Culture solution for inducing proliferation and differentiation of mesenchymal stem cells and preparation method thereof

Abstract

The invention discloses a culture solution for inducing proliferation and differentiation of mesenchymal stem cells and a preparation method of the culture solution, the culture solution comprises the following raw materials in parts by weight: 40-60 parts of a basal culture medium, 5-10 parts of a serum substitute, 3-5 parts of growth factors, 1-3 parts of hormones, 1-3 parts of vitamins, 0.5-1 part of a small molecule compound and 1-3 parts of other additives, and the invention relates to the technical field of stem cell culture. According to the culture solution for inducing proliferation and differentiation of the mesenchymal stem cells and the preparation method of the culture solution, by optimizing basic components and adding specific growth factors and small molecule compounds, the proliferation capacity and directional differentiation efficiency of the MSCs are remarkably improved, and the culture solution is suitable for in-vitro induction of various tissues such as bones, cartilages and nerves. The culture solution adopts an animal-origin-free formula, has the advantages of low cost, high safety, strong applicability and the like, and provides an important technical tool for the field of regenerative medicine.

Description

Technical Field

[0001] This invention relates to the field of stem cell culture technology, specifically to a culture medium for inducing the proliferation and differentiation of mesenchymal stem cells and its preparation method. Background Technology

[0002] Mesenchymal stem cells (MSCs) are widely used in the regeneration and repair of bone, cartilage, and neural tissues due to their multipotent differentiation potential, immunomodulatory properties, and low immunogenicity. However, current technologies for the in vitro expansion and directed differentiation of MSCs still face the following challenges: 1. Low proliferation efficiency: Traditional culture media (such as DMEM containing 10% fetal bovine serum) are difficult to maintain the long-term proliferation capacity of MSCs and are prone to cell senescence or phenotypic changes.

[0003] 2. Unstable differentiation induction effect: Existing differentiation media (such as media containing TGF-β3, dexamethasone, etc.) have low differentiation induction efficiency for MSCs and the differentiation time is long (usually 21-28 days).

[0004] 3. Complex composition and high cost: Some culture media rely on high concentrations of serum or expensive growth factors (such as FGF-2, IGF-1), which increases production costs and may introduce the risk of exogenous contamination.

[0005] Therefore, there is an urgent need to develop a novel culture medium that can efficiently induce MSCs to proliferate and differentiate into target cell types in vitro, while simplifying the formulation, reducing costs, and ensuring safety and reproducibility. Summary of the Invention

[0006] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a culture medium for inducing mesenchymal stem cell proliferation and differentiation, and its preparation method. By optimizing the composition of the basal culture medium and adding specific growth factors and small molecule compounds, the proliferation capacity and directed differentiation efficiency of MSCs are significantly improved.

[0007] (II) Technical Solution To achieve the above objectives, the present invention is implemented through the following technical solution: a culture medium for inducing the proliferation and differentiation of mesenchymal stem cells, the raw materials of which include, by weight: 40-60 parts of basal culture medium, 5-10 parts of serum substitute, 3-5 parts of growth factor, 1-3 parts of hormone, 1-3 parts of vitamin, 0.5-1 parts of small molecule compound and 1-3 parts of other additives.

[0008] Preferably, the raw materials include, by weight: 50 parts basal culture medium, 7 parts serum substitute, 4 parts growth factor, 2 parts hormone, 2 parts vitamin, 0.7 parts small molecule compound and 2 parts other additives.

[0009] Preferably, the raw materials include, by weight: 40 parts basal culture medium, 5 parts serum substitute, 3 parts growth factor, 1 part hormone, 1 part vitamin, 0.5 parts small molecule compound and 1 part other additives.

[0010] Preferably, the raw materials include, by weight: 60 parts basal culture medium, 10 parts serum substitute, 5 parts growth factor, 3 parts hormone, 3 parts vitamin, 1 part small molecule compound and 3 parts other additives.

[0011] Preferably, the basal culture medium is either low-glucose DMEM or α-MEM, serving as the basic nutrient source for cell growth, and the serum substitute is 5-20% (v / v) of animal-free serum substitute (such as StemPro® XF Serum-Free Medium).

[0012] Preferably, the growth factors are transforming growth factor TGF-β3 at 5-50 ng / mL and fibroblast growth factor FGF-2 at 10-50 ng / mL, and the hormone is dexamethasone at 10-100 nM.

[0013] Preferably, the vitamin is 50-100 μg / m L ascorbic acid, and the small molecule compounds are 5-10 μM ROCK inhibitor MY-27632 and 1-5 μM TGF-β receptor inhibitor SB431542 (used to regulate differentiation direction).

[0014] Preferably, the other additives include 1-5 μg / mL of ITS complex and 0.1-1 mM of proline, and the ITS complex is an insulin, transferrin and selenium complex.

[0015] This invention also provides a method for preparing a culture medium for inducing the proliferation and differentiation of mesenchymal stem cells, specifically including the following steps: S1. Preparation, filtration and sterilization of basal culture medium: Dissolve the basal culture medium in deionized water, filter it through a 0.22μm filter membrane for sterilization, then dispense it and store it at -20℃ for later use. S2. Component addition: Under aseptic conditions, add serum substitute, growth factor, hormone, small molecule compound and other additives in sequence and mix well; S3. Final solution preparation: Sterilize the mixture by double filtration through a 0.22μm filter membrane, dispense into sterile bottles, and store at 4℃ protected from light. The shelf life is 1 month. After double filtration for sterilization, dispense and store.

[0016] This invention also provides a method for using a culture medium to induce the proliferation and differentiation of mesenchymal stem cells, specifically including the following steps: T1. Cell pretreatment: MSCs (such as those derived from bone marrow, umbilical cord, or adipose tissue) were seeded into gelatin-coated culture dishes and expanded to the logarithmic growth phase using basal medium containing 10% serum substitute. T2, Induction of Differentiation: Cells were seeded at a density of 1×10^6 cells / mL into 3D scaffold material (such as calcium alginate microspheres), replaced with the culture medium of this invention, and incubated at 37°C and 5%... Incubate in an incubator; T3. Dynamic culture: A rotary bioreactor is used for dynamic culture at a speed of 50-100 rpm to promote uniform cell distribution and material exchange. T4. Differentiation Termination and Harvesting: After 14-21 days of culture, the scaffold material is dissolved in sodium citrate solution, and the differentiated target cells (such as chondrocytes, osteoblasts, or neural progenitor cells) are collected.

[0017] (III) Beneficial Effects This invention provides a culture medium for inducing the proliferation and differentiation of mesenchymal stem cells and its preparation method. Compared with the prior art, it has the following advantages: (1) The culture medium for inducing mesenchymal stem cell proliferation and differentiation and its preparation method significantly improve the proliferation rate of MSCs by adding Y-27632 and FGF-2 (2-3 times higher than the traditional method), and precisely regulate the differentiation direction through the synergistic effect of TGF-β3 and SB431542. The use of serum-free substitutes avoids exogenous virus contamination and batch differences, and meets the requirements of GMP production.

[0018] (2) The culture medium for inducing mesenchymal stem cell proliferation and differentiation and its preparation method reduce the culture cost by optimizing the concentration of growth factors and small molecule compounds (40-50% lower than the cost of commercially available products), and are suitable for in vitro induction of differentiation of various tissues such as bone, cartilage, and nerves, and are compatible with 3D culture and dynamic culture systems. Attached Figure Description

[0019] Figure 1 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see Figure 1 The present invention provides five technical solutions: a culture medium for inducing the proliferation and differentiation of mesenchymal stem cells and its preparation method, specifically including the following embodiments: Example 1: A culture medium for inducing the proliferation and differentiation of mesenchymal stem cells, comprising the following raw materials by weight: 50 parts basal culture medium, 7 parts serum substitute, 4 parts growth factors, 2 parts hormones, 2 parts vitamins, 0.7 parts small molecule compounds, and 2 parts other additives. The basal culture medium is low-glucose DMEM, serving as the basic nutrient source for cell growth. The serum substitute is 12% (v / v) animal-free serum substitute (such as StemPro® XF Serum-Free Medium). The growth factors are 30 ng / mL transforming growth factor TGF-β3 and 30 ng / mL fibroblast growth factor FGF-2. The hormone is 50 nM dexamethasone. The vitamin is 70 μg / mL L ascorbic acid. The small molecule compounds are 7 μM ROCK inhibitor MY-27632 and 3 μM TGF-β receptor inhibitor SB431542 (used to regulate differentiation direction). Other additives include 3 μg / mL ITS complex and 0.5 mM proline. Furthermore, the ITS complex is a complex of insulin, transferrin, and selenium.

[0022] This invention also provides a method for preparing a culture medium for inducing the proliferation and differentiation of mesenchymal stem cells, specifically including the following steps: S1. Preparation, filtration and sterilization of basal culture medium: Dissolve the basal culture medium in deionized water, filter it through a 0.22μm filter membrane for sterilization, then dispense it and store it at -20℃ for later use. S2. Component addition: Under aseptic conditions, add serum substitute, growth factor, hormone, small molecule compound and other additives in sequence and mix well; S3. Final solution preparation: Sterilize the mixture by double filtration through a 0.22μm filter membrane, dispense into sterile bottles, and store at 4℃ protected from light. The shelf life is 1 month. After double filtration for sterilization, dispense and store.

[0023] This invention also provides a method for using a culture medium to induce the proliferation and differentiation of mesenchymal stem cells, specifically including the following steps: T1. Cell pretreatment: MSCs (such as those derived from bone marrow, umbilical cord, or adipose tissue) were seeded into gelatin-coated culture dishes and expanded to the logarithmic growth phase using basal medium containing 10% serum substitute. T2, Induction of Differentiation: Cells were seeded at a density of 1×10^6 cells / mL into 3D scaffold material (such as calcium alginate microspheres), replaced with the culture medium of this invention, and incubated at 37°C and 5%... Incubate in an incubator; T3. Dynamic culture: A rotary bioreactor at 70 rpm is used for dynamic culture to promote uniform cell distribution and material exchange. T4. Differentiation Termination and Harvesting: After 17 days of culture, the scaffold material was dissolved in sodium citrate solution, and the differentiated target cells (such as chondrocytes, osteoblasts, or neural progenitor cells) were collected.

[0024] Example 2: A culture medium for inducing mesenchymal stem cell proliferation and differentiation, comprising the following components by weight: 40 parts basal culture medium, 5 parts serum substitute, 3 parts growth factors, 1 part hormone, 1 part vitamin, 0.5 parts small molecule compound, and 1 part other additives. The basal culture medium is α-MEM, serving as the basic nutrient source for cell growth. The serum substitute is 5% (v / v) animal-free serum substitute (such as StemPro® XF Serum-Free Medium). The growth factors are 5 ng / mL transforming growth factor TGF-β3 and 10 ng / mL fibroblast growth factor FGF-2, and the hormone is 10 ng / mL. The product contains nM dexamethasone, 50 μg / mL L ascorbic acid as a vitamin, and 5 μM of the ROCK inhibitor MY-27632 and 1 μM of the TGF-β receptor inhibitor SB431542 (used to regulate differentiation direction). Other additives include 1 μg / mL of ITS complex and 0.1 mM of proline, and the ITS complex is a complex of insulin, transferrin and selenium.

[0025] This invention also provides a method for preparing a culture medium for inducing the proliferation and differentiation of mesenchymal stem cells, specifically including the following steps: S1. Preparation, filtration and sterilization of basal culture medium: Dissolve the basal culture medium in deionized water, filter it through a 0.22μm filter membrane for sterilization, then dispense it and store it at -20℃ for later use. S2. Component addition: Under aseptic conditions, add serum substitute, growth factor, hormone, small molecule compound and other additives in sequence and mix well; S3. Final solution preparation: Sterilize the mixture by double filtration through a 0.22μm filter membrane, dispense into sterile bottles, and store at 4℃ protected from light. The shelf life is 1 month. After double filtration for sterilization, dispense and store.

[0026] This invention also provides a method for using a culture medium to induce the proliferation and differentiation of mesenchymal stem cells, specifically including the following steps: T1. Cell pretreatment: MSCs (such as those derived from bone marrow, umbilical cord, or adipose tissue) were seeded into gelatin-coated culture dishes and expanded to the logarithmic growth phase using basal medium containing 10% serum substitute. T2, Induction of Differentiation: Cells were seeded at a density of 1×10^6 cells / mL into 3D scaffold material (such as calcium alginate microspheres), replaced with the culture medium of this invention, and incubated at 37°C and 5%... Incubate in an incubator; T3. Dynamic culture: A rotary bioreactor at 50 rpm is used for dynamic culture to promote uniform cell distribution and material exchange. T4. Differentiation Termination and Harvesting: After 14 days of culture, the scaffold material is dissolved in sodium citrate solution, and the differentiated target cells (such as chondrocytes, osteoblasts, or neural progenitor cells) are collected.

[0027] Example 3: A culture medium for inducing mesenchymal stem cell proliferation and differentiation, comprising the following components by weight: 60 parts basal culture medium, 10 parts serum substitute, 5 parts growth factors, 3 parts hormones, 3 parts vitamins, 1 part small molecule compound, and 3 parts other additives. The basal culture medium is low-glucose DMEM, serving as the basic nutrient source for cell growth. The serum substitute is 20% (v / v) animal-free serum substitute (such as StemPro® XF Serum-Free Medium). The growth factors are 50 ng / mL transforming growth factor TGF-β3 and 50 ng / mL fibroblast growth factor FGF-2, and the hormone is 100... The product contains nM dexamethasone, 100 μg / mL L ascorbic acid as a vitamin, and 10 μM of the ROCK inhibitor MY-27632 and 5 μM of the TGF-β receptor inhibitor SB431542 (used to regulate differentiation direction). Other additives include 5 μg / mL of ITS complex and 1 mM of proline. The ITS complex is a complex of insulin, transferrin, and selenium.

[0028] This invention also provides a method for preparing a culture medium for inducing the proliferation and differentiation of mesenchymal stem cells, specifically including the following steps: S1. Preparation, filtration and sterilization of basal culture medium: Dissolve the basal culture medium in deionized water, filter it through a 0.22μm filter membrane for sterilization, then dispense it and store it at -20℃ for later use. S2. Component addition: Under aseptic conditions, add serum substitute, growth factor, hormone, small molecule compound and other additives in sequence and mix well; S3. Final solution preparation: Sterilize the mixture by double filtration through a 0.22μm filter membrane, dispense into sterile bottles, and store at 4℃ protected from light. The shelf life is 1 month. After double filtration for sterilization, dispense and store.

[0029] This invention also provides a method for using a culture medium to induce the proliferation and differentiation of mesenchymal stem cells, specifically including the following steps: T1. Cell pretreatment: MSCs (such as those derived from bone marrow, umbilical cord, or adipose tissue) were seeded into gelatin-coated culture dishes and expanded to the logarithmic growth phase using basal medium containing 10% serum substitute. T2, Induction of Differentiation: Cells were seeded at a density of 1×10^6 cells / mL into 3D scaffold material (such as calcium alginate microspheres), replaced with the culture medium of this invention, and incubated at 37°C and 5%... Incubate in an incubator; T3. Dynamic culture: A rotary bioreactor at 100 rpm is used for dynamic culture to promote uniform cell distribution and material exchange. T4. Differentiation Termination and Harvesting: After 21 days of culture, the scaffold material was dissolved in sodium citrate solution, and the differentiated target cells (such as chondrocytes, osteoblasts, or neural progenitor cells) were collected.

[0030] Example 4: Inducing MSCs to differentiate into chondrocytes: Preparation of culture medium: Mix the components in the following proportions: Low-sugar DMEM: 80 mL; Animal-derived serum substitute: 15 mL; TGF-β3 (100 ng / mL stock solution): 50 μL; FGF-2 (10 μg / mL stock solution): 50 μL; Dexamethasone (1 mM stock solution): 10 μL; Ascorbic acid (10 mg / mL stock solution): 500 μL; Y-27632 (10 mM stock solution): 5 μL; ITS complex (100× stock solution): 50 μL.

[0031] Human bone marrow MSCs were inoculated into calcium alginate microspheres, the above-mentioned culture medium was added, and the mixture was cultured in a rotary bioreactor for 21 days.

[0032] Results: Alcian Blue staining showed significantly enhanced cartilage matrix secretion, with type II collagen expression rate exceeding 95%.

[0033] Example 5: Inducing MSCs to differentiate into neural progenitor cells: Replace TGF-β3 with BDNF (brain-derived neurotrophic factor, 50 ng / mL) in the culture medium and add SB431542 (2 μM).

[0034] Human adipose-derived MSCs were seeded into gelatin-coated 6-well plates and cultured statically for 14 days as usual.

[0035] Results: Nestin and β-III-tubulin positive cells accounted for 80%, indicating that they were successfully induced into neural progenitor cells.

[0036] Furthermore, any content not described in detail in this specification is existing technology known to those skilled in the art.

[0037] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0038] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A culture medium for inducing the proliferation and differentiation of mesenchymal stem cells, characterized in that: Its raw materials, by weight, include: 40-60 parts basal culture medium, 5-10 parts serum substitute, 3-5 parts growth factor, 1-3 parts hormone, 1-3 parts vitamin, 0.5-1 part small molecule compound and 1-3 parts other additives.

2. The culture medium for inducing the proliferation and differentiation of mesenchymal stem cells according to claim 1, characterized in that: Its raw materials, by weight, include: 50 parts basal culture medium, 7 parts serum substitute, 4 parts growth factor, 2 parts hormone, 2 parts vitamin, 0.7 parts small molecule compound and 2 parts other additives.

3. The culture medium for inducing the proliferation and differentiation of mesenchymal stem cells according to claim 1, characterized in that: Its raw materials, by weight, include: 40 parts basal culture medium, 5 parts serum substitute, 3 parts growth factor, 1 part hormone, 1 part vitamin, 0.5 parts small molecule compound and 1 part other additives.

4. The culture medium for inducing the proliferation and differentiation of mesenchymal stem cells according to claim 1, characterized in that: Its raw materials, by weight, include: 60 parts basal culture medium, 10 parts serum substitute, 5 parts growth factor, 3 parts hormone, 3 parts vitamin, 1 part small molecule compound, and 3 parts other additives.

5. The culture medium for inducing the proliferation and differentiation of mesenchymal stem cells according to any one of claims 1-4, characterized in that: The basal culture medium is either low-glucose DMEM or α-MEM, and the serum substitute is 5-20% (v / v) of animal-free serum substitute.

6. The culture medium for inducing the proliferation and differentiation of mesenchymal stem cells according to any one of claims 1-4, characterized in that: The growth factors are transforming growth factor TGF-β3 at 5-50 ng / mL and fibroblast growth factor FGF-2 at 10-50 ng / mL, and the hormone is dexamethasone at 10-100 nM.

7. The culture medium for inducing the proliferation and differentiation of mesenchymal stem cells according to any one of claims 1-4, characterized in that: The vitamin is 50-100 μg / m L ascorbic acid, and the small molecule compounds are 5-10 μM ROCK inhibitor MY-27632 and 1-5 μM TGF-β receptor inhibitor SB431542.

8. The culture medium for inducing the proliferation and differentiation of mesenchymal stem cells according to any one of claims 1-4, characterized in that: The other additives include 1-5 μg / mL of ITS complex and 0.1-1 mM of proline, and the ITS complex is a complex of insulin, transferrin and selenium.

9. A method for preparing the culture medium for inducing the proliferation and differentiation of mesenchymal stem cells according to any one of claims 1-4, characterized in that, Specifically, the following steps are included: S1. Preparation, filtration and sterilization of basal culture medium: Dissolve the basal culture medium in deionized water, filter it through a 0.22μm filter membrane for sterilization, then dispense it and store it at -20℃ for later use. S2. Component addition: Under aseptic conditions, add serum substitute, growth factor, hormone, small molecule compound and other additives in sequence and mix well; S3. Final solution preparation: Sterilize the mixture by double filtration through a 0.22μm filter membrane, dispense into sterile bottles, and store at 4℃ protected from light. The shelf life is 1 month. After double filtration and sterilization, dispense and store.

10. A method for using the culture medium for inducing mesenchymal stem cell proliferation and differentiation according to any one of claims 1-4, characterized in that, Specifically, the following steps are included: T1. Cell pretreatment: MSCs were seeded in gelatin-coated culture dishes and expanded to the logarithmic growth phase using basal medium containing 10% serum substitute. T2, Induction of Differentiation: Cells were seeded at a density of 1×10^6 cells / mL into the 3D scaffold material, replaced with the culture medium of this invention, and incubated at 37°C and 5%... Incubate in an incubator; T3. Dynamic culture: A rotary bioreactor is used for dynamic culture at a speed of 50-100 rpm to promote uniform cell distribution and material exchange. T4. Differentiation Termination and Harvest: After 14-21 days of culture, the scaffold material is dissolved in sodium citrate solution, and the differentiated target cells are collected.

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