Skin organ chip and preparation thereof, skin model construction method and application

Abstract

This invention discloses a skin organ-on-a-chip, relating to the fields of biomedical engineering and microfluidics. The chip includes a drive module integrating a reservoir with a fixed height difference, a culture layer with arrayed independent skin culture units, and a fluid layer separated by a porous membrane. The drive module utilizes the hydrostatic pressure generated by the liquid level difference to drive the culture medium to form a stable laminar flow in an optimized fluid channel network, achieving pump-free perfusion. This invention achieves stable pump-free drive through a reservoir with a fixed height difference; the integrated sealed structure eliminates the risk of contamination and ensures complete isolation between units; the simulation-optimized uniform flow channel network, in synergy with the drive, ensures high consistency in high-throughput parallel culture, enabling the chip to provide a long-term stable biomimetic perfusion microenvironment. A single experiment can efficiently complete multiple replicates and multi-condition transdermal drug screening, toxicity testing, and disease model construction, improving experimental throughput, data reliability, and ease of operation.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and microfluidics, and particularly to a skin organ-on-a-chip, its preparation, skin model construction method, and applications. Background Technology

[0002] With the deepening of biomedical research, traditional two-dimensional cell culture and animal models are insufficient to simulate the dynamic fluid microenvironment, mechanical stimulation, and complex interactions between tissues within the human body. Organ-on-a-chip technology has emerged to address this need. By integrating microfluidic channels and cell culture chambers at the microscale and constructing them using biocompatible materials, it can reproduce key physiological characteristics of organs, such as tissue interfaces, mechanical stimulation, and biochemical gradients, providing a revolutionary in vitro model platform for drug screening, toxicity testing, and disease mechanism research.

[0003] Skin organ-on-a-chip technology, an important branch of this field, aims to simulate the multi-layered structure of human skin (such as the stratum corneum, epidermis, and dermis) and its barrier function. Typical models construct physiologically relevant skin tissues by co-culturing keratinocytes and fibroblasts on a chip, which are then used to study transdermal drug absorption, irritation assessment, wound healing, and inflammatory responses. However, current skin organ-on-a-chip technology still faces several significant bottlenecks, hindering its transformation into standardized, high-throughput applications.

[0004] First, in terms of the driving method, most systems rely on external active micropumps (such as peristaltic pumps and syringe pumps) to provide perfusion power. This method results in complex system structure, large size, and a high degree of specialization in operation. Furthermore, the pulsating fluid generated by the pump introduces non-physiological shear force disturbances, disrupting the stability of the microenvironment and hindering the long-term culture and functional maintenance of skin tissue. The tubing connections also increase the risk of contamination and air bubbles.

[0005] Secondly, in terms of experimental throughput, common chips typically integrate only a single or a few (e.g., 2-4) culture units, making it difficult to set up sufficient biological replicates or multi-condition gradient tests in parallel in a single experiment, thus failing to meet the needs of high-efficiency drug screening and systematic research.

[0006] To simplify the system, some studies have attempted to employ pump-free drive strategies, such as using liquid evaporation or oscillating gravity-driven agitation to generate fluid motion. However, these alternatives still have certain limitations. For example, evaporation-based agitation is greatly affected by ambient temperature and humidity, resulting in unstable flow rates and potential concentration of culture medium components; while oscillating agitation generates periodic oscillating flow, which cannot provide the stable, homogeneous laminar flow environment required for cell growth and differentiation. More importantly, such unstable flow fields are difficult to maintain consistent hydrodynamic stimulation across multiple parallel culture units, thus failing to support high-throughput, high-data-consistency screening experiments.

[0007] Therefore, there is an urgent need for a skin organ chip that can simultaneously achieve pump-free operation, stable drive, and support high-throughput biomimetic culture. Summary of the Invention

[0008] In order to overcome the above-mentioned defects of the prior art, the present invention provides a skin organ chip and its preparation method to solve the problems existing in the background art.

[0009] This invention provides the following technical solution: a skin organ chip, comprising: The chip body has a culture layer and a fluid layer arranged sequentially from top to bottom, and the culture layer and the fluid layer are separated by a porous membrane. The culture layer contains multiple independent skin culture units arranged in an array. Each skin culture unit includes a culture chamber that penetrates the culture layer and communicates with the upper surface of the porous membrane. A fluid channel network is provided within the fluid layer, the fluid channel network being located below the porous membrane and covering the arrangement area of ​​the plurality of skin culture units; The chip body is also provided with a driving module for driving the flow of culture medium. The driving module includes at least one high-level reservoir and a low-level collection section. The outlet of the high-level reservoir is connected to the inlet of the fluid channel network, and the inlet of the low-level collection section is connected to the outlet of the fluid channel network. There is a height difference between the liquid surface of the high-level reservoir and the liquid surface of the low-level collection section, so that the static pressure generated by the height difference drives the culture medium to form a stable laminar flow through the fluid channel network and perfuse the cells in the culture chamber through the porous membrane.

[0010] Preferably, the height difference Δh The value range is 10~100mm.

[0011] Preferably, the number of multiple skin culture units is 9 to 24, and they are arranged in a rectangular array of 3×3 or 4×6.

[0012] Preferably, the porous membrane is made of one of polycarbonate, polyethylene terephthalate or polydimethylsiloxane, and its pore size ranges from 0.4 to 8.0 μm.

[0013] Preferably, the high-level liquid storage tank and the low-level collection section are both integrated on the chip body, with the high-level liquid storage tank located above one end of the chip body.

[0014] Preferably, the fluid channel network includes multiple parallel main channels and multiple branch channels communicating with each of the main channels. The branch channels are arranged in a direction parallel to the main channels. Each branch channel is also provided with a widening section, and the projection of the widening section under the porous membrane covers the corresponding culture chamber.

[0015] Preferably, the chip body is made of polydimethylsiloxane, and the culture layer, the fluid layer, and the porous membrane are formed into an integral structure by plasma bonding.

[0016] A method for preparing a skin organ-on-a-chip includes the following steps: S21: Based on the design drawing optimized by simulating the gravity-driven laminar flow state using COMSOL software, 3D printing technology was used to process the molds of the top layer liquid storage tank and culture chamber structure, as well as the bottom layer fluid channel structure. S22: The polydimethylsiloxane PDMS prepolymer is poured into the mold, cured, and demolded to obtain the corresponding culture layer and fluid layer; the polycarbonate porous membrane with a hydrophilic surface is precisely aligned and placed in the preset area of ​​the fluid layer; S23: The culture layer, porous membrane and fluid layer are bonded by oxygen plasma treatment to form a chip body with a complete sealed microfluidic network inside; S24: Sterilize the assembled chip and wet the internal channels of the chip with sterile PBS or culture medium before use.

[0017] A method for constructing a skin model using a skin organ-on-a-chip includes the following steps: S31: A skin organ chip is provided, and a stable static pressure for driving the culture medium is established by adjusting the fixed height difference between the high-level reservoir and the low-level collection part. S32: Based on the chip body prepared in step S31, a stable static pressure for driving the culture medium is established by adjusting the fixed height difference between the high-level liquid storage tank and the low-level collection section. S33: In the culture chamber of the chip, skin tissue structures are constructed respectively; the construction of skin tissue structures includes: seeding dermal cells on the porous membrane and culturing to form a dermal layer, and then seeding epidermal cells on the dermal layer; S34: Add culture medium to the high-level storage tank, and under the drive of the stable static pressure, make the culture medium flow through the fluid channel network, and perform parallel dynamic perfusion culture of the skin tissue structures in the multiple skin culture units through the porous membrane; at the same time, expose the epidermal cells to the air for gas-liquid interface culture to induce their differentiation to form the stratum corneum.

[0018] The application of a skin model constructed using a skin model construction method in the construction of skin disease models, drug screening, and / or evaluation of cosmetic irritation.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses a high-level storage tank with a fixed height difference and a low-level collection section as driving modules. It utilizes the principle of hydrostatic pressure to provide power to the system, achieving stable perfusion without the need for a complex external pump. Compared with traditional peristaltic pumps, syringe pumps and their control circuits, it not only simplifies the system structure but also reduces equipment costs and the barriers to operation and maintenance. At the same time, the constant static pressure difference drives continuous, stable and pulse-free laminar flow. This laminar flow closely simulates the physiological environment of slow infiltration of interstitial fluid in the body, effectively eliminating the periodic mechanical interference of pulse shear force on skin cells, thereby improving the long-term stable mechanical microenvironment of the skin barrier function. Meanwhile, the structural design without any mechanical moving parts ensures near-zero failure silent operation during long-term culture, thus improving the reliability of the system. 2. This invention employs an integrated sealed structure in which the culture layer, porous membrane, and fluid layer are permanently bonded by plasma, forming a complete closed microfluidic network. This effectively avoids leakage that may occur due to pipe connections or detachable assembly interfaces, preventing external contamination during long-term skin tissue culture. At the same time, complete sealing and isolation ensure that each skin culture unit and its underlying flow channel form an independent microenvironment. When conducting high-throughput parallel testing with multiple conditions and samples, it avoids cross-contamination caused by different drugs or test substances sharing flow paths or gas phase spaces, thereby ensuring the absolute independence and high reliability of the data from each experimental group. 3. This invention combines a uniform flow channel network optimized by hydrodynamic simulation with a static pressure drive source with a fixed height difference. Under constant static pressure, the flow channel network ensures that the culture medium flows through the bottom of each culture chamber at the same flow rate and pressure. This guarantees that the nutrient exchange and fluid shear force stimulation obtained by all parallel culture units are consistent. As a result, multiple skin models cultured simultaneously on the same chip exhibit high synchronicity and comparability in their developmental status, differentiation degree, and barrier function, thereby achieving the goal of high-throughput screening. 4. This invention integrates multiple independent skin culture units arranged in an array on a single-chip substrate, and works in conjunction with a stable drive and uniform flow channel system, which not only improves the overall experimental efficiency but also reduces the cost. At the same time, all units share a unified infusion port and sample loading interface, simplifying the operation process. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the skin organ chip structure of the present invention.

[0021] Figure 2 This is a schematic diagram of the flow velocity in the fluid channel network of the present invention.

[0022] Figure 3 This is a schematic diagram of the skin tissue structure according to the present invention.

[0023] The attached figures are labeled as follows: 100, culture layer; 101, culture chamber; 102, high-level storage tank; 103, low-level collection section; 200, fluid layer; 201, fluid channel network; 2011, parallel main flow channel; 2012, branch flow channel; 2013, widening section; 300, porous membrane. Detailed Implementation

[0024] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0025] Example 1

[0026] A pump-free, gravity-driven high-throughput skin organ-on-a-chip, such as Figures 1 to 3 As shown, this embodiment provides a pump-free, gravity-driven high-throughput skin organ-on-a-chip.

[0027] The skin organ chip includes a chip body, a driving module, a culture layer 100, a fluid layer 200, and a porous membrane 300.

[0028] The chip body serves as the supporting structure for the entire device and is preferably fabricated using microfabrication technology with good biocompatibility and optical transparency, polydimethylsiloxane (PDMS). PDMS material possesses excellent breathability, elasticity, and reproducible moldability, making it suitable for constructing microfluidic structures.

[0029] The chip body contains a culture layer 100 and a fluid layer 200 arranged sequentially from top to bottom. The culture layer 100 and the fluid layer 200 are separated by a porous membrane 300.

[0030] The culture layer 100, fluid layer 200, and porous membrane 300 are permanently bonded through oxygen plasma treatment, forming an integrated, non-removable, and completely sealed microfluidic network. This integrated sealed structure fundamentally eliminates potential leaks, contamination, and uncontrollable differences in fluid paths between culture units during long-term culture, ensuring the consistency of high-throughput experimental data.

[0031] The culture layer 100 contains multiple independent skin culture units arranged in an array. Each skin culture unit is a culture chamber 101 that extends through the culture layer 100. The lower part of this culture chamber 101 is directly connected to the upper surface of the porous membrane 300. The independent design of multiple skin culture units allows for multiple parallel experiments to be performed simultaneously on a single chip, thereby increasing experimental throughput. As a preferred embodiment, the number of skin culture units is 9 or 24, arranged in a 3×3 or 4×6 rectangular array accordingly. The diameter of the culture chamber 101 can be designed as needed, for example, 3-6 mm, and the depth is 1-2 mm, to accommodate a sufficient volume of three-dimensional skin tissue.

[0032] The fluid layer 200 contains a sophisticated fluid channel network 201 located beneath the porous membrane 300. This network design, optimized through fluid dynamics simulation, ensures that the stable flow field generated by the drive module uniformly covers the entire arrangement area of ​​the skin culture units. Specifically, the fluid channel network 201 includes multiple parallel main channels 2011 and multiple branch channels 2012 connected to each main channel 2011. The branch channels 2012 are arranged parallel to the main channels 2011. Specifically, each branch channel 2012 has a widened section 2013 at a location corresponding to a culture chamber 101. The projection of this widened section 2013 beneath the porous membrane 300 completely covers the corresponding culture chamber 101 area. This reduces the local fluid velocity, creating a diffusion-dominated, uniform nutrient supply zone beneath the culture chamber 101, while simultaneously ensuring that the fluid shear force applied to the cells on the porous membrane remains within a low, biomimetic physiological range.

[0033] The porous membrane 300 serves as the interface connecting static culture and dynamic perfusion, separating the epidermal and dermal microenvironments. The porous membrane 300 can be selected from polycarbonate, polyethylene terephthalate, or polydimethylsiloxane with a hydrophilicated surface. The pore size range is preferably 0.4–8.0 μm. This pore size range allows for the free diffusion of nutrients, growth factors, and metabolic waste in the culture medium, while effectively supporting the attachment, growth, and extracellular matrix deposition of dermal fibroblasts, and preventing cells from migrating downwards from the culture chamber 101 into the fluid channel. The porous membrane 300 undergoes hydrophilication treatment before use to ensure that the culture medium within the PDMS channel can smoothly wet and pass through the membrane pores.

[0034] The drive module includes at least one high-level liquid storage tank 102 and a low-level collection section 103. The outlet of the high-level liquid storage tank 102 is connected to the inlet of the fluid channel network 201, and the inlet of the low-level collection section 103 is connected to the outlet of the fluid channel network 201.

[0035] The high-level liquid storage tank 102 and the low-level collection section 103 are both integrated on the chip body. The high-level liquid storage tank 102 is located above one end of the chip body, thus naturally forming a liquid level difference with the low-level collection section 103 located at the other end or below. Δ h .

[0036] By precisely adjusting the initial liquid addition volume of the high-level liquid storage tank 102 or its installation height relative to the chip body, a fixed height difference can be set and maintained. Δh According to fluid mechanics principles, this fixed height difference will generate a constant and calculable static pressure. Driven by this static pressure, the culture medium will follow a path from high to low, forming a smooth, continuous, and pulse-free laminar flow within the fluid channel network 201. The preferred value range for the height difference Δh is 10~100mm.

[0037] Brief description of working principle: Fresh culture medium is added to the high-level storage tank 102, and the culture medium is kept at a fixed height difference. Δh Driven by the generated stable hydrostatic pressure, the culture medium flows at a constant low flow rate through the fluid channel network 201 covering all culture units. Nutrients diffuse through the porous membrane 300 into the skin tissue within the culture chamber 101 above it, and finally flow into the low-level collection section 103. The entire process requires no mechanical pumps, achieving long-term, silent, and stable dynamic perfusion.

[0038] Example 2

[0039] A method for preparing a skin organ-on-a-chip, this embodiment describes in detail the method for preparing the skin organ-on-a-chip described in Example 1, the method comprising the following steps: S21: Simulation Optimization and Mold Fabrication. First, based on the chip's 3D design, fluid dynamics simulations are performed using finite element analysis software such as COMSOL Multiphysics. The simulated target height difference... Δh The dimensions, length, and arrangement of the fluid channel network 201 were simulated and optimized, with the goal of obtaining a fluid shear force distribution with a difference rate of less than 10% in the porous membrane regions corresponding to each culture chamber 101. Based on the optimized design model, high-precision 3D printing technology was used to fabricate male molds for forming the culture layer 100 and male molds for forming the fluid layer 200.

[0040] S22: PDMS Casting and Membrane Preparation. The PDMS prepolymer and curing agent are mixed in a standard ratio and thoroughly degassed. The mixture is poured into two positive molds obtained in step S1 and placed in an oven for curing. After curing, the molds are carefully demolded to obtain the corresponding PDMS components with culture layer 100 and fluid layer 200. Simultaneously, a polycarbonate porous membrane 300 is prepared, and its surface is subjected to oxygen plasma hydrophilication treatment to enhance its hydrophilicity.

[0041] S23: Precision Alignment and Permanent Bonding. With the aid of a microscope or a precision alignment fixture, the hydrophilically treated porous membrane 300 is precisely aligned and placed in a predetermined area of ​​the fluid layer 200, ensuring that the membrane completely covers the widened sections 2013 of all branch channels 2012. Then, the culture layer 100 is aligned and placed on top of the porous membrane 300. The stacked culture layer 100, porous membrane 300, and fluid layer 200 are then placed in a plasma cleaner for oxygen plasma treatment. Immediately after treatment, the layers are tightly bonded together, utilizing the active silanol groups generated on the PDMS surface to undergo a condensation reaction, forming strong and permanent chemical bonds at room temperature, thereby constructing a completely sealed integrated microfluidic network.

[0042] S24: Sterilization and Pretreatment. The bonded, intact chip is placed in a sterilization bag and sterilized using ethylene oxide gas or gamma rays. Before use, sterile phosphate-buffered saline (PBS) or basal culture medium is injected through a syringe from the inlet of the high-level reservoir 102, allowing the liquid to naturally fill the entire fluid channel network 201 under gravity and wet the porous membrane 300, thus removing all air bubbles and preparing for cell seeding.

[0043] Example 3

[0044] A method for constructing a skin model, which is described in detail in this embodiment using the chip described in embodiment 1 or 2, includes the following steps: S31: Chip Preparation and Driver Setup. A sterilized chip prepared as in Example 2 is provided. By injecting a certain volume of culture medium into the high-level storage tank 102, or by fine-tuning the height of its support frame, a preset fixed height difference is precisely established between the liquid surface of the high-level storage tank 102 and the liquid surface of the low-level collection section 103. Δh It is 20mm.

[0045] S32: Dermal Layer Construction. The dermal layer is constructed in each individual culture chamber 101 of the chip. Specifically, a mixture of collagen or matrix gel containing human dermal fibroblasts (HDFs) is carefully seeded onto the porous membrane 300 within each culture chamber 101. The chip is placed in a cell culture incubator and allowed to stand for 1-2 hours until the gel solidifies. Subsequently, dermal fibroblast culture medium is added from the high-level reservoir 102, and under the hydrostatic pressure established in step S10, low-speed dynamic perfusion culture of the dermal layer begins, continuing for 3-7 days to form an activated dermal equivalent layer rich in cells and matrix.

[0046] S33: Epidermal inoculation and air-liquid interface culture. Human keratinocyte (HaCaT) suspension was inoculated into each culture chamber 101 on the surface of the mature dermis. Dynamic perfusion culture was continued using a keratinocyte-specific culture medium containing growth factors for approximately 2-3 days to allow the keratinocytes to fully adhere, proliferate, and fuse into a monolayer.

[0047] S34: Parallel Dynamic Perfusion and Stratum Corneum Differentiation. This step is crucial for achieving high-throughput parallel construction and functional maturation. First, the perfusion of liquid from the elevated reservoir 102 is stopped, and the culture medium level in the culture chamber 101 is lowered until only the dermis is wetted, exposing the epidermis, thus establishing an air-liquid interface. Then, the culture medium in the elevated reservoir 102 is replaced with a dedicated gas-liquid interface differentiation medium. Driven by the stable hydrostatic pressure generated by a constant height difference Δh, the differentiation medium flows continuously and smoothly through the fluid channel network 201 below, providing nutrients to the dermis through the porous membrane 300 and maintaining its humidity environment; while the epidermal cells exposed to air proliferate and differentiate upwards under the stimulation of air and the induction of signals from the dermis, successively forming the spinous layer, the granular layer, and finally keratinizing to form a dense stratum corneum. This differentiation process typically takes 7-14 days. Because all culture units share the same stable-driven perfusion system, and the flow channels are optimized for homogeneity, the skin models within each unit can mature at a highly synchronized and consistent rate.

[0048] Example 4

[0049] A skin model construction method is presented, and the skin model is applied to the construction of skin inflammation models and drug screening. This embodiment demonstrates the use of the chip described in Embodiment 1 and the skin model construction method in Embodiment 3 to construct a skin inflammation model and use it for parallel screening of anti-inflammatory drugs. The specific steps are as follows: S41: Preparation of the dermis. Human dermal fibroblasts, rat tail collagen, etc. are mixed in a certain proportion and injected into the central culture chamber 5 of the chip placed on the upper channel 1; cell culture medium is added to the culture chamber and fluid channel 6 through the inlet 3 on the chip, and cultured for a certain period of time to form the dermis C; S42: Preparation of the epidermal layer. Add a mixture containing keratinocytes and culture medium to the central culture chamber 5; continue culturing for a certain period of time to form the epidermal layer B above the dermis; S43: Preparation of the stratum corneum. Air is introduced into the central culture chamber 5 through channels 1 and 2 on the chip to promote the keratinization of the epidermis and form the stratum corneum A; culture is continued for a certain period of time to complete the construction of the skin tissue model; S44: Inflammation Induction and Drug Screening. The constructed skin model was exposed to inflammatory inducing factors (such as tumor necrosis factor-α, TNF-α); the recruitment and differentiation of circulating monocytes into M1 macrophages and their infiltration in the skin model were observed; the anti-inflammatory drugs to be screened were applied topically to the skin model, and the anti-inflammatory effect of the drugs was evaluated by monitoring the release of cytokines (such as interleukin-6, IL-6).

[0050] Example 5

[0051] A skin model construction method is presented, and the skin model is applied to skin cancer model construction and drug screening. This embodiment demonstrates the use of the chip described in Embodiment 1 and the skin model construction method in Embodiment 3 to construct a three-dimensional skin cancer model and simulate the tumor microenvironment for anti-tumor drug testing. The specific steps are as follows: S51: Preparation of a skin tissue model. The preparation method for the skin model is the same as that in Example 1: prepare the dermis (C); prepare the epidermis (B); prepare the stratum corneum (A); S52: Inoculation of skin cancer cells. Skin cancer cells (such as melanoma cells) are inoculated into the epidermal layer B or dermal layer C of a skin model; they are then cultured for a certain period of time (e.g., 3 days) to allow the cancer cells to grow and spread within the skin model; S53: Tumor Microenvironment Simulation and Drug Screening. By adjusting parameters such as fluid flow and oxygen concentration in the chip, hypoxic and acidic conditions in the tumor microenvironment are simulated to promote the growth and invasion of tumor cells. The anti-tumor drugs to be screened are added to a skin cancer model, and the anti-tumor effects of the drugs are evaluated by monitoring the proliferation, apoptosis, and invasion capabilities of tumor cells.

[0052] Example 6

[0053] A skin model construction method is provided for skin toxicity testing of cosmetics or chemicals. This embodiment demonstrates the use of the chip described in Embodiment 1 and the skin model construction method of Embodiment 3 to construct a skin irritation or corrosion test. The specific steps are as follows: S61: Preparation of a skin tissue model. The preparation method for the skin model is the same as that in Example 1: prepare the dermis (C); prepare the epidermis (B); prepare the stratum corneum (A); S62: Toxicity test substance exposure. The substance to be tested (such as cosmetics, drugs, etc.) is applied topically to a skin model, for example, the substance is immersed in a filter paper plate, and then the filter paper plate is placed on the stratum corneum A of the skin model; the control group uses a filter paper plate without the test substance or an unexposed culture; S63: Toxicity assessment. The skin toxicity of the test substance is assessed by monitoring indicators such as cellular metabolic status (e.g., MTT assay), cytokine release (e.g., interleukin-1β, IL-1β), and cellular morphological changes; the differences in indicators between the experimental group and the control group are compared to determine the degree of skin toxicity of the test substance.

[0054] Several points should be noted: First, in the description of this application, it should be noted that, unless otherwise specified and limited, the terms "installation", "connection" and "linkage" should be interpreted broadly, and can be mechanical or electrical connection, or internal connection between two components, or direct connection. "Up", "down", "left", "right", etc. are only used to indicate relative positional relationship. When the absolute position of the described object changes, the relative positional relationship may change.

[0055] The above description is only a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. Any equivalent modifications or changes made by those skilled in the art based on the content disclosed in the present invention should be included within the scope of protection set forth in the claims.

Claims

1. A skin organ chip, characterized in that, include: The chip body has a culture layer (100) and a fluid layer (200) arranged sequentially from top to bottom, and the culture layer (100) and the fluid layer (200) are separated by a porous membrane (300). The culture layer (100) contains a plurality of independent skin culture units arranged in an array. Each skin culture unit includes a culture chamber (101) that penetrates the culture layer (100) and communicates with the upper surface of the porous membrane (300). A fluid channel network (201) is provided within the fluid layer (200), the fluid channel network (201) is located below the porous membrane (300) and covers the arrangement area of ​​the plurality of skin culture units; The chip body is also provided with a driving module for driving the flow of culture medium. The driving module includes at least one high-level reservoir (102) and a low-level collection section (103). The outlet of the high-level reservoir (102) is connected to the inlet of the fluid channel network (201), and the inlet of the low-level collection section (103) is connected to the outlet of the fluid channel network (201). There is a height difference between the liquid surface of the high-level reservoir (102) and the liquid surface of the low-level collection section (103) so that the static pressure generated by the height difference drives the culture medium to form a stable laminar flow through the fluid channel network (201) and perfuse the cells in the culture chamber (101) through the porous membrane (300).

2. The skin organ chip according to claim 1, characterized in that: The height difference Δh The value range is 10~100mm.

3. The skin organ chip according to claim 1, characterized in that: The number of skin culture units is 9 to 24, arranged in a rectangular array of 3×3 or 4×6.

4. A skin organ chip according to claim 1, characterized in that: The porous membrane (300) is made of one of polycarbonate, polyethylene terephthalate or polydimethylsiloxane, and its pore size ranges from 0.4 to 8.0 μm.

5. A skin organ chip according to claim 1, characterized in that: The high-level liquid storage tank (102) and the low-level collection section (103) are both integrated on the chip body, with the high-level liquid storage tank (102) located above one end of the chip body.

6. A skin organ chip according to claim 1, characterized in that: The fluid channel network (201) includes multiple parallel main channels and multiple branch channels (2012) connected to each of the main channels. The branch channels (2012) are arranged in a direction parallel to the main channels (2011). Each branch channel (2012) is also provided with a widening section (2013), and the projection of the widening section (2013) below the porous membrane (300) covers the corresponding culture chamber (101).

7. A skin organ chip according to claim 1, characterized in that: The chip body is made of polydimethylsiloxane, and the culture layer (100), the fluid layer (200), and the porous membrane (300) are formed into an integral structure by plasma bonding.

8. A method for preparing a skin organ-on-a-chip according to any one of claims 1-7, characterized in that, Includes the following steps: S21: Based on the design drawing optimized by simulating the gravity-driven laminar flow state using COMSOL software, 3D printing technology was used to process the molds of the top layer liquid storage tank and culture chamber structure, as well as the bottom layer fluid channel structure. S22: The polydimethylsiloxane PDMS prepolymer is poured into the mold, cured and demolded to obtain the corresponding culture layer (100) and fluid layer (200); the polycarbonate porous membrane (300) with hydrophilic treatment is precisely aligned and placed in the preset area of ​​the fluid layer (200); S23: The culture layer (100), porous membrane (300) and fluid layer (200) are bonded by oxygen plasma treatment to form a chip body with a complete sealed microfluidic network inside; S42: Sterilize the assembled chip and wet the internal channels of the chip with sterile PBS or culture medium before use.

9. A method for constructing a skin model, based on a skin organ-on-a-chip according to any one of claims 1-7, characterized in that, Includes the following steps: S31: A skin organ chip is provided, and a stable static pressure for driving the culture medium is established by adjusting the fixed height difference between the high-level reservoir (102) and the low-level collection part (103); S32: Based on the chip body prepared in step S31, a stable static pressure for driving the culture medium is established by adjusting the fixed height difference between the high-level liquid storage tank (102) and the low-level collection part (103). S33: Skin tissue structures are constructed in the culture chamber (101) of the chip; The construction of the skin tissue structure includes: seeding dermal cells onto the porous membrane (300) and culturing to form a dermal layer, and then seeding epidermal cells onto the dermal layer; S34: Add culture medium to the high-level storage tank (102), and under the drive of the stable static pressure, make the culture medium flow through the fluid channel network (201), and perform parallel dynamic perfusion culture of the skin tissue structure in the multiple skin culture units through the porous membrane (300); at the same time, expose the epidermal cells to the air for gas-liquid interface culture to induce their differentiation to form the stratum corneum.

10. The application of the skin model constructed by the skin model construction method according to claim 9 in the construction of skin disease models and drug screening and / or evaluation of cosmetic irritation.

Images