Three-dimensional cell culture bioreactor and culture method

Abstract

The application belongs to the technical field of biomedical engineering, and particularly relates to a three-dimensional cell culture bioreactor and a culture method, which comprises a membrane aeration oxygen supply module, a cell culture module, a culture solution module and a monitoring and control module; the membrane aeration oxygen supply module comprises a plurality of hollow fiber membranes and is configured to allow oxygen-containing gas to flow in the membranes and diffuse oxygen outside the membranes to a culture area; the cell culture module is configured to allow cells to be cultured in three dimensions outside the hollow fiber membranes; the culture solution module is configured to provide cell culture solution to the culture area; and the monitoring and control module comprises a plurality of sensors for real-time monitoring of culture parameters and feedback control based on the monitoring results. Through the synergistic effect of the bubble-free oxygen supply by the gas-permeable membrane and the reverse diffusion mass transfer structure, the oxygen supply efficiency is significantly improved, there is no shear damage, a real bionic microenvironment is constructed, the mass transfer process is independently controllable, the culture environment is long-term stable, and a practical technical path is provided for the transition of three-dimensional cell culture from laboratory scale to industrial application.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and more specifically, to a three-dimensional cell culture bioreactor and culture method. Background Technology

[0002] With the rapid development of tissue engineering, organoid construction, regenerative medicine, and biopharmaceuticals, three-dimensional cell culture technology has become a key supporting means for simulating the tissue microenvironment in vitro, drug screening, and large-scale production of cell therapy products. However, how to achieve high-density three-dimensional cell culture, especially ensuring the long-term stable maintenance of cell function and activity in the culture system, has become a core technological bottleneck restricting the transformation of these fields from laboratory research to industrial applications.

[0003] Currently, conventional three-dimensional cell culture systems mainly include static culture systems, stirred bioreactor culture systems, and perfusion culture systems. Static culture (such as T-flasks and multilayer cell factories) is simple to operate, but due to the lack of convection and active mass transfer, nutrients and oxygen mainly rely on diffusion, resulting in extremely low mass transfer efficiency and making it difficult to support high-density cell growth. Stirred bioreactors (such as stirred tank reactors and rotating wall reactors) create culture medium flow through mechanical stirring or rotation, enhancing mixing and mass transfer within the culture system. However, the shear force generated by stirring can easily cause irreversible damage to cells, especially shear-sensitive cells such as stem cells and hepatocytes, limiting its applicability. Perfusion culture systems continuously or intermittently pump fresh culture medium into the reactor and drain the used medium, which can improve nutrient supply and metabolic waste removal to some extent. However, the consumption of culture medium is large, the utilization rate is low, and the equipment is complex and the operating cost is high, making it difficult to meet economic requirements.

[0004] In the aforementioned culture systems, oxygen supply remains one of the most critical issues limiting high-density three-dimensional cell culture. Existing systems typically rely on liquid-phase oxygen transfer within the culture medium to meet cellular respiration requirements. However, when cell density increases to a certain level (e.g., 10⁻⁶), oxygen supply becomes increasingly critical. 6 When the oxygen consumption rate per unit volume of cells exceeds a certain threshold (above a certain value), the oxygen diffusion coefficient in the culture medium is relatively low (approximately 2 × 10⁻⁶). -5 The effective diffusion distance is typically only a few hundred micrometers (cm² / s). This physical limitation leads to severe oxygen concentration gradients inside the reactor, especially in the central region of the three-dimensional scaffold or cell aggregates, creating a localized hypoxic microenvironment. This, in turn, triggers a series of problems such as cell necrosis, mitochondrial dysfunction, abnormal metabolic reprogramming, and downregulation of key functional protein expression, severely affecting culture quality and the reliability of experimental data.

[0005] To address the problem of insufficient oxygen supply, several improvement strategies have been proposed in this field. Increasing the circulation rate of the culture medium can enhance convective transport to some extent, but the potential for further increases is limited by the shear tolerance threshold of cells. Direct bubbling aeration into the culture medium can significantly increase dissolved oxygen concentration, but the shear force generated by bubble rupture and the surface tension at the gas-liquid interface have a significant damaging effect on adherent cells in suspended cell and microcarrier culture systems. Furthermore, the presence of numerous bubbles can interfere with light microscopy observation and online monitoring. While increasing the gas-liquid contact area by using a permeable culture flask bottom or a permeable membrane can improve oxygen supply under static culture conditions, its oxygen supply rate is limited by the membrane area and the oxygen partial pressure difference across the membrane. Moreover, as the culture scale increases, the specific surface area decreases sharply, failing to meet the oxygen supply requirements of large-scale, high-density culture. In addition, all the above-mentioned aeration methods suffer from large local fluctuations in oxygen concentration, failing to stably simulate the physiological process of continuous and uniform oxygen supply from the capillary network to surrounding tissues in vivo, which is detrimental to the construction of a biomimetic oxygen microenvironment.

[0006] It is worth noting that the membrane aerated biofilm reactor (MABR) is a relatively mature new bioreactor technology in the field of wastewater treatment. Its core working principle is as follows: oxygen diffuses through the permeable hollow fiber membrane wall into the biofilm attached to the outer surface, while organic substrates and nutrients are transferred from the bulk liquid phase into the biofilm. The mass transfer directions of oxygen and substrate are opposite, forming a typical reverse diffusion mass transfer structure. In this mass transfer mode, the concentration gradients of oxygen and nutrients are opposite, with a higher oxygen concentration and a lower substrate concentration near the membrane wall, and vice versa near the liquid phase, thus achieving a zoned distribution of active components within the biofilm. MABR has significant technical advantages such as high oxygen utilization (up to 80%~100%), stable oxygen supply, no need for bubbling, low energy consumption, and a large membrane specific surface area. However, to date, the application scenarios of MABR technology have been mainly limited to microbial nitrification, denitrification, and organic pollutant degradation in wastewater treatment. There are no systematic design schemes to transfer its reverse diffusion oxygen supply and mass transfer mechanism to three-dimensional culture of mammalian cells, large-scale expansion of organoids, or culture of tissue engineering constructs. Due to fundamental differences between mammalian cells and microorganisms in culture conditions (temperature, pH, osmotic pressure, shear tolerance), metabolic characteristics (oxygen demand, nutrient requirements, types of metabolites), and culture cycles, existing system configurations and operating parameters of MABR in wastewater treatment cannot be directly transferred. This technological gap means that proposing this application direction is far from an obvious and simple conversion.

[0007] Furthermore, in existing three-dimensional cell culture processes, the culture medium supply strategy mostly employs batch culture or semi-continuous medium exchange methods. During culture, cells continuously consume key nutrients such as glucose, amino acids, vitamins, and growth factors, while simultaneously releasing lactic acid, ammonia, and other acidic metabolic byproducts into the culture system. As the culture time extends, nutrients are gradually depleted while metabolic waste accumulates, leading to a decrease in pH, an increase in osmotic pressure, and nutrient deficiency in the culture environment, severely inhibiting cell proliferation and functional expression. To maintain cell viability, operators typically need to frequently replace large amounts of culture medium, which not only significantly increases culture costs (especially serum-containing media or serum-free media containing specific growth factors are expensive), but also the instantaneous and drastic fluctuations in temperature, pH, and biochemical composition caused by medium exchange operations can easily cause stress to cells, which is detrimental to long-term stable culture. Although perfusion culture systems have been developed to achieve continuous inflow and outflow of culture medium, most existing perfusion systems are only "one-way" or "partially circulating," lacking the function of selectively removing metabolic waste and accurately replenishing key nutrients in the outflowing culture medium for recycling and reuse. A large amount of culture medium containing usable components is directly discarded, resulting in low overall utilization of the culture medium and making it difficult to achieve the multiple goals of high-density culture, stable oxygen supply, and efficient utilization of culture medium.

[0008] In summary, the current field of three-dimensional cell culture technology urgently needs a novel bioreactor system and corresponding culture method that can simultaneously solve the problems of stable oxygen supply under high-density culture conditions, avoid shear damage, construct a biomimetic oxygen microenvironment, and realize the recycling of culture medium and dynamic removal of metabolic waste, in order to overcome the current technical bottlenecks restricting the industrialization of tissue engineering, organoids, and biomanufacturing. This invention is proposed based on the above-mentioned needs. Summary of the Invention

[0009] In view of this, the present invention addresses the deficiencies of the existing technology by providing a three-dimensional cell culture bioreactor and culture method that can simultaneously solve the problems of stable oxygen supply under high-density culture conditions, avoid shear damage, construct a biomimetic oxygen microenvironment, and realize the recycling of culture medium and dynamic removal of metabolic waste.

[0010] To solve the above problems, this application adopts the following technical solution: One objective of this application is to provide a three-dimensional cell culture bioreactor, comprising: A membrane aeration and oxygen supply module, comprising a plurality of hollow fiber membranes configured to allow oxygen-containing gas to flow within the membrane and to allow oxygen to diffuse through the membrane wall to the culture area outside the membrane. A cell culture module configured to allow cells to be cultured in three dimensions in the external space of the hollow fiber membrane; A culture medium module configured to provide cell culture medium to the culture region; The reactor is configured as a reverse diffusion mass transfer structure, wherein the direction of oxygen transfer from the interior of the hollow fiber membrane to the culture region is opposite to the direction of nutrient transfer from the culture medium to the culture region. The monitoring and control module includes several sensors for real-time monitoring of culture parameters and feedback control based on the monitoring results.

[0011] In some embodiments, the hollow fiber membrane is made of at least one of poly(4-methyl-1-pentene), polydimethylsiloxane, and polyurethane.

[0012] In some embodiments, the oxygen-containing gas is oxygen, air, or a mixture of oxygen and carbon dioxide.

[0013] In some embodiments, the cell culture medium contains at least one of glucose, amino acids, vitamins, growth factors, cytokines, serum, or serum-free culture medium components.

[0014] In some embodiments, the cells are selected from at least one of stem cells, hepatocytes, cardiomyocytes, endothelial cells, tumor cells, and nerve cells.

[0015] In some embodiments, in the cell culture module, cells grow by direct adhesion to the outer surface of the hollow fiber membrane, or by adhesion to the surface of microcarriers suspended in the culture medium.

[0016] In some embodiments, the sensors of the monitoring and control module include at least one of a dissolved oxygen sensor, a pH sensor, a temperature sensor, and a flow sensor.

[0017] In some embodiments, a culture medium recycling module is also included, which is connected to the cell culture module and is used to recycle the culture medium flowing out of the reactor and return it to the reactor.

[0018] In some embodiments, the culture medium recycling module includes: A cell / microcarrier retention module is used to retain cells or microcarriers in the reactor and allow the culture medium to enter the circulation loop; The metabolic waste removal and purification module is used to remove metabolic byproducts from the culture medium in the circulation loop. The nutrient solution replenishment module is used to replenish nutrients to the culture medium in the circulation loop.

[0019] In some embodiments, the metabolic byproducts include lactic acid and / or ammonia.

[0020] In some embodiments, the nutrients include at least one of glucose, amino acids, and growth factors.

[0021] A second objective of this application is to provide a three-dimensional cell culture method for the aforementioned reactor, comprising the following steps: Cells are seeded into the external space of the hollow fiber membrane of the reactor and supplied with cell culture medium for three-dimensional culture. Oxygen-containing gas is introduced into the hollow fiber membrane, allowing the oxygen to diffuse through the membrane wall into the external space; During the cultivation process, the monitoring and control module monitors the cultivation parameters in real time and performs feedback control. In this process, oxygen is transferred from inside the membrane to the culture area in the opposite direction to the transfer of nutrients from the culture medium to the culture area, forming a reverse diffusion mass transfer.

[0022] In some embodiments, a culture medium recycling step is also included: The culture medium flowing out of the reactor is drawn off, metabolic waste is removed and / or nutrients are added, and then it is returned to the reactor to continue to participate in the culture.

[0023] In some embodiments, the metabolic waste removal includes the removal of lactic acid and / or ammonia.

[0024] In some embodiments, the nutrient supplementation includes supplementation with glucose, amino acids, and / or growth factors.

[0025] The present application adopts the above technical solution, and its beneficial effects are as follows: The three-dimensional cell culture bioreactor provided in this application includes: a membrane aeration and oxygen supply module comprising several hollow fiber membranes configured to allow oxygen-containing gas to flow within the membranes and to diffuse oxygen through the membrane walls to the culture area outside the membranes; a cell culture module configured to allow cells to undergo three-dimensional culture in the external space of the hollow fiber membranes; a culture medium module configured to supply cell culture medium to the culture area; and a monitoring and control module comprising several sensors for real-time monitoring of culture parameters and feedback control based on the monitoring results. This application, through the synergistic effect of a breathable membrane with bubble-free oxygen supply and a reverse diffusion mass transfer structure, significantly improves oxygen supply efficiency, eliminates shear damage, realistically constructs a biomimetic microenvironment, provides independent and controllable mass transfer processes, and ensures long-term stability of the culture environment, providing a practical and feasible technical path for the industrial application of three-dimensional cell culture from laboratory scale. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic flowchart of the culture method for a three-dimensional cell culture bioreactor provided in an embodiment of the present invention.

[0028] Figure 2 This is a schematic diagram of the structure of a three-dimensional cell culture bioreactor provided in an embodiment of the present invention. Detailed Implementation

[0029] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified. To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0030] The three-dimensional cell culture bioreactor provided in this application includes: A membrane aeration and oxygen supply module, comprising a plurality of hollow fiber membranes configured to allow oxygen-containing gas to flow within the membrane and to allow oxygen to diffuse through the membrane wall to the culture area outside the membrane.

[0031] A cell culture module configured to allow cells to be cultured in three dimensions in the external space of the hollow fiber membrane.

[0032] A culture medium module configured to provide cell culture medium to the culture area.

[0033] The reactor is configured with a reverse diffusion mass transfer structure, which allows oxygen to be transferred from the interior of the hollow fiber membrane to the culture region in the opposite direction to the transfer of nutrients from the culture medium to the culture region.

[0034] The monitoring and control module includes several sensors for real-time monitoring of culture parameters and feedback control based on the monitoring results.

[0035] In this embodiment, the hollow fiber membrane is configured to allow oxygen-containing gas to flow within the membrane and diffuse through the membrane wall to the culture area outside the membrane. This allows oxygen to diffuse through the membrane wall into the culture medium and cell layer via molecular diffusion, rather than being directly bubbled into the culture system. This directly avoids the mechanical damage to cells caused by the severe shear force and surface tension of the gas-liquid interface resulting from bubble rupture in traditional bubbling aeration methods. The reverse diffusion mass transfer structure ensures that the direction of oxygen transfer from the inside of the hollow fiber membrane to the culture area is opposite to the direction of nutrient transfer from the culture medium to the culture area. This allows oxygen to be supplied from the membrane wall outwards, and nutrients to be supplied from the liquid phase to the cells, creating a complementary concentration gradient distribution within the cell layer. This mass transfer mechanism accurately simulates the physiological process of continuous oxygen supply from the capillary network to surrounding tissues in vivo. It can maintain a stable oxygen concentration gradient even in high-density cell aggregation areas (such as inside a three-dimensional scaffold or at the center of cell aggregates), effectively overcoming the "central hypoxic necrosis" problem caused by the low diffusion coefficient of oxygen in the culture medium and an effective diffusion distance of only a few hundred micrometers in traditional culture systems, making high-density three-dimensional culture possible.

[0036] Furthermore, the reverse diffusion structure allows the oxygen supply pathway (gas side → membrane side → cell side) and the nutrient supply pathway (liquid side → cell side) to form an orthogonal or reverse complementary layout in space. The technical advantage of this layout is that the oxygen supply rate no longer depends on the culture medium flow rate and dissolved oxygen concentration, but is independently regulated by the membrane material, membrane area, and intramembrane oxygen partial pressure; nutrient supply is independently regulated by the culture medium composition and circulation flow rate. After decoupling, these two aspects can be independently optimized according to the actual needs of the cells, avoiding the shear stress increase problem caused by the forced increase in flow rate to improve oxygen supply in traditional cultures. This achieves a "mild yet efficient" dual mass transfer objective, significantly improving the cell carrying capacity per unit volume of the reactor.

[0037] Furthermore, the monitoring and control module includes several sensors for real-time monitoring of culture parameters and feedback control based on the monitoring results. This enables the system to continuously monitor and dynamically adjust key culture conditions such as dissolved oxygen, pH, and temperature. When cell density increases, leading to increased oxygen consumption, the system can automatically increase the intramembrane oxygen partial pressure or gas flow rate. When pH changes, it can adjust the CO2 concentration or culture medium replenishment strategy accordingly. This technology ensures that the culture environment remains stable over long-term culture periods of several days or even weeks, avoiding the stress on cells caused by sudden and drastic fluctuations in temperature, pH, and biochemical components due to manual operation in batch culture or semi-continuous medium change methods. This provides a process control basis for the large-scale, standardized production of cell therapy products and tissue engineering constructs.

[0038] The three-dimensional cell culture bioreactor provided in the above embodiments of this application achieves the breakthrough technical effect of "long-term stable high-density cell culture" through the synergistic effect of two core features: "breathable membrane bubble-free oxygen supply" and "reverse diffusion mass transfer structure". Specifically, this effect manifests as: significantly improved oxygen supply efficiency (oxygen utilization rate can reach over 80%), no shear damage, realistic construction of a biomimetic microenvironment, independent and controllable mass transfer process, and long-term stable culture environment. This effectively solves the core technical challenges pointed out in the background art, such as "insufficient oxygen supply, shear damage, inability to simulate physiological oxygen supply, and fluctuations in the culture environment," providing a practical and feasible technical path for the transition of three-dimensional cell culture from laboratory scale to industrial application.

[0039] Specifically, the hollow fiber membrane is selected from at least one of poly(4-methyl-1-pentene), polydimethylsiloxane, and polyurethane. This embodiment uses a hydrophobic membrane material with high air permeability and good biocompatibility to ensure high oxygen mass transfer flux, no leakage during long-term operation, and no cytotoxicity.

[0040] Specifically, the oxygen-containing gas is oxygen, air, or a mixture of oxygen and carbon dioxide. By adjusting the gas composition (such as the CO2 ratio), the pH of the culture medium can be controlled simultaneously with oxygen supply, achieving "acid control while supplying gas".

[0041] Furthermore, the cell culture medium contains at least one of the following components: glucose, amino acids, vitamins, growth factors, cytokines, serum, or serum-free culture medium.

[0042] This is understandable, as it covers both serum-containing and serum-free systems, adapts to the nutritional needs of different cell types, and expands the system's versatility.

[0043] Furthermore, the cells are selected from at least one of stem cells, hepatocytes, cardiomyocytes, endothelial cells, tumor cells, and nerve cells.

[0044] Furthermore, in the cell culture module, cells either adhere directly to the outer surface of the hollow fiber membrane or adhere to the surface of a microcarrier suspended in the culture medium.

[0045] It is understood that the reactor provided in this embodiment is widely applicable to adherent and suspension cell lines, as well as primary and immortalized cell lines. It also takes into account different three-dimensional culture scenarios (such as tissue engineering scaffolds vs. microcarrier amplification) through two modes: "direct adhesion outside the membrane" or "microcarrier suspension".

[0046] Furthermore, the sensors of the monitoring and control module include at least one of a dissolved oxygen sensor, a pH sensor, a temperature sensor, and a flow sensor.

[0047] It is understandable that using multi-sensor integration to achieve multi-dimensional process monitoring provides key data support for precise feedback control and ensures long-term automated and stable operation of the system.

[0048] Furthermore, it also includes a culture medium recycling module, which is connected to the cell culture module and is used to recycle the culture medium flowing out of the reactor and return it to the reactor.

[0049] It is understood that this embodiment introduces a closed-loop circulation architecture, which allows the culture medium to be reused repeatedly, significantly reducing the total amount of culture medium consumed, especially for culture media containing expensive serum / growth factors, resulting in significant cost savings; it also provides a physical loop basis for the targeted removal of metabolic waste and the dynamic replenishment of nutrients.

[0050] Furthermore, the culture medium recycling module includes: A cell / microcarrier retention module is used to retain cells or microcarriers within the reactor and allow the culture medium to enter the circulation loop; thereby ensuring that high-value cells or microcarriers are always retained within the reactor during culture medium circulation, preventing cell loss and supporting continuous high-density culture.

[0051] The metabolic waste removal and purification module is used to remove metabolic byproducts from the culture medium in the circulation loop; thereby selectively removing inhibitory metabolic byproducts such as lactic acid and ammonia, effectively alleviating acidification of the culture environment and ammonia toxicity, delaying cell apoptosis and functional decline, and maintaining a clean culture environment without frequent medium changes.

[0052] The nutrient solution replenishment module is used to replenish nutrients in the culture medium in the circulation loop, thereby precisely replenishing glucose (energy substrate), amino acids (basic protein synthesis) and growth factors (signal regulation) according to the real-time consumption rate of cells, avoiding nutrient depletion and maintaining cells in the optimal state of growth and functional expression.

[0053] Furthermore, the metabolic byproducts include lactic acid and / or ammonia.

[0054] Furthermore, the nutrients include at least one of glucose, amino acids, and growth factors.

[0055] Furthermore, the cell / microcarrier retention module employs one or more combinations of a sieve separation structure, a membrane filtration structure, a cyclone separation structure, or a centrifugal separation structure; the metabolic waste removal module employs one or more combinations of an ultrafiltration membrane, a tangential flow filtration membrane (TFF), an adsorption packing material, an ion exchange material, or a dialysis membrane; the nutrient solution replenishment module includes a culture medium storage bottle, a metering pump, and an automatic control valve, which adjusts the amount of culture medium replenished according to parameters such as glucose, lactic acid, pH, and dissolved oxygen in the culture medium, thereby achieving culture medium recycling.

[0056] The three-dimensional cell culture bioreactor provided in this application transfers the mature reverse diffusion oxygen supply and mass transfer mechanism from the field of membrane aeration biofilm reactors to the three-dimensional culture system of mammalian cells. Oxygen is supplied directly to the culture area through the hollow fiber permeable membrane wall in the form of molecular diffusion, without generating any bubbles throughout the process. Compared with traditional bubbling aeration or high-flow-rate perfusion oxygen supply methods, this invention completely eliminates the risk of mechanical damage to cells caused by bubble rupture and turbulent shear force. It is particularly suitable for large-scale, high-density culture of shear-sensitive cells such as stem cells, hepatocytes, nerve cells, and cardiomyocytes, significantly improving cell viability and functional integrity.

[0057] This invention utilizes reverse diffusion through the membrane wall to supply oxygen, creating a stable oxygen concentration gradient that gradually decreases from the membrane surface outwards within the culture region. This precisely simulates the physiological process of continuous and uniform oxygen supply to surrounding tissues from the capillary network in vivo. This biomimetic oxygen microenvironment ensures that cells receive a sufficient and stable oxygen supply even within high-cell-density and thick-layered three-dimensional tissue constructs, effectively avoiding the central hypoxic necrosis and metabolic function degradation problems commonly found in traditional culture systems. Furthermore, this oxygen supply method is not limited by the flow rate or dissolved oxygen saturation of the culture medium, and can operate stably and continuously over long-term culture periods of several days to several weeks, providing a reliable culture platform for long-term experiments such as organoid maturation, tissue construct functionalization, and long-term drug safety evaluation.

[0058] This invention integrates a culture medium recycling system comprising a cell / microcarrier retention module, a metabolic waste removal and purification module, and a nutrient solution replenishment module. After the culture medium flows out of the reactor, the cells and microcarriers are effectively retained within the reactor, while the culture medium enters an external circulation loop for purification and regeneration, and is then returned to the reactor for continued culture. This closed-loop circulation mode allows the culture medium to be reused multiple times. Compared to traditional batch culture or unidirectional perfusion culture methods, the total consumption of culture medium can be significantly reduced. Especially for culture media containing expensive components such as serum, growth factors, and cytokines, it can significantly save culture costs and improve the overall economic feasibility of the process.

[0059] This invention selectively removes lactic acid, ammonia, and other inhibitory metabolic byproducts from the culture medium via a metabolic waste removal and purification module, while simultaneously replenishing key nutrients such as glucose, amino acids, and growth factors precisely based on the actual consumption rate of the cells via a nutrient replenishment module. This two-way regulation mechanism of "real-time waste removal and dynamic nutrient replenishment" ensures that key parameters of the culture environment within the reactor, such as pH, osmotic pressure, and nutrient concentration, are maintained within the optimal range over the long term, avoiding environmental fluctuations caused by frequent manual medium changes. Therefore, this invention possesses the capability to support long-term expansion of ultra-high-density cells, and its process exhibits strong controllability and batch-to-batch consistency, fully meeting the quality requirements for large-scale GMP production of cell therapy products, and providing crucial equipment support for the industrialization of regenerative medicine and cell therapy.

[0060] Please see Figure 1 This application also provides a three-dimensional cell culture method for the reactor, comprising the following steps: Cells are seeded into the external space of the hollow fiber membrane of the reactor and supplied with cell culture medium for three-dimensional culture. Oxygen-containing gas is introduced into the hollow fiber membrane, allowing the oxygen to diffuse through the membrane wall into the external space; During the cultivation process, the monitoring and control module monitors the cultivation parameters in real time and performs feedback control. In this process, oxygen is transferred from inside the membrane to the culture area in the opposite direction to the transfer of nutrients from the culture medium to the culture area, forming a reverse diffusion mass transfer.

[0061] In some embodiments, a culture medium recycling step is also included: The culture medium flowing out of the reactor is drawn off, metabolic waste is removed and / or nutrients are added, and then it is returned to the reactor to continue to participate in the culture.

[0062] Understandably, this scheme introduces an external intermittent circulating purification and regeneration process for the culture medium during the cultivation process. This involves the culture medium periodically flowing out of the reactor, undergoing metabolic waste removal and nutrient replenishment, and then being reintroduced into the culture system. This method avoids the environmental parameter fluctuations and pollution risks associated with frequent manual medium changes in traditional batch culture, while also overcoming the resource waste caused by single-use and large-scale disposal of the culture medium in unidirectional perfusion culture. It enables multiple recycling and reuse of the culture medium within the batch culture cycle, significantly reducing culture medium consumption costs while maintaining long-term chemical stability in the culture environment.

[0063] In some embodiments, the metabolic waste removal includes the removal of lactic acid and / or ammonia.

[0064] In some embodiments, the nutrient supplementation includes supplementation with glucose, amino acids, and / or growth factors.

[0065] It is understandable that by precisely removing specific metabolic wastes (lactic acid / ammonia) and replenishing specific nutrients, the chemical homeostasis (pH, osmotic pressure, key nutrient concentrations) of the reactor's microenvironment can be maintained for a significantly longer period, thereby supporting higher densities (e.g., >10). 7 Cell-scale expansion at the cell / mL level and over longer periods (weeks to months) directly meets the stringent requirements of GMP-compliant large-scale production of cell therapy products for batch-to-batch consistency and high viability.

[0066] The method described in this application fundamentally eliminates bubble shear force and protects shear-sensitive cells (corresponding to the bubbling damage problem in the background art) through "breathable membrane bubble-free oxygen supply"; it forms a stable oxygen gradient outside the membrane through "reverse diffusion structure", simulating the capillary oxygen supply mechanism in vivo, overcoming the physical limitation of short diffusion distance of oxygen in the culture medium (only a few hundred micrometers), and eliminating central hypoxic necrosis under high-density culture; it achieves independent regulation of oxygen supply rate and nutrient supply rate through spatial decoupling of oxygen supply path and nutrient supply path, avoiding shear damage caused by forced increase of flow rate to increase dissolved oxygen; and it maintains the dynamic stability of culture environment (DO, pH, temperature) in long-term culture through "real-time monitoring and feedback control", avoiding environmental fluctuations caused by manual medium change.

[0067] Please see Figure 2 One embodiment of this application provides a three-dimensional cell culture bioreactor, comprising: a reactor shell 1, a membrane aeration and oxygen supply module 2 (a hollow fiber membrane bundle), a gas supply system / gas source 3, a peristaltic pump / culture medium drive pump 4, a culture medium storage bottle 5, and a culture medium circulation and recovery module 6. The probes extending into the reactor on the right side of the figure are labeled with their monitoring functions using English abbreviations: DO - dissolved oxygen sensor, pH - pH sensor, t - temperature sensor. The following provides a detailed description of the reactor's operation: Target cells (stem cells, hepatocytes, etc. as described in claim 5) are seeded into the external space of the hollow fiber membrane bundle (2) inside the reactor shell (1). Depending on the culture method, the cells can directly adhere to the outer surface of the membrane or attach to the surface of microcarriers suspended in the culture medium. Fresh culture medium in the culture medium storage bottle (5) is pumped into the reactor by a peristaltic pump (4) to fill the outer space of the membrane with culture medium, thus establishing an initial three-dimensional culture system.

[0068] The gas supply system (3) is activated to introduce oxygen-containing gas (pure oxygen, air, or O2 / CO2 mixture) into the inner cavity of the hollow fiber membrane bundle (2). During the flow of oxygen inside the membrane, relying on the oxygen partial pressure difference across the membrane, oxygen diffuses through the dense or microporous membrane wall in the form of molecular diffusion and is continuously and bubble-free released to the culture area outside the membrane. At the same time, the peristaltic pump (4) drives the culture medium to flow from the culture medium circulation recovery module to the culture medium storage bottle, and nutrients (glucose, amino acids, etc.) in the culture medium are transferred from the liquid phase bulk to the cell surface.

[0069] The key mass transfer characteristics are as follows: oxygen is transferred in the direction of "inside the membrane → membrane wall → cell side" (from inside to outside), while nutrients are transferred in the direction of "liquid phase → cell side" (from outside to inside). These two directions are opposite, forming the aforementioned "reverse diffusion mass transfer structure." This structure simulates the oxygen supply mode of capillaries in vivo, forming a stable oxygen concentration gradient on the outer surface of the membrane (higher oxygen near the membrane and lower oxygen far from the membrane), ensuring that even the high-density cell layer can obtain sufficient oxygen.

[0070] During the cultivation process, the peristaltic pump (4) operates continuously, driving the culture medium to flow from the culture medium circulation recovery module to the culture medium storage bottle. The DO (dissolved oxygen) sensor, pH sensor, and temperature sensor (t) extending into the reactor on the right side monitor the cultivation environment parameters in real time and transmit the signals to the control system (not shown in the figure). Based on the real-time monitoring data, the control system automatically performs feedback regulation: DO sensor: Feedback to regulate the oxygen partial pressure or gas flow rate of the gas supply system (3) to maintain dissolved oxygen within the set range; pH sensor: can be used to adjust the CO2 ratio in the gas or trigger the addition of acid-base balance solution in the nutrient supplementation module. Temperature sensor (t): Linked to heating / cooling device to maintain constant temperature (e.g., 37℃).

[0071] The culture medium flowing out of the reactor shell (1) is not discarded directly, but enters the culture medium recycling module (6). This module performs the following processes in sequence: Cell / microcarrier retention: First, the cells or microcarriers in the culture medium are retained by a retention device (such as a screen or sedimentation chamber) and returned to the reactor to ensure that high-value nutrients are not lost, while the clarified culture medium enters the subsequent circulation loop. Metabolic waste removal and purification: The clarified culture medium enters the removal and purification unit, where inhibitory metabolic byproducts such as lactic acid and ammonia are selectively removed through membrane filtration, adsorption, or enzymatic hydrolysis, thereby reducing their toxicity to cells; Dynamic nutrient solution replenishment: The purified culture medium enters the replenishment unit, and based on real-time monitoring and a preset consumption model, it accurately replenishes the key depleted components such as glucose, amino acids, and growth factors.

[0072] The "regenerated culture medium" that has completed the above steps is then driven by a peristaltic pump (4) and returned to the inside of the reactor shell (1) to continue participating in cell culture, forming a closed loop of "outflow → cell retention → toxin removal → nutrient replenishment → reinfusion".

[0073] Understandably, throughout the process, reverse diffusion mass transfer ensures stable biomimetic oxygen supply under high-density culture, closed-loop recycling achieves efficient utilization of culture medium and maintenance of chemical homeostasis, and multi-sensor feedback control ensures automation and batch-to-batch consistency in long-term culture.

[0074] The technical solutions described above in this application will be explained in detail below with reference to specific embodiments.

[0075] Example 1: 1. In this embodiment, poly(4-methyl-1-pentene) (PMP) hollow fiber membrane is selected as the oxygen supply membrane material. The hollow fiber membrane has an outer diameter of 400 μm and an inner diameter of 200 μm. The hollow fiber membranes are arranged in parallel to form a membrane bundle structure and fixed inside the reactor shell. The oxygen supply module is connected to the gas supply system through a gas inlet.

[0076] 2. Before inoculation, the outer surface of the hollow fiber membrane was pretreated with rat tail type I collagen to improve cell adhesion. Then, a suspension of human hepatocytes was added to the culture chamber at a cell seeding density of 1×10⁻⁶ cells / mL. 6 cells / cm 2 After inoculation, the cells were incubated statically for 12 hours to allow them to fully adhere to the outer surface of the hollow fiber membrane. After 48 hours of culture, a continuous cell layer formed on the outer surface of the hollow fiber membrane.

[0077] 3. A mixed gas, consisting of 5% carbon dioxide and 95% air, is continuously introduced into the hollow fiber membrane. The gas flows inside the membrane and diffuses continuously outward through the PMP membrane wall, providing gas supply to the cells attached to the membrane surface while maintaining the acid-base balance of the culture system.

[0078] 4. DMEM medium was used as the cell culture medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture, as well as the corresponding growth factors required for cell culture. The culture temperature was maintained at 37℃, and the pH of the culture medium was maintained at 7.2-7.4.

[0079] 5. During the culture process, oxygen diffuses from the hollow fiber lumen to the outer cell layer, while glucose, amino acids, growth factors, and other nutrients in the culture medium diffuse from the bulk of the culture medium to the cell layer, thus forming a reverse diffusion mass transfer environment. This mass transfer mode simulates the physiological process of capillaries supplying oxygen to surrounding tissues in the body.

[0080] 6. The reactor integrates dissolved oxygen, pH, temperature, and flow sensors, and the system automatically collects operating data. When the dissolved oxygen concentration in the culture medium is lower than the set value, the oxygen supply flow rate is automatically increased; when the pH value is too low, the culture medium circulation and recovery flow rate is increased to maintain a stable culture environment.

[0081] 7. After 14 days of continuous culture, a uniform and continuous cell layer formed on the outer surface of the hollow fiber membrane, and the cell proliferation activity was significantly higher than that of the traditional static culture group. Compared with conventional culture methods, the membrane aeration reverse diffusion oxygen supply system constructed in this invention can significantly improve cell oxygen utilization efficiency and metabolic activity, achieving long-term stable culture of adherent cells.

[0082] Example 2: 1. In this embodiment, poly(4-methyl-1-pentene) (PMP) hollow fiber membrane is selected as the oxygen supply membrane material. The hollow fiber membrane has an outer diameter of 600 μm and an inner diameter of 400 μm. The hollow fiber membranes are arranged in parallel to form a membrane bundle structure and fixed inside the reactor shell. The oxygen supply module is connected to the gas supply system through a gas inlet.

[0083] 2. Before inoculation, the outer surface of the hollow fiber membrane was pretreated with rat tail type I collagen to improve cell adhesion. Then, a suspension of human hepatocytes was added to the culture chamber at a cell seeding density of 1×10⁻⁶ cells / mL. 6 cells / cm 2 After inoculation, the cells were incubated statically for 12 hours to allow them to fully adhere to the outer surface of the hollow fiber membrane. After 48 hours of culture, a continuous cell layer formed on the outer surface of the hollow fiber membrane.

[0084] 3. A mixed gas, consisting of 5% carbon dioxide and 95% air, is continuously introduced into the hollow fiber membrane. The gas flows inside the membrane and diffuses continuously outward through the PMP membrane wall, providing gas supply to the cells attached to the membrane surface while maintaining the acid-base balance of the culture system.

[0085] 4. DMEM medium was used as the cell culture medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture, as well as the corresponding growth factors required for cell culture. The culture temperature was maintained at 37℃, and the pH of the culture medium was maintained at 7.2-7.4.

[0086] 5. During the culture process, oxygen diffuses from the hollow fiber lumen to the outer cell layer, while glucose, amino acids, growth factors, and other nutrients in the culture medium diffuse from the bulk of the culture medium to the cell layer, thus forming a reverse diffusion mass transfer environment. This mass transfer mode simulates the physiological process of capillaries supplying oxygen to surrounding tissues in the body.

[0087] 6. The reactor integrates dissolved oxygen, pH, temperature, and flow sensors, and the system automatically collects operating data. When the dissolved oxygen concentration in the culture medium is lower than the set value, the oxygen supply flow rate is automatically increased; when the pH value is too low, the culture medium circulation and recovery flow rate is increased to maintain a stable culture environment.

[0088] 7. After 14 days of continuous culture, a uniform and continuous cell layer formed on the outer surface of the hollow fiber membrane, and the cell proliferation activity was significantly higher than that of the traditional static culture group. Compared with conventional culture methods, the membrane aeration reverse diffusion oxygen supply system constructed in this invention can significantly improve cell oxygen utilization efficiency and metabolic activity, achieving long-term stable culture of adherent cells.

[0089] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A three-dimensional cell culture bioreactor, characterized in that, include: A membrane aeration and oxygen supply module, comprising a plurality of hollow fiber membranes configured to allow oxygen-containing gas to flow within the membrane and to allow oxygen to diffuse through the membrane wall to the culture area outside the membrane. A cell culture module configured to allow cells to be cultured in three dimensions in the external space of the hollow fiber membrane; A culture medium module configured to provide cell culture medium to the culture region; The reactor is configured as a reverse diffusion mass transfer structure, wherein the direction of oxygen transfer from the interior of the hollow fiber membrane to the culture region is opposite to the direction of nutrient transfer from the culture medium to the culture region. The monitoring and control module includes several sensors for real-time monitoring of culture parameters and feedback control based on the monitoring results.

2. The reactor according to claim 1, characterized in that, The hollow fiber membrane is made of at least one of poly(4-methyl-1-pentene), polydimethylsiloxane, and polyurethane.

3. The reactor according to claim 1, characterized in that, The oxygen-containing gas is oxygen, air, or a mixture of oxygen and carbon dioxide.

4. The reactor according to claim 1, characterized in that, The cell culture medium contains at least one of the following components: glucose, amino acids, vitamins, growth factors, cytokines, serum, or serum-free culture medium.

5. The reactor according to claim 1, characterized in that, The cells are selected from at least one of stem cells, hepatocytes, cardiomyocytes, endothelial cells, tumor cells, and nerve cells.

6. The reactor according to claim 1, characterized in that, In the cell culture module, cells either adhere directly to the outer surface of the hollow fiber membrane or adhere to the surface of a microcarrier suspended in the culture medium.

7. The reactor according to claim 1, characterized in that, The sensors in the monitoring and control module include at least one of a dissolved oxygen sensor, a pH sensor, a temperature sensor, and a flow sensor.

8. The reactor according to any one of claims 1 to 7, characterized in that, It also includes a culture medium recycling module, which is connected to the cell culture module and is used to recycle the culture medium flowing out of the reactor and return it to the reactor.

9. The reactor according to claim 8, characterized in that, The culture medium recycling module includes: A cell / microcarrier retention module is used to retain cells or microcarriers in the reactor and allow the culture medium to enter the circulation loop; The metabolic waste removal and purification module is used to remove metabolic byproducts from the culture medium in the circulation loop. The nutrient solution replenishment module is used to replenish nutrients to the culture medium in the circulation loop.

10. The reactor according to claim 9, characterized in that, The metabolic byproducts include lactic acid and / or ammonia.

11. The reactor according to claim 9, characterized in that, The nutrients include at least one of glucose, amino acids, and growth factors.

12. A three-dimensional cell culture method using the reactor described in any one of claims 1 to 11, characterized in that, Includes the following steps: Cells are seeded into the external space of the hollow fiber membrane of the reactor and supplied with cell culture medium for three-dimensional culture. Oxygen-containing gas is introduced into the hollow fiber membrane, allowing the oxygen to diffuse through the membrane wall into the external space; During the cultivation process, the monitoring and control module monitors the cultivation parameters in real time and performs feedback control. In this process, oxygen is transferred from inside the membrane to the culture area in the opposite direction to the transfer of nutrients from the culture medium to the culture area, forming a reverse diffusion mass transfer.

13. The method according to claim 12, characterized in that, It also includes a culture medium recycling step: The culture medium flowing out of the reactor is drawn off, metabolic waste is removed and / or nutrients are added, and then it is returned to the reactor to continue to participate in the culture.

14. The method according to claim 13, characterized in that, The removal of metabolic waste includes the removal of lactic acid and / or ammonia.

15. The method according to claim 13, characterized in that, The nutrient supplementation includes the addition of glucose, amino acids, and / or growth factors.

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