A low-concentration serum culture medium for human umbilical cord mesenchymal stem cells and a culture method for human umbilical cord mesenchymal stem cells

Abstract

The application discloses a low-concentration serum culture medium of human umbilical cord mesenchymal stem cells and a culture method of human umbilical cord mesenchymal stem cells. The serum dosage is reduced from 10%-20% to 2%-5% in the application, which greatly reduces the risk of animal source pathogen pollution and immunogenicity problems; meanwhile, the application utilizes six factors of vitronectin, EGF, bFGF, IGF-1, SCF and NAD+ to form a synergistic network with low-concentration serum, so that the cell proliferation is rapid, and the yield and purity are significantly improved. In addition, the culture medium has clear components, low and fixed serum dosage, and the key functional factors are all recombinant proteins or small molecule compounds, so that the batch difference is small, the culture result is good in repeatability, and the quality control requirements of clinical grade cell preparation are met.

Description

Technical Field

[0001] This invention belongs to the field of cell engineering technology, specifically relating to a low-concentration serum culture medium for human umbilical cord mesenchymal stem cells and a method for culturing human umbilical cord mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a class of tissue stem cells with self-renewal capacity and multi-lineage differentiation potential that have attracted much attention. They can be obtained from various tissues such as umbilical cord, adipose tissue, bone marrow, placenta, fetal blood, and liver. Although MSCs originate from the mesoderm, they can differentiate into various cell types from different germ layers under specific conditions, such as osteocytes, chondrocytes, adipocytes, cardiomyocytes, neuron-like cells, endothelial cells, and hepatocytes, providing broad application prospects for clinical cell therapy and tissue engineering.

[0003] Traditional MSC culture typically uses a medium containing 10%-20% fetal bovine serum (FBS). However, high-concentration serum has the following inherent drawbacks: (1) its composition is complex and unclear, with significant batch-to-batch variations, making it difficult to replicate culture results; (2) there is a risk of contamination by animal-derived pathogens (such as mycoplasma, viruses, etc.); (3) high concentrations of foreign proteins in serum are immunogenic and may trigger immune rejection responses in receptors; and (4) a high-serum environment may induce premature cell differentiation or senescence. These drawbacks severely limit the application of MSCs in clinical trials and large-scale production.

[0004] To address the aforementioned issues, serum-free culture media have become a research hotspot in recent years. Existing serum-free culture media typically replace serum functions by adding recombinant proteins, growth factors, and adhesion factors. However, the serum-free system has significant shortcomings in practical applications: (1) cell adhesion efficiency is unstable, especially with low adhesion rates in primary tissue block culture; (2) cell proliferation rate is limited, and doubling time is long; (3) cells are prone to senescence and decreased differentiation potential after long-term passage; (4) recombinant proteins and growth factors are expensive, resulting in poor economic efficiency for large-scale production.

[0005] Therefore, it is crucial to provide a low-serum mesenchymal stem cell culture medium that significantly reduces animal serum usage and culture costs while ensuring efficient MSC adhesion, rapid proliferation, and purity. Summary of the Invention

[0006] To address the shortcomings of existing serum-free culture media, such as unstable adhesion efficiency, limited proliferation rate, and high culture cost, as well as the technical deficiencies of traditional high-serum culture media, including high animal-derived risk, large batch-to-batch variability, and unclear composition, the present invention aims to provide a mesenchymal stem cell culture medium containing low serum and a multi-factor synergistic combination.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a culture medium additive composition for culturing mesenchymal stem cells.

[0008] Furthermore, the composition includes SCF and NAD+.

[0009] Furthermore, the composition also includes vitrin, EGF, bFGF, and IGF-1.

[0010] In this invention, mesenchymal stem cells (MSCs) are a type of pluripotent stem cells originating from the mesoderm. They have the characteristics of self-renewal, self-replication, unlimited proliferation, and multi-lineage differentiation potential. Under specific induction conditions in vivo or in vitro, they can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, and endothelium. They still have multi-lineage differentiation potential after continuous passage culture and cryopreservation.

[0011] The stem cell factor (SCF), also known as c-Kit ligand (KITLG), steel factor, or mast cell growth factor, is the natural ligand of the tyrosine kinase receptor c-Kit (CD117). The nicotinamide adenine dinucleotide (NAD) + FGFR is one of the most important coenzymes in cells, participating in energy metabolism processes such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Vitronectin (VN), also known as vitreous adhesion protein or serum diffusion factor, is an adhesive glycoprotein found in plasma and the extracellular matrix. Epidermal growth factor (EGF) is a prototype member of the epidermal growth factor family. Basic fibroblast growth factor (bFGF), also known as fibroblast growth factor-2 (FGF-2), can bind to FGFR and activate the MAPK / ERK and PI3K / AKT pathways. Insulin-like growth factor-1 (IGF-1), also known as somatomedin C, is a member of the insulin-like growth factor family.

[0012] In this invention, the applicant discovered that the aforementioned six factors can form a synergistic network with low-concentration serum, shortening the primary cell emergence time from 10-12 days in traditional methods to 6 days, and increasing the P0 generation cell yield to 1.0 × 10⁻⁶. 6 -1.3×10 6 / dish (traditional 10% FBS is approximately 5.0 × 10 5 -6.0×10 5 / pan).

[0013] Furthermore, in the culture medium prepared from the composition, the concentration of SCF is 10-20 ng / mL, the concentration of NAD+ is 1-5 μM, the concentration of fibronectin is 1-5 μg / mL, the concentration of EGF is 5-10 ng / mL, the concentration of bFGF is 10-20 ng / mL, and the concentration of IGF-1 is 10-20 ng / mL.

[0014] Preferably, in the culture medium prepared from the composition, the concentration of SCF is 15 ng / mL, the concentration of NAD+ is 3 μM, the concentration of fibronectin is 3 μg / mL, the concentration of EGF is 10 ng / mL, the concentration of bFGF is 10 ng / mL, and the concentration of IGF-1 is 15 ng / mL.

[0015] Furthermore, the mesenchymal stem cells may be derived from umbilical cord, bone marrow, adipose tissue, placenta, amnion, dental pulp, or synovium.

[0016] Preferably, the mesenchymal stem cells are derived from the umbilical cord.

[0017] In some embodiments, the composition is present in the form of a kit.

[0018] In some implementations, the kit may also include, but is not limited to, kit instructions, containers, buffers, preservatives, or antibiotics.

[0019] In some embodiments, suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container for holding one component, and preferably, for appropriate aliquoting. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container for separately holding the additional components. However, different combinations of components may be contained in a single vial.

[0020] A second aspect of the present invention provides a low-serum culture medium for culturing mesenchymal stem cells.

[0021] Furthermore, the low serum culture medium includes a basal culture medium, the composition described in the first aspect of the present invention, and additive factors.

[0022] Furthermore, the added factors include fetal bovine serum, recombinant human albumin, recombinant human transferrin, recombinant human insulin, sodium selenite, linoleic acid, cholesterol, PDGF-BB, transforming growth factor-β1, recombinant human fibronectin, ascorbic acid-2-phosphate, β-mercaptoethanol, and hydrocortisone.

[0023] Furthermore, the basal culture medium is selected from one of DMEM / F12 medium, DMEM medium, MEM medium, F10 medium, F12 medium, and IMDM medium.

[0024] Preferably, the basal culture medium is DMEM / F12 medium.

[0025] Furthermore, the concentration of the added factor in the low serum culture medium is: The concentrations of fetal bovine serum were 2%-5%, recombinant human albumin was 5 g / L, recombinant human transferrin was 10 mg / L, recombinant human insulin was 5 mg / L, sodium selenite was 30 nM, linoleic acid was 5 mg / L, cholesterol was 2 mg / L, PDGF-BB was 5 ng / mL, transforming growth factor-β1 was 2 ng / mL, recombinant human fibronectin was 5 μg / mL, ascorbic acid-2-phosphate was 50 μg / mL, β-mercaptoethanol was 50 μM, and hydrocortisone was 10 nM.

[0026] Preferably, the concentration of the fetal bovine serum is 3%.

[0027] Furthermore, the mesenchymal stem cells may be derived from umbilical cord, bone marrow, adipose tissue, placenta, amnion, dental pulp, or synovium.

[0028] Preferably, the mesenchymal stem cells are derived from the umbilical cord.

[0029] The term "culture medium" is recognized in the art and generally refers to any substance or preparation used to culture living cells. As used in relation to cell culture, the term "culture medium" includes components of the environment surrounding the cells. Culture media can be solid, liquid, gaseous, or a mixture of phases and substances. Culture media include liquid growth media and liquid media that do not sustain cell growth. Culture media also include gel-like media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous culture media include the gaseous phase in which cells grown on covered petri dishes or other solid or semi-solid supports are exposed. The term "culture medium" also refers to a substance intended for use in cell culture, even if it has not yet come into contact with cells. In other words, a nutrient-rich liquid prepared for bacterial culture is a culture medium.

[0030] The third aspect of this invention provides a method for the in vitro isolation and culture of umbilical cord mesenchymal stem cells.

[0031] Furthermore, the method includes: inoculating an umbilical cord tissue block into the low serum culture medium described in the second aspect of the present invention for culturing.

[0032] Furthermore, the method also includes a tissue pretreatment step, which includes taking umbilical cord tissue, removing residual umbilical blood, cutting it into small segments, disinfecting and cleaning the umbilical cord tissue with disinfectant and buffer solution, removing blood vessels and amnion from the umbilical cord tissue, and cutting the remaining tissue into tissue blocks.

[0033] Furthermore, the disinfectant is 75% ethanol, and the umbilical cord tissue is soaked in the 75% ethanol and agitated for 3-5 minutes.

[0034] Furthermore, the buffer solution is D-PBS, and the umbilical cord tissue is washed in the D-PBS until the washing solution is clear.

[0035] Furthermore, the length of the segment is 4-8 cm.

[0036] Furthermore, the blood vessels include the umbilical vein and the umbilical artery, and the remaining tissue after removing the blood vessels and amnion is Wharton's glue.

[0037] Furthermore, the volume of the tissue block is 0.1-1 mm³.

[0038] Furthermore, the method also includes a step of replacing half the medium every 2-4 days, and harvesting primary cells when the cell confluence reaches 80%-90%.

[0039] Furthermore, the amount of the low serum culture medium used is 8-10 mL per 150 mm culture dish.

[0040] Furthermore, the cultivation conditions are a temperature of 37±1℃, a CO2 volume fraction of 5±0.5%, and saturated humidity.

[0041] Furthermore, the method also includes a passage culture step, which includes digesting and collecting the primary cells, filtering to remove residual tissue clumps, centrifuging and resuspending them, and then inoculating them into the low serum culture medium for 3-4 days to harvest P1 generation umbilical cord mesenchymal stem cells.

[0042] Furthermore, the inoculation density for the subculture is 6000-8000 cells / cm².

[0043] Furthermore, the inoculation density for the subculture is 8000 cells / cm².

[0044] The fourth aspect of the present invention provides any one of the following products: 1) An umbilical cord mesenchymal stem cell, wherein the umbilical cord mesenchymal stem cell is prepared by the method described in the third aspect of the present invention; 2) A cell population comprising umbilical cord mesenchymal stem cells prepared by the method described in the third aspect of the present invention; 3) A pharmaceutical composition comprising the umbilical cord mesenchymal stem cells described in 1) or the cell population described in 2); Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

[0045] The cell-based pharmaceutical compositions involved in this invention are suitable for administration via intravenous, intra-arterial, portal venous, or local tissue administration. The number of cells administered is not particularly limited and can be appropriately determined based on the patient's age, weight, disease, symptoms, and method of administration.

[0046] When the cell-based pharmaceutical composition of the present invention is formulated into an injectable formulation, additives commonly used in the art can be appropriately used. Examples of additives include: isotonic agents, stabilizers, buffers, preservatives, chelating agents, antioxidants, and solubilizers. Examples of isotonic agents include: sugars such as glucose, sorbitol, and mannitol; sodium chloride; glycerol; propylene glycol; and polyethylene glycol. Examples of stabilizers include: sodium sulfite. Examples of buffers include: borate buffers, phosphate buffers, citrate buffers, tartaric acid buffers, and acetate buffers. Examples of preservatives include: parabens, benzyl alcohol, chlorocresol, phenethyl alcohol, and benzyl chloride. Examples of chelating agents include: sodium edetate and sodium citrate. Examples of antioxidants include: sodium sulfite, sodium bisulfite, sodium ascorbate, and sodium thiosulfate. Examples of cosolvents include: dextran, polyvinylpyrrolidone, sodium benzoate, ethylenediamine, salicylamide, nicotinamide, and polyoxyethylene hydrogenated castor oil derivatives.

[0047] The fifth aspect of the present invention provides the use of the composition described in the first aspect of the present invention in the preparation of products for culturing umbilical cord mesenchymal stem cells.

[0048] The sixth aspect of the present invention provides the use of the product described in the fourth aspect of the present invention in the preparation of a medicament for promoting tissue repair, promoting tissue regeneration, promoting angiogenesis and / or reducing inflammatory response.

[0049] In some implementation schemes, umbilical cord mesenchymal stem cells can differentiate into various cell types, such as osteocytes, chondrocytes, adipocytes, nerve cells, and cardiomyocytes, under suitable conditions. These cells can replace damaged tissue cells, promoting fracture healing, articular cartilage repair, and myocardial regeneration after myocardial infarction. Simultaneously, by secreting growth factors and cytokines, they stimulate the proliferation and differentiation of surrounding cells, accelerating tissue repair.

[0050] In some implementations, umbilical cord mesenchymal stem cells secrete antioxidant enzymes, anti-apoptotic factors, and telomerase activators, which delay cell aging, improve skin elasticity, cardiovascular function, and nervous system function, and can also regulate endocrine function, improve sleep quality, and enhance the body's vitality.

[0051] In some implementations, umbilical cord mesenchymal stem cells have the ability to migrate to sites of injury or inflammation, enabling them to precisely target lesions and exert a repair effect, thus improving the targeting of treatment.

[0052] In some implementations, umbilical cord mesenchymal stem cells can reverse liver fibrosis and promote hepatocyte regeneration.

[0053] In some implementations, umbilical cord mesenchymal stem cells can combat pulmonary fibrosis and improve respiratory function.

[0054] In some implementations, umbilical cord mesenchymal stem cells can protect nerve cells and promote nerve regeneration. Advantages and beneficial effects of the present invention: 1) Serum dosage is significantly reduced, balancing safety and cost-effectiveness: This invention reduces the amount of serum used from the traditional 10%-20% to 2%-5%, significantly reducing the risk of animal-derived pathogen contamination and immunogenicity issues. At the same time, the low concentration of serum provides natural active factor support that is difficult to replace in a completely serum-free system, reducing the cost of the culture medium by more than 30% compared to a completely serum-free commercial culture medium, making it more suitable for large-scale production.

[0055] 2) Synergistic effect of multiple factors significantly improves adhesion efficiency and proliferation rate: Six factors—glassnein, EGF, bFGF, IGF-1, SCF, and NAD+—form a synergistic network with low-concentration serum, reducing the primary cell emergence time from 10-12 days in traditional methods to 6 days, and increasing the yield of P0 generation cells to 1.0 × 10⁻⁶. 6 -1.3×10 6 / dish (traditional 10% FBS is approximately 5.0 × 10 5 -6.0×10 5 / pan).

[0056] 3) High cell viability and purity: The viability of P0 and P1 generation cells obtained by the method of this invention is over 97%. Flow cytometry analysis shows that the expression rates of positive cell surface markers CD73, CD90, and CD105 are ≥99.5%, while the expression rates of negative cell markers CD11b, CD19, CD34, CD45, and HLA-DR are all less than 1%, and the cell purity is over 99%.

[0057] 4) Effectively maintains cell stemness: The culture medium of this invention, through the combination of bFGF, TGF-β1, SCF and NAD+, effectively maintains the stemness characteristics and multi-lineage differentiation potential (osteogenic, adipogenic and chondrogenic differentiation ability) of MSCs during long-term passage, and avoids premature cell senescence or spontaneous differentiation.

[0058] 5) The cultivation system is stable and controllable: The culture medium of this invention has clearly defined components, low and fixed serum dosage, and key functional factors are all recombinant proteins or small molecule compounds. The batch-to-batch differences are small, the culture results are highly reproducible, and it meets the quality control requirements for clinical-grade cell preparation. Attached Figure Description

[0059] Figure 1The P0 generation MSCs in Example 1 exhibit adherent morphology, typical spindle shape, fibroblast-like cell morphology, orderly cell arrangement, and uniform density.

[0060] Figure 2 This displays the number of cells that have crawled out of the umbilical cord per centimeter.

[0061] Figure 3 This shows the cell viability of P1-P5 cells under different culture conditions.

[0062] Figure 4 This shows the doubling time of P1-P5 cells under different culture conditions.

[0063] Figure 5 This indicates the detection of positive markers on the cell surface.

[0064] Figure 6 This indicates the detection of negative markers on the cell surface.

[0065] Figure 7 Display of differentiation capacity detection.

[0066] Figure 8 The growth curves of MSCs P5 cells cultured in culture medium components 1, 2, 3 and 4 in Example 2 are shown. Detailed Implementation

[0067] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0068] Unless otherwise specified in the following examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0069] Example 1: Preparation of Preferred Culture Medium and Culture of Umbilical Cord MSCs 1.1 Preparation of the culture medium of the present invention Take DMEM / F12 basal culture medium, add each component according to the final concentration shown in Table 1, mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and store at 4℃ for later use.

[0070] Table 1 Concentration of each component in the culture medium

[0071] 1.2 Control Group Setup Control group culture medium: DMEM / F12 + 10% FBS (traditional culture method, without the above factors added except for serum).

[0072] 1.3 Experimental Methods Umbilical cord tissue from the same source was divided into two portions and cultured in parallel using the tissue block adherence method.

[0073] (1) Remove the umbilical cord with sterile toothed forceps, cut off both ends, wipe away residual blood, and cut into 4-8 cm segments; (2) Wash with 75% ethanol for 3-5 min, and wash repeatedly with D-PBS more than 3 times until clear; (3) Remove the arteries, veins and amnion, and cut into pieces <1 mm³; (4) Take 40 tissue blocks and spread them evenly on a 150 mm culture dish, and air dry at 25℃ for 40 min; (5) Add 8 mL of the culture medium of this invention (experimental group) or 10% FBS culture medium (control group) respectively, and culture in a 37℃, 5% CO2 incubator; (6) Add 15 mL of the corresponding culture medium on the 2nd and 3rd days; (7) On the 6th day, a large number of spindle-shaped adherent cells can be seen crawling out under a microscope (around the 8th-9th day for the control group), remove tissue fragments and old culture medium, and replace with 30 mL of the corresponding fresh culture medium; (8) On the 10th day, the cell fusion rate reaches 80-90% (around the 12th day for the control group), and digest and collect P0 generation cells.

[0074] 1.4 Results P0 yield: Experimental group 1.15×10 6 cells / cm; control group 5.8×10 5 pcs / cm ( Figure 2 ) P0 survival rate: 97.2% in the experimental group; 95.0% in the control group. P0 harvest time: Day 10 in the experimental group; Day 12 in the control group. P0 generation cells were seeded and passaged at 8000 cells / cm², and P1 generation cells were harvested after 3 days for P1 cell bank construction. P1 cells were then resuscitated for small-scale P1-P5 cell culture, and cell viability and doubling time during passage were calculated. Figure 3 and Figure 4 ).

[0075] Flow cytometry: CD73 + CD90 + CD105 + Expression rate ≥99.5%; CD11b - CD19 - CD34 - CD45 - HLA-DR - Expression rates were all <1%. Then, at a rate of 8000 cells / cm²... 2The inoculation density was re-inoculated into culture flasks, and after culturing for another 3 days, high-purity P1 generation umbilical cord mesenchymal stem cells were obtained. Figure 6 (As shown).

[0076] Differentiation capacity assay: P5 cells were induced to differentiate into osteogenic, adipogenic, and chondrogenic lineages using osteogenic, adipogenic, and chondrogenic differentiation induction media, respectively. After culture for the specified time, staining was performed using appropriate methods (osteogenic: Alizarin Red staining; adipogenic: Oil Red O staining; chondrogenic: Alixin Blue staining). The staining results were observed to assess cell differentiation capacity. The results showed that cells could differentiate into bone, adipogenic, and cartilage lineages. Figure 7 (As shown).

[0077] Example 2: Multifactor Detection 2.1 Combined promoting effect of NAD+ and SCF 2.1.1 Experimental Grouping To clarify the combined promoting effect of NAD+ and SCF on MSC proliferation, three groups of culture media were set up as shown in Table 2 (the basal culture medium was DMEM / F12 + 3% FBS + the additives described in Example 1 (except for NAD+ and SCF)).

[0078] Table 2 Culture medium groups

[0079] 2.1.2 Detection methods and results The P5 generation umbilical cord MSCs obtained in Example 1 were seeded at 8000 cells / cm² in 96-well plates and cultured using four different culture media: component 1, component 2, component 3, and component 4. At 24h, 48h, 72h, 96h, and 168h after culture, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37℃ for 2h. The absorbance at 450 nm was then measured. The results are as follows: Figure 8 As shown, NAD+ and SCF have a synergistic promoting effect on MSC proliferation, and the cell proliferation effect is significantly better when both are present than when a single factor is used.

[0080] Example 3: Preparation and cultivation of high-dose factor culture medium 3.1 Culture medium preparation: The basal culture medium was DMEM / F12, supplemented with: fetal bovine serum: 5% (v / v), porphyrin: 5 μg / mL, NAD+: 5 μM, EGF: 20 ng / mL, bFGF: 20 ng / mL, IGF-1: 20 ng / mL, SCF: 20 ng / mL, and other additives were the same as those in the culture medium of the present invention in Example 1.

[0081] 3.2 Experimental Methods Same as Example 1 1.3.

[0082] 3.3 Results P0 output: 1.25 × 10 6 pcs / cm P0 viability: 97.5% P1 viability: 98.8% Flow cytometry purity: positive markers ≥99.5%, negative markers <1%.

[0083] Example 4: Low serum levels but without multifactor combination 4.1 Culture medium preparation DMEM / F12 + 3% FBS + basic additives (albumin, transferrin, insulin, sodium selenite, linoleic acid, cholesterol, PDGF-BB, TGF-β1, ascorbic acid-2-phosphate, β-mercaptoethanol, hydrocortisone, at the same concentration as in Example 1), free of hydrin, EGF, bFGF, NAD+, IGF-1, and SCF.

[0084] 4.2 Experimental Methods Same as Example 1 1.3.

[0085] 4.3 Results P0 output: 3.2 × 10 5 pcs / cm P0 viability: 93.0% Emergence time: A small number of cells can be seen on day 9. P1 viability: 95.5% Flow cytometry purity: positive markers ≥96%, but CD90 expression rate (approximately 96.5%) was slightly lower than that of the culture medium of this invention.

[0086] The results showed that simply reducing the serum dosage without adding the combination of multiple factors including fibronectin, EGF, bFGF, NAD+, IGF-1, and SCF significantly reduced cell adhesion and proliferation.

[0087] Example 5: Traditional high serum culture medium 5.1 Culture medium preparation DMEM / F12 + 10% FBS (traditional culture method, without adding the above factors other than serum).

[0088] 5.2 Experimental Methods Same as Example 1 1.3.

[0089] 5.3 Results P0 output: 5.8 × 10 5 pcs / cm P0 viability: 95.0% Crawling out time: Day 8 Achieving Fusion: Day 12 P1 viability: 96.8% Flow cytometry purity: Positive marker ≥98% The results showed that the yield and efficiency of the traditional high-serum culture medium were lower than those of the culture medium of the present invention.

[0090] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A culture medium additive composition for culturing mesenchymal stem cells, characterized in that, The composition includes SCF and NAD+; Preferably, the composition further includes vitrin, EGF, bFGF and IGF-1.

2. The composition according to claim 1, characterized in that, In the culture medium prepared from the composition, the concentration of SCF is 10-20 ng / mL, the concentration of NAD+ is 1-5 μM, the concentration of fibronectin is 1-5 μg / mL, the concentration of EGF is 5-10 ng / mL, the concentration of bFGF is 10-20 ng / mL, and the concentration of IGF-1 is 10-20 ng / mL. Preferably, in the culture medium prepared from the composition, the concentration of SCF is 15 ng / mL, the concentration of NAD+ is 3 μM, the concentration of fibronectin is 3 μg / mL, the concentration of EGF is 10 ng / mL, the concentration of bFGF is 10 ng / mL, and the concentration of IGF-1 is 15 ng / mL.

3. A low-serum culture medium for culturing mesenchymal stem cells, characterized in that, The low-serum culture medium comprises a basal culture medium, the composition of claim 1 or 2, and additive factors; Preferably, the added factors include fetal bovine serum, recombinant human albumin, recombinant human transferrin, recombinant human insulin, sodium selenite, linoleic acid, cholesterol, PDGF-BB, transforming growth factor-β1, recombinant human fibronectin, ascorbic acid-2-phosphate, β-mercaptoethanol, and hydrocortisone. Preferably, the basal culture medium is selected from one of DMEM / F12 medium, DMEM medium, MEM medium, F10 medium, F12 medium, and IMDM medium; Preferably, the basal culture medium is DMEM / F12 medium.

4. The low-serum culture medium according to claim 3, characterized in that, The concentration of the added factor in the low serum culture medium is: The concentrations of fetal bovine serum were 2%-5%, recombinant human albumin was 5 g / L, recombinant human transferrin was 10 mg / L, recombinant human insulin was 5 mg / L, sodium selenite was 30 nM, linoleic acid was 5 mg / L, cholesterol was 2 mg / L, PDGF-BB was 5 ng / mL, transforming growth factor-β1 was 2 ng / mL, recombinant human fibronectin was 5 μg / mL, ascorbic acid-2-phosphate was 50 μg / mL, β-mercaptoethanol was 50 μM, and hydrocortisone was 10 nM. Preferably, the concentration of the fetal bovine serum is 3%.

5. The composition according to claim 1 or the low-serum culture medium according to claim 3, characterized in that, The mesenchymal stem cells mentioned can be derived from umbilical cord, bone marrow, adipose tissue, placenta, amnion, dental pulp, or synovium. Preferably, the mesenchymal stem cells are derived from the umbilical cord.

6. A method for in vitro isolation and culture of umbilical cord mesenchymal stem cells, characterized in that, The method includes: inoculating an umbilical cord tissue block into the low serum culture medium of any one of claims 3-5, and culturing it.

7. The method according to claim 6, characterized in that, The method further includes a tissue pretreatment step, which includes taking umbilical cord tissue, removing residual umbilical blood, cutting it into small segments, disinfecting and cleaning the umbilical cord tissue with disinfectant and buffer solution, removing blood vessels and amnion from the umbilical cord tissue, and cutting the remaining tissue into tissue blocks. Preferably, the disinfectant is 75% ethanol, and the umbilical cord tissue is soaked in the 75% ethanol and agitated for 3-5 minutes. Preferably, the buffer solution is D-PBS, and the umbilical cord tissue is washed in the D-PBS until the washing solution is clear; Preferably, the length of the segment is 4-8 cm; Preferably, the blood vessels include the umbilical vein and the umbilical artery, and the remaining tissue after removing the blood vessels and amnion is Wharton's glue; Preferably, the volume of the tissue block is 0.1-1 mm³; Preferably, the method further includes the step of replacing half the medium every 2-4 days, and harvesting primary cells when the cell confluence reaches 80%-90%; Preferably, the amount of the low serum culture medium used is 8-10 mL per 150 mm culture dish; Preferably, the culture conditions are a temperature of 37±1℃, a CO2 volume fraction of 5±0.5%, and saturated humidity; Preferably, the method further includes a passage culture step, wherein the passage culture includes digesting and collecting the primary cells, filtering to remove residual tissue clumps, centrifuging and resuspending them, and then inoculating them into the low serum culture medium for 3-4 days to harvest P1 generation umbilical cord mesenchymal stem cells. Preferably, the inoculation density for the subculture is 6000-8000 cells / cm²; Preferably, the inoculation density for the subculture is 8000 cells / cm².

8. Any one of the following products: 1) An umbilical cord mesenchymal stem cell, characterized in that, The umbilical cord mesenchymal stem cells are prepared by the method according to claim 6 or 7; 2) A cell population, characterized in that the cell population includes umbilical cord mesenchymal stem cells prepared by the method of claim 6 or 7; 3) A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the umbilical cord mesenchymal stem cells described in 1) or the cell population described in 2); Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.

9. Use of the composition of claim 1 or 2 in the preparation of products for culturing umbilical cord mesenchymal stem cells.

10. The use of the product of claim 8 in the preparation of a medicament for promoting tissue repair, promoting tissue regeneration, promoting angiogenesis and / or reducing inflammatory response.

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