Lung organ chip based on stretchable biological membrane and preparation method thereof

Abstract

The invention discloses a lung organ chip based on a stretchable biological membrane and a preparation method of the lung organ chip, and belongs to the field of biomedicine and pharmacology. The chip comprises a top cell culture layer, a bottom vacuum negative pressure layer and a stretchable porous membrane layer, the top cell culture layer comprises a cell culture chamber, a fluid micro-channel and a fluid micro-channel outlet / inlet, and the cell culture chamber, the fluid micro-channel and the fluid micro-channel outlet / inlet are communicated with one another; the bottom vacuum negative-pressure layer comprises a vacuum negative-pressure chamber, a gas micro-channel and a gas micro-channel outlet / inlet, and the vacuum negative-pressure chamber, the gas micro-channel and the gas micro-channel outlet / inlet are communicated with one another; the stretchable porous film layer is a deformable single-layer elastic film, and a plurality of through round holes simulating the alveolar structure are formed in the middle of the stretchable porous film layer. The method has the characteristics of low cost, easiness in manufacturing, easiness in operation and the like, and application and popularization of the lung organ chip are facilitated.

Description

Technical Field

[0001] The present invention relates to a lung organ chip based on a stretchable biomembrane and a preparation method thereof, and belongs to the fields of biomedicine and pharmacology. Background Art

[0002] The lung is an important respiratory organ in the human body. Its main function is to transport oxygen from the outside to the blood and to expel stored carbon dioxide from the body. In recent years, due to the aging population, high smoking rates and air pollution, the incidence and mortality of lung diseases have continued to rise worldwide. Common chronic lung diseases, such as COPD, have become a major health issue worldwide due to their high incidence and mortality.

[0003] Lung transplantation is currently an effective treatment for patients with end-stage lung disease, but only about 20% of patients can wait for suitable donor lungs for transplantation. In addition to the lack of suitable donor lungs, lung transplantation is further complicated by the high incidence of acute and chronic rejection.

[0004] 2D cell culture is an important experimental method for studying the occurrence and development of diseases and early drug research. However, this single, flat cell culture model causes cells to lose their original interactions with other cells and extracellular matrix in the in vivo environment. After the cells lose these extracellular information transmission modes, their original state and gene expression patterns also undergo tremendous changes. Therefore, the positive data obtained in cell experiments often cannot obtain ideal results after in vivo experimental verification.

[0005] Although the results of animal experiments are more reliable, the cost of experimental animals, especially non-human primates, is often very high. In recent years, government management agencies in many countries have put forward higher requirements for animal welfare and ethics, which has greatly hindered the development of animal experiments. More importantly, the physiological environment of mice and even primates is not completely similar to that of humans, which will also lead to huge differences in in vitro and in vivo experimental results. Therefore, it is very necessary to develop an in vitro experimental platform that can better simulate the human body environment. Before clinical testing, drugs need to be tested on disease experimental models in in vitro models (for example: cell culture) and in vivo models (i.e. on animals). 3D culture technology is a complex in vitro cell culture method that integrates multiple types of cells and extracellular matrix. It can greatly enrich the types of external stimuli that cells can accept during in vitro culture, so it can more realistically reflect the human body environment. Microfluidics refers to the science and technology involved in the system that uses microchannels (with a size of tens to hundreds of microns) to process or manipulate tiny fluids (volumes of picoliters to nanoliters). It is an emerging interdisciplinary subject involving chemistry, fluid physics, microelectronics, new materials, biology and biomedical engineering. On this basis, the working platform formed by mounting the 3D culture system on an automatically controllable microfluidic chip is called an organ chip, which is expected to replace traditional cell culture and become the main form of cell experiment in the subsequent drug development process.

[0006] Alveoli are the main site for gas exchange and the main structure of the lungs. The alveoli have alveolar walls and alveolar septa. The alveolar walls are mainly composed of type I alveolar epithelial cells and type II alveolar epithelial cells. Type I alveolar epithelial cells are slightly thicker than the nucleus-containing part, and the rest are flat. Type II alveolar epithelial cells are rectangular, cubic or nearly spherical, and are scattered among type I alveolar epithelial cells. Type I accounts for about 95% of the alveolar area and is the main respiratory site. Type II alveolar epithelial cells mainly secrete surfactant required by the alveoli. The alveolar septa are rich in capillaries and elastic fibers. Its anatomical and physiological functions, on the one hand, constitute the air-blood-barrier (ABB) and the alveolar fluid-air interface; on the other hand, under rhythmic respiratory movements, it ensures the connectivity between alveoli. At present, most alveolar model studies use organ chip technology to simulate the physiological structure of alveoli, construct the gas-cell-liquid interface, and then simulate respiratory movements.

[0007] Currently, there is no lung organ-on-a-chip that can simulate the simultaneous expansion of alveoli and alveolar septa, that is, the expansion of the entire alveolar sac.

[0008] Ingber et al. (1. Huh, D.; Matthews, BD; Mammoto, A.; Montoya-Zavala, M.; Hsin, HY; Ingber, DE, Reconstituting Organ-Level Lung Functions ona Chip. Science 2010, 328 (5986), 1662-1668.) reported a PDMS microdevice to construct a breathable alveolar model in 2010. This method has great potential in in vitro toxicology and drug development, but several key factors cannot effectively simulate the in vivo characteristics of the lung organ. The mechanical properties of the porous PDMS membrane coated with collagen are far different from those of the physiological alveolar matrix, and its pore size is about 10 µm, which is much smaller than the average diameter of the alveoli (about 200 µm), which is quite different from the interaction between cells and matrix reflected in vivo. Crucially, this lung organ model is difficult to simulate the three-dimensional porous and interpenetrating physiological structure of human alveoli.

[0009] Huang Di et al (Huang, D.; Liu, TT; Liao, JL; Maharjan, S.; YS, Reversed-engineered human alveolar lung-on-a-chip model. P Natl Acad Sci USA 2021, 118 (19). In 2021, a physiologically relevant alveolar chip model was reported, consisting of a three-dimensional (3D) porous hydrogel made of methacryloyl gelatin (GelMA) with an inverse opal structure, bonded to a chip device that provides an air-liquid interface and circulatory respiratory movement. It is worth noting that this GelMA structure has a high degree of similarity to human alveolar sacs and also has a hardness similar to that of the human distal lung. However, it also has certain limitations, namely that the model does not introduce a vascular system to construct an air-blood barrier.

[0010] Danielle Baptista et al (Baptista, D.; Moreira Teixeira, L.; Barata, D.; Tahmasebi Birgani, Z.; King, J.; van Riet, S.; Pasman, T.; Poot, AA; Stamatialis, D.; Rottier, RJ; Hiemstra, PS; Carlier, A.; vanBlitterswijk, C.; Habibović, P.; Giselbrecht, S.; Truckenmüller, R., 3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes. ACS Biomaterials Science & Engineering 2022, 8 (6), 2684-2699.) reported in 2021 a three-dimensional lung organ chip model based on a biomimetic micro-curved culture membrane, which reconstructed the main spherical geometry of the cell's natural microenvironment. However, the membrane is fixed and cannot be mechanically stretched to simulate respiratory movements.

[0011] Therefore, there is a need to provide an effective and reliable cell tissue model for in vitro testing in the above-mentioned field to overcome the shortcomings of the known technology. More specifically, there is a need to provide a method and device for drug screening and development at the preclinical level, which is effective and economical while overcoming the shortcomings of the known technology. Summary of the invention

[0012] In order to overcome the above-mentioned shortcomings, the present invention aims to provide a lung organ chip based on a stretchable biomembrane and a preparation method thereof, which can be used effectively, reliably, simply and economically, and the lung organ chip has the advantages of low cost and low difficulty in production.

[0013] In order to reproduce the micro-alveolar array, the bionic membrane in the organ chip provided by the present invention is designed as a porous structure, each hole represents an independent alveolus, and the bionic membrane is composed of a porous membrane scaffold and a thin film in the hole, wherein the porous membrane scaffold is made of a chemically inert material with excellent mechanical elasticity, and the thin film is formed in the hole by a hydrogel material with good biocompatibility under the combined action of gravity and liquid surface tension. The excellent mechanical properties of the bionic membrane can realize its stretching in three-dimensional space, and due to the different mechanical elasticity of the chemically inert material and the hydrogel material, under the same negative pressure, the porous membrane scaffold and the thin film in the hole will produce different degrees of stretching, which can better simulate the expansion of the alveoli during breathing. The film made of hydrogel material provides a platform and space for cell co-culture due to its good biocompatibility, thereby constructing an air-blood barrier in vitro.

[0014] The present invention provides a lung organ chip based on a stretchable biomembrane. The lung organ chip is an in vitro experimental platform that simulates the structure and function of the human lung. It can simulate the structure and microenvironment of the human lung organ on a microfluidic device through the combination of microfluidic technology and cell biology. The microfluidic device includes: a top cell culture layer, a bottom vacuum negative pressure layer and a stretchable porous membrane layer; lung epithelial cells and endothelial cells are co-cultured in the cell culture layer to form a lung organ chip.

[0015] The top cell culture layer comprises a cell culture chamber, a fluid microchannel and a fluid microchannel inlet / outlet, and the cell culture chamber, the fluid microchannel and the fluid microchannel inlet / outlet are all connected; The bottom vacuum negative pressure layer comprises a vacuum negative pressure chamber, a gas microchannel and a gas microchannel inlet / outlet, and the vacuum negative pressure chamber, the gas microchannel and the gas microchannel inlet / outlet are all connected; The fluid microchannel inlet / outlet and the gas microchannel inlet / outlet, the fluid or gas microchannel outlet and inlet are the same pipeline, and can be freely switched according to application needs; The stretchable porous membrane layer is a deformable single-layer elastic film with a plurality of transparent circular holes simulating alveolar structures in the middle, each circular hole has the same size, and the spacing between the circular holes is the same; The bottom vacuum negative pressure layer, the stretchable porous membrane layer and the top cell culture layer are assembled and connected in sequence from bottom to top.

[0016] Furthermore, the top cell culture layer is an octagonal structure, with two long sides arranged in parallel, two short sides arranged in parallel, and the long sides and the short sides are connected by four oblique sides respectively. As a microfluidic device, the size of the entire chip should not be too large. Preferably, the length of the long side is 30-50 mm, the length of the short side is 10-20 mm, and the angles between the long side and the oblique side and between the short side and the oblique side are 120°-150°; more preferably, the lengths of the two long sides are both 35 mm, the lengths of the two short sides are both 15 mm, and the angles between the long side and the oblique side and between the short side and the oblique side are both 135°.

[0017] Furthermore, the bottom vacuum negative pressure layer has the same external structure as the top cell culture layer.

[0018] Preferably, the thickness of the top cell culture layer is 3-5 mm, the cell culture chamber of the top cell culture layer is a cylindrical groove, the cross-sectional diameter of the groove is 10-12 mm, and the depth is 2-4 mm; more preferably, the thickness of the top cell culture layer is 4 mm, the cross-sectional diameter of the groove is 10 mm, and the depth is 3 mm.

[0019] Further, the fluid microchannel of the top cell culture layer is a pipe with a circular or square cross section. Preferably, a pipe with a circular cross section is selected, and the diameter of the circular cross section is 1-1.2 mm; more preferably, the diameter of the circular cross section is 1 mm; Furthermore, the fluid microchannel of the top cell culture layer is composed of two parts, which are distributed on both sides of the cell culture chamber. The two parts have the same structure and are divided into a horizontal section and an inclined section. The horizontal section connects the cell culture chamber and the inclined section, and the inclined section connects the horizontal section and the fluid microchannel outlet / inlet; preferably, the horizontal section is 6-8 mm long, the inclined section is 7-9 mm long, and the angle between the horizontal section and the inclined section is 120°-150°; more preferably, the horizontal section is 7 mm long, the inclined section is 8 mm long, and the angle between the horizontal section and the inclined section is 135°.

[0020] Preferably, the thickness of the bottom vacuum negative pressure layer is 3-5 mm, the vacuum negative pressure chamber of the bottom vacuum negative pressure layer is a cylindrical groove, the cross-sectional diameter of the groove is 10-12 mm, and the depth is 2-4 mm; more preferably, the thickness of the bottom vacuum negative pressure layer is 4 mm, the cross-sectional diameter of the groove is 10 mm, and the depth is 3 mm.

[0021] Further, the gas microchannel of the bottom vacuum negative pressure layer is a pipe with a circular or square cross section. Preferably, a pipe with a circular cross section is selected, and the diameter of the circular cross section is 1-1.2 mm; more preferably, the diameter of the circular cross section is 1 mm; Furthermore, the gas microchannel of the bottom vacuum negative pressure layer is composed of two parts, which are distributed on both sides of the vacuum negative pressure chamber. The two parts have the same structure and are divided into a horizontal section and an inclined section. The horizontal section connects the vacuum negative pressure chamber and the inclined section, and the inclined section connects the horizontal section and the gas microchannel outlet / inlet; preferably, the horizontal section is 6-8 mm long, the inclined section is 7-9 mm long, and the angle between the horizontal section and the inclined section is 120°-150°; more preferably, the horizontal section is 7 mm long, the inclined section is 8 mm long, and the angle between the horizontal section and the inclined section is 135°.

[0022] Preferably, the inlet / outlet diameters of the fluid microchannels of the top cell culture layer and the gas microchannels of the bottom vacuum negative pressure layer are both 1200-1600 μm; more preferably, the diameter is 1400 μm.

[0023] Preferably, the stretchable porous membrane layer has a thickness of 50-200 μm, more preferably, a thickness of 100 μm; Preferably, the total number of the intermediate circular holes of the stretchable porous membrane layer is 19-61, the diameter of the circular holes is 200-1000 μm, and the spacing between adjacent circular holes is 100-300 μm. More preferably, the total number of the intermediate circular holes is 61, the diameter of the circular holes is 700 μm, and the spacing between adjacent circular holes is 200 μm. Preferably, all circular holes are defined as a regular geometric arrangement, more preferably, a regular hexagonal, square or circular arrangement, and even more preferably, a regular hexagonal arrangement, and the diameter of the circumscribed circle of the hexagonal structure obtained by the 61-hole arrangement is 7.9 mm.

[0024] Furthermore, the cell culture chamber and the vacuum negative pressure chamber are separated by a stretchable porous membrane layer, and the two are not connected.

[0025] The present invention provides a method for preparing the above-mentioned lung organ chip based on a stretchable biomembrane, comprising the following steps: (1) Preparing a top cell culture layer, a bottom vacuum negative pressure layer, and a stretchable porous membrane layer through a mold; (2) dripping a hydrogel cell resuspension containing vascular endothelial cells onto the circular hole structure of the stretchable porous membrane scaffold to form a thin film in the circular hole and then performing photocuring; (3) The three-layer structure is assembled to form a microfluidic device. Lung epithelial cells are planted on the membrane by perfusion. After a dense cell monolayer is formed, the chip is completed.

[0026] Preferably, the device is obtained by micro-nano processing technology, more preferably using CNC engraving, laser engraving and soft lithography technology, wherein it is preferably obtained in layers and then combined into a whole by a bonding method.

[0027] In a preferred embodiment, the device is assembled after the production of the various components obtained by micro-nanofabrication techniques.

[0028] Preferably, the microfluidic device is obtained by demolding the casting mold, and the mold material is selected from transparent, strong, and heat-resistant materials, more preferably, one of polystyrene (PS), polycarbonate (PC), polybutylene terephthalate (PBT), polyetherimide (PEI), polytetrafluoroethylene (PTFE), and polymethyl methacrylate (PMMA); even more preferably, polymethyl methacrylate (PMMA) is selected; laser cutting PMMA sheets, and the sheets are combined and fixed to obtain the mold. The above mold needs to be sprayed with a release agent before pouring.

[0029] Preferably, the microfluidic device is made of a single chemically inert material or a composite of multiple materials, and the chemically inert material is selected from silicone rubber, fluorinated rubber, polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate (PC), glass, silicon, polyethylene glycol (PEG), polydimethylsiloxane (PDMS), etc., and more preferably, it is made of polydimethylsiloxane (PDMS).

[0030] Preferably, the hydrogel is selected from fibrin gel, collagen, collagen gelatin, hyaluronic acid, elastin, agarose, chitosan, alginate, etc., and combinations thereof, and more preferably, GelMA hydrogel is selected.

[0031] Preferably, the vascular endothelial cells include human umbilical vein endothelial cells (HUVECs), human lung microvascular cell line (HULEC-5a), human lung microvascular endothelial cells (HPMEC), human umbilical vein cell fusion cell line (EA.hy926), etc.; preferably, human umbilical vein endothelial cells (HUVECs) are selected; Preferably, the light curing time should be controlled within 20 s; more preferably, the light curing time is 10 s.

[0032] Preferably, assembly refers to the process of aligning, stacking and fixing different layers of the microfluidic device in a certain order, including bonding process, thermal / chemical connection, external clamp fixation, etc.; more preferably, a bonding process is selected.

[0033] Preferably, the bottom vacuum negative pressure layer, the stretchable porous membrane layer and the top cell culture layer of the microfluidic device are subjected to plasma treatment and silanization treatment and bonded in sequence from bottom to top; Preferably, the lung epithelial cells include type I alveolar epithelial cells (Alveolar Type I Cells, AT1), type II alveolar epithelial cells (Alveolar Type II Cells, AT2), human non-small cell lung cancer cell line (A549), human alveolar epithelial cells (HPAEpiC), etc.; preferably, human alveolar epithelial cells (HPAEpiC) are selected.

[0034] The present invention provides an application of the above-mentioned lung organ chip based on a stretchable biomembrane in respiratory simulation. Specifically, cell co-culture and respiratory simulation are performed in the lung organ chip, including the following steps: (1) Introducing culture medium into the cell culture chamber to provide nutrients to the cells on the membrane and remove metabolic waste; (2) Stretching the porous membrane by introducing negative pressure into the vacuum negative pressure chamber; The negative pressure frequency is 0.1-1 Hz, and the negative pressure intensity is within 20 kPa; through cell co-culture, an air-blood barrier is constructed in vitro, and combined with membrane stretching, a three-dimensional dynamic lung model is constructed in vitro.

[0035] Beneficial effects of the present invention: (1) The porous structure of the stretchable porous membrane of the present invention can better simulate the alveoli in the human body; (2) The present invention constructs a thinner air-blood barrier by co-culturing cells inside and on the surface of GelMA hydrogel; (3) The present invention simulates the breathing process in vitro by cyclic mechanical stretching of the biomimetic membrane under negative pressure conditions; (4) The equipment of the present invention is easy to manufacture; (5) The present invention uses PMMA molds and laser engraving to make them, without the need for traditional photolithography templates, and is low-cost; (6) The present invention can be made of two materials, and the manufacturing process of the materials used is mature and has a wide range of applications; (7) The present invention has the characteristics of low cost, easy production and easy operation, which is conducive to the application and promotion of lung organ chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the overall structure of a lung organ chip based on a stretchable biomembrane provided in an embodiment of the present invention.

[0037] Figure 2 Schematic diagram of an explosion of a lung organ chip based on a stretchable biomembrane provided in an embodiment of the present invention.

[0038] Figure 3A top view of a stretchable porous membrane layer of a lung organ chip based on a stretchable biomembrane provided in an embodiment of the present invention.

[0039] In the figure: 1 is a lung organ chip, 2 is a top cell culture layer, 3 is a middle stretchable porous membrane layer, 4 is a bottom vacuum negative pressure layer, 5 is a cell culture chamber, 6 is a fluid microchannel, 7 is a fluid microchannel inlet / outlet, 8 is a vacuum negative pressure chamber, 9 is a gas microchannel, 10 is a porous structure, and 11 is a gas microchannel inlet / outlet. DETAILED DESCRIPTION

[0040] The present invention is further illustrated by the following examples, but is not limited to the following examples. Example

[0041] like Figures 1 to 3 As shown, the present invention provides a lung organ chip 1 based on a stretchable biomembrane, comprising: a top cell culture layer 2, a middle stretchable porous membrane layer 3 and a bottom vacuum negative pressure layer 4. Lung epithelial cells and endothelial cells are co-cultured in the cell culture layer to form a lung organ chip.

[0042] The top cell culture layer 2 comprises a cell culture chamber 5, a fluid microchannel 6 and a fluid microchannel inlet / outlet 7, and the cell culture chamber, the fluid microchannel and the fluid microchannel inlet / outlet are all connected; The bottom vacuum negative pressure layer 4 comprises a vacuum negative pressure chamber 8, a gas microchannel 9 and a gas microchannel inlet / outlet 11, and the vacuum negative pressure chamber, the gas microchannel and the gas microchannel inlet / outlet are all connected; The stretchable porous membrane layer is a deformable single-layer elastic film with a plurality of transparent circular holes simulating alveolar structures in the middle, each circular hole has the same size, and the spacing between the circular holes is the same; The bottom vacuum negative pressure layer, the stretchable porous membrane layer and the top cell culture layer are assembled and connected in sequence from bottom to top.

[0043] The top cell culture layer is an octagonal structure, with two long sides arranged in parallel, two short sides arranged in parallel, and the long sides and the short sides are respectively connected by four oblique sides; the bottom vacuum negative pressure layer has the same octagonal structure as the top cell culture layer; The fluid microchannel of the top cell culture layer is composed of two parts, which are distributed on both sides of the cell culture chamber. The two parts have the same structure and are divided into a horizontal section and an inclined section. The horizontal section connects the cell culture chamber and the inclined section, and the inclined section connects the horizontal section and the fluid microchannel inlet / outlet. The pipe cross-section of the fluid microchannel of the top cell culture layer is circular or square. The gas microchannel of the bottom vacuum negative pressure layer is composed of two parts, which are distributed on both sides of the vacuum negative pressure chamber. The two parts have the same structure and are divided into a horizontal section and an inclined section. The horizontal section connects the vacuum negative pressure chamber and the inclined section, and the inclined section connects the horizontal section and the gas microchannel outlet / inlet; the pipeline cross-section of the gas microchannel of the bottom vacuum negative pressure layer is circular or square.

[0044] The dimensions of the top cell culture layer are as follows: the lengths of the two long sides are both 35 mm, the lengths of the two short sides are both 15 mm, and the angles between the long side and the hypotenuse, and between the short side and the hypotenuse are both 135°; the thickness of the top cell culture layer is 4 mm, the diameter of the groove cross section is 10 mm, and the depth is 3 mm; the horizontal section of the fluid microchannel is 7 mm long, the inclined section is 8 mm long, and the angle between the horizontal section and the inclined section is 135°; The thickness of the bottom vacuum negative pressure layer is 4 mm, the cross-sectional diameter of the groove is 10 mm, and the depth is 3 mm; the horizontal section of the gas microchannel is 7 mm long, the inclined section is 8 mm long, and the angle between the horizontal section and the inclined section is 135°; the inlet / outlet diameters of the fluid microchannel of the top cell culture layer and the gas microchannel of the bottom vacuum negative pressure layer are both 1400 μm.

[0045] The gas microchannel of the bottom vacuum negative pressure layer is a pipe with a circular cross-section, and the diameter of the circular cross-section is 1-1.2 mm; preferably, the diameter of the circular cross-section is 1 mm.

[0046] The total number of middle circular holes in the stretchable porous membrane layer is 61, the diameter of the circular holes is 700 μm, and the spacing between adjacent circular holes is 200 μm; all the circular holes are arranged in a regular regular hexagon, and the diameter of the circumscribed circle of the hexagonal structure obtained by arranging 61 holes is 7.9 mm.

[0047] Furthermore, the cell culture chamber and the vacuum negative pressure chamber are separated by a stretchable porous membrane layer, and the two are not connected.

[0048] The present invention also provides a method for preparing the above-mentioned lung organ chip, comprising: (1) Preparing a top cell culture layer, a bottom vacuum negative pressure layer, and a stretchable porous membrane layer through a mold; (2) dripping a hydrogel cell resuspension containing vascular endothelial cells onto the circular hole structure of the stretchable porous membrane scaffold to form a thin film in the circular hole and then performing photocuring; (3) The three-layer structure is assembled to form a microfluidic device. Lung epithelial cells are planted on the membrane by perfusion. After a dense cell monolayer is formed, the chip is completed.

[0049] The top cell culture layer 2 and the bottom vacuum negative pressure layer 4 are made of polydimethylsiloxane (PDMS), and the stretchable porous membrane layer is made of polydimethylsiloxane (PDMS) and methacrylated gelatin (GelMA).

[0050] The microfluidic device fabrication process is described as follows: Each part of the device is made in layers, wherein the top cell culture layer 2 and the bottom vacuum negative pressure layer 4 are made by pouring polydimethylsiloxane (PDMS) into a PMMA mold and then demolding it.

[0051] The mold is made by laser engraving PMMA sheets and pasting them together.

[0052] Preferably, PDMS and curing agent are mixed in a ratio of 10:1, briefly degassed and debubbled in a vacuum drying oven, poured on a mold, and cured at 80°C for 6 h.

[0053] Preferably, the mold needs to be sprayed with a release agent before pouring PDMS. After demoulding, the fluid microchannel inlet / outlet 7 and the gas microchannel inlet / outlet 11 are punched, washed with deionized water and placed in a drying oven for standby use.

[0054] The stretchable porous membrane layer 3 is manufactured in two steps; Step 1: By spin coating, preferably, directly purchase a 100 um thick PDMS membrane and make it into a stretchable porous membrane support by laser engraving and punching, and then wash it with deionized water and put it into a drying oven for use.

[0055] Step 2: add a methacrylylated gelatin (GelMA) cell suspension containing human umbilical vein endothelial cells (HUVECs) onto the porous structure 10 of the stretchable porous membrane scaffold, and the suspension forms a thin film inside the circular hole through the action of gravity and liquid surface tension, followed by 10 s of ultraviolet curing to obtain a complete stretchable porous membrane layer 3.

[0056] Preferably, the bottom vacuum negative pressure layer 4, the middle stretchable porous membrane layer 3 and the top cell culture layer 2 are subjected to plasma treatment and silanization treatment, and are bonded in sequence from bottom to top through positioning pins.

[0057] The culture medium containing human alveolar epithelial cells (HPAEpiC) is slowly injected into the cell culture chamber through the fluid microchannel inlet / outlet. After the human alveolar epithelial cells (HPAEpiC) adhere to the surface of the hydrogel film and form a cell monolayer, the residual culture medium is sucked off, and the lung organ chip is completed.

[0058] A preferred embodiment is provided in which cell co-culture and respiratory simulation are performed in the present lung organ chip, comprising the following steps: (1) introducing culture medium into the cell culture chamber to provide nutrients to the cells on the membrane and to clear metabolic waste.

[0059] (2) The porous membrane is stretched by introducing negative pressure into the vacuum negative pressure chamber.

[0060] The term "co-culture" refers to cell-to-cell communication between different tissues in the multiple compartments through direct contact or culture medium.

[0061] Preferably, the above-mentioned culture medium refers to a mixed culture medium consisting of a culture medium dedicated to vascular endothelial cells and a culture medium dedicated to lung epithelial cells; Preferably, stretching of the membrane is achieved by actuation of a type selected from pneumatic, hydraulic, mechanical, electrical, magnetic, thermal and combinations thereof, more preferably pneumatic actuation.

[0062] Preferably, the negative pressure frequency should be selected within the range of 0.1-1 Hz; more preferably, 0.2 Hz is selected.

[0063] Preferably, the negative pressure intensity should be controlled within 20 kPa; more preferably, 10 kPa is selected.

[0064] The lung organ core based on the stretchable biomembrane provided by the present invention constructs an air-blood barrier in vitro through cell co-culture, and combines the stretching of the membrane to construct a three-dimensional dynamic lung model in vitro.

[0065] The applications of the above-mentioned lung organ chip based on the stretchable biomembrane provided by the present invention include but are not limited to toxicity testing and drug screening. Various stimuli such as nanoparticles, inhaled drugs, etc. can be directly introduced into the chip, especially for PM2.5, nanoparticles in the air, cigarette smoke and other inhaled particles, toxicity testing and drug screening can be performed. At the same time, the stretching of the membrane in three-dimensional space is more conducive to lung disease modeling and helps to discover drugs for corresponding lung diseases.

[0066] The present invention effectively overcomes various shortcomings of the prior art and has high industrial utilization value. The present invention well simulates the micrometer-level 3D microcapsule structure of human alveoli, constructs an air-blood barrier in vitro by realizing three-dimensional cell culture inside the hydrogel, and well simulates respiratory movement by stretching the membrane. The different stretching degrees of the membrane scaffold and the film better simulate the deformation of real alveoli during breathing.

[0067] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A lung organ chip based on a stretchable biomembrane, characterized in that include: A top cell culture layer, a bottom vacuum negative pressure layer and a stretchable porous membrane layer; lung epithelial cells and endothelial cells are co-cultured in the cell culture layer to form a lung organ chip; The top cell culture layer comprises a cell culture chamber, a fluid microchannel and a fluid microchannel inlet / outlet, and the cell culture chamber, the fluid microchannel and the fluid microchannel inlet / outlet are all connected; The bottom vacuum negative pressure layer comprises a vacuum negative pressure chamber, a gas microchannel and a gas microchannel inlet / outlet, and the vacuum negative pressure chamber, the gas microchannel and the gas microchannel inlet / outlet are all connected; The stretchable porous membrane layer is a deformable single-layer elastic film with a plurality of transparent circular holes simulating alveolar structures in the middle, each circular hole has the same size, and the spacing between the circular holes is the same; The bottom vacuum negative pressure layer, the stretchable porous membrane layer and the top cell culture layer are assembled and connected in sequence from bottom to top.

2. The lung organ chip based on a stretchable biomembrane according to claim 1, characterized in that: The top cell culture layer is an octagonal structure, with two long sides arranged in parallel, two short sides arranged in parallel, and the long sides and the short sides are respectively connected by four oblique sides; the bottom vacuum negative pressure layer has the same octagonal structure as the top cell culture layer; The fluid microchannel of the top cell culture layer is composed of two parts, which are distributed on both sides of the cell culture chamber. The two parts have the same structure and are divided into a horizontal section and an inclined section. The horizontal section connects the cell culture chamber and the inclined section, and the inclined section connects the horizontal section and the fluid microchannel inlet / outlet. The pipe cross-section of the fluid microchannel of the top cell culture layer is circular or square. The gas microchannel of the bottom vacuum negative pressure layer is composed of two parts, which are distributed on both sides of the vacuum negative pressure chamber. The two parts have the same structure and are divided into a horizontal section and an inclined section. The horizontal section connects the vacuum negative pressure chamber and the inclined section, and the inclined section connects the horizontal section and the gas microchannel outlet / inlet; the pipeline cross-section of the gas microchannel of the bottom vacuum negative pressure layer is circular or square.

3. The lung organ chip based on a stretchable biomembrane according to claim 2, characterized in that: The dimensions of the top cell culture layer are as follows: the long side length is 30-50 mm, the short side length is 10-20 mm, and the angles between the long side and the hypotenuse and between the short side and the hypotenuse are 120°-150°; the thickness of the top cell culture layer is 3-5 mm, the cell culture chamber is a cylindrical groove, the cross-sectional diameter of the groove is 10-12 mm, and the depth is 2-4 mm; the channel cross-section of the fluid microchannel is circular, and the diameter of the circular cross-section is 1-1.2 mm; the horizontal section of the fluid microchannel is 6-8 mm long, the inclined section is 7-9 mm long, and the angle between the horizontal section and the inclined section is 120°-150°; The thickness of the bottom vacuum negative pressure layer is 3-5 mm, the vacuum negative pressure chamber of the bottom vacuum negative pressure layer is a cylindrical groove, the cross-sectional diameter of the groove is 10-12 mm, and the depth is 2-4 mm; the cross-section of the gas microchannel is circular, and the diameter of the circular cross-section is 1-1.2 mm; the horizontal section of the gas microchannel is 6-8 mm long, the inclined section is 7-9 mm long, and the angle between the horizontal section and the inclined section is 120°-150°; the inlet / outlet diameters of the fluid microchannel of the top cell culture layer and the gas microchannel of the bottom vacuum negative pressure layer are both 1200-1600 μm.

4. The lung organ chip based on a stretchable biomembrane according to claim 3, characterized in that: The dimensions of the top cell culture layer are as follows: the lengths of the two long sides are both 35 mm, the lengths of the two short sides are both 15 mm, and the angles between the long side and the hypotenuse, and between the short side and the hypotenuse are both 135°; the thickness of the top cell culture layer is 4 mm, the diameter of the groove cross section is 10 mm, and the depth is 3 mm; the horizontal section of the fluid microchannel is 7 mm long, the inclined section is 8 mm long, and the angle between the horizontal section and the inclined section is 135°; The thickness of the bottom vacuum negative pressure layer is 4 mm, the cross-sectional diameter of the groove is 10 mm, and the depth is 3 mm; the horizontal section of the gas microchannel is 7 mm long, the inclined section is 8 mm long, and the angle between the horizontal section and the inclined section is 135°; the inlet / outlet diameters of the fluid microchannel of the top cell culture layer and the gas microchannel of the bottom vacuum negative pressure layer are both 1400 μm.

5. The lung organ chip based on a stretchable biomembrane according to claim 1, characterized in that: The stretchable porous membrane layer has a thickness of 50-200 μm, a total number of 19-61 intermediate circular holes, a circular hole diameter of 200-1000 μm, a spacing between adjacent circular holes of 100-300 μm, and all circular holes are arranged in a regular geometric shape, such as a regular hexagon, square or circular arrangement.

6. A method for preparing a lung organ chip based on a stretchable biomembrane according to any one of claims 1 to 5, characterized in that The following steps are involved: (1) Preparing a top cell culture layer, a bottom vacuum negative pressure layer, and a stretchable porous membrane layer through a mold; (2) dripping a hydrogel cell resuspension containing vascular endothelial cells onto the circular hole structure of the stretchable porous membrane scaffold to form a thin film in the circular hole and then performing photocuring; (3) The three-layer structure is assembled to form a microfluidic device. Lung epithelial cells are planted on the membrane by perfusion. After a dense cell monolayer is formed, the chip is completed.

7. The method for preparing a lung organ chip based on a stretchable biomembrane according to claim 6, characterized in that: The top cell culture layer, the bottom vacuum negative pressure layer and the stretchable porous membrane layer are made of polydimethylsiloxane; the hydrogel is GelMA hydrogel; The vascular endothelial cells include one of human umbilical vein endothelial cells, human lung microvascular cell line, human lung microvascular endothelial cells, and human umbilical vein cell fusion cell line; the light curing time is controlled within 20 s; The lung epithelial cells include one of type I alveolar epithelial cells, type II alveolar epithelial cells, human non-small cell lung cancer cell lines, and human alveolar epithelial cells.

8. The method for preparing a lung organ chip based on a stretchable biomembrane according to claim 7, characterized in that: Human umbilical vein endothelial cells were selected as vascular endothelial cells; human alveolar epithelial cells were selected as lung epithelial cells; and the light curing time was 10 s.

9. Use of a lung organ chip based on a stretchable biomembrane according to any one of claims 1 to 5 in respiratory simulation.

10. The use according to claim 9, characterized in that: Cell co-culture and respiratory simulation were performed in this lung organ chip, including the following steps: (1) Introducing culture medium into the cell culture chamber to provide nutrients to the cells on the membrane and remove metabolic waste; (2) Stretching the porous membrane by introducing negative pressure into the vacuum negative pressure chamber; The negative pressure frequency is 0.1-1 Hz, and the negative pressure intensity is within 20 kPa; through cell co-culture, an air-blood barrier is constructed in vitro, and combined with membrane stretching, a three-dimensional dynamic lung model is constructed in vitro.

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