiation cannot be achieved.

Table 2Example 3 Mesenchymal Stem Cell Differentiation Detection

serial number Basal: SUP1 Abundance of fat droplets at day 12 of differentiation M1 100:0.1 8 M2 100: 0.5 twenty two M3 100:1 33 M4 100:3 15 M5 100:5 apoptosis

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention, All should be included within the protection scope of the present invention.

Claims (6)

1. a mesenchymal stem cell adipogenic differentiation medium, is characterized in that, is made up of basal medium and added component, and described basal medium is composed of inorganic salt component, amino acid component, vitamin component, nucleotide Components, trace element components and carbohydrates; the added components are composed of recombinant human serum albumin, sodium carboxymethyl cellulose, 3-isobutyl-1-methylxanthine, recombinant human insulin, dexamethasone Metasone, pioglitazone hydrochloride, palmitic acid, oleic acid, linoleic acid, cholesterol. 2. The mesenchymal stem cell adipogenic differentiation medium according to claim 1, wherein the added components are in volume, and the content of each component is: recombinant human serum albumin 100g/L, carboxymethyl Cellulose sodium 100g/L, 3-isobutyl-1-methylxanthine 50mM, recombinant human insulin 500uM, dexamethasone 50uM, pioglitazone hydrochloride 1mM, palmitic acid 3mM, oleic acid 1mM, sub Oleic acid 3mM, cholesterol 1mM; The volume ratio of the basic medium to the added components is 100:0.1-100:3. 3 . The mesenchymal stem cell adipogenic differentiation medium according to claim 2 , wherein the volume ratio of the basal medium to the added components is 100:1. 4 . 4. according to the arbitrary described mesenchymal stem cell adipogenic differentiation medium of claim 1-3, it is characterized in that, described basal medium, composition is as follows:

. 5. Use of the mesenchymal stem cell adipogenic differentiation medium of any one of claims 1-4 in the in vitro adipogenic differentiation of mesenchymal stem cells. 6 . The use of mesenchymal stem cells in adipogenic differentiation in vitro according to claim 5 , wherein the mesenchymal stem cells are derived from any tissue in fat, bone marrow, umbilical cord, and skeletal muscle. 7 .

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