Skin organ chip based on multi-condition simulation detection

Abstract

The invention belongs to the technical field of organoid culture, and provides a skin organoid chip based on multi-condition simulation detection. The device comprises a gas flowing layer, a culture layer, a porous membrane layer and a liquid flowing layer which are sequentially stacked from top to bottom, the gas flowing layer comprises a gas pump unit and a gas distribution structure connected with the gas pump unit in series, the gas pump unit is used for stabilizing gas flow, and the gas distribution structure is used for distributing the gas flow and buffering gas flow fluctuation; the culture layer comprises a plurality of culture holes communicated with the gas flow layer and the porous membrane layer; the porous membrane layer is used for supporting skin organs and allowing exchange of small molecular substances; the liquid flowing layer comprises a parallel liquid pump unit and a liquid distribution mechanism connected with the parallel liquid pump unit in series, the parallel liquid pump unit is used for stabilizing liquid flow, and the liquid distribution mechanism is used for distributing liquid and buffering liquid fluctuation. The invention aims to realize high-throughput culture and detection of skin organs and meet the application in large-scale drug screening and toxicological evaluation.

Description

Technical Field

[0001] This invention belongs to the field of organoid culture technology and provides a skin organoid chip based on multi-condition simulation detection. Background Technology

[0002] Skin, the largest organ in the human body, has multiple functions, including barrier protection, immune defense, and metabolic regulation. With environmental pollution, population aging, and the rapid development of cosmetics and topical medications, skin-related diseases and drug safety issues are becoming increasingly prominent. To better study the physiological functions of skin, disease mechanisms, and the toxicological effects of drugs and cosmetics, establishing in vitro skin models has gradually become an important alternative to animal experiments. Compared with traditional two-dimensional culture and animal models, skin organoids have significant advantages in simulating the structure and function of human skin and have been widely used in disease modeling, drug screening, and personalized medicine research. However, the application of skin organoids still faces the following technical bottlenecks: 1. Insufficient high-throughput culture and detection capabilities: Traditional culture methods are mostly based on petri dishes or well plates, making it difficult to achieve large-scale, diversified parallel culture and detection, severely limiting their application in drug screening and toxicological evaluation. 2. Limited detection conditions: Existing organoid chip platforms often can only operate in a single environment or detection mode, failing to simultaneously meet the multi-condition detection needs such as drug gradients, physical factor stimulation, and multi-component microenvironments. 3. Poor standardization and compatibility: Some chip structures are complex and have high processing costs, making them difficult to integrate with existing automated imaging and analysis systems, which hinders promotion and large-scale application. 4. Insufficient microenvironment control: Skin organoids are highly sensitive to the culture environment, such as oxygen concentration, nutrient supply, and mechanical stress. However, existing culture systems have limited ability to dynamically regulate and monitor these parameters in situ, affecting the accuracy of organoid maturity and functional evaluation. Summary of the Invention

[0003] The purpose of this invention is to achieve high-throughput culture and multi-environmental condition simulation of skin organoids, meeting the application requirements in large-scale drug screening and toxicological evaluation. Specifically, it can adjust the multi-condition detection requirements of the skin organoid culture environment, such as oxygen concentration, nutrient supply, drug gradient, mechanical stress, temperature gradient, physical factor stimulation, and multi-component microenvironment, to achieve multi-condition detection of skin organoids.

[0004] A skin organoid chip based on multi-condition simulation detection comprises, from top to bottom, a gas flow layer, a culture layer, a porous membrane layer, and a liquid flow layer. The gas flow layer includes a gas pump unit and a gas distribution structure connected in series with the gas pump unit. The gas pump unit stabilizes the airflow, and the gas distribution structure distributes the airflow and buffers airflow fluctuations. The culture layer contains multiple culture wells connecting the gas flow layer and the porous membrane layer. The porous membrane layer supports the skin organoid and allows for the exchange of small molecules. The liquid flow layer includes a parallel liquid pump unit and a liquid distribution mechanism connected in series with the parallel liquid pump unit. The parallel liquid pump unit stabilizes the liquid flow, and the liquid distribution structure distributes the liquid and buffers airflow fluctuations. The porous membrane layer also includes through-holes that cooperate with the porous membrane. Piezoelectric elements are provided on the sidewalls of the through-holes, and the two ends of the piezoelectric elements are connected to the edge of the porous membrane and the through-holes respectively using a biocompatible adhesive. The multi-layer chip structure allows for the replacement of one or more chips as needed, adapting to various application scenarios; the culture layer can be expanded as required to increase the number of culture wells in the array, enabling high-throughput culture and meeting the needs of large-scale drug screening and toxicological evaluation.

[0005] Preferably, the parallel pump unit of the liquid flow layer consists of multiple pump units connected in parallel. The liquid distribution structure includes fluid channels, with the number of pump units equal to the number of fluid channels in the liquid flow layer. Each pump unit has a corresponding fluid channel. The liquid channels can be connected using multi-port valves with an equal number of outlets as the number of liquid channels. For example, a four-port valve with three outlets is selected for three liquid channels, with each valve port connected to a liquid channel. The operator selects independent operation according to the experimental conditions. For example, when multiple reagents need to be tested, each operates independently. Through the parallel pump and independent channel design, the independent channels ensure that the liquid flow in each culture well does not interfere with each other, achieving stable liquid flow and parallel experiments under multiple conditions, significantly improving experimental accuracy and system reliability. Furthermore, multi-port valves can be set to control and connect the independent fluid channels, enabling switching between culture under the same liquid conditions and culture under different liquid conditions. For example, the inlet of a four-port valve can be connected to a pump unit to divide the inflowing liquid into three parts, which flow evenly into the liquid channels. For example, when there is only one reagent to be tested in flow culture, a four-port valve can be selected to split the liquid flow, reducing the number of pumps used.

[0006] Preferably, the liquid pump unit includes miniature metering pumps installed at both ends of the fluid channel. The electrical connection of the miniature metering pumps ensures that the volume of liquid flowing out and into the fluid channel is equal. Through precise matching of the metering pumps at both ends, stable control of the liquid level is achieved, perfectly simulating the microenvironment of the gas-liquid interface in the skin. The metering pumps employ closed-loop control, with the inflow and outflow volume error controlled within ±0.1μL. The liquid level is always maintained on the surface of the porous membrane layer, avoiding barrier function disorder caused by liquid submerging the epidermal layer. In addition, the stable liquid level avoids the impact of osmotic pressure fluctuations on the morphology of skin organoids, such as the hair follicle formation rate of skin organoids, solving the problem of structural distortion of skin organoids caused by uncontrolled liquid level on the chip.

[0007] Preferably, the fluid channel includes a liquid region in the middle, the top of which contacts the porous membrane layer and passes through the bottom of the culture well. The liquid region and both ends of the fluid channel are horizontally positioned in the liquid flow layer, with the height of the liquid region higher than the ends of the fluid channel. Through structural optimization of the liquid region, fluid flow buffering and controlled contact between the liquid and the skin organoids are achieved, ensuring that the liquid level is controlled within the dermis layer and does not submerge the epidermis layer, significantly improving the quality and experimental stability of organoid culture.

[0008] Preferably, the area of ​​the liquid flow cross-section in the liquid region is larger than that at both ends of the fluid channel, and the depth of the liquid region is less than 1 mm, which satisfies the requirement for liquid to flow within the liquid flow layer. The larger cross-sectional area of ​​the liquid region compared to the ends of the channel significantly reduces the flow velocity in the micro-flow state, minimizing damage to skin organoids caused by fluid shear forces, and maximizing the gradual infiltration of the dermis from bottom to top. The height difference design creates a natural buffer, effectively absorbing pressure fluctuations during pump start-up and shutdown.

[0009] Preferably, a temperature control element is located at the bottom of the liquid region, corresponding to the culture well. The surface of the temperature control element is covered with a thermally conductive silicone layer, which transfers temperature to the cultured skin organoid through the liquid in the liquid region. The temperature control element enables temperature control of the culture region, achieving temperature gradient differences between different layers of the skin by heating from the bottom of the skin organoid, such as a gradient distribution of 33°C for the epidermis and 37°C for the dermis. Simultaneously, the temperature control element enables controlled release of heat-responsive polymers and chemotherapeutic drugs, expanding the scenarios for temperature-dependent drug screening, such as studies on the synergistic effects of thermotherapy combined with chemotherapy.

[0010] Preferably, the gas flow layer includes gas channels with downward-sloping bends at both ends. The bottom of the gas channels contacts the porous membrane layer and passes through the top of the culture wells. The downward-sloping bends design achieves airflow buffering and uniform distribution, significantly improving the stability of the gas microenvironment and the quality of organoid culture. The downward-sloping bends effectively counteract the airflow impact during the start-up and shutdown of the gas pump unit, reducing the airflow velocity and preventing the epidermis of skin organoids from drying out.

[0011] Preferably, the gas channel depth is less than 1 mm, ensuring sufficient gas flow within the gas flow layer. This shallow channel design enables micro-flow and efficient gas exchange, significantly improving chip energy efficiency and the respiratory function of skin organoids. A shallow channel depth of 0.5 mm promotes rapid gas diffusion and enhances CO2 removal efficiency, preventing apoptosis in skin organoids caused by acidosis. Furthermore, the compact shallow channel structure keeps the overall thickness of the chip's upper layer below 3 mm, allowing for compatibility with existing high-magnification microscope imaging systems and fluorescence imaging systems. This enables real-time dynamic observation of skin organoids, such as continuous imaging of hair follicle formation, avoiding the imaging blurring issues associated with thicker chips.

[0012] Preferably, the porous membrane layer includes a porous membrane at the bottom of each culture well. The diameter of the porous membrane at the bottom of each culture well is the same as that of the culture well, and its edge extends to the bottom of the sidewall of the culture well. The porous membrane is made of at least one of polymethyl methacrylate, polydimethylsiloxane, or polycarbonate, and the pore size of the porous membrane can be selected in the range of 0.4-5 μm. This flexible selection of material and pore size allows for adaptation to various scenarios and material exchange with different culture media, significantly improving the experimental adaptability of the chip. PDMS membranes have good biocompatibility and are suitable for long-term culture; PC membranes have high strength and are suitable for mechanical stimulation experiments with piezoelectric elements; PMMA membranes have good light transmittance and are suitable for fluorescence imaging.

[0013] Preferably, the piezoelectric element is a fan-shaped piezoelectric ceramic sheet. This sheet can be driven by voltage to expand and contract, causing the porous membrane edges to stretch synchronously. The piezoelectric element generates voltage signals under pressure and tension. An external control circuit applies periodic voltage signals to the piezoelectric element, causing it to expand and contract, further stretching the porous membrane edges. The skin-like organoids on the porous membrane, located in the central region, experience uniform tensile strain, simulating various daily mechanical activities of human skin, such as limb movements and facial muscle contractions. The piezoelectric element is embedded in the porous membrane layer, without obstructing gas-liquid flow channels or affecting the stability of the gas-liquid microenvironment. Simultaneously, utilizing the positive piezoelectric effect, the piezoelectric element generates voltage signals under pressure and tension, enabling it to detect the force exerted by the skin-like organoids on the porous membrane and acquire real-time data on the expansion and contraction changes of the skin-like organoids under external stimuli such as drug stimulation.

[0014] This invention offers the following advantages: through a multi-layered replaceable structure and high-throughput scalability design, it adapts to various scenarios and meets the needs of large-scale drug screening and toxicological evaluation; the gas-liquid dual microfluidic system achieves precise liquid surface stability, temperature gradient simulation, and airflow buffering, and reproduces the gas-liquid interface and interlayer microenvironment of skin organoids as closely as possible; the piezoelectric element integrates mechanical stimulation and mechanical monitoring in a closed loop, enhancing the physiological relevance of skin organoids; and the porous membrane material and pore size can be flexibly selected to adapt to the needs of long-term culture, mechanical experiments, and imaging. Attached Figure Description

[0015] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of a skin organoid chip based on multi-condition simulation detection.

[0017] Figure 2 This is a top view of a skin organoid chip based on multi-condition simulation detection.

[0018] Figure 3 For a skin organoid chip based on multi-condition simulation detection Figure 2 AA section view in the image.

[0019] Figure 4 For a skin organoid chip based on multi-condition simulation detection Figure 2 BB section view in the middle.

[0020] Figure 5 This is a schematic diagram of the gas flow layer of the present invention.

[0021] Figure 6 This is a perspective view of the structural schematic diagram of the gas flow layer of the present invention.

[0022] Figure 7 This is a bottom view of the gas flow layer of the present invention.

[0023] Figure 8 This is a top view of the porous membrane layer in Embodiment 2 of the present invention.

[0024] Figure 9 This is a schematic diagram of the liquid flow layer structure of the present invention.

[0025] Figure 10 This is a top view of the liquid flow layer of the present invention.

[0026] Legend: 1 Gas flow layer; 11 Gas channel; 2 Culture layer; 21 Culture well; 3 Porous membrane layer; 31 Piezoelectric element; 32 Porous membrane; 4 Liquid flow layer; 41 Liquid region; 42 Temperature control element; 5 Fixing hole. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0028] Example 1 like Figure 1 and Figure 2 As shown, a skin organoid chip based on multi-condition simulation detection includes a gas flow layer 1, a culture layer 2, a porous membrane layer 3, and a liquid flow layer 4 stacked sequentially from top to bottom. The gas flow layer 1 includes a gas pump unit and a gas distribution structure connected in series with the gas pump unit. The gas pump unit stabilizes the airflow, and the gas distribution structure distributes the airflow and buffers airflow fluctuations. The culture layer 2 includes multiple culture wells 21 connecting the gas flow layer 1 and the porous membrane layer 3. The porous membrane layer 3 supports the skin organoid and allows the exchange of small molecules. In this embodiment, the porous membrane layer 3 is a polycarbonate membrane, also known as a PC membrane. The liquid flow layer 4 includes a parallel liquid pump unit and a liquid distribution mechanism connected in series with the parallel liquid pump unit. The parallel liquid pump unit stabilizes the liquid flow, and the liquid distribution structure distributes the liquid and buffers fluid fluctuations. Fixing holes 5 are uniformly punched at the four corners of the skin organoid chip, penetrating the four layers for fixation. Teflon tape is wrapped around the four-layer chip structure, and a silicone membrane is placed between two layers to prevent gas and liquid leakage and ensure the system's airtightness. Raw material tape also serves to prevent tissue contamination and fix the chip structure.

[0029] The four-layer chip structure allows for the replacement of one or more layers of chips as needed, adapting to various application scenarios. The culture layer 2 can be expanded as needed to increase the number of culture wells 21, enabling high-throughput culture and meeting the needs of large-scale drug screening and toxicological evaluation.

[0030] like Figure 3 , Figure 5 , Figure 6 and Figure 7 As shown, the gas flow layer 1 includes a gas channel 11 with downward-sloping bends at both ends. The bottom of the gas channel 11 contacts the porous membrane layer 3 and passes through the top of the culture well 21. The downward-sloping bends design achieves airflow buffering and uniform distribution, significantly improving the stability of the gas microenvironment and the quality of organoid culture. The downward-sloping bends effectively counteract the airflow impact during the start-up and shutdown of the gas pump unit, reducing the airflow velocity and preventing the epidermis of the skin organoids from drying out.

[0031] like Figure 6 and Figure 7 As shown, the depth of gas channel 11 is less than 1 mm, which satisfies the requirement for gas flow within gas flow layer 1. This shallow channel design enables micro-flow and efficient gas exchange, significantly improving chip energy efficiency and the respiratory function of skin organoids. The shallow channel depth of 0.5 mm promotes rapid gas diffusion and improves CO2 removal efficiency, preventing apoptosis in skin organoids caused by acidosis. Furthermore, the compact shallow channel structure keeps the overall thickness of the upper chip layer within 3 mm, allowing for compatibility with existing high-magnification microscope imaging systems and fluorescence imaging systems. This enables real-time dynamic observation of skin organoids, such as continuous imaging of the hair follicle formation process, avoiding the imaging blurring issues associated with thicker chips.

[0032] like Figure 8 As shown, the porous membrane layer 3 includes a porous membrane 32 at the bottom of each culture well 21. The diameter of the porous membrane 32 at the bottom of each culture well 21 is the same as that of the culture well 21, and its edge extends to the bottom of the sidewall of the culture well 21. The material of the porous membrane 32 is selected from at least one of polymethyl methacrylate, polydimethylsiloxane, or polycarbonate, and the pore size of the porous membrane 32 can be selected in the range of 0.4-5 μm. Through the flexible selection of materials and pore sizes, multi-scenario adaptation and material exchange with different culture media can be achieved, significantly improving the experimental adaptability of the chip. Polymethyl methacrylate (PDMS) membranes have good biocompatibility and are suitable for long-term culture; polycarbonate (PC) membranes have high strength and are suitable for mechanical stimulation experiments with piezoelectric elements 31; polydimethylsiloxane (PMMA) membranes have good light transmittance and are suitable for fluorescence imaging.

[0033] like Figure 9 and Figure 10 As shown, the parallel pump unit of the liquid flow layer 4 consists of multiple pump units connected in parallel. The liquid distribution structure includes fluid channels, and the number of pump units is equal to the number of fluid channels in the liquid flow layer 4. Each pump unit has an independent fluid channel. The liquid channels can be connected using multi-port valves with an equal number of outlets as the number of liquid channels. For example, three liquid channels can use a four-port valve with three outlets, with each valve port connected to a liquid channel. The operator can select independent operation modes according to experimental conditions. For example, when multiple reagents need to be detected, each can operate independently. Through the parallel pump and independent channel design, the independent channels ensure that the liquid flow in each culture well does not interfere with each other, achieving stable liquid flow and parallel experiments under multiple conditions, significantly improving experimental accuracy and system reliability.

[0034] Furthermore, multi-way valves can be set up to control and connect independent fluid channels, enabling switching between cultivation under the same liquid conditions and cultivation under different liquid conditions. For example, a liquid pump unit can be connected to the inlet of a four-way valve to divide the inflowing liquid into three parts, which then flow into the liquid channel evenly. For example, when there is only one reagent to be tested in flow culture, a four-way valve can be selected to divert the liquid to reduce the number of pumps used.

[0035] The liquid pump unit includes miniature metering pumps installed at both ends of the fluid channel. The electrical connection of these pumps ensures that the volume of liquid flowing out and into the fluid channel is equal. Precise matching of the two metering pumps achieves stable control of the liquid level, perfectly simulating the microenvironment of the gas-liquid interface within the skin. The metering pumps employ closed-loop control, keeping the inflow and outflow volume error within ±0.1 μL. The liquid level is consistently maintained on the surface of the porous membrane layer 3, preventing barrier function disruption caused by liquid submersion of the epidermis. Furthermore, the stable liquid level avoids the impact of osmotic pressure fluctuations on the morphology of skin organoids, such as the hair follicle formation rate, thus resolving the problem of structural distortion in skin organoids caused by uncontrolled liquid level on the chip.

[0036] The fluid channel includes a central liquid region 41. The top of the liquid region 41 contacts the porous membrane layer 3 and extends past the bottom of the culture well 21. The liquid region 41 and both ends of the fluid channel are horizontally positioned within the liquid flow layer 4, with the height of the liquid region 41 exceeding that of the fluid channel's ends. Through structural optimization of the liquid region 41, fluid flow buffering and controlled contact between the liquid and the skin organoids are achieved, ensuring the liquid level remains within the dermis and does not submerge the epidermis, significantly improving organoid culture quality and experimental stability.

[0037] The cross-sectional area of ​​the liquid flow in liquid region 41 is larger than that at both ends of the fluid channel. The depth of liquid region 41 is less than 1 mm, which satisfies the requirement for liquid to flow within the liquid flow layer 4. The larger cross-sectional area of ​​liquid region 41 compared to the ends of the channel significantly reduces the flow velocity of liquid region 41 in a micro-flow state, minimizing damage to skin organoids caused by fluid shear force and maximizing the gradual infiltration of the dermis from bottom to top. The height difference design forms a natural buffer, effectively absorbing pressure fluctuations during pump start-up and shutdown.

[0038] Example 2 The difference from Example 1 is that, as Figure 4As shown, a temperature control element 42 is located at the bottom of the liquid region 41, corresponding to the culture well 21. The surface of the temperature control element 42 is covered with a thermally conductive silicone layer, which transfers temperature to the skin organoid in the culture layer 2 through the liquid in the liquid region 41. The temperature control element 42 can achieve temperature control of the culture region, heating from the bottom of the skin organoid to achieve temperature gradient differences between different layers of the skin, such as a gradient distribution of 33°C for the epidermis and 37°C for the dermis. At the same time, the temperature control element can achieve controlled release of heat-responsive polymers and chemotherapeutic drugs, expanding the scenarios for temperature-dependent drug screening, such as the synergistic effect study of thermotherapy combined with chemotherapy.

[0039] Combination Figure 4 and Figure 8 As shown, the porous membrane layer 3 also includes through-holes that cooperate with the porous membrane 32. A piezoelectric element 31 is provided on the sidewall of the through-hole, and the two ends of the piezoelectric element 31 are connected to the edge of the porous membrane 32 and the through-hole respectively using a biocompatible adhesive. In this embodiment, the piezoelectric element 31 is a fan-shaped piezoelectric ceramic sheet, and its two ends are connected to the edge of the porous membrane and the sidewall of the through-hole using PLGA adhesive to maximize the uniformity of the force exerted by the piezoelectric element 31 on the porous membrane 32. An external control circuit applies a periodic voltage signal to the piezoelectric element 31. Driven by the voltage, the piezoelectric element 31 undergoes expansion and contraction deformation, causing the edge of the porous membrane 32 to stretch synchronously. The skin-like organoids on the porous membrane 32, located in the central region of the porous membrane 32, can be subjected to uniform tensile strain, simulating different daily mechanical activities of human skin, such as limb movements and facial muscle contractions. The piezoelectric element 31 is embedded in the porous membrane layer 3, without occupying the gas-liquid flow channel and without affecting the stability of the gas-liquid microenvironment. At the same time, utilizing the positive piezoelectric effect, the piezoelectric element 31 generates a voltage signal under pressure and tension. The piezoelectric element 31 can detect the force exerted by the skin-like organoids on the porous membrane 32 and obtain the expansion and contraction changes of the skin-like organoids under external stimuli such as drug stimulation in real time.

[0040] The synergistic regulation of mechanical stimulation, temperature gradients, and chemical factors can more realistically simulate the microenvironment of the skin in vivo, enhancing the physiological relevance of organoids. The synergistic effect of multiple factors can promote the structural maturation, functional expression, and pathological state simulation of organoids, providing a more reliable experimental model for disease modeling and drug screening.

[0041] Example 3 In this embodiment, the porous membrane layer 3 is a porous membrane 32 made of polycarbonate (PC), which, together with the piezoelectric element 31, realizes mechanical stimulation and mechanical monitoring; the liquid flow layer 4 adopts a parallel liquid pump unit and an independent liquid channel to support parallel experiments of multiple drug concentrations; the temperature control element 42 is a platinum temperature control element with a platinum heating electrode and a platinum temperature measuring electrode on the same substrate, which is located at the bottom of the liquid area 41 to realize local temperature regulation and temperature monitoring of the inflamed area; the gas flow layer 1 ensures a stable supply of inflammation-related gases through the design of a sunken bend and shallow channel.

[0042] The gas flow layer 1 is introduced with a mixed gas flow containing inflammation-related gases to simulate the skin gas microenvironment under inflammation; the independent channels of the liquid flow layer 4 introduce culture medium containing inflammatory mediators into the culture well 21 to induce an inflammatory response in the skin organoids; the piezoelectric element 31 applies periodic tensile strain to simulate the abnormal mechanical tension of the skin during inflammation; the platinum temperature control element adjusts the temperature of the liquid region 41 to maintain the physiological temperature gradient of the inflamed region.

[0043] Through the independent channels of the liquid flow layer 4, anti-inflammatory drugs of varying concentrations are introduced into different culture wells 21 to achieve parallel screening under multiple conditions; the positive piezoelectric effect of the piezoelectric element 31 is used to monitor the changes in the mechanical response of organoids under the action of drugs in real time; combined with the shallow channel design of the gas flow layer 1, the morphological and structural changes of organoids are observed through a high-power microscope to evaluate the anti-inflammatory effect of the drugs.

[0044] This invention has the following beneficial effects: through a multi-layer replaceable structure and high-throughput scalability design, it can adapt to the needs of multiple scenarios and meet the requirements of large-scale drug screening and toxicological evaluation; the gas-liquid dual microfluidic system achieves precise and stable liquid surface, temperature gradient simulation and airflow buffering, and reproduces the gas-liquid interface and interlayer microenvironment of skin organoids as much as possible; the piezoelectric element 31 integrates mechanical stimulation and mechanical monitoring closed loop, improving the physiological relevance of skin organoids; the porous membrane 32 allows for flexible selection of material and pore size to adapt to the needs of long-term culture, mechanical experiments and imaging.

[0045] The above embodiments and / or implementation methods are merely illustrative of preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art may make some modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but these should still be regarded as the technology or embodiments that are substantially the same as the present invention.

Claims

1. A skin organoid chip based on multi-condition simulation detection, characterized in that, The skin organoid chip comprises, from top to bottom, a gas flow layer (1), a culture layer (2), a porous membrane layer (3), and a liquid flow layer (4). The gas flow layer (1) includes a gas pump unit and a gas distribution structure connected in series with the gas pump unit. The gas pump unit is used to stabilize the airflow, and the gas distribution structure is used to distribute the airflow and buffer airflow fluctuations. The culture layer (2) includes multiple culture wells (21) connecting the gas flow layer (1) and the porous membrane layer (3). The porous membrane layer (3) is used to support the skin organoid and allow small molecules to pass through. Mass exchange; the liquid flow layer (4) includes a parallel liquid pump unit and a liquid distribution mechanism connected in series with the parallel liquid pump unit. The parallel liquid pump unit is used to stabilize the liquid flow, and the liquid distribution structure is used to distribute the liquid and buffer fluid fluctuations; the porous membrane layer (3) includes a porous membrane (32) at the bottom of the culture well (21) and a through hole that cooperates with the porous membrane (32). The sidewall of the through hole is provided with a piezoelectric element (31). The two ends of the piezoelectric element (31) are connected to the edge of the porous membrane (32) and the sidewall of the through hole respectively by a biocompatible adhesive.

2. The skin organoid chip based on multi-condition simulation detection according to claim 1, characterized in that, The parallel pump unit of the liquid flow layer (4) consists of multiple pump units connected in parallel. The liquid distribution structure includes a liquid channel. The number of pump units is equal to the number of liquid channels in the liquid flow layer (4). Each pump unit has a corresponding liquid channel. The liquid channel can be connected by a multi-port valve with the same number of outlets as the number of liquid channels.

3. The skin organoid chip based on multi-condition simulation detection according to claim 2, characterized in that, The liquid pump unit includes miniature metering pumps installed at both ends of the liquid channel. The electrical connection of the miniature metering pumps ensures that the volume of liquid flowing into and out of the liquid channel is equal.

4. The skin organoid chip based on multi-condition simulation detection according to claim 3, characterized in that, The liquid channel includes a liquid region (41) located in the middle, the top of the liquid region (41) is in contact with the porous membrane layer (3) and passes through the bottom of the culture pore (21), the two ends of the liquid region (41) and the liquid channel are horizontally arranged in the liquid flow layer (4), and the height of the liquid region (41) is higher than the two ends of the liquid channel.

5. A skin organoid chip based on multi-condition simulation detection according to claim 4, characterized in that, The area of ​​the liquid flow cross section of the liquid region (41) is greater than the area of ​​the liquid flow cross section at both ends of the liquid channel, the depth of the liquid region (41) is less than 1 mm, and the depth of the liquid region (41) satisfies the requirement that the liquid flows within the liquid flow layer (4).

6. A skin organoid chip based on multi-condition simulation detection according to claim 5, characterized in that, The bottom of the liquid region (41) is provided with a temperature control element (42), which corresponds to the culture well (21). The surface of the temperature control element (42) is covered with a thermally conductive silicone layer, which transfers the temperature to the skin organoid of the culture layer (2) through the liquid in the liquid region (41).

7. The skin organoid chip based on multi-condition simulation detection according to claim 1, characterized in that, The gas flow layer (1) includes a gas channel (11), with downward bends at both ends of the gas channel (11). The bottom of the gas channel (11) is in contact with the porous membrane layer (3) and passes through the top of the culture pore (21).

8. A skin organoid chip based on multi-condition simulation detection according to claim 7, characterized in that, The depth of the gas channel (11) is less than 1 mm, and the depth of the gas channel (11) is sufficient for the gas to flow in the gas flow layer (1).

9. A skin organoid chip based on multi-condition simulation detection according to claim 1, characterized in that, The diameter of the porous membrane (32) at the bottom of each culture well (21) is the same as that of the culture well (21), and the edge extends to the bottom of the sidewall of the culture well (21). The material of the porous membrane (32) is selected from at least one of polymethyl methacrylate, polydimethylsiloxane or polycarbonate, and the pore size of the porous membrane (32) can be selected in the range of 0.4-5 μm.

10. A skin organoid chip based on multi-condition simulation detection according to claim 1, characterized in that, The piezoelectric element (31) is a fan-shaped piezoelectric ceramic sheet. The piezoelectric ceramic sheet can be driven by voltage to generate expansion and contraction deformation, which drives the edge of the porous membrane to stretch synchronously. The piezoelectric element can generate voltage signals under pressure and tension.

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