Culture medium for promoting high-efficiency expansion of adipose-derived stem cells and culture method thereof

Abstract

This invention relates to the field of large-scale adipose-derived stem cell culture technology, and discloses a culture medium and method for promoting the efficient expansion of adipose-derived stem cells. The method includes: seeding adipose-derived stem cells in a serum-free basal culture medium and maintaining a normoxic environment; adding an expansion additive composed of a Rho-associated kinase inhibitor and a redox buffer component; during the window period when the ambient oxygen partial pressure decreases from normoxic to hypoxic, using the Rho-associated kinase inhibitor to relieve cytoskeleton tension to induce a physically relaxed cell membrane; and assisting the redox buffer component in constructing a reducing environment on the cell membrane surface in situ. This invention utilizes the dynamic synergy of membrane tension regulation and oxygen stress shielding to neutralize transient damage caused by sudden changes in ambient oxygen partial pressure, avoid transmembrane receptor conformational inactivation, inhibit premature cell aging at the mechanistic level, and ensure long-term stable expansion of adipose-derived stem cells without the need for heterologous protein protection.

Description

Technical Field

[0001] This invention belongs to the field of large-scale culture technology of adipose stem cells, and particularly relates to a culture medium and culture method for promoting the efficient expansion of adipose stem cells. Background Technology

[0002] Currently, the in vitro expansion of adipose-derived stem cells mainly uses culture media containing fetal bovine serum. However, considering the large batch-to-batch variations, immunogenicity risks, and pathogen transmission risks associated with fetal bovine serum, serum-free culture systems with clearly defined chemical compositions have become the current direction of technological evolution.

[0003] In actual amplification processes, the industry commonly uses methods such as lowering the ambient oxygen concentration to simulate the in vivo microenvironment, thereby promoting cell proliferation and maintaining an undifferentiated state. However, after stripping away the physical shielding and biochemical buffering of serum proteins, the tolerance of adipose-derived stem cells in serum-free systems to changes in oxygen partial pressure is significantly reduced. When the culture environment undergoes a phased transition from normoxic to hypoxic conditions, the sudden drop in oxygen concentration generates transient oxidative stress. This mutation in the physical environment induces peroxidation of cell membrane lipid molecules, resulting in microscopic physicochemical coupling damage. This damage leads to tension distortion of the cell membrane, which in turn causes conformational invagination or inactivation of key receptors that maintain stemness. This makes the hypoxic environment a trigger for inducing autophagy and premature aging. Existing technologies attempt to improve cell growth by adding conventional antioxidants or signaling pathway inhibitors, but the mechanisms of action of these chemical components are related to the physical process of oxygen partial pressure switching. The lack of temporal alignment makes it impossible to construct an effective physical buffer interface at the critical window of membrane structure damage. When the biochemical activity of the protective component cannot synergize with the step process of the physical environment, the existing biochemical intervention strategies and process timing coordination are insufficient. For example, Chinese invention patent CN109517872B discloses the application of rhodioloside in protecting stem cell activity. It uses the antioxidant properties of rhodioloside to reduce oxygen damage. This is a static chemical component addition and does not address the underlying physical cause of the surge in cell membrane tension during the step switch of oxygen partial pressure. During the window of sudden drop in oxygen partial pressure, if the mechanical stress of the cytoskeleton is not relieved in advance, the cell membrane is in a high-tension compact conformation, which produces a steric hindrance effect, hindering the deep anchoring of antioxidant molecules and failing to prevent irreversible inactivation of transmembrane receptors due to membrane distortion. Simply relying on biochemical component compensation is insufficient to cope with the dynamic physical stress under serum-free conditions.

[0004] Therefore, the technical problem to be solved by this invention is how to achieve efficient expansion and maintenance of stemness of adipose stem cells by coupling the culture medium components with environmental physical parameters through the mechanism, while avoiding oxygen partial pressure step damage. Summary of the Invention

[0005] This invention aims to solve the problem of membrane damage and premature aging of adipose stem cells caused by oxygen partial pressure step changes in a serum-free system.

[0006] In this technical solution, a method for culturing a culture medium to promote the efficient expansion of adipose-derived stem cells includes the following steps: Step S101: Adipose stem cells are seeded in serum-free basal culture medium and cultured under normoxic adherent conditions at a temperature of 37°C, a carbon dioxide volume percentage of 5%, and an oxygen volume percentage of 21% for 1 to 2 days. Step S102: Add an amplification additive consisting of a Rho-associated kinase inhibitor, a lipid-soluble reducing component vitamin E, and a water-soluble reducing component composed of 2-mercaptoethanol and ethanolamine to the serum-free basal medium, and make the final concentration of the Rho-associated kinase inhibitor in the serum-free basal medium reach 5 μmol / L to 20 μmol / L. Step S103: Within a time window of 2 to 6 hours after adding the amplification additive, the inhibitory effect of Rho-related kinase inhibitors on the cross-linking contraction of actin and myosin in the adipose stem cell cytoskeleton is utilized to relieve cytoskeleton tension, thereby placing the cell membrane in a physically relaxed conformation with reduced surface tension, and the oxygen volume percentage in the culture environment is reduced from 21% to 1% to 10%. Step S104: Utilizing the hydrophobic tail of the cell membrane lipid bilayer in a physically relaxed conformation, vitamin E is induced to intercalate to the hydrophobic tail. In conjunction with water-soluble reducing components, a continuous reduction potential barrier is constructed around the cell to eliminate transient reactive oxygen species induced by a step decrease in oxygen partial pressure. The cells are then cultured continuously under hypoxic conditions with an oxygen volume percentage of 1% to 10% until the cell confluence reaches 80% to 90%.

[0007] Preferably, the final concentration of vitamin E in serum-free basal medium involved in step S102 is 10 μg / mL to 50 μg / mL, the final concentration of 2-mercaptoethanol is 2 mmol / L to 4 mmol / L, and the final concentration of ethanolamine is 10 μmol / L to 20 μmol / L, thereby inducing reduction potential and blocking lipid peroxidation chain reaction in serum-free basal medium.

[0008] Preferably, the seeding density of adipose-derived stem cells involved in step S101 is 2500 / 2500. Up to 10,000 / Furthermore, the oxygen volume percentage reduction process involved in step S103 is completed within 30 minutes to form a step physical stimulus of oxygen partial pressure targeting adipose stem cells and induce the activation of the hypoxia-inducible factor pathway.

[0009] Preferably, the Rho-related kinase inhibitor is Y-27632; and in step S103, inhibiting the Rho-related kinase pathway through Y-27632 directly relieves the mechanical tension of the cytoskeleton, assisting vitamin E to overcome steric hindrance and deeply anchor to the hydrophobic tail of the exposed cell membrane lipid bilayer.

[0010] Preferably, after step S104, a passage step is included: when the cell fusion reaches 90%, the adipose stem cells are dissociated using a digestive solution without animal-derived components, and passaged at an expansion ratio of 1:3 to 1:5. Then, steps S101 to S104 are repeated until a predetermined number of adipose stem cells are obtained.

[0011] Preferably, the serum-free basal culture medium is further supplemented with CHIR99021 at a concentration of 1 μmol / L to 5 μmol / L and SB431542 at a concentration of 5 μmol / L to 20 μmol / L. By acting on functional cell surface receptors, it enhances the fidelity of stemness maintenance signals transmitted into the cell and blocks the differentiation pathway of adipose stem cells.

[0012] Preferably, step S104 involves the bioreactor culture conditions, including the following sub-steps: monitoring the real-time oxygen consumption rate of adipose stem cells, and adjusting the mixing ratio of nitrogen and air introduced into the culture container according to the oxygen consumption rate, so that the fluctuation range of the oxygen volume percentage involved in step S104 is within 0.5%.

[0013] Preferably, the amplification additive also includes recombinant human serum albumin at a concentration of 1 g / L to 5 g / L and recombinant human fibronectin at a concentration of 1 mg / L to 10 mg / L, for physically supporting the microenvironment of adipose-derived stem cells in vivo and providing osmotic pressure buffering in serum-free basal culture medium.

[0014] Preferably, during the continuous culture process in step S104, the glucose concentration in the serum-free basal medium is collected every 24 hours. When the glucose concentration is lower than 5 mmol / L, half of the serum-free basal medium is replaced, and the corresponding proportion of amplification additive is added simultaneously to maintain the chemical potential energy stability of the culture system.

[0015] A culture medium for promoting the efficient expansion of adipose-derived stem cells, wherein the culture medium is prepared by a culture method for promoting the efficient expansion of adipose-derived stem cells.

[0016] Compared with existing technologies, the culture method of the culture medium for promoting the efficient expansion of adipose-derived stem cells of the present invention has the following advantages: 1. In the culture medium for the efficient expansion of adipose-derived stem cells, through the deep coupling of small molecule regulatory components with the phased oxygen partial pressure switching, and by utilizing the decongestant effect of Rho-related kinase inhibitors on cytoskeleton contraction, the cell membrane maintains a physically relaxed conformation at the critical window of oxygen partial pressure step cleavage. This assists the antioxidant components in the culture medium to construct a redox buffer interface in situ within the lipid bilayer. This physical-redox coupling shielding mechanism eliminates transient oxidative stress and membrane tension distortion induced by sudden changes in environmental parameters in serum-free systems, avoids irreversible invagination of the transmembrane receptor spatial conformation, and provides a stable material structure prerequisite for subsequent signal transduction.

[0017] 2. This invention establishes a strong correlation between the regulation of oxygen partial pressure in the culture environment and the timing of action of specific signaling pathway inhibitors. By utilizing a pre-constructed membrane surface protective barrier to block the lipid peroxidation chain reaction, the GSK-3β inhibitor and TGF-β inhibitor can continuously act on functional cell surface receptors, improving the fidelity of stem cell maintenance signals transmitted into the cell. This synergistic mechanism solves the receptor passivation problem caused by the lack of serum protein buffer in traditional serum-free hypoxia culture processes, ensuring that adipose stem cells are precisely blocked from differentiation pathways during the high-speed expansion window. Attached Figure Description

[0018] Figure 1 This is a flowchart of the efficient culture method for inhibiting premature aging of adipose stem cells according to the present invention; Figure 2 This is a system architecture diagram of a stem cell reactor with dynamic environmental parameter compensation function according to the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0020] It should be noted that all directional and positional terms used in this invention, such as: up, down, left, right, front, back, vertical, horizontal, inner, outer, top, bottom, transverse, longitudinal, center, etc., are only used to explain the relative positional relationship and connection between components in a specific state (as shown in the accompanying drawings). They are only for the convenience of describing this invention and do not require that this invention be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention. In addition, the descriptions of "first," "second," etc., in this invention are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0021] In the description of this invention, unless otherwise explicitly specified and limited, the terms installation, connection, and linking should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections; they can refer to direct connections or indirect connections through an intermediate medium; they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0022] In the description of this specification, references to the terms "an embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example, and the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0023] A method for culturing a culture medium that promotes the efficient expansion of adipose-derived stem cells includes the following steps: Step S101: Adipose stem cells are seeded in serum-free basal culture medium and cultured under normoxic adherent conditions at a temperature of 37°C, a carbon dioxide volume percentage of 5%, and an oxygen volume percentage of 21% for 1 to 2 days. Step S102: Add an amplification additive consisting of a Rho-associated kinase inhibitor, a lipid-soluble reducing component vitamin E, and a water-soluble reducing component composed of 2-mercaptoethanol and ethanolamine to the serum-free basal medium, and make the final concentration of the Rho-associated kinase inhibitor in the serum-free basal medium reach 5 μmol / L to 20 μmol / L. Step S103: Within a time window of 2 to 6 hours after adding the amplification additive, the inhibitory effect of Rho-related kinase inhibitors on the cross-linking contraction of actin and myosin in the adipose stem cell cytoskeleton is utilized to relieve cytoskeleton tension, thereby placing the cell membrane in a physically relaxed conformation with reduced surface tension, and the oxygen volume percentage in the culture environment is reduced from 21% to 1% to 10%. Step S104: Utilizing the hydrophobic tail of the cell membrane lipid bilayer in a physically relaxed conformation, vitamin E is induced to intercalate to the hydrophobic tail. In conjunction with water-soluble reducing components, a continuous reduction potential barrier is constructed around the cell to eliminate transient reactive oxygen species induced by a step decrease in oxygen partial pressure. The cells are then cultured continuously under hypoxic conditions with an oxygen volume percentage of 1% to 10% until the cell confluence reaches 80% to 90%.

[0024] Preferably, the final concentration of vitamin E in serum-free basal medium involved in step S102 is 10 μg / mL to 50 μg / mL, the final concentration of 2-mercaptoethanol is 2 mmol / L to 4 mmol / L, and the final concentration of ethanolamine is 10 μmol / L to 20 μmol / L, thereby inducing reduction potential and blocking lipid peroxidation chain reaction in serum-free basal medium.

[0025] Preferably, the seeding density of adipose-derived stem cells involved in step S101 is 2500 / 2500. Up to 10,000 / Furthermore, the oxygen volume percentage reduction process involved in step S103 is completed within 30 minutes to form a step physical stimulus of oxygen partial pressure targeting adipose stem cells and induce the activation of the hypoxia-inducible factor pathway.

[0026] Preferably, the Rho-related kinase inhibitor is Y-27632; and in step S103, inhibiting the Rho-related kinase pathway through Y-27632 directly relieves the mechanical tension of the cytoskeleton, assisting vitamin E to overcome steric hindrance and deeply anchor to the hydrophobic tail of the exposed cell membrane lipid bilayer.

[0027] Preferably, after step S104, a passage step is included: when the cell fusion reaches 90%, the adipose stem cells are dissociated using a digestive solution without animal-derived components, and passaged at an expansion ratio of 1:3 to 1:5. Then, steps S101 to S104 are repeated until a predetermined number of adipose stem cells are obtained.

[0028] Preferably, the serum-free basal culture medium is further supplemented with CHIR99021 at a concentration of 1 μmol / L to 5 μmol / L and SB431542 at a concentration of 5 μmol / L to 20 μmol / L. By acting on functional cell surface receptors, it enhances the fidelity of stemness maintenance signals transmitted into the cell and blocks the differentiation pathway of adipose stem cells.

[0029] Preferably, step S104 involves the bioreactor culture conditions, including the following sub-steps: monitoring the real-time oxygen consumption rate of adipose stem cells, and adjusting the mixing ratio of nitrogen and air introduced into the culture container according to the oxygen consumption rate, so that the fluctuation range of the oxygen volume percentage involved in step S104 is within 0.5%.

[0030] Preferably, the amplification additive also includes recombinant human serum albumin at a concentration of 1 g / L to 5 g / L and recombinant human fibronectin at a concentration of 1 mg / L to 10 mg / L, for physically supporting the microenvironment of adipose-derived stem cells in vivo and providing osmotic pressure buffering in serum-free basal culture medium.

[0031] Preferably, during the continuous culture process in step S104, the glucose concentration in the serum-free basal medium is collected every 24 hours. When the glucose concentration is lower than 5 mmol / L, half of the serum-free basal medium is replaced, and the corresponding proportion of amplification additive is added simultaneously to maintain the chemical potential energy stability of the culture system.

[0032] A culture medium for promoting the efficient expansion of adipose-derived stem cells, wherein the culture medium is prepared by a culture method for promoting the efficient expansion of adipose-derived stem cells.

[0033] Example 1: In industrial deployment scenarios involving large-scale serum-free expansion of adipose-derived stem cells, due to the absence of the physical buffering and free radical scavenging mechanisms provided by albumin and lipoproteins in fetal bovine serum, the culture system experiences abrupt changes in culture environment parameters during the step transition window from normoxic to hypoxic environments. For example, when the ambient oxygen partial pressure decreases from 21%,... A sharp drop to 1% to 10% When the preset range is reached, adipose stem cells in a serum-free system experience a surge in transient hypoxic oxidative stress. This mutation in the physical environment induces passive contraction and lipid peroxidation of the cell membrane lipid bilayer, which in turn triggers conformational invagination and inactivation of transmembrane receptors. This transforms the hypoxia-proliferation strategy into an inducer of autophagy and premature aging proliferation arrest after the removal of serum protein protection.

[0034] To address the membrane tension and oxidative stress coupled damage caused by the oxygen partial pressure step, the method provided by this invention, in practical operation, involves adding a specific ingredient to the serum-free basal culture medium within the critical window of 2 to 6 hours during the cell transition from normoxic to hypoxic phase. An amplification additive consisting of a related kinase inhibitor and a redox buffer component was used to inhibit Y-27632. Related kinase pathways unlink the cross-linking contractions of actin and myosin in the cytoskeleton, enabling adipose-derived stem cells to maintain a physically relaxed cell membrane conformation in a serum-free state. Under this physically relaxed membrane surface topology, lipid-soluble vitamin E overcomes steric hindrance and deeply anchors to the hydrophobic tail of the exposed cell membrane lipid bilayer. Combined with water-soluble 2-mercaptoethanol and ethanolamine, a continuous reduction potential barrier is constructed on the cell periphery. This synergistic effect of Y-27632-mediated membrane physical relaxation and targeted anchoring of reducing components constructs a dynamic physical-redox coupling buffer interface in situ on the surface of adipose-derived stem cells. This interface counteracts transient oxidative stress and membrane tension distortion during oxygen partial pressure step decreases, maintaining the cell membrane's... receptors and The spatial conformational integrity of the pathway receptors is not affected by this physical relaxation mechanism, which does not cause macroscopic membrane rupture. Instead, it is due to the mechanical uncoupling of cortical actin with the inner lobe of the plasma membrane, which induces a surge in the lateral fluidity of local lipid molecules and generates nanoscale transient free volume expansion. Thus, while maintaining the macroscopic mechanical structure of the cell membrane, hydrophobic anchor microregions for the free embedding of antioxidant lipid-soluble molecules are briefly exposed at the microscale.

[0035] By blocking the lipid peroxidation chain reaction through the aforementioned physical-redox coupling buffer interface, CHIR99021 and SB431542 in the culture medium continuously act on cell surface receptors, transducing stemness maintenance signals into the cell and blocking the differentiation pathway. This approach shifts the mechanism for dealing with reactive oxygen species from passive intracellular scavenging to extracellular physical shielding based on membrane tension regulation, enabling the system to withstand 1% to 10% of reactive oxygen species. Step physical stimulation activates the hypoxia-inducible factor pathway, thus avoiding telomere shortening and organelle autophagy; after five consecutive generations of amplification, it is in a state of... The experimental group cells of the generation exhibited highly uniform phenotypic characteristics, and their surface markers , and The expression rate remained above 98%, and the positive rate in aging detection was at a low value of 5.5%, which reflects the effect of improving the proliferation rate and generational stability of adipose stem cells under the condition of no heterologous protein constraint. Its core lies in reconstructing the cell's adaptability to environmental mutations by strengthening the underlying physical barrier function of the cell.

[0036] Example 2: In the experimental scenario verifying the impact of the culture method on the expansion stability of adipose-derived stem cells, a three-gas incubator was used to construct an oxygen partial pressure step-switching environment. The human adipose-derived stem cells used in the experiment were obtained from ex vivo adipose tissue through authorized channels and isolated to the third generation via collagenase digestion. The temperature control accuracy of the experimental platform was better than ±0.1℃, and the concentration adjustment resolution of carbon dioxide and oxygen was not less than 0.1%, to simulate the environmental parameter fluctuation characteristics of an industrial-grade bioreactor during large-scale culture. When determining the addition window period of the expansion additive, the addition window period was set in the range of 2h to 6h by balancing the stress adaptation time of cells to physical environmental mutations and the accumulation rate of intracellular reactive oxygen species. If the addition window period was earlier than 2h, the cells had not yet completed the transcriptome pre-regulation from normoxic adhesion to hypoxic metabolism. If it was later than 6h, the lipid peroxidation damage induced by oxidative stress was in an irreversible state. A gas flow rate fluctuation perturbation with a signal-to-noise ratio of 25dB was actively introduced at the input end of the culture environment to verify the stability of the method under non-ideal conditions.

[0037] The experiment involved setting up multiple control groups to confirm the synergistic effects and parameter boundaries between components. The experimental group used serum-free basal medium supplemented with 10 The complete formulation consisted of 1 mol / L Y-27632, 2 μmol / L vitamin E, 50 μmol / L 2-mercaptoethanol, and 10 μmol / L ethanolamine. Control group 1 removed Y-27632, control group 2 removed the redox buffer components (vitamin E, 2-mercaptoethanol, and ethanolamine), control group 3 had an oxygen partial pressure (OPP) of 0.5%, and control group 4 had an OPP of 15%. At 4 hours after the OPP step, the level of cell membrane lipid peroxidation was measured using a malondialdehyde (MDA) assay kit as an intermediate indicator. The MDA content in control group 1 increased from 1.12 nmol / mgprot in the normoxic state to 4.56 nmol / mgprot, while in the experimental group, with the assistance of Y-27632-induced membrane physical relaxation, the lipid-soluble reducing components achieved efficient intercalation, and the MDA content remained at 1.78 nmol / mgprot. This indicates... When the cell membrane is in a state of high tension contraction, the single reducing component cannot block the peroxidation chain reaction due to the steric hindrance of the phospholipid head. The physical shielding channel is opened by relying on Y-27632 to relieve the tension of the cytoskeleton. More importantly, the large amount of high-density industrial flow data in the early stage clearly defined the boundary: when facing the oxidative pressure of large-scale system, vitamin E can only cover the continuously generated microscopic membrane defect sites by increasing and maintaining it to a threshold of more than 10 μg / mL. However, once it exceeds the limit of 50 μg / mL, it induces abnormal differentiation due to the deep intervention of the physical rigidity of the lipid raft region. Similarly, water-soluble 2-mercaptoethanol needs to be maintained in the high range of 2 mmol / L to 4 mmol / L in a dynamically stirred medium to have sufficient electron donor redundancy to resist dissipation. This is different from the extremely small molar amount required for the early culture of small bodies. The effective working parameter window of this invention is anchored from the process tolerance scale.

[0038] In the validation scenario of optimizing the concentration range of signaling pathway regulatory components in the culture medium, since CHIR99021's inhibitory effect on GSK-3β kinase and SB431542's blocking effect on TGF-β receptor signaling have concentration-sensitive thresholds, the effective range of the components was established by setting a gradient experiment. When the concentration of CHIR99021 is below 2... At concentrations of 15 mol / L, the activation intensity of signaling pathways is insufficient to inhibit cell differentiation into non-stem cells, while at concentrations higher than 15 mol / L... At concentrations of 1 μmol / L, SB431542 induces cell cycle arrest and reduces amplification efficiency. Correspondingly, regarding the dosage of SB431542, concentrations below 1 μmol / L cannot maintain the polygonal phenotype of the cell population, while concentrations above 10 μmol / L exert inhibitory pressure on cell metabolism. Experimental results confirmed that CHIR99021 concentrations were between 2 and 15 μmol / L and SB431542 concentrations were between 1 and 10 μmol / L. The mol / L ratio range represents the optimal window for maintaining the balance between stemness and proliferation rate of adipose-derived stem cells.

[0039] The validation data regarding the oxygen partial pressure boundary exhibited non-linear characteristics. When the oxygen partial pressure was 0.5%, it fell below the physical limit for adipose stem cells to maintain aerobic respiration, inhibiting pyruvate dehydrogenase activity and leading to lactate accumulation. The cell doubling time increased from 32.5 h in the experimental group to 58.2 h, indicating a metabolic stall state. When the oxygen partial pressure was 15%, the stable level of hypoxia-inducible factor HIF-1α decreased, preventing the initiation of downstream proliferative gene expression. The cell colony formation rate was 18.5%, lower than the 36.2% in the experimental group. After five consecutive generations of expansion, the doubling time of cells in the experimental group at the P8 generation stabilized within the range of 31.8 h to 33.5 h, and the cell diameter uniformity remained at 15.6 mm. Furthermore, the positive rate of staining for aging markers was 5.2%, while the positive rate of control group 2 was 22.4% in the absence of a reduction barrier. This demonstrated that the physical-redox coupling shielding compensated for the buffer deficiency in the serum-free system during the environmental mutation window.

[0040] Example 3: In a 10L stirred bioreactor used for serum-free expansion of adipose-derived stem cells, when the system faces a situation where 21% Normal oxygen environment to 5% When switching to a low oxygen target value, the increase in the culture system volume causes a physical time lag in the oxygen partial pressure gradient in the liquid phase. To address this condition, the real-time density of adipose-derived stem cells is obtained using an online cell counter. And according to density Determine the target concentration of Y-27632 in the amplification additive. The concentration of Y-27632 With real-time density Satisfies a linear relationship: ,in, This refers to the molar concentration of Y-27632 in serum-free basal medium. This represents the real-time density of adipose-derived stem cells. This is the cell membrane stress regulation coefficient, in a 10L reactor scale. The value is 2.5 mol mL / ( cells).

[0041] After the amplification additive was added to the culture system, a microfluidic impedance analysis unit integrated into the sampling loop was used to monitor the physical state of the cell membrane. The normalized membrane capacitance value of the cells at a frequency of 100 kHz was measured to characterize the change in membrane tension. This capacitance value was defined as the membrane relaxation index. When the membrane relaxation index When three consecutive sampling points show an increase and reach 1.25 times the initial value, the adipose-derived stem cells are determined to have entered a preset physical relaxation conformation. The oxygen partial pressure step procedure is then initiated. The system adjusts the output voltage of the inlet mass flow controller based on the real-time oxygen partial pressure change rate fed back from the liquid phase dissolved oxygen electrode, driving the oxygen partial pressure to decrease at a constant slope of 1% to 2% per minute. This allows the reduced component, vitamin E, to in-situ integrate with the lipid bilayer topology, and maintains a continuous electron donor pool for 2-mercaptoethanol in the extracellular fluid. The microfluidic impedance analysis unit measurement process includes the system opening a bypass valve according to a set sampling cycle to introduce a suspension containing adipose-derived stem cell culture into the impedance detection microcavity. Within the microcavity, a microcolumn array physically intercepts suspended cells, and a micropump continuously flushes and replaces the original high-salt culture medium with isotonic, low-conductivity sucrose buffer. When the background conductivity within the microcavity drops below a preset baseline, an AC excitation signal with a frequency of 100 kHz is applied to the intercepted cell population to collect raw membrane capacitance data. Based on the raw membrane capacitance data, the membrane relaxation index is calculated and output. After a single acquisition cycle is completed, the basal culture medium is pumped in reverse to return the test cells to the main chamber of the stirred bioreactor. Because the absolute volume of the test cell suspension for bypass interception analysis is limited to an extremely low nanoliter level, the residual sucrose molecules brought in after the return operation are instantly diluted to a massive scale upon entering the 10L main chamber, far below the physical critical concentration that would induce hyperosmolar autophagy or ion depletion in the main system cells. Furthermore, this extremely small quantum group can spontaneously rebuild the transmembrane potential within seconds after entering the main culture medium rich in macromolecular resident proteins, avoiding the risk of local disturbances being transmitted to the global reactor environment.

[0042] Under the above calibration procedure, the density of adipose-derived stem cells Depend on cells / mL increased to During the cells / mL process Subsequently from 5 Dynamically adjust to 20 mol / L To achieve a balance between stress shielding capacity and cell biomass, the experimental group maintained a cell damage index of 4.2% within 24 hours after oxygen partial pressure step cut, as monitored by the lactate dehydrogenase release rate in the culture medium. In contrast, the control group (without dynamic parameter adjustment) showed a damage index of 18.6%. This 10L reactor system, without fetal bovine serum support, maintained a doubling rate of 5.2 times for adipose-derived stem cell populations, with a total cell output per batch reaching [missing value]. One, and cell surface markers The positive rate was 99.2%, thus confirming the effectiveness of establishing a membrane relaxation index-based system. The feedback regulation loop and component ratio method enable serum-free culture protocols to maintain cell quality stability in large-scale industrial equipment.

[0043] Example 4: In industrial applications involving heterologous adaptation of human adipose stem cells from different donor sources, due to biological differences in initial cell activation state and membrane fluidity, it is necessary to perform membrane tension benchmark calibration on each batch of raw materials. This involves initial seeding density calibration at a constant temperature of 37°C. cells / mL to Cell populations of cells / mL were analyzed using impedance scanning at 100 kHz to obtain the normalized membrane capacitance reference value in the initial state. The baseline fluctuation rate was monitored for three cycles with a sampling period of 15 minutes to confirm that it was less than 2%. After confirming that the cells were in a stable adherent phase, a concentration gradient of 2 was introduced into the culture system. mol / L, 5 mol / L and 10 A preliminary experimental group of Y-27632 at mol / L was used to record the membrane relaxation index at each gradient. The response times corresponding to 1.10, 1.20, and 1.30 are achieved. The stress regulation coefficient of this batch of cells was obtained by fitting the data using the least squares method. This will increase the concentration in subsequent amplification procedures. The command output covers the conformational transition requirements of this batch of cells. The membrane relaxation index, when internally calculated, follows a dimensionality reduction criterion: the absolute value of the transient high-frequency capacitive response of the same population after real-time inhibitor addition is used as the numerator, divided by the normoxic static initial baseline value obtained from the batch's data acquisition, which is used as the denominator. This outputs a single dimensionless ratio characteristic that eliminates interference from the initial cell population's background size, thus determining the stress adjustment coefficient. The data processing steps include: The data module acquires three pre-experimental sample groups, sets the inhibitor concentration, and establishes the corresponding conformational change response time at each concentration to construct a two-dimensional data matrix. Using a response time shorter than fifteen minutes as a boundary condition, the least squares method is applied to obtain a linearly fitted line for the two-dimensional data matrix. The slope parameter of the fitted line is extracted, and its reciprocal is calculated. This reciprocal slope is then assigned as the specific stress regulation coefficient for the current batch of adipose-derived stem cells. The calculation unit uses the control bus to set the reciprocal value as a quantitative physical parameter for adjusting the pumping rate of the feed device. Before executing the final assignment instruction, the calculation unit calls the single-cell volume reference constant calibrated in the system as a conversion factor to perform multiplication compensation processing on the reciprocal value. Through this dimensional normalization step, the interference caused by the time attribute extracted from the fitted curve is eliminated, and based on the standard mathematical dimension of its concentration to cell density ratio, the dimensional symmetry of the equation is ensured when it is substituted into the subsequent flow rate derivation formula.

[0044] When the system faces fluctuations in culture medium osmotic pressure or changes in mass transfer efficiency due to batch component adjustments, in order to maintain the structural integrity of the physical-redox coupling buffer interface, it is necessary to determine the dissolved oxygen transfer coefficient at the specific culture medium viscosity using a standardized dissolved oxygen electrode response time calibration method before field deployment. and according to The measured values ​​are used to compensate the adjustment parameters of the mass flow controller, so that the root mean square error between the measured oxygen concentration change curve in the liquid phase during the oxygen partial pressure step cut-off and the preset linear slope curve is within 0.05%. Under this preset calibration procedure, the membrane physical relaxation induced by Y-27632 and the interfacial anchoring of reducing components vitamin E, 2-mercaptoethanol and ethanolamine are in a state of temporal overlap. After 120 hours of continuous culture, the cell body diameter of adipose stem cells is distributed in the range of 14.2μm to 16.8μm, and the doubling time difference of the cell population is within 1.5 hours under control. When implementing operating condition compensation, the built-in algorithm of the system compares the measured transfer coefficient with the reference aqueous phase mass transfer constant and extracts the logarithmic offset value. This quantified offset value is set as the basic proportional gain amplification multiplier of the feedforward controller, thereby adaptively amplifying the transient air inlet flux to forcibly overcome the mass transfer time lag caused by liquid phase viscosity.

[0045] In the standardized preparation process for the finished culture medium, the above-mentioned amplification additive components are added in proportion to serum-free basal medium preheated to 37°C, and the pH of the system is controlled within the range of 7.2 to 7.4. Homogenization is then performed for 10 minutes at 50 rpm using a magnetic stirring system, allowing the fat-soluble vitamin E to form stable micron-sized dispersed micelles with the assistance of the surfactant ethanolamine. A pore size of 0.22 mm is used. The mixture is filtered by a sterile filter to remove particulate interference. The prepared culture medium is used within 24 hours to maintain the stability of the reduction potential of 2-mercaptoethanol in the liquid phase. This pre-treatment physical homogenization and sterilization procedure provides a material basis for the subsequent construction of a physical-redox coupling buffer interface during the oxygen partial pressure step cleavage window. Under this method, the adipose stem cells produced exhibit stable phenotypic characteristics.

[0046] Example 5: In an expansion scenario involving metabolic synchronization of adipose-derived stem cells from different donor batches, differences in initial cellular metabolic rates can cause deviations in the triggering timing of the transition from normoxic to hypoxic environments. An online biochemical sensor is used to monitor the residual glucose concentration in the serum-free basal culture medium in real time and to adjust the glucose concentration based on real-time cell density. The glucose consumption rate (GCR) per unit cell is derived from the conversion; the glucose consumption rate (GCR) per unit cell is determined by the formula: ,in, This refers to the rate of glucose consumption per unit cell. For the first glucose concentration at the time of the second sampling The glucose concentration at the (i+1)th sampling time. For real-time cell density, The time interval between two consecutive sampling periods is defined as the period when the glucose consumption rate (GCR) per unit cell remains stable at 0.18 for three consecutive sampling periods. When the cell confluence reaches 80% within the specified range, the process is initiated by... The procedure for transitioning from normoxic conditions to a hypoxic target value.

[0047] When the culture method is applied to a stirred bioreactor and faces component diffusion lag caused by high-viscosity serum-free media, the kinematic viscosity of the culture medium, as pre-calibrated, is used to determine the optimal conditions. Establish the injection compensation method, if the kinematic viscosity Greater than 1.2 The addition of Y-27632 to the amplification additive was set at 180 min before the oxygen partial pressure cut command, and the dynamic release rate of lactate dehydrogenase in the culture medium was monitored simultaneously. If the lactate dehydrogenase release rate exceeded 5% within the first 10 min of the oxygen partial pressure step, the addition ratio of 2-mercaptoethanol was increased by 10% until the lactate dehydrogenase release rate returned to a controlled range below 2%. Adipose-derived stem cells entering the P8 generation showed a doubling time of 31.5 h and 98.5% [percentage missing]. Surface marker expression rate.

[0048] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. A culture method of a culture medium for promoting efficient expansion of adipose-derived stem cells, characterized by, Includes the following steps: Step S101: Adipose stem cells are seeded in serum-free basal culture medium and cultured under normoxic adherent conditions at a temperature of 37°C, a carbon dioxide volume percentage of 5%, and an oxygen volume percentage of 21% for 1 to 2 days. Step S102: Add an amplification additive consisting of a Rho-associated kinase inhibitor, a lipid-soluble reducing component vitamin E, and a water-soluble reducing component composed of 2-mercaptoethanol and ethanolamine to the serum-free basal medium, and make the final concentration of the Rho-associated kinase inhibitor in the serum-free basal medium reach 5 μmol / L to 20 μmol / L. Step S103: Within a time window of 2 to 6 hours after adding the amplification additive, the inhibitory effect of Rho-related kinase inhibitors on the cross-linking contraction of actin and myosin in the adipose stem cell cytoskeleton is utilized to relieve cytoskeleton tension, thereby placing the cell membrane in a physically relaxed conformation with reduced surface tension, and the oxygen volume percentage in the culture environment is reduced from 21% to 1% to 10%. Step S104: Utilizing the hydrophobic tail of the cell membrane lipid bilayer in a physically relaxed conformation, vitamin E is induced to intercalate to the hydrophobic tail. In conjunction with water-soluble reducing components, a continuous reduction potential barrier is constructed around the cell to eliminate transient reactive oxygen species induced by a step decrease in oxygen partial pressure. The cells are then cultured continuously under hypoxic conditions with an oxygen volume percentage of 1% to 10% until the cell confluence reaches 80% to 90%.

2. The method for culturing a culture medium to promote the efficient expansion of adipose-derived stem cells according to claim 1, characterized in that, In step S102, the final concentration of vitamin E in the serum-free basal medium is 10 μg / mL to 50 μg / mL, the final concentration of 2-mercaptoethanol is 2 mmol / L to 4 mmol / L, and the final concentration of ethanolamine is 10 μmol / L to 20 μmol / L, thereby inducing a reduction potential and blocking the lipid peroxidation chain reaction in the serum-free basal medium.

3. The culture method of claim 1, wherein the culture medium is a medium for promoting efficient expansion of adipose tissue-derived stem cells. The seeding density of adipose-derived stem cells involved in step S101 is 2500 / Up to 10,000 / Furthermore, the oxygen volume percentage reduction process involved in step S103 is completed within 30 minutes to form a step physical stimulus of oxygen partial pressure targeting adipose stem cells and induce the activation of the hypoxia-inducible factor pathway.

4. The method for culturing a culture medium to promote the efficient expansion of adipose-derived stem cells according to claim 1, characterized in that, The Rho-related kinase inhibitor used is Y-27632; and in step S103, the inhibition of the Rho-related kinase pathway by Y-27632 directly relieves the mechanical tension of the cytoskeleton, helping vitamin E to overcome steric hindrance and deeply anchor to the hydrophobic tail of the exposed cell membrane lipid bilayer.

5. The method for culturing a culture medium to promote the efficient expansion of adipose-derived stem cells according to claim 1, characterized in that, Step S104 is followed by a passage step: when the cell fusion reaches 90%, the adipose stem cells are dissociated using a digestive solution without animal-derived components and passaged at an expansion ratio of 1:3 to 1:

5. Then, steps S101 to S104 are repeated until a predetermined number of adipose stem cells are obtained.

6. The method for culturing a culture medium to promote the efficient expansion of adipose-derived stem cells according to claim 1, characterized in that, The serum-free basal medium also contains CHIR99021 at concentrations of 1 μmol / L to 5 μmol / L and SB431542 at concentrations of 5 μmol / L to 20 μmol / L. By acting on functional cell surface receptors, it enhances the fidelity of stemness maintenance signals transmitted into the cell and blocks the differentiation pathway of adipose stem cells.

7. The method for culturing a culture medium to promote the efficient expansion of adipose-derived stem cells according to claim 1, characterized in that, Step S104 relates to the bioreactor culture conditions and includes the following sub-steps: monitoring the real-time oxygen consumption rate of adipose stem cells and adjusting the mixing ratio of nitrogen and air introduced into the culture container according to the oxygen consumption rate, so that the fluctuation range of the oxygen volume percentage involved in step S104 is within 0.5%.

8. The method for culturing a culture medium for promoting the efficient expansion of adipose-derived stem cells according to claim 1, characterized in that, The amplification additive also includes recombinant human serum albumin at concentrations of 1 g / L to 5 g / L and recombinant human fibronectin at concentrations of 1 mg / L to 10 mg / L, which are used to simulate the microenvironmental physical support of adipose-derived stem cells in vivo and provide osmotic pressure buffering in serum-free basal culture medium.

9. The method for culturing a culture medium to promote the efficient expansion of adipose-derived stem cells according to claim 1, characterized in that, During the continuous culture process in step S104, the glucose concentration in the serum-free basal medium was collected every 24 hours. When the glucose concentration was lower than 5 mmol / L, half of the serum-free basal medium was replaced, and the corresponding proportion of amplification additives was added simultaneously to maintain the chemical potential energy stability of the culture system.

10. A medium for promoting efficient expansion of adipose-derived stem cells, characterized by, The culture medium is prepared by the culture method of the culture medium for promoting the efficient expansion of adipose stem cells as described in claim 1.

Images