Dynamic oxygen concentration regulation micro-fluidic chip for organoid culture and control method

Abstract

The invention belongs to the technical field of organoid chips, and discloses a dynamic oxygen concentration regulation micro-fluidic chip for organoid culture and a control method.The dynamic oxygen concentration regulation micro-fluidic chip comprises an upper layer structure, the upper layer structure comprises a plurality of independently-arranged culture modules, and the upper portions of the culture modules are attached and communicated with first channels; the bottom end of the culture module is attached to and communicated with a second channel; the lower-layer structure comprises a plurality of gas distribution channels attached to the lower end of the second channel, and the gas distribution channels correspond to and communicate with the culture modules; the gas supply structure comprises a gas supply module, the gas supply module is communicated with a plurality of independently arranged distribution channels, and the plurality of distribution channels are respectively communicated with the gas distribution channel; and the distribution channel is communicated with an adjusting channel for adjusting the oxygen concentration. The device is compact in structure and high in modularization degree, free culture of various different organoids can be achieved, dynamic oxygen concentration regulation and control can be achieved, oxygen environments in different physiological or pathological states in the body can be accurately simulated, and the physiological correlation and plasticity of the organoid model are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid chips, and in particular to a dynamic oxygen concentration control microfluidic chip and a control method thereof. Background Art

[0002] In recent years, organoids, as three-dimensional in vitro models that simulate the structure and function of human tissues, have been widely used in disease research, drug screening, and regenerative medicine. Compared with traditional two-dimensional cells, organoids are closer to the microenvironment of real organs, and the requirements for culture conditions are more refined, especially they are highly sensitive to oxygen concentration. There are significant differences in oxygen partial pressure in different organs of the human body. For example, organs such as the liver and intestines are in a relatively low oxygen environment, while the lungs and renal cortex are in a higher oxygen environment. When constructing a multi-organoid co-culture system in vitro, if a unified oxygen supply condition is used, it is often impossible to meet the personalized needs of different organoids for oxygen concentration, which may lead to abnormal organoid function or restricted development.

[0003] Most of the current organoid microfluidic chips use a constant gas ratio or passive diffusion method to maintain the oxygen concentration in the culture module; however, this type of organoid microfluidic control chip lacks a real-time feedback control mechanism and cannot adjust the oxygen concentration in time according to the metabolic changes of the organoids during the culture process; the control response speed is slow and the accuracy is low, making it difficult to adapt to the needs of the dynamic culture environment; at the same time, existing organoid chips are difficult to achieve multi-channel and differentiated regulation, which limits the development of multi-organ joint culture platforms.

[0004] Therefore, it is urgent to design a dynamic oxygen concentration regulation microfluidic chip and control method for organoid culture to solve the above technical problems. Summary of the Invention

[0005] The purpose of the present invention is to provide a microfluidic chip and control method for dynamic oxygen concentration regulation in organoid culture, so as to achieve differentiated, adjustable, and dynamically responsive oxygen concentration control of different organoid culture modules, simulate the complex in vivo oxygen gradient environment, and solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a microfluidic chip for dynamic oxygen concentration regulation in organoid culture, comprising:

[0007] an upper structure comprising a plurality of independently arranged culture modules for organoid culture, wherein the upper ends of the culture modules are fitted and connected to a first channel, and the lower ends of the culture modules are fitted and connected to a second channel;

[0008] a lower structure, the lower structure comprising a plurality of air distribution channels disposed at the lower end of the second channel, the plurality of air distribution channels corresponding to and communicating with the culture modules;

[0009] The air supply structure includes an air supply module, the air supply module is connected to a plurality of independently arranged distribution channels, and the plurality of distribution channels are respectively connected to the air distribution channels; the distribution channels are connected to a regulating channel for adjusting the oxygen concentration.

[0010] Preferably, the gas supply module includes a mixing chamber, in which a mixing assembly for mixing gases is provided; an air inlet of the mixing chamber is connected to a plurality of air inlet pipes, and the distribution channel is connected to an air outlet of the mixing chamber.

[0011] Preferably, the distribution channel includes a distribution pipe connected to the outlet of the mixing chamber, and the regulating channel is connected to the distribution pipe; the distribution pipe is connected to the corresponding air distribution channel through a distribution module, and the distribution module is connected to the regulating channel.

[0012] Preferably, the regulating channel includes a regulating pipe connected to the distribution pipe, and a regulating valve is provided on the regulating pipe, and the opening of the regulating valve is controlled by the control center.

[0013] Preferably, the distribution module includes a flow controller connected to the distribution pipe, and an oxygen concentration sensor is connected between the flow controller and the air distribution channel. The flow through the flow controller is controlled by the control center, and the oxygen concentration sensor measures the oxygen concentration in the distribution pipe, and the oxygen concentration sensor feeds back the oxygen concentration detection result to the control center.

[0014] Preferably, the culture module includes a culture frame made of biocompatible material, and a culture chamber isolated from the outside world is provided in the culture frame; the first channel, the second channel and the air distribution channel are respectively attached to the culture frame and connected to the culture chamber.

[0015] Preferably, an isolation membrane is provided between the gas distribution channel and the culture chamber, and the mixed gas in the gas distribution channel can pass through the isolation membrane and enter the culture chamber.

[0016] Preferably, a plurality of connecting channels are provided between the first channel and the culture chamber, and the connecting channels and the culture chamber are staggered to avoid direct flushing.

[0017] Preferably, vascular endothelial cells are planted in the second channel to form a microvascular barrier, and the substance in the second channel is connected to the culture chamber through the microvascular barrier.

[0018] The present invention also discloses a method for controlling dynamic oxygen concentration of a microfluidic chip based on dynamic oxygen concentration regulation for organoid culture, comprising the following steps:

[0019] Assembling the microfluidic chip for dynamic oxygen concentration control of organoid culture according to any one of claims 9;

[0020] The gas supply module is connected to the external gas source and introduces gases of different types and components to form the main gas;

[0021] Implanting the organoids to be cultured in the culture module, and injecting and circulating the culture medium into the culture module through the first channel and the second channel;

[0022] The main gas is supplied to the gas distribution channel through the distribution channel to provide the air required for organoid culture at a set flow rate, and the flow rate and oxygen content of the main gas are monitored in real time;

[0023] According to the oxygen content required for the culture of different organoids, inert gas is injected into the distribution channel through the regulating channel to adjust the oxygen content and achieve synchronous culture of different organoids.

[0024] Compared with the existing technology, the present invention has the following advantages and technical effects: the present invention discloses a dynamic oxygen concentration control microfluidic chip and a control method thereof, which realizes effective control of the oxygen concentration in the organoid culture environment through the coordinated design of the upper structure, the lower structure and the air supply structure; the upper structure is provided with a culture module, and the culture module is connected to the first channel and the second channel, allowing the culture of multiple different organoids to be carried out simultaneously on the same chip. Different organoids have different requirements for culture conditions such as oxygen concentration and nutrients. Independent culture modules, combined with their own channels, can realize the separate regulation of the culture environment of different organoids, meet diverse culture requirements, and help to carry out multi-organ co-culture experiments and simulate more complex in vivo environments; the gas supply module in the gas supply structure is connected to multiple independent distribution channels, and the distribution channels are connected to the regulating channels, which can adjust the oxygen concentration of the gas entering the gas distribution channel. Combined with the design of the corresponding connection between the gas distribution channels and the culture modules of the lower structure, the oxygen concentration of different areas in the culture module can be accurately adjusted according to the real-time needs of different organoid culture processes. At different stages of organoid growth, or when simulating different physiological and pathological states, it can provide a suitable oxygen concentration environment in time, providing more precise condition control for the growth, development and research of organoids; the lower structure The gas distribution channel is fitted at the lower end of the second channel and is connected to the culture module. This layout helps to evenly and efficiently transmit gas to the culture module, and can make the gas evenly distributed at the bottom of the culture module, avoiding uneven oxygen concentration, and ensuring that all parts of the organoid can obtain sufficient and stable oxygen supply during the culture process, thereby improving the consistency and reliability of the culture effect; the chip adopts a modular design, and each structure is relatively independent and cooperates with each other; independently set culture modules, distribution channels, etc., facilitate flexible adjustment of the type and quantity of cultured organoids and gas supply plans according to experimental needs; if the number of culture modules needs to be increased or the type of cultured organoids needs to be changed, only the corresponding modules and channels need to be adjusted, without the need for large-scale redesign of the entire chip, which greatly improves the flexibility of the experiment and the scalability of the chip, and reduces R&D and experimental costs.

[0025] The present invention has a compact structure and a high degree of modularity, which enables the free cultivation of a variety of different organoids, and can realize dynamic oxygen concentration regulation, accurately simulating the oxygen environment under different physiological or pathological conditions in the body, and significantly improving the physiological relevance and plasticity of the organoid model. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of this application. The exemplary embodiments and descriptions of this application are intended to explain this application and do not constitute an improper limitation on this application. In the accompanying drawings:

[0027] Figure 1 Schematic diagram of the microfluidic chip for dynamic oxygen concentration control of the present invention;

[0028] Figure 2 Schematic diagram of the upper structure of the present invention;

[0029] Figure 3 This is a schematic diagram of the gas supply structure of the present invention;

[0030] Figure 4 This is a control signal transmission diagram of the control center of the present invention;

[0031] In the figure: 1. Upper structure; 2. Lower structure; 3. Air supply structure; 4. Control center; 11. First channel; 12. Second channel; 13. Culture frame; 14. Culture chamber; 15. Connecting channel; 16. Microvascular barrier; 21. Gas distribution channel; 22. Isolation membrane; 31. Mixing chamber; 32. Mixing assembly; 33. Inlet pipe; 34. Inlet valve; 35. Distribution pipe; 36. Regulating pipe; 37. Regulating valve; 38. Flow controller; 39. Oxygen concentration sensor. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Reference Figure 1-Figure 4 As shown, this embodiment provides a dynamic oxygen concentration control microfluidic chip, including: an upper structure 1, the upper structure 1 includes several independently arranged culture modules for organoid culture, the upper part of the culture module is fitted and connected with a first channel 11, and the bottom end of the culture module is fitted and connected with a second channel 12; a lower structure 2, the lower structure 2 includes several gas distribution channels 21 fitted at the lower end of the second channel 12, and the several gas distribution channels 21 correspond to and are connected with the culture module; an air supply structure 3, the air supply structure 3 includes an air supply module, and the air supply module is connected with several independently arranged distribution channels, and the several distribution channels are respectively connected with the air distribution channels 21; the distribution channels are connected with a regulating channel for adjusting the oxygen concentration.

[0035] The present invention discloses a dynamic oxygen concentration control microfluidic chip and a control method thereof. Through the coordinated design of an upper structure 1, a lower structure 2 and an air supply structure 3, effective control of the oxygen concentration in an organoid culture environment is achieved; the upper structure 1 is provided with a culture module, which is connected to a first channel 11 and a second channel 12, allowing the simultaneous culture of multiple different organoids on the same chip. Different organoids have different requirements for culture conditions such as oxygen concentration and nutrients. Independent culture modules, combined with their own channels, can realize the separate regulation of the culture environment of different organoids, meet diverse culture requirements, and help to carry out multi-organ joint culture experiments and simulate more complex in vivo environments; the gas supply module in the gas supply structure 3 is connected to multiple independent distribution channels, and the distribution channels are connected to the adjustment channels, which can adjust the oxygen concentration of the gas entering the gas distribution channel 21. Combined with the design of the corresponding connection between the gas distribution channel 21 of the lower structure 2 and the culture module, the oxygen concentration in different areas of the culture module can be accurately adjusted according to the real-time needs of different organoid culture processes. At different stages of organoid growth, or when simulating different physiological and pathological states, a suitable oxygen concentration environment can be provided in time, providing more precise condition control for the growth, development and research of organoids; the lower structure 2 The gas distribution channel 21 is fitted at the lower end of the second channel 12 and is connected to the culture module. This layout helps to evenly and efficiently transmit the gas to the culture module, and can evenly distribute the gas at the bottom of the culture module, avoiding the situation of uneven oxygen concentration, ensuring that all parts of the organoid can obtain sufficient and stable oxygen supply during the culture process, and improving the consistency and reliability of the culture effect; the chip adopts a modular design, and each structure is relatively independent and cooperates with each other; the independently set culture modules, distribution channels, etc. are convenient for flexible adjustment of the cultured organoid types, quantities and gas supply plans according to experimental requirements; if the number of culture modules needs to be increased or the type of cultured organoid needs to be changed, it is only necessary to adjust the corresponding modules and channels, without the need for large-scale redesign of the entire chip, which greatly improves the flexibility of the experiment and the scalability of the chip, and reduces the cost of research and development and experiments. The present invention has a compact structure and a high degree of modularity. It can realize the free culture of a variety of different organoids, and can realize dynamic oxygen concentration regulation, accurately simulate the oxygen environment under different physiological or pathological conditions in the body, and significantly improve the physiological relevance and plasticity of the organoid model.

[0036] In a further optimization scheme, the gas supply module includes a mixing chamber 31, within which is located a mixing assembly 32 for mixing gases. The inlet of the mixing chamber 31 is connected to a number of inlet pipes 33, and the distribution channel is connected to the outlet of the mixing chamber 31. Different types of gases are supplied from an external source and introduced into the mixing chamber 31 through the inlet pipes 33. Mixed uniformly by the mixing assembly 32, they form the primary gas. This ensures uniform mixing of gases entering the distribution channel, maintains oxygen concentration stability, and improves the reliability of the culture environment. The uniformly mixed primary gas is then supplied to the gas distribution channel 21 at a constant rate and quantity through the distribution channel.

[0037] In one embodiment of the present application, the mixing chamber 31 is provided with three air inlets connected to different air inlet pipes 33 , respectively for supplying oxygen, carbon dioxide and nitrogen, respectively.

[0038] In one embodiment of the present application, an intake valve 34 is provided on the intake pipe 33 corresponding to oxygen, for controlling the intake flow rate of oxygen, thereby adjusting the oxygen content of the main gas.

[0039] In a further optimization, the distribution channel includes a distribution tube 35 connected to the outlet of the mixing chamber 31, and the regulating channel is connected to the distribution tube 35. The distribution tube 35 is connected to the corresponding gas distribution channel 21 through a distribution module, and the distribution module is connected to the regulating channel. The distribution tube 35 is connected to the gas distribution channel 21 through the distribution module, and the main gas enters the distribution tube 35. The distribution module monitors and controls the gas flow rate and oxygen content in the main gas in real time, achieving precise distribution and regulation of gas flow rate and oxygen concentration. The regulating channel is connected to the distribution tube 35, and inert gas or oxygen can be introduced into the distribution tube 35 according to culture requirements, thereby changing the oxygen content of the main gas and ensuring a more precise oxygen concentration environment for each culture module.

[0040] In a further optimization, the regulating channel includes a regulating tube 36 connected to the distribution tube 35, on which a regulating valve 37 is installed. The regulating channel, consisting of regulating tube 36 and regulating valve 37, precisely controls the flow of inert gas through regulating valve 37, thereby achieving fine adjustment of the oxygen concentration. Regulating valve 37 is an automatic valve automatically controlled by the distribution module.

[0041] In a further optimization, the distribution module includes a flow controller 38 connected to the distribution pipe 35. An oxygen concentration sensor 39 is connected between the flow controller 38 and the gas distribution channel 21. The oxygen concentration sensor 39, flow controller 38, and regulating valve 37 are interconnected with the control center 4 for signal exchange. The distribution module includes the interconnected flow controller 38 and oxygen concentration sensor 39. The oxygen concentration sensor monitors the oxygen content in the main gas in real time. When a gas with a different oxygen content is required, the monitored oxygen content is compared with the required oxygen content, and then the opening and closing of the intake valve 34 or the regulating valve 37 are controlled to achieve precise regulation.

[0042] In summary, the gas supply module serves as the main gas source of the system, and has a built-in mixing component 32 and a pressure stabilizer. The external gas source enters the mixing chamber 31 and is mixed to continuously output a certain concentration of mixed gas. The mixed gas is output to different branches of the gas distribution channel 21 through multiple distribution channels, supporting parallel multi-organ culture; each distribution channel is equipped with an independent flow controller 38, which is used to accurately adjust the flow rate of the mixed gas in the distribution channel and control the volume flow of the gas entering the chip; a high-sensitivity micro oxygen concentration sensor 39 is set between each distribution channel and the culture module to detect and The oxygen concentration entering the culture module is collected and transmitted to the control center 4 for feedback calculation by the control center 4. At the same time, each distribution channel is connected to the regulation channel and merged with the distribution channel before entering the chip. The oxygen concentration is diluted by adjusting the inert gas flow rate; the control center 4 obtains the real-time oxygen concentration readings of each channel from the oxygen concentration sensor 39, and compares it with the set target value. If the oxygen concentration is too high, the regulation channel is opened to introduce inert gas, increase its flow rate, and reduce the oxygen concentration through inert gas dilution; if the oxygen concentration is too low, reduce the inert gas flow rate or increase the main gas flow rate to increase the oxygen concentration.

[0043] In one embodiment of the present application, nitrogen is selected as the inert gas, which has little impact on biological organs and is low in cost.

[0044] In a further optimization, the culture module includes a culture frame 13 made of a biocompatible material, within which is housed a culture chamber 14, isolated from the outside world. A first channel 11, a second channel 12, and an air distribution channel 21 are attached to the culture frame 13 and communicate with the culture chamber 14. The culture module forms a culture chamber 14, isolated from the outside world, through the culture frame 13. The biocompatible material facilitates organoid growth and can be used to culture multiple organoids simultaneously. The first channel 11 and the second channel 12 are each used to supply liquid culture medium to the culture chamber 14, while the air distribution channel 21 independently diffuses gases into the culture chamber 14, allowing a mixed gas containing oxygen to enter the chamber.

[0045] As a further optimization, an isolation membrane 22 is provided between the gas distribution channel 21 and the culture chamber 14. The mixed gas in the gas distribution channel 21 can pass through the isolation membrane 22 and enter the culture chamber 14. The isolation membrane 22 is provided between the culture chamber 14 and the gas distribution channel 21, allowing only the mixed gas to pass through. It also serves to isolate the liquid and maintain the stability of the microenvironment, preventing cross-contamination between gas and liquid, ensuring a clean culture environment, and ensuring that the gas can enter the culture chamber 14 smoothly, maintaining a stable culture microenvironment.

[0046] In one embodiment of the present application, the isolation membrane 22 of the present application is sandwiched between the gas distribution channel and the culture chamber 14 using a PDMS film, allowing gas to diffuse into the culture chamber 14 through the isolation membrane 22, thereby achieving ventilation of the lower layer and suspending the upper organoids in the culture medium.

[0047] In one embodiment of the present application, an independent air inlet channel is provided under each culture chamber 14 and is connected to the air distribution channel 21 to achieve layered regulation of the local microenvironment.

[0048] A further optimization scheme features several connecting channels 15 between first channel 11 and culture chamber 14, staggered to prevent direct erosion. Second channel 12 is seeded with endothelial cells to form a microvascular barrier 16, through which substances within second channel 12 communicate with culture chamber 14. Liquid culture medium is injected into first and second channels 11, 12 via a peristaltic pump, providing a dynamic culture environment that promotes vascularization and functional maturation of various organoids, while also enhancing material exchange and promoting uniform oxygen distribution in the upper layers of the various organoids at different stages of culture.

[0049] In one embodiment of the present application, the first channel 11 is connected to the culture chamber 14 through a connecting channel 15 composed of multiple 20-50 μm micropores. The connecting channel 15 is staggered with the chamber to avoid direct erosion; vascular endothelial cells are planted on the inner wall of the second channel 12 to form a simulated microvascular barrier 16, which simulates the microvascular environment in the body, promotes material exchange, and provides an environment closer to the physiological state for organoid culture.

[0050] In one embodiment of the present application, multiple culture modules and culture chambers 14 can be provided for simultaneously culturing different organoids, such as four culture chambers with a length of 1200 μm, a width of 800 μm, and a depth of 600 μm; in the culture chamber 14, human embryonic stem cells or induced pluripotent stem cells can grow by self-assembly in matrix gel to form organoids.

[0051] In one embodiment of the present application, the oxygen concentration sensor 39 is installed on the wall of the gas distribution channel 21 near the gas inlet of the microfluidic chip, supports an O2 concentration range of 0-100%, has a fast response (T90<1s), high repeatability (<±0.1%), and monitors the oxygen concentration before entering the chip in real time, providing highly sensitive data as feedback control input.

[0052] In one embodiment of the present application, the control center 4 is a PID controller that uses a fuzzy parameter adjustment algorithm to optimize parameters, has the ability to adjust PID control parameters online, and has good adaptability to nonlinear factors in the operating environment of the microfluidic chip, such as inert gas diffusion delay and sudden changes in gas concentration caused by the small internal volume of the chip; the control center 4 realizes independent control of multi-channel oxygen concentration in organoid co-culture, and can support the setting of independent oxygen concentration environments for organoid culture chambers of different organoids and at different growth stages; in addition, the oxygen concentration control accuracy of the PID controller can reach an error within ±0.5%. When the oxygen concentration needs to be changed due to external disturbances such as sudden changes in organ oxygen consumption and micro-fluctuations in the gas source, it can stabilize within 30 seconds after the concentration set value is changed, and has the characteristics of fast response speed and strong adaptability.

[0053] In one embodiment of the present application, the control hub 4 utilizes an embedded microcontroller structure (e.g., STM32) and integrates multi-channel high-precision analog signal acquisition, an OLED real-time display, and a Bluetooth communication interface, capable of autonomously running feedback control programs. By collaborating with the oxygen concentration sensor 39, it supports a real-time closed-loop control algorithm based on PID and fuzzy logic, dynamically adjusting the output gas flow rate of the mass flow control module to achieve precise oxygen control in the multi-channel organoid culture chamber 14.

[0054] The present invention integrates an air supply structure 3 in the microfluidic chip, which can set the time domain oxygen concentration gradient (such as periodic hypoxia / reoxygenation to simulate stroke reperfusion) and the spatial oxygen concentration gradient (such as the oxygen concentration difference in the real growth environment of organs such as the intestine, liver, and kidney) according to experimental requirements; the main airflow and inert gas flow are used to adjust and control the oxygen concentration to achieve non-contact precise control of the local oxygen environment; a high-sensitivity micro oxygen concentration sensor 39 is used to monitor the oxygen concentration in real time, and the oxygen concentration signal is transmitted to the PID controller, and the PID controller controls the main gas and inert gas flow to achieve independent and real-time feedback adjustment of the oxygen concentration of multiple organ culture channels in the microfluidic chip, support the simultaneous culture of multiple organ types in the same microfluidic chip system, have the advantages of real-time regulation of oxygen concentration, independent control of multiple channels, and improved oxygen concentration uniformity, support long-term and stable culture of multiple organ types, and provide support for organoid disease modeling, developmental research, and drug efficacy testing.

[0055] The present invention is an integrated and expandable platform that adopts a modular design concept and supports the expansion of multiple different organoid culture chambers 14; the culture chamber 14 is connected to a peristaltic pump and an inert gas channel; and real-time control of oxygen concentration is achieved through PID controller program setting.

[0056] The multi-organoid co-culture microfluidic chip proposed in this paper can simultaneously co-cultivate and freely combine two to four different organoids at the micrometer scale. It also allows for dynamic oxygen concentration regulation during the culture process, accurately simulating the oxygen environment in vivo under different physiological or pathological conditions, significantly enhancing the physiological relevance and plasticity of organoid models. This platform not only facilitates in-depth understanding of the pathogenesis of complex diseases such as ischemic disorders, tumors, and neurodegenerative disorders, but also serves as an important tool for novel drug screening and precision treatment evaluation, with broad application value.

[0057] The present invention also discloses a method for controlling dynamic oxygen concentration of a microfluidic chip based on dynamic oxygen concentration regulation for organoid culture, comprising the following steps:

[0058] Assemble the dynamic oxygen concentration control microfluidic chip for organoid culture; the gas supply mechanism of the present invention is composed of a gas supply module, a distribution channel, a regulation channel and a control center 4 electrically connected to the first three to meet the needs of fine control of the oxygen concentration of the microfluidic chip for co-culture of multiple organoids. It is integrated with the microfluidic channel and the signal circuit, and the control center 4 realizes automatic regulation and feedback closed-loop control of the entire process.

[0059] The gas supply module is connected to an external gas source and introduces gases of different types and components to form a main gas. The gas supply module has a volume of about 50-140 mL, is connected to an external gas bottle, and has a built-in pressure stabilizer and mixing assembly 32. The mixed gas is uniformly output to the downstream distribution channel.

[0060] The organoids to be cultured are implanted in the culture module, and the culture medium is injected into the culture module through the first channel 11 and the second channel 12 and circulated;

[0061] The main gas is supplied to the gas distribution channel 21 through the distribution channel to provide the air required for organoid culture at a set flow rate, and the flow rate and oxygen content of the main gas are monitored in real time;

[0062] Depending on the oxygen content required for culturing different organoids, inert gas or oxygen is introduced into the distribution channel through the regulating channel, thereby changing the oxygen content and achieving synchronous cultivation of different organoids. The distribution channel is divided into 4-8 independent channels, each equipped with an inert gas branch; the distribution channel is approximately 300μm wide, the regulating channel is approximately 200μm wide, and the height is approximately 150μm. The spacing between each channel is approximately 1.5mm, and the channels are designed to be distributed according to laminar flow to ensure pressure balance. The outlet of the distribution pipe 35 is connected to the distribution module, precisely controlling the flow rate of the main gas and inert gas in each channel to achieve dynamic adjustment of oxygen concentration.

[0063] Example 1: Simulating physiological hypoxia in cortical organoids

[0064] The physiological hypoxic state of the cortical organoids requires that the cortical organoids be maintained in a physiological hypoxic environment containing 90% N2, 5% CO2 and 5% O2 for 30 days starting from the first day of culture. First, the oxygen concentration control conditions that change with time are input into the PID controller, and culture is carried out in a physiological hypoxic environment of 90% N2, 5% CO2 and 5% O2 for 30 days. At the beginning of the culture, the N2, CO2 and O2 gas cylinders are opened, and the three gases are mixed evenly in the mixing chamber 31 according to the proportion. Then, the corresponding gas distribution channel 21 of the cortical organoid is opened, and the mixed gas passes through the channel into the oxygen regulation interface layer connected to the cortical organoid culture chamber 14. The mixed gas dissolves in the perfused culture medium to provide the organoid with the set hypoxic state.

[0065] Example 2: Simulating the hypoxia-oxygen enrichment model of endometrial organoids

[0066] The hypoxia-enriched oxygen model of endometrial organoids requires 12 hours of hypoxia under the conditions of 92% N2, 5% CO2, and 3% O2 before returning to normoxia (21% O2). First, input the oxygen concentration control conditions that change with time into the PID controller, culture for 12 hours under the hypoxic conditions of 92% N2, 5% CO2, and 3% O2, and then restore to normoxia (21% O2). At the beginning of the culture, open the N2, CO2, and O2 gas cylinders, mix the three gases in proportion in the mixing chamber 31, and then open the corresponding gas distribution channel 21 of the endometrial organoid. The mixed gas passes through the channel into the oxygen regulation interface layer connected to the endometrial organoid culture chamber 14. The mixed gas dissolves in the perfused culture medium to provide the set hypoxic state for the organoid. After this state is maintained for 12 hours, the PID controller adjusts the opening of the intake valve 34. At this time, the oxygen flow rate increases, while the nitrogen and carbon dioxide flow rates remain unchanged. The oxygen concentration in the mixing chamber 31 rises, and the high-oxygen-concentration mixed gas passes through the same channel to the oxygen concentration sensor 39 at the gas inlet of the microfluidic chip. The sensor detects the oxygen concentration and feeds the detection result back to the PID controller. If the oxygen concentration is lower than the target value, the PID controller controls to increase the oxygen valve opening and increase the oxygen concentration. Otherwise, the oxygen valve opening is reduced and the oxygen concentration is reduced. Through iterative feedback control, the oxygen concentration reaches the target value.

[0067] Example 3: Dynamic optimization of the oxygen microenvironment in multi-organ culture using a PID control method integrated with fuzzy parameter adjustment

[0068] In the process of co-culturing multiple organoids, in order to simulate the changes in oxygen demand under different microenvironments, the system needs to set dynamic oxygen control strategies according to the organ type. First, input the oxygen concentration control conditions of each organ into the PID controller, such as the hypoxic conditions of brain organoids and endometrial organoids in Example 1 and Example 2, and the ALB +high High oxygen conditions (40% O2) of HNF4α liver organoids, etc.

[0069] In order to meet the heterogeneous requirements of different organoids for oxygen microenvironment, a fuzzy parameter adjustment mechanism is introduced on the basis of PID closed-loop control. By setting up a fuzzy rule base, the PID parameters are adjusted online according to the oxygen concentration error and error change rate fed back by the sensor, so that the system has stronger adaptability and robustness. When entering the normoxia recovery stage, the PID controller cooperates with the fuzzy parameter adjustment algorithm to dynamically adjust the opening of the intake valve 34, the oxygen flow rate increases, and the oxygen concentration in the mixed gas rises. The oxygen concentration sensor 39 monitors the oxygen concentration at the gas inlet in real time and feeds the data back to the PID controller. If the oxygen concentration deviates from the target value, the fuzzy parameter adjustment mechanism will adjust the PID parameters to optimize the adjustment efficiency, and ultimately achieve precise control and dynamic stability of the oxygen concentration in different organ areas.

[0070] In the description of the present invention, it should be understood that the terms "longitudinal", "transverse", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0071] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A microfluidic chip for dynamic oxygen concentration control in organoid culture, characterized in that: include: An upper structure (1), the upper structure (1) comprising a plurality of independently arranged culture modules for organoid culture, wherein the upper ends of the culture modules are fitted and connected to a first channel (11), and the bottom ends of the culture modules are fitted and connected to a second channel (12); A lower structure (2), the lower structure (2) comprising a plurality of air distribution channels (21) arranged in close contact with the lower end of the second channel (12), the plurality of air distribution channels (21) corresponding to and communicating with the culture module; An air supply structure (3) includes an air supply module, wherein the air supply module is connected to a plurality of independently arranged distribution channels, and the plurality of distribution channels are respectively connected to the air distribution channel (21); the distribution channels are connected to a regulating channel for regulating the oxygen concentration.

2. The microfluidic chip for dynamic oxygen concentration control for organoid culture according to claim 1, characterized in that: The air supply module comprises a mixing chamber (31), wherein a mixing assembly (32) for mixing gas is provided in the mixing chamber (31); an air inlet of the mixing chamber (31) is connected to a plurality of air inlet pipes (33), and the distribution channel is connected to an air outlet of the mixing chamber (31).

3. The microfluidic chip for dynamic oxygen concentration control for organoid culture according to claim 2, characterized in that: The distribution channel includes a distribution pipe (35) connected to the outlet of the mixing chamber (31), and the regulating channel is connected to the distribution pipe (35); the distribution pipe (35) is connected to the corresponding air distribution channel (21) through a distribution module, and the distribution module is connected to the regulating channel.

4. The microfluidic chip for dynamic oxygen concentration control for organoid culture according to claim 3, characterized in that: The regulating channel comprises a regulating pipe (36) communicating with the distribution pipe (35), a regulating valve (37) is provided on the regulating pipe (36), and the opening of the regulating valve (37) is controlled by the control center (4).

5. The dynamic oxygen concentration control microfluidic chip according to claim 4, characterized in that: The distribution module includes a flow controller (38) connected to the distribution pipe (35), an oxygen concentration sensor (39) is connected between the flow controller (38) and the air distribution channel (21), the flow through the flow controller (38) is controlled by the control center (4), the oxygen concentration sensor (39) measures the oxygen concentration in the distribution pipe (35), and the oxygen concentration sensor (39) feeds back the oxygen concentration detection result to the control center (4).

6. The microfluidic chip for dynamic oxygen concentration control for organoid culture according to claim 1, characterized in that: The culture module comprises a culture frame (13) made of a biocompatible material, wherein a culture chamber (14) isolated from the outside is provided in the culture frame (13); the first channel (11), the second channel (12) and the air distribution channel (21) are respectively attached to the culture frame (13) and communicated with the culture chamber (14).

7. The microfluidic chip for dynamic oxygen concentration control for organoid culture according to claim 6, characterized in that: An isolation membrane (22) is provided between the gas distribution channel (21) and the culture chamber (14), and the mixed gas in the gas distribution channel (21) can pass through the isolation membrane (22) and enter the culture chamber (14).

8. The microfluidic chip for dynamic oxygen concentration control for organoid culture according to claim 6, characterized in that: A plurality of connecting channels (15) are provided between the first channel (11) and the culture chamber (14), and the connecting channels (15) and the culture chamber (14) are staggered to avoid direct flushing.

9. The dynamic oxygen concentration control microfluidic chip according to claim 6, characterized in that: The second channel (12) is planted with vascular endothelial cells to form a microvascular barrier (16), and the substances in the second channel (12) are connected to the culture chamber (14) through the microvascular barrier (16).

10. A method for dynamic oxygen concentration control in organoid culture, characterized in that The following steps are involved: Assembling the microfluidic chip for dynamic oxygen concentration control of organoid culture according to any one of claims 1 to 9; The gas supply module is connected to the external gas source and introduces gases of different types and components to form the main gas; The organoid to be cultured is implanted in the culture module, and the culture medium is injected into the culture module through the first channel (11) and the second channel (12) and circulated; The main gas supplies the air required for organoid culture at a set flow rate to the gas distribution channel (21) through the distribution channel, and the flow rate and oxygen content of the main gas are monitored in real time; According to the oxygen content required for the culture of different organoids, inert gas is injected into the distribution channel through the regulating channel to adjust the oxygen content and achieve synchronous culture of different organoids.

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