Culture medium for closed culture of mesenchymal stem cells and culture method and application thereof

Abstract

This invention provides a culture medium for closed-system culture of mesenchymal stem cells, comprising the following components: 1% (v / v) human platelet lysate, 10-15 g / L basal culture medium powder, 15-20 mM HEPES, 1.0-2.0 g / L sodium bicarbonate, and 0.1-1.0 g / L sodium chloride; wherein the basal culture medium powder includes non-essential amino acids, D-glucose, L-glutamine, specific growth factors, inorganic salts, and vitamins. Using the culture medium provided by this invention for mesenchymal stem cell culture eliminates the need for exogenous CO2, reducing equipment costs and process complexity; furthermore, it results in cell viability ≥97%, more concentrated particle size distribution, and more stable expression of surface markers, which is beneficial for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of human mesenchymal stem cell culture technology, specifically relating to a culture medium for closed culture of mesenchymal stem cells, its culture method, and its application. Background Technology

[0002] Currently, large-scale culture of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) mainly relies on static culture systems using T175 culture flasks and cell factory culture flasks. This system has the following main problems: 1. The culture relies on a CO2 incubator to maintain the pH of the culture medium, which has high requirements for equipment and environment and poor flexibility; 2. Using a breathable cover poses a risk of contamination, and fluctuations in temperature, humidity, and CO2 concentration within the incubator can affect the consistency of cell state. 3. Static culture leads to uneven distribution of oxygen and nutrients in the culture medium, resulting in an inconsistent cell growth microenvironment; 4. The temperature of the culture system is affected by the airflow inside the incubator and the placement of the incubator, which can easily lead to uneven local temperature distribution; 5. Limited culture area and low expansion efficiency make it difficult to meet clinical-grade cell dosage requirements; 6. The operation is highly open, but batch-to-batch consistency is poor, and cell phenotype and functional stability are insufficient.

[0003] Therefore, there is an urgent need to develop a large-scale culture process for hUC-MSCs that does not require a CO2 incubator to provide exogenous CO2, has a closed and controllable environment, and provides stable cell quality. Summary of the Invention

[0004] In view of this, the present invention provides a method for large-scale culture of hUC-MSCs based on a closed roller bottle dynamic culture system. By using an airtight cap to seal the culture container, combined with an optimized culture medium buffer system and dynamic mixing process, autonomous and stable control of the culture pH is achieved without the need for an external CO2 supply from a CO2 incubator. At the same time, the system optimizes the combination of culture parameters (temperature, rotation speed, inoculation density) to achieve high-quality and highly uniform cell expansion in 3L or 15L roller bottles, thus meeting the requirements for large-scale culture.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A culture medium for closed culture of mesenchymal stem cells, the culture medium comprising the following components: human platelet lysate at a volume concentration of 1%, basal culture medium powder at a concentration of 10-15 g / L, HEPES at a concentration of 15-20 mM, sodium bicarbonate at a concentration of 1.0-2.0 g / L, and sodium chloride at a concentration of 0.1-1.0 g / L. The basal culture medium powder includes non-essential amino acids, D-glucose, L-glutamine, specific growth factors, inorganic salts, and vitamins.

[0006] In some specific embodiments, preferably, the culture medium comprises the following components: 1% human platelet lysate, 12.65 g / L basal culture medium powder, 20 mM HEPES buffer, 1.5 g / L sodium bicarbonate, and 0.7 g / L sodium chloride.

[0007] A method for culturing mesenchymal stem cells using the above-mentioned culture medium, the method comprising the following steps: first, preparing the culture medium according to the above-described group allocations, and then sterilizing and filtering it; then, taking mesenchymal stem cells and inoculating them into a roller bottle containing the culture medium, and tightening the airtight cap; finally, rotating and culturing them at a temperature of 37±0.2℃.

[0008] Furthermore, the mesenchymal stem cells include mesenchymal stem cells derived from human umbilical cord, adipose tissue, and bone marrow. The inoculation density is (1~2.5)×10 4 cells / cm 2 .

[0009] In some specific embodiments, preferably, the inoculation density is 2×10⁻⁶. 4 cells / cm 2 .

[0010] Furthermore, the rotation speed of the rotary bottle culture is 10~20 rpm.

[0011] In some specific embodiments, preferably, the rotation speed of the rotary flask culture is 15 rpm.

[0012] Furthermore, the volume of the rotating bottle is 3~15L.

[0013] Application of the above-mentioned culture medium or method in the large-scale closed culture of human umbilical cord-derived mesenchymal stem cells.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The cultivation system is simplified and no CO2 incubator is required: the use of an airtight cover and an optimized buffer system eliminates the dependence on CO2 gas, reduces equipment costs and process complexity, and improves production flexibility and site applicability; (2) Closed system with low risk of contamination: The airtight cover effectively reduces the risk of external contamination and is more in line with GMP requirements; (3) pH control is autonomous and stable: pH stability is achieved without external gas regulation through the optimization of the culture medium buffer system and the synergistic effect of dynamic mixing, thus overcoming the pH management problem of closed culture. (4) Significantly improved cell quality: Under optimized parameter combinations, cell viability is ≥97%, particle size distribution is more concentrated, and surface marker expression is more stable; (5) Uniform and controllable culture environment: The combination of dynamic temperature control (37±0.2℃) and rotation speed (10~20rpm) in the rotating bottle effectively avoids local temperature and nutrient unevenness. (6) Good process repeatability and easy to scale up: the parameters are highly controllable, the batch-to-batch differences are small, the 3L and 15L roller bottle systems can be directly scaled up, supporting large-scale production. Attached Figure Description

[0015] Figure 1 This is a schematic diagram comparing the traditional T-bottle (CO2 incubator) with the closed roller bottle culture system of the present invention in Example 1 and Comparative Example 1 of the present invention.

[0016] Figure 2 This invention provides an example of exploring the results of culture at different rotation speeds in Example 1; where A is a 3L rotating bottle and B is a 15L rotating bottle.

[0017] Figure 3 This study investigates the culture results of human platelet lysate at different concentrations in Example 1 of the present invention.

[0018] Figure 4 This is a bar chart comparing cell viability in Example 1 (3L, 15L) and Comparative Example 1 (T175) of the present invention.

[0019] Figure 5 This is a bar chart comparing cell size in Example 1 (3L, 15L) and Comparative Example 1 (T175) of the present invention.

[0020] Figure 6 , 7 The expression levels of different biomarkers in dynamically cultured cells in a 3L flask in Example 1 of this invention.

[0021] Figure 8 This is a comparison of cell morphology in Example 1 (3L, 15L) and Comparative Example 1 (T175) of the present invention. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0023] Example 1 This embodiment provides a method for large-scale culture of hUC-MSCs based on a closed roller bottle dynamic culture system (see physical image of the culture system). Figure 1), as detailed below: Preparation of culture medium: Accurately measure 50 mL of human platelet lysate (brand: AventaCell Biomedical Co., Ltd., Helios Bioscience), 63.25 g of basal culture medium powder (brand: Shanghai Yuanpei Biotechnology Co., Ltd.), 100 mL of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES, 1M concentration), 7.5 g of sodium bicarbonate, and 3.5 g of sodium chloride, dissolve them in water for injection, and then bring the volume to 5 L. Filter the solution sterilely through a 0.22 µm microporous membrane to obtain the culture medium.

[0024] Culture conditions: First, add 0.3 L and 0.6 L of the prepared culture medium to 3 L and 15 L roller bottles respectively; then, take P4 generation human umbilical cord mesenchymal stem cells and seed them at a density of 2 × 10⁻⁶. 4 cells / cm 2 The inoculation was carried out sequentially in the 3L and 15L roller flasks mentioned above; finally, the airtight caps were tightly closed, and the flasks were placed at a temperature of 37℃ and a rotation speed of 15rpm for incubation.

[0025] Furthermore, based on Example 1, this application investigated the rotation speed and human platelet lysate concentration while keeping other conditions unchanged. The specific results are as follows: 1.1 Investigation of Cultivation Rotation Speed The rotation speed was adjusted to 10 rpm, 15 rpm, and 20 rpm respectively for 3 days. The final culture results are shown in Table 1 below. Figure 2 .

[0026] Table 1. Culture results under different rotation speeds

[0027] In the culture of mesenchymal stem cells, dynamic culture is superior to static culture (dynamic culture is more conducive to nutrient utilization and local dilution of metabolites during cell growth). However, the choice of rotation method in dynamic culture directly affects the cell growth status and culture efficiency. For example, dynamic culture relying on agitation with a stirring paddle drives liquid flow through the rotation of the paddle blades in the culture medium, thereby moving the suspended carrier or cells and achieving mixing and material transfer of the culture medium. However, this method is prone to generating local high shear stress at the paddle edges and in the eddy current region, causing mechanical damage to cells attached to the carrier surface, which may lead to cell detachment, decreased viability, or even death, especially for shear-sensitive mesenchymal stem cells.

[0028] In contrast, the bottle rotation method used in this scheme does not rely on the built-in agitator (as shown in Table 1). Figure 2It can be seen that the technology performs well at lower rotation speeds (10-20 rpm), with the best results achieved at 15 rpm; while at excessively high rotation speeds (40 rpm), the culture results deteriorate significantly. When the flask rotates, the culture medium flows periodically along the inner surface of the flask wall, and the cells attached to the flask wall move accordingly, sequentially contacting the culture medium and gas interface to achieve nutrient uptake and gas exchange. During this process, the shear force experienced by the cells mainly originates from the viscous boundary layer between the liquid and the flask wall, and its stress level is much lower than that of the agitator method, and it is more evenly distributed throughout the flask. Therefore, the rotary flask method provides a gentler mechanical environment, reduces mechanical damage to cells, maintains the adhesion stability and undifferentiated state of mesenchymal stem cells, and is suitable for large-scale mesenchymal stem cell culture and related process applications that are sensitive to shear forces.

[0029] 1.2 Investigation of platelet lysate concentrations in different individuals The concentrations of human platelet lysate in the culture medium were adjusted to 1% and 2%, respectively, and cultured for 3 days. The final culture results are shown in Table 2 below. Figure 3 The T175 culture flasks were prepared by adding 30 mL of culture medium (which is exactly the same as the medium used for roller flask culture) containing 1% and 2% human platelet lysate to two T175 culture flasks, respectively, covering them with breathable caps, and placing them in a static environment at 37°C for incubation.

[0030] Table 2. Culture results at different concentrations of human platelet lysate.

[0031] From Table 2, Figure 3 The results show that both 1% and 2% concentrations of human platelet lysate culture in 3L roller bottles are superior to conventional T175 culture. Although the effect is better at a concentration of 2%, considering the overall cost, a 1% concentration of human platelet lysate can achieve the same technical effect.

[0032] Example 2 This embodiment provides a method for large-scale culture of hUC-MSCs based on a closed roller bottle dynamic culture system, as detailed below: Preparation of culture medium: Accurately measure 50 mL of human platelet lysate (brand: AventaCell Biomedical, Helios Bioscience), 63.25 g of basal culture medium powder (brand: Shanghai Yuanpei Biotechnology Co., Ltd.), 75 mL of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES, 1M), 10 g of sodium bicarbonate, and 2.5 g of sodium chloride, dissolve them in water for injection, and then bring the volume to 5 L. Filter the solution through a 0.22 µm microporous membrane for sterilization to obtain the culture medium.

[0033] Culture conditions: First, add 0.3 L and 0.6 L of the prepared culture medium to 3 L and 15 L roller bottles respectively; then, take P4 generation human adipose-derived mesenchymal stem cells and seed them at a density of 2.5 × 10⁻⁶. 4 cells / cm 2 The inoculation was carried out sequentially in the 3L and 15L roller flasks mentioned above; finally, the airtight caps were tightly closed, and the flasks were placed at a temperature of 37℃ and a rotation speed of 10rpm for incubation.

[0034] Comparative Example 1 This comparative example uses conventional culture methods for human umbilical cord mesenchymal stem cell culture (i.e., conventional T-flask culture; see the image of the culture system). Figure 1 ), as detailed below: Culture medium preparation: Take an accurate amount of 50 mL of human platelet lysate, 12.65 g of basal culture medium powder (brand source: Shanghai Yuanpei Biotechnology Co., Ltd.), and 3 g of sodium bicarbonate, dissolve them in water for injection, and then make up to 1 L. Filter the solution through a 0.22 µm microporous membrane for sterilization to obtain the culture medium.

[0035] Culture conditions: Add 30 mL of the above culture medium to a T175 culture flask, cover with a breathable cap, and place at 37℃ for static culture.

[0036] Comparative Example 2 This comparative example provides a method for large-scale culture of hUC-MSCs based on a closed roller bottle dynamic culture system. The basic steps and methods are basically the same as those in Example 1, except that: the culture medium contains 50 mL of HEPES, 15 g of sodium bicarbonate, and 8 g of sodium chloride, and is cultured in a 3 L roller bottle, while the rest remain unchanged.

[0037] Comparative Example 3 This comparative example provides a method for large-scale culture of hUC-MSCs based on a closed roller bottle dynamic culture system. The basic steps and methods are essentially the same as in Example 1, except that the inoculation density is 0.8 × 10⁻⁶. 4 cells / cm 2 The rotation speed was 25 rpm, and the culture was carried out in a 3L transfer bottle, with all other parameters remaining unchanged.

[0038] Comparative Example 4 This comparative example provides a method for large-scale culture of hUC-MSCs based on a closed roller bottle dynamic culture system. The basic steps and methods are basically the same as those in Example 1, except that sodium chloride is removed from the culture medium, while the rest remain unchanged.

[0039] Furthermore, to understand the specific culture effects of the above embodiments and comparative examples, relevant tests were performed on the cultured cells after 3 days of culture, as follows: Cell viability test: 20 μL of cell suspension and 0.4% trypan blue staining solution were mixed thoroughly in a 1:1 ratio in a 1.5 mL centrifuge tube. Then, 20 μL of the mixture was placed in a counting chamber. The cell viability in the counting chamber was detected using a fully automated cell counter (CountStar, IE 1000). The results were averaged in three segments.

[0040] Cell size assay: 20 μL of cell suspension was mixed thoroughly with 0.4% trypan blue in a 1.5 mL centrifuge tube at a 1:1 ratio. Then, 20 μL of the mixture was placed in a counting chamber. The cell size in the counting chamber was measured using a fully automated cell counter (CountStar, IE 1000). The results were averaged in three segments.

[0041] Assay for expression levels of different cell biomarkers: Collect cultured cells and prepare 1×10⁻⁶ cells using buffer. 6 Single-cell suspensions of cells / mL were prepared. Four control groups were set up: blank control, single-label compensation control, isotype control mixed tube, and sample detection tube. 200 μL of cell suspension was added to the blank control tube without any antibody; 200 μL of cell suspension was added to the single-label tube, with one fluorescently labeled target antibody added to each tube; 200 μL of cell suspension was added to the isotype control tube, along with a pre-prepared volume of fluorescently labeled isotype control antibody mixture according to the kit instructions, and gently mixed; 200 μL of cell suspension was added to the sample detection tube, along with a pre-prepared volume of target antibody mixture according to the kit instructions, and gently mixed; all tubes were incubated at room temperature in the dark for 15 min. 5 mL of buffer was added to each tube, centrifuged at 200g for 5 min, the supernatant was discarded, and the cells were resuspended and fixed in 200 μL of 2% paraformaldehyde. The cells were stored at 4°C in the dark and analyzed within 30 min.

[0042] The results for cell number, viability, and cell size are shown in Table 3. Figure 4 , 5 .

[0043] Table 3. Details of cell viability and cell size in different systems

[0044] From Table 3, Figure 4 , 5 It can be seen that the roller bottle system of the present invention simplifies culture conditions and reduces equipment dependence while achieving a comprehensive improvement in cell quality.

[0045] Compared to Comparative Example 1: After closed roller bottle dynamic culture, the cell viability of both 3L and 15L roller bottles was higher than that of the conventional static T175 flask; after closed roller bottle dynamic culture, the cell size of both 3L and 15L roller bottles was smaller than that of the conventional static T175 flask. Compared to the static culture of T175 flask, the dynamic culture of 3L and 15L roller bottles resulted in a better local cell culture microenvironment (lower local concentration of metabolic waste) and better cell condition, manifested as smaller and more uniform cell size.

[0046] Compared with Comparative Example 2: During dynamic culture, Comparative Example 2 increased the sodium bicarbonate concentration and decreased the amount of HEPES, which led to an imbalance in the buffer capacity of the culture medium, resulting in greater pH fluctuations. In addition, the increase in sodium chloride led to higher osmotic pressure, which slowed down the growth rate of cells in Comparative Example 2 in 3L roller bottles. These changes worked together to result in a lower total number of cells harvested, and the cells showed a higher stress level in the later stages of expansion.

[0047] Compared with Comparative Example 3: The low seeding density in Comparative Example 3 resulted in insufficient cell-cell contact in the early stage of expansion, delaying the initiation of proliferation, while the higher rotation speed introduced excessive shear force; the combination of the two led to a decrease in the cell adhesion efficiency in the 3L roller bottle of Comparative Example 3, a longer culture period, and cells were more likely to detach from the carrier surface, resulting in a relatively low yield per unit volume.

[0048] Compared to Comparative Example 4: In a closed, dynamic culture environment, the lower sodium bicarbonate concentration already weakened the chemical buffering capacity, while the severe sodium ion deficiency in Comparative Example 4 further disrupted the electrolyte balance necessary for maintaining intracellular and extracellular ion equilibrium and pH homeostasis. During the culture process, the cells in Comparative Example 4 exhibited significant morphological swelling and metabolic abnormalities in the 3L roller flasks due to severe edema and ion disturbances, with significantly lower cell viability and proliferation capacity throughout the entire culture period.

[0049] Furthermore, by Figure 6 , 7 The results are from flow cytometry analysis in a 3L roller bottle. Figure 6 For the control flow cytometry, A represents cells within the gate that account for 45.9% of the total events; B represents single cells that account for 94.8% of the cells within the gate; C represents HLR-DR positivity rate of 0.12%; D represents CD45 positivity rate of 0.91%; E represents CD34 positivity rate of 0.081%; F represents CD14 positivity rate of 0.22%; G represents CD19 positivity rate of 0.54%; H represents CD73 positivity rate of 99.8%; and I represents CD90 positivity rate of 99.8%. Figure 7The flow cytometry results are as follows: A shows that after dynamic culture in a CO2-free roller bottle, hUC-MSCs cells within the gate of the 3L bottle accounted for 42.7% of the total events, which was not significantly different from the control group; B shows that the proportion of single cells in the sample was 91.7%, indicating a low proportion of adherent cells and good cell quality; C shows that the CD105 positivity rate was as high as 99.800%, which is consistent with the phenotypic characteristics of MSCs; D shows that the CD11b positivity rate was only 0.500%, indicating extremely low contamination by other cells.

[0050] Depend on Figure 8 (After 48 hours of culture) it was found that the stem cell density in the T175 flask was lower than that in the 15L and 3L roller bottles, and the cell size was larger; the cell size in the 15L roller bottle was similar to that in the 3L roller bottle, but the density in the 3L bottle was higher.

[0051] The reason this invention eliminates the need for exogenous CO2 supplied by a CO2 incubator lies in its radical change to the pH buffering system: 1. The HEPES chemical buffer system replaces the traditional CO2-dependent system: Traditional culture relies on the balance between CO2 gas and high-concentration sodium bicarbonate (approximately 3.7 g / L) to stabilize pH. This formulation uses a sufficient concentration (15~20 mM) of HEPES as the primary buffer, whose buffering capacity is independent of a specific external gas environment and can independently maintain pH stability within a closed container. Simultaneously, the concentration of human platelet lysate (hPL) in the culture medium is optimized to 1% (v / v), far lower than the amount used in traditional culture methods.

[0052] 2. Eliminate unstable factors: Significantly reduce the sodium bicarbonate concentration (to 1.0-3.0 g / L), fundamentally avoiding the problem of culture medium alkalization caused by the lack of external CO2 balance in closed systems.

[0053] 3. System self-stabilization: The optimized ion environment and dynamic mixing in the roller flask further ensure the uniform and stable operation of the buffer system. Through the design of "highly effective chemical buffer (HEPES) + removal of risk factors (low NaHCO3)," an intrinsically self-stabilizing pH environment is constructed, thereby eliminating absolute dependence on CO2 gas from the external environment.

[0054] In summary, the culture medium formula and culture conditions of this application can achieve a good balance, resulting in high survival rate and small cell size of cultured mesenchymal stem cells.

[0055] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0056] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A culture medium for closed culture of mesenchymal stem cells, characterized in that, The culture medium comprises the following components: human platelet lysate at a volume concentration of 1%, basal culture medium dry powder at a concentration of 10-15 g / L, HEPES at a concentration of 15-20 mM, sodium bicarbonate at a concentration of 1.0-2.0 g / L, and sodium chloride at a concentration of 0.1-1.0 g / L. The basal culture medium powder includes non-essential amino acids, D-glucose, L-glutamine, specific growth factors, inorganic salts, and vitamins.

2. The culture medium according to claim 1, characterized in that, The culture medium comprises the following components: 1% human platelet lysate, 12.65 g / L basal culture medium powder, 20 mM HEPES buffer, 1.5 g / L sodium bicarbonate, and 0.7 g / L sodium chloride.

3. A method for culturing mesenchymal stem cells using the culture medium described in claim 1, characterized in that, The method includes the following steps: first, prepare culture medium according to the allocation of each group as specified in claim 1, and filter it for sterilization; then, take mesenchymal stem cells and inoculate them into a roller bottle containing culture medium, and tighten the airtight cap; finally, rotate and culture them at a temperature of 37±0.2℃.

4. The method according to claim 3, characterized in that, The mesenchymal stem cells include mesenchymal stem cells derived from human umbilical cord, adipose tissue, and bone marrow. The inoculation density is (1~2.5)×10 4 cells / cm 2 .

5. The method according to claim 4, characterized in that, The inoculation density is 2×10 4 cells / cm 2 .

6. The method according to claim 3, characterized in that, The rotation speed of the rotary bottle culture is 10~20 rpm.

7. The method according to claim 6, characterized in that, The rotation speed of the rotary bottle culture was 15 rpm.

8. The method according to claim 3, characterized in that, The volume of the rotating bottle is 3~15L.

9. The application of the culture medium of claim 1 or the method of any one of claims 3-8 in the large-scale culture of human umbilical cord-derived mesenchymal stem cells in a closed culture.

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