Preparation and application of 3D dispersion culture agent for mesenchymal stem cells

Abstract

The invention relates to the field of biological materials, in particular to a 3D dispersion culture agent for mesenchymal stem cells, the 3D dispersion culture agent contains a microscopic network scaffold structure, and epidermal growth factors can promote proliferation of the mesenchymal stem cells. The 3D dispersion culture agent can be used for large-scale dispersion culture of the mesenchymal stem cells in a cell complete culture medium under physiological conditions, is suitable for in-vitro large-scale culture of the mesenchymal stem cells, and has wide application prospects and research values in the biological medicine fields of tissue engineering, regenerative medicine and the like.

Description

Technical field:

[0001] The present invention relates to the field of biomaterials, and in particular to a 3D dispersion culture medium for mesenchymal stem cells. Background technology:

[0002] Mesenchymal stem cells (MSCs) have received great attention for their use in cell-based therapies. MSCs are highly proliferative multipotent stromal cells that can differentiate into mesodermal cell lineages. Due to the therapeutic potential of MSCs, many studies have focused on their characterization and identification. Traditional two-dimensional (2D) cell culture has been the main method used in medical and biological research and drug development for decades due to its simplicity. However, the static and planar conditions of two-dimensional culture cannot simulate the physiological environment of cells, causing mesenchymal stem cells using this culture model to decrease in cell viability as they are cultured for multiple passages, making it difficult to achieve stable proliferation in vitro. It is unable to meet the requirements for cell scale-up production for clinical applications.

[0003] In view of the above difficulties, the stem cell 3D dispersed culture medium proposed in the present invention provides an effective solution. Summary of the invention:

[0004] The present invention designs a 3D dispersed culture medium for mesenchymal stem cells, which contains a polypeptide compound, preferably with epidermal growth factor added; the concentration of the polypeptide compound is between 0.5 mg / ml and 6 mg / ml, preferably 1 mg / ml to 3 mg / ml, and is optimal under neutral conditions of pH greater than 6. When there is a positive charge source substance in the environment, a stable hydrogel containing a 3D micro-nano scaffold structure is formed; the positive charge source substance contains a substance with a positively charged group, a metal cation, or a substance containing a positively charged group, such as a biomacromolecule or drug containing an electron-withdrawing group such as an amino group, a functional molecule, a positively charged ion / group source substance commonly found in the body or in a cell culture medium, etc. It can be considered that under in vivo conditions, the polypeptide can respond to the cell culture medium or the positively charged ions / groups commonly found in the body to form a hydrogel containing a cross-linked network structure. No other substances need to be added or environmental conditions need to be changed. It is beneficial to cell culture, sustained drug release, and other applications that require physiological conditions.

[0005] One of the technical solutions provided by the present invention is that the concentration of the polypeptide compound is between 0.5mg / ml and 6mg / ml, and the concentration of the epidermal growth factor is 5ng / ml-60ng / ml, preferably 10ng / ml to 30ng / ml. In the presence of a positive charge source substance, a pH greater than 6 or under neutral conditions can form a dispersed culture agent that is stable in the macroscopic state, forms nano-scale fibers in the microscopic state, and presents a three-dimensional network structure, which can effectively support cells for 3D culture.

[0006] The self-assembly behavior of polypeptide compounds is mainly driven by weak interaction forces such as hydrophobic interaction, ionic bond, van der Waals force, hydrogen bond, etc. Through the aforementioned driving forces, the amino acid chain is folded to form a stable and effective secondary structure. Therefore, by increasing the concentration of polypeptides, the number of amino acid chains acting on the self-assembly behavior increases, and the stable structure formed is also improved accordingly, which is manifested in the formation of a stable solution hydrogel system on a macroscopic level.

[0007] The self-assembly behavior of polypeptide compounds is affected by the pH of the external environment. The polypeptide chain contains multiple ionizable amino acids, including but not limited to RGD sequences with specific biological activity. The charge characteristics of these amino acids are affected by the pH value in the external environment, and corresponding ionization occurs as the pH changes. When the ionizable amino acids are ionized, the electrostatic interactions between the polypeptide chains will change, leading to significant changes in the conformation and folding state of the polypeptide chains. These intermolecular interactions and conformational adjustments at the microscopic level reflect at the macroscopic level that the dispersion culture agent of the present invention can form stable nano-scale fibers and present a three-dimensional network structure under a neutral pH environment.

[0008] The second technical solution provided by the present invention is a method for preparing the above-mentioned mesenchymal stem cell 3D dispersed culture agent, wherein the culture agent dry powder is mixed with a positive charge source material solution, fully dissolved, and the pH is adjusted to 6, and a stable polypeptide hydrogel is formed by standing at room temperature. The preferred concentration range is between 0.5 mg / ml and 6 mg / ml.

[0009] The third technical solution provided by the present invention is a method for preparing a 3D dispersed culture medium of mesenchymal stem cells containing epidermal growth factor at different concentrations by adding epidermal growth factor to the above polypeptide: the epidermal growth factor is dissolved in a complete cell culture medium to prepare a complete culture medium solution containing growth factor at concentrations of 10 ng / ml, 20 ng / ml, and 30 ng / ml, respectively. The dispersed culture medium dry powder is mixed with the above culture medium, fully dissolved, and allowed to stand at room temperature to form a stable hydrogel containing human recombinant epidermal growth factor polypeptide at different concentrations. The preferred concentration is 6 mg / ml.

[0010] The fourth technical solution provided by the present invention is the application of the above-mentioned mesenchymal stem cell 3D dispersed culture agent in the field of biomedicine, especially as a synthetic extracellular matrix, 3D suspended culture cells, and the role of promoting cell proliferation. Description of the drawings:

[0011] Figure 1 Complete cell culture medium is used to prepare polypeptide hydrogel;

[0012] Figure 2 Scanning electron microscope image of mesenchymal stem cell 3D dispersed culture medium;

[0013] Figure 3Biocompatibility of 3D dispersed culture medium of mesenchymal stem cells;

[0014] Figure 4 Mesenchymal stem cells 3D dispersed culture medium with multiple cell densities (A) Cell viability at multiple cell densities; (B) Inverted microscope image of multiple cell density culture;

[0015] Figure 5 Macroscopic characterization of 3D dispersed culture medium containing epidermal growth factor for mesenchymal stem cells (A) Growth factor concentration 10 ng / ml, polypeptide concentration 6 mg / ml; (B) Growth factor concentration 20 ng / ml, polypeptide concentration 6 mg / ml; (C) Growth factor concentration 30 ng / ml, polypeptide concentration 6 mg / ml;

[0016] Figure 6 Cell culture of 3D dispersed culture medium containing epidermal growth factor-containing mesenchymal stem cells (A) Cell proliferation capacity cultured in hydrogels with different growth factor peptides (B) Inverted microscope image of cell culture; Specific implementation method:

[0017] The present invention is further described below in conjunction with specific examples, which are provided only in an illustrative manner and do not limit the scope of protection of the present invention. The following examples are specifically carried out using the polypeptide sequence of SEQ ID 1.

[0018] Based on the problems in the background technology, the main component of the dispersion culture agent of the present invention is polypeptide, which is a biologically active substance involved in various cell functions in the body. It is a class of compounds between amino acids and proteins, which are formed by combining multiple amino acids in a certain arrangement order through peptide bonds and have good biocompatibility. In addition, since the amino acid residues on the polypeptide chain segment have different chemical structures, the polypeptide can effectively realize molecular self-assembly by utilizing the hydrogen bonding between its peptide bonds and the hydrogen bonding between amino acid residues, electrostatic action, hydrophobic action and π-π stacking action, forming nano-scale fibers and a three-dimensional network structure.

[0019] In addition, epidermal growth factor is added to the dispersed culture medium of the present invention. This is a multifunctional cell growth factor that exerts its physiological effects by binding to the epidermal growth factor receptor on the cell membrane, prompting a series of complex biochemical cascade reactions inside the cell, increasing the synthesis of RNA, DNA and protein, and ultimately promoting cell growth and reproduction and accelerating cell metabolism.

[0020] The microscopic three-dimensional network structure is composed of a mesenchymal stem cell 3D culture dispersion culture agent. Due to its high water content and porous structure, it simulates the natural extracellular matrix (ECM) environment. Therefore, in in vitro culture, the three-dimensional network structure can support the cells in the solution so that they are evenly dispersed and three-dimensionally distributed in the culture medium, achieving the effect of 3D cell culture. This culture mode is closer to the environment in which cells grow in the body. Compared with traditional 2D culture, it is more conducive to cell nutrition, waste absorption and discharge, as well as cell-to-cell interaction and biological signal transmission, which is conducive to the stable life activities of cells in vitro. The addition of epidermal growth factor can effectively promote the proliferation ability of mesenchymal stem cells, thereby realizing the use of a 3D culture mode and stable and efficient in vitro expansion and culture of mesenchymal stem cells.

[0021] The present invention is explained in detail below by way of examples.

[0022] Example 1 Complete cell culture medium for preparing mesenchymal stem cell 3D dispersion culture medium

[0023] Experimental method: Dissolve 6 mg of peptide dry powder in 1 ml of complete cell culture medium to prepare a stock solution with a concentration of 6 mg / ml, add it to a 1.5 ml EP tube, vortex thoroughly, and let it stand for five minutes.

[0024] Experimental results: Stable transparent hydrogel was obtained, and the peptide concentration was 6 mg / ml. Figure 1 )

[0025] Example 2 Scanning electron microscope image of mesenchymal stem cell 3D dispersion culture medium

[0026] Experimental method: Seq1 was used as an example in this experiment. According to the method in Example 1, 1 ml of the culture medium with a polypeptide concentration of 6 mg / ml was prepared, frozen in liquid nitrogen for 15 minutes, and freeze-dried using a vacuum freeze dryer to prepare a powder sample, and its microstructure was observed using a scanning electron microscope (JSM-IT300LV, Japan Electronics Co., Ltd.).

[0027] Experimental results: Scanning electron microscope microscopic images show that polypeptide fibers are intertwined and intersected into a sheet-like structure, and the sheet-like structure is further combined to form a three-dimensional network structure composed of nano-scale fibers, proving that the dispersed culture agent has a microstructure that can stably support cells for 3D culture. ( Figure 2 )

[0028] Example 3 Biocompatibility of 3D Dispersed Culture Medium for Mesenchymal Stem Cells

[0029] Experimental method: Seq1 was used as an example in this experiment. According to the method in Example 1, 6 mg / ml of dispersion culture medium was prepared, and mouse bone marrow mesenchymal stem cells (BMSCs) were 3D cultured in a 96-well plate without TC treatment. The cell seeding density was 5×103 cells / ml. On the 1st, 4th, and 7th days after inoculation, 10ul of CCK-8 solution was added to the experimental group every day, mixed and incubated in a cell culture incubator for 30 minutes. The microplate reader was turned on 30 minutes before the experiment, and the absorbance of the experimental group containing CCK-8 was measured at 450nm using the cell microplate reader. The data was processed to calculate the relative cell viability and determine the biocompatibility of the culture agent.

[0030] Experimental results: It is generally believed that if the relative cell viability is higher than 70%, the biomaterial is considered to be non-cytotoxic and biocompatible. From the overall results of CCK-8, it can be seen that the cell survival rate in the 7-day culture period is higher than 70%, and the relative cell viability increases with the increase of culture time. Therefore, it can be proved that the culture agent has good biocompatibility and can promote cell growth. Figure 3 )

[0031] Example 4: Multi-cell density culture of mesenchymal stem cells in 3D dispersed culture medium

[0032] Experimental method: Seq2 was used as an example in this experiment. According to the method in Example 1, 6 mg / ml of dispersion medium was prepared, and mouse bone marrow mesenchymal stem cells (BMSCs) were 3D cultured in a 96-well plate without TC treatment. The cell seeding density was 5×10 5 cells / ml,1x10 6 cells / ml,2x10 6 cells / ml,3x10 6 cells / ml; on the 1st, 4th, and 7th days after inoculation, add 10ul of CCK-8 solution to the experimental group every day, mix well, and incubate in a cell culture incubator for 30 minutes. Turn on the microplate reader 30 minutes before the experiment, use the cell microplate reader to measure the absorbance of the experimental group containing CCK-8 at 450nm, process the data, calculate the relative viability of cells at different cell concentrations, compare them, and determine the optimal culture cell concentration.

[0033] Experimental results: The relative viability of cells cultured at different cell concentrations for 7 days can be obtained from the overall CCK-8 results. Figure 4 A) It can be clearly seen that during the 7-day culture period, the cell density was 2x10 6 The experimental group with cells / ml had the highest relative cell viability, which was significantly higher than that of other cell concentrations. Combined with the inverted microscope images ( Figure 4 B), can be considered as 2x10 6 cells / ml is the optimal culture density.

[0034] Example 5 Preparation of 3D Dispersed Culture Medium Containing Epidermal Growth Factor and Mesenchymal Stem Cells

[0035] Experimental method: Seq2 was used as an example in this experiment. Epidermal growth factor (purchased from Shanghai Haohai Biotechnology Co., Ltd.) with a specification of 50,000 IU / bottle was dissolved in a complete cell culture medium gradient to prepare 10 ng / ml, 20 ng / ml, and 30 ng / ml growth factor-containing complete culture medium solutions, 1 ml each, and added to 1.5 ml EP tubes.

[0036] 6 mg of dry powder was added to the aforementioned EP tubes containing complete cell culture medium solutions with different concentrations of growth factors, vortexed thoroughly, and allowed to stand for five minutes. The mesenchymal stem cell 3D dispersed culture medium powder CulXII was synthesized and purified by Matrix (Tianjin) New Materials Technology Co., Ltd.

[0037] Experimental results: The dispersion culture medium containing growth factor concentrations of 10 ng / ml, 20 ng / ml, and 30 ng / ml and the polypeptide concentration of 6 mg / ml was obtained. Figure 5 )

[0038] Example 6 Cultivation of cells in 3D dispersed culture medium containing epidermal growth factor and mesenchymal stem cells

[0039] Experimental method: Seq4 was used as an example in this experiment. According to the methods of Example 1 and Example 5, 6 mg / ml of mesenchymal stem cell 3D dispersion culture medium and mesenchymal stem cell 3D dispersion culture medium containing growth factor concentrations of 10 ng / ml, 20 ng / ml, 30 ng / ml, and peptide concentration of 6 mg / ml were prepared respectively. Mouse bone marrow mesenchymal stem cells (BMSCs) were 3D cultured in a 96-well plate without TC treatment, and the cell seeding density was 2x10 6 cells / mL; on the 1st, 4th, and 7th days after inoculation, add 10uL of CCK-8 solution to the experimental group every day, mix well, and incubate in a cell culture incubator for 30 minutes. Turn on the microplate reader 30 minutes before the experiment, use the cell microplate reader to measure the absorbance of the experimental group containing CCK-8 at 450nm, process the data, calculate the cell proliferation capacity of different experimental groups, and compare them.

[0040] Experimental results: The relative viability of cells cultured at different cell concentrations for 7 days can be obtained from the overall CCK-8 results. Figure 5 A) It can be clearly seen that in the 7-day culture period, the cell proliferation ability was significantly improved by adding the culture medium containing epidermal growth factor, and the cell proliferation ability increased with the increase of growth factor concentration. The experimental group with a growth factor concentration of 30ng / ml had the highest cell proliferation ability, which could proliferate 2.4 times in 7 days. Combined with the inverted microscope image ( Figure 5B), it can be considered that the addition of growth factors improves the cell culture ability of the culture medium and effectively promotes cell proliferation, among which the epidermal growth factor concentration of 30ng / ml is the best.

[0041] Without departing from the inventive concept, several modifications and improvements may be made, all of which fall within the protection scope of the present invention.

Claims

1. A 3D dispersed culture medium for mesenchymal stem cells, characterized in that: It contains polypeptide compounds and epidermal growth factor. The polypeptide compounds are not chemically modified and can form nano-scale fibers and present a three-dimensional network structure in a complete cell culture medium to support 3D culture of cells. Preferably, the addition of epidermal growth factor can synergize with the polypeptide compounds to enhance the cell proliferation promoting effect.

2. The 3D dispersed culture medium according to claim 1, characterized in that: The polypeptide compound is selected from the following polypeptides: SEQ ID 1, IIIIIGSIIGPGGEGPGGV SEQ ID 2, FLIVIGSIIGPGGDGPGGV SEQ ID 3, FLIVIGSIIGPGGKGPGGV SEQ ID 4, FLIVIGSIIGPGGDGPGGD.

3. The 3D dispersed culture medium according to any one of claims 1 to 3, characterized in that: The final concentration of the polypeptide compound in the cell culture medium is 0.5 mg / ml to 6 mg / ml, preferably 1 mg / ml to 3 mg / ml; the final concentration of epidermal growth factor in the cell culture medium is 5 ng / ml-60 ng / ml, preferably 10 ng / ml to 30 ng / ml.

4. A method for preparing the 3D dispersed culture medium according to any one of claims 1 to 3, namely, preparing the dispersed culture medium by using a polypeptide compound dry powder, characterized in that 。 5. A preparation comprising the 3D dispersed culture agent according to any one of claims 1 to 4, characterized in that: The preparation is a solution preparation, a freeze-dried powder preparation, or a suspension preparation.

6. The 3D dispersed culture medium according to claim 5, characterized in that The solution in the solution preparation or suspension is water, or cell culture medium, or phosphate buffer.

7. The 3D dispersed culture medium according to claims 1-6, characterized in that: The cell density of mesenchymal stem cells used was 1×10 4 -1×10 7 / ml, preferably 5×10 5 -4×10 6 / ml.

8. Use of the preparation of the mesenchymal stem cell 3D dispersion culture agent according to any one of claims 1 to 2 or the mesenchymal stem cell 3D dispersion culture agent according to claim 5, characterized in that: The 3D dispersed culture medium is used for in vitro expansion of mesenchymal stem cells in a three-dimensional pattern.

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