Porous membrane-based multi-stage drug metabolism organ-on-chip and application thereof

Abstract

The present invention provides a porous membrane-based multi-stage metabolism organ-on-chip, comprising a porous membrane layer, and upper and lower chip layers, wherein a number N of non-intersected upper fluid channels are provided in the lower surface of the upper chip layer. A number N-1 of metabolic chambers are provided on the upper surface of the lower chip layer, projections of the metabolic chambers on the upper chip layer respectively overlap with two upper fluid channels adjacent thereto, and each metabolic chamber is in communication with the two upper fluid channels adjacent thereto by means of the porous membrane layer. The porous membrane layer is made of a material which is impermeable to cells but permeable to cellular metabolic secretions, wherein N is an integer greater than or equal to 2. By means of providing the porous membrane layer allowing the transfer of metabolites, the metabolites generated by the cells and test drugs in the first upper fluid channels enter the metabolic chambers on the lower chip layer, and then the metabolites in the metabolic chambers enter the next fluid channel of the upper chip layer, the cells in the next fluid channel can continue to interact with the metabolites generated in the previous stage, and the influences of multi-stage drug-cell metabolism can thus be researched.

Description

A drug multi-stage metabolism organ chip based on porous membrane and its application Technical Field

[0001] The present invention relates to the fields of biomedical engineering and microfluidics, and in particular to a porous membrane-based drug multi-stage metabolism organ chip and applications thereof. Background Art

[0002] Organ-on-a-chips are cell culture devices developed from microfluidic chips. By integrating technologies such as microfabrication, stem cells, materials, and tissue engineering, they create in vitro biomimetic models with physiological functions and structures close to those in vivo, while also providing a stable and controllable microenvironment. Consequently, these devices are attracting increasing attention in areas such as disease simulation, personalized medicine, and drug development.

[0003] Drug metabolism refers to one of the basic processes from the point of administration into the body to the production of drug effects until the body excretes the drug. The drug metabolism process begins with the absorption of the drug from the site of administration into the bloodstream; then, after the drug enters the bloodstream, it is distributed throughout the body through the blood circulation; then, the drug is transported from various blood vessels into the liver and, under the action of drug metabolizing enzymes, the drug is oxidized, reduced, decomposed, or combined, thereby changing the structure of the drug to varying degrees; finally, the drug is excreted from the body through excretory or secretory organs in the form of the original prototype or metabolites. Therefore, the drug metabolism process can help people better understand the mechanism of action and effects of drugs and metabolites. This process is related to various cellular microenvironments and the physical and chemical properties of drugs, and is also of great significance to new drug development, drug evaluation, clinical drug use, and other aspects.

[0004] At present, in vitro drug metabolism experiments are mainly carried out through cell models and animal models. Cell models have good repeatability and operability, which can easily add drug dosages and control the duration of action. However, most cell models are two-dimensional cell cultures. Even three-dimensional organoid cultures find it difficult to reconstruct the microenvironment of cells within the organ, and each culture unit is relatively independent, making it impossible to study cellular metabolic products after drugs interact with different cells at multiple levels under the same environment. In order to better reproduce the microenvironment of drugs in the body, the use of animal models in drug metabolism experiments is an important supplement to cell models. However, animal models cannot observe the drug metabolism process in real time, and after all, there are species differences between animal models and humans, which often leads to the inability to exert drug testing effects in humans.

[0005] In view of the shortcomings of the above experimental methods, the inventors of the present application aim to develop the multi-stage metabolic organ chip based on porous membranes of the present invention after a long period of research and practice.

[0006] Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the main purpose of the present invention is to provide a multi-stage metabolic organ chip based on a porous membrane. The organ chip of the present application comprises, from top to bottom, an upper chip layer, a porous film layer and a lower chip layer. The multiple independent upper fluid channels on the upper layer of the chip have high efficiency in culturing a single cell or realizing simultaneous culture of multiple cells. By arranging a porous film layer for the transfer of metabolites between the upper layer and the lower layer of the chip, the metabolites generated by the upper fluid channel enter the metabolic chamber of the lower layer of the chip, and the metabolites in the metabolic chamber can enter the next fluid channel on the upper layer of the chip, so that the effect of drugs on the multi-stage metabolism of cells can be studied.

[0008] To achieve the above-mentioned purpose and other related purposes, the present invention is implemented by including the following technical solutions.

[0009] In a first aspect, the present invention provides a multi-stage metabolic organ chip based on a porous membrane, wherein the organ chip comprises, from top to bottom, an upper chip layer, a porous film layer, and a lower chip layer; N upper fluid channels are provided on the lower surface of the upper chip layer, and the upper fluid channels do not intersect with each other; N-1 metabolic chambers are provided on the upper surface of the lower chip layer, and the projection of each metabolic chamber on the upper chip layer is respectively located between two adjacent upper fluid channels and overlaps with the two adjacent upper fluid channels, and each metabolic chamber is connected to its two adjacent upper fluid channels through the porous film layer;

[0010] The porous film layer is made of a material that is impermeable to cells but permeable to cell metabolic secretions; and N is an integer greater than or equal to 2.

[0011] In some preferred embodiments, the upper layers of the chip corresponding to both ends of the upper fluid channel are respectively provided with perfusion through-holes penetrating the upper layer of the chip; one side of the perfusion through-hole is exposed outside the organ chip, and the other side is connected to the upper fluid channel.

[0012] In some preferred embodiments, a liquid storage tube is provided outside each of the perfusion through holes.

[0013] In some preferred embodiments, the liquid storage tube is made of glass or high molecular polymer material.

[0014] In some preferred embodiments, a transfer port is provided at one end of the metabolic chamber; the transfer port overlaps with a projection of a perfusion through hole corresponding to one end of the fluid channel on the upper layer of the chip.

[0015] In some preferred embodiments, the upper fluid channels are parallel; and / or the intervals between the upper fluid channels are the same.

[0016] In some preferred embodiments, the inner width of the upper fluid channel is 0.4-0.8 mm.

[0017] In some preferred embodiments, the metabolic chambers are parallel; and / or, the intervals between the metabolic chambers are the same.

[0018] In some preferred embodiments, the metabolic chamber has an inner width of 0.5 to 1.5 mm.

[0019] In some preferred embodiments, the angle between the upper fluid channel and the metabolic chamber is 30-120°.

[0020] In some preferred embodiments, the upper fluid channel is perpendicular to the metabolic chamber.

[0021] In some preferred embodiments, in a state of use, the chip upper layer, the porous film layer and the chip lower layer are sequentially attached, connected and sealed.

[0022] In some preferred embodiments, the porous film layer is made of at least one porous film; and / or the pore size of the porous film layer is 0.4-30 μm; and / or the porous film layer is made of an organic polymer material; and / or the porous film layer is infiltrated and modified with collagen;

[0023] In some preferred embodiments, the thickness of the porous film layer is 0.1 to 0.3 mm; and / or the material of the porous film layer is polyethylene terephthalate or polycarbonate.

[0024] In some preferred embodiments, the thickness of the lower layer of the chip is less than the thickness of the upper layer of the chip.

[0025] In some preferred embodiments, the thickness of the upper layer of the chip is 2 to 3 mm; and / or the thickness of the lower layer of the chip is 1 to 1.5 mm.

[0026] In some preferred embodiments, the porous membrane layer covers each of the upper fluid channels and each of the metabolic chambers.

[0027] In some preferred embodiments, the materials of the chip upper layer and the chip lower layer are both organic polymer materials.

[0028] In some preferred embodiments, the materials of the chip upper layer and the chip lower layer are both polydimethylsiloxane or COC plastic.

[0029] In some specific embodiments, the lower surface of the upper layer of the chip is provided with a first fluid channel, a second fluid channel, a third fluid channel and a fourth fluid channel which are parallel to each other and whose lengths decrease in sequence;

[0030] The upper surface of the lower layer of the chip is provided with a first metabolic chamber, a second metabolic chamber and a third metabolic chamber;

[0031] The projection of the first metabolic chamber on the upper layer of the chip overlaps with the first fluid channel and the second fluid channel, the projection of the second metabolic chamber on the upper layer of the chip overlaps with the second fluid channel and the third fluid channel, and the projection of the third metabolic chamber on the upper layer of the chip overlaps with the third fluid channel and the fourth fluid channel.

[0032] In some specific embodiments, the first fluid channel is provided with a first channel groove and a left perfusion hole and a right perfusion hole located at both ends of the first channel groove;

[0033] The second fluid channel is provided with a second channel groove and two left perfusion holes and two right perfusion holes located at both ends of the second channel groove; the first metabolic chamber is provided with a first middle chamber and a first transmission port located at one end of the first middle chamber, the first transmission port and the projection of the two left perfusion holes on the upper layer of the chip overlap, and the projection of the first middle chamber and the first channel groove on the upper layer of the chip intersect;

[0034] Wherein, the structures of the third fluid channel and the fourth fluid channel are the same as those of the first channel and the second channel;

[0035] The structures and configurations of the second metabolic chamber and the third metabolic chamber are the same as those of the first metabolic chamber.

[0036] The second aspect of the present invention provides the application of the porous membrane-based multi-stage metabolic organ chip in disease simulation, drug development and nutrition research.

[0037] In some preferred embodiments, a porous membrane-based multi-stage metabolic organ chip is used to study the multi-stage metabolism of drugs in vitro.

[0038] As described above, the porous membrane-based multi-stage metabolic organ chip of the present invention has the following beneficial effects:

[0039] 1) The efficiency of culturing a single cell species in multiple independent upper fluid channels on the chip's upper layer, or the simultaneous culturing of multiple cells, can be improved. By placing a porous film layer between the upper and lower layers of the chip, allowing for the transfer of metabolites, the metabolites produced by the cells and the test drug in the first upper fluid channel can enter the metabolic chamber in the lower layer of the chip. The metabolites in the metabolic chamber can then enter the next fluid channel on the upper layer of the chip. The cells in the next fluid channel can continue to interact with the metabolites produced in the previous level, performing multi-stage processing in sequence, thereby studying the effects of drugs on multi-level cellular metabolism.

[0040] 2) Perfusion holes are provided on the upper layer of the chip at either end of the fluidic channel. One side of the perfusion hole is exposed to the outside of the Organ Chip, while the other side is connected to the upper fluidic channel. This allows for sampling of metabolites at any time during their interaction, shortening drug metabolism experiments and improving the reliability of metabolite results from drug-cell interactions.

[0041] 3) Liquid storage tubes are provided outside the perfusion through-holes, and the pressure is adjusted by the amount of liquid in each liquid storage tube, thereby realizing the power of movement of liquid or metabolites in each flow channel and each metabolic chamber of the chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG1 is a schematic diagram showing the overall structure of a porous membrane-based multi-stage metabolic organ chip according to the present invention.

[0043] FIG2 shows an exploded view of the porous membrane-based multi-stage metabolic organ chip without the liquid storage tube of the present invention.

[0044] FIG3 is a schematic diagram showing the structure of the upper layer of the multi-stage metabolic organ chip based on a porous membrane according to the present invention.

[0045] FIG4 is a schematic structural diagram of the lower layer of the multi-stage metabolic organ chip based on a porous membrane according to the present invention.

[0046] FIG5 shows a top view of the upper layer of the chip of the porous membrane-based multi-stage metabolic organ chip of the present invention (front side of the upper layer of the chip).

[0047] FIG6 shows a bottom view of the upper layer of the chip of the porous membrane-based multi-stage metabolic organ chip of the present invention (the back side of the upper layer of the chip).

[0048] FIG7 shows a top view of the chip lower layer (front side of the chip lower layer) of the porous membrane-based multi-stage metabolic organ chip of the present invention.

[0049] 1 to 7 are denoted as follows: 1 Chip upper layer 100 Upper fluid channel 101 First fluid channel 1011 First channel groove 1012 Left first perfusion hole 1013 Right first perfusion hole 102 Second fluid channel 1021 Second channel groove 1022 Left second perfusion holes 1023 Right second perfusion holes 103 Third fluid channel 1031 Third channel groove 1032 Left third perfusion holes 1033 Right third perfusion holes 104 Fourth fluid channel 1041 Fourth channel groove 1042 Left fourth perfusion holes 1043 Right fourth perfusion holes 2 Porous film layer 200 Perfusion through hole 3 Chip lower layer 300 Metabolic chamber 301 First metabolic chamber 3011 First middle chamber 3012 First transfer port 302 Second metabolic chamber 3021 Second middle chamber 3022 Second transfer port 303 Third metabolic chamber 3031 Third middle chamber 3032 The third transmission port DETAILED DESCRIPTION

[0050] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents of this specification.

[0051] Please refer to Figures 1 to 7. It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the efficacy and purpose that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" cited in this specification are only for the convenience of description and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0052] The present invention provides a multi-stage metabolic organ chip based on a porous membrane. Referring to FIG1 , the organ chip comprises, from top to bottom, an upper chip layer 1, a porous film layer 2, and a lower chip layer 3. The lower surface of the upper chip layer 1 is provided with N upper fluid channels 100, and each upper fluid channel 100 does not cross each other. The upper surface of the lower chip layer 3 is provided with N-1 metabolic chambers 300. The projection of each metabolic chamber 300 on the upper chip layer 1 is located between two adjacent upper fluid channels 100 and overlaps with the two adjacent upper fluid channels 100, and each metabolic chamber 300 is connected to its two adjacent upper fluid channels 100 through the porous film layer 2. Wherein, the porous film layer 2 is made of a material that is impermeable to cells but permeable to cell metabolic secretions. N is an integer greater than or equal to 2.

[0053] Further explanation of the multi-stage metabolic organ chip of this example: 1) The multi-stage metabolic organ chip of this example is composed of the chip upper layer 1, the porous film layer 2 and the chip lower layer 3, which are sequentially bonded and sealed. Conventional biochip bonding and pasting methods can be used to ensure the firmness and sealing of the chip. 2) The lower surface of the chip upper layer 1 is provided with N upper fluid channels 100, and each upper fluid channel 100 does not cross each other. The N non-crossing upper fluid channels 100 are used to culture cells separately. First, multiple independent culture channels can effectively improve the efficiency of cell culture, and second, different types of cells can be cultured simultaneously. 3) The upper surface of the chip lower layer 3 is provided with N-1 metabolic chambers 300. The function of the metabolic chamber is to transfer the metabolic products (secretions) in one upper fluid channel to the next upper fluid channel. After the transfer of multiple metabolic chambers, the experimental verification of multi-stage metabolism can be achieved. 4) Regarding the number of upper fluid channels 100, it can be set according to the requirements of the metabolic level. For example, if you need to study tertiary metabolism, then N is 3; if you need to study quaternary metabolism, then N is 4. For example, if studying quaternary metabolism, four upper flow channels are set up, and intestinal cells, vascular endothelial cells, liver endothelial cells, and hepatocytes are cultured in each of these channels in order to study the multi-step metabolism of the same drug in these four cell types. Of course, two or more drugs can also be tested.

[0054] In a preferred embodiment, the upper layer 1 of the chip corresponding to each end of the upper fluid channel 100 is provided with a perfusion through-hole 200 that penetrates the upper layer 1 of the chip. One side of the perfusion through-hole 200 is exposed to the outside of the organ chip, and the other side is connected to the upper fluid channel 100. That is, cells or culture fluid are injected into the upper fluid channel by opening a through-hole in the upper layer 1 of the chip. Preferably, the head of each upper fluid channel at each end corresponds to a through-hole 300, and the through-hole 300 is perpendicular to the upper fluid channel. By controlling the different heights of the liquid in the through-hole 300 corresponding to the two ends of the upper fluid channel, a pressure difference can be generated, thereby driving the liquid in the upper fluid channel to flow according to the design requirements. Furthermore, as described above, each upper fluid channel 100 has a through-hole 300 at each end of the head, which is also conducive to sampling at any time during the metabolite reaction process. It can analyze the effect of each metabolic level separately, shorten the time of drug metabolism experiments, and improve the reliability of the metabolite results of drug-cell interaction.

[0055] More preferably, each perfusion through-hole 200 is provided with a liquid reservoir tube 400. The liquid reservoir tube 400 may be integrally formed with the through-hole 300 or affixed to the through-hole. The liquid reservoir tube 400 may be made of glass or a polymer material, forming a storage space for storing cell culture fluid and adjusting the pressure difference to drive the liquid flow in the upper fluid channel. Of course, the liquid reservoir tube 400 is preferably made of a transparent material, and graduations may also be marked on the liquid reservoir tube 400.

[0056] In a preferred embodiment, a transfer port is provided at one end of the metabolic chamber 300. This transfer port overlaps with the projection of the perfusion through-hole 200 corresponding to one end of the upper fluid channel 100 on the chip upper layer 1. This ensures that metabolic product secretions can smoothly flow from the metabolic chamber 300 into the corresponding upper fluid channel.

[0057] Continuing to refer to FIG1 and FIG6, in some specific embodiments, the following specific solutions are also provided:

[0058] 1) The upper fluid channels 100 are parallel and spaced uniformly apart. Preferably, the inner width of the upper fluid channels 100 is 0.4 to 0.8 mm, for example, 0.4 to 0.6 mm or 0.6 to 0.8 mm. Preferably, the depth of the upper fluid channels 100 is 80 to 150 μm, for example, 80 to 100 μm, 100 to 120 μm, or 120 to 150 μm.

[0059] Specifically, multi-stage metabolism involves the synergistic effects between multiple organs. For example, in terms of blood vessels formed by endothelial cells, the blood flow of each tissue and organ is different. Since the blood flow of each tissue and organ is inconsistent, in order to better simulate the action process, the width and depth of the fluid channels are set to different levels. More specifically, the width and depth of the first fluid channel 101, the second fluid channel 102, the third fluid channel 103, and the fourth fluid channel 104 can be set to different levels as needed. For example, when a drug first enters the capillaries in the body, the blood flow is small, the blood flow into the liver and kidneys is large, and the blood flow during the excretion and elimination process is small. Therefore, the groove width of the first fluid channel 101 and the fourth fluid channel 104 can be set to 0.4mm to 0.6mm; the groove width of the second fluid channel 102 and the third fluid channel 103 can be set to 0.6 to 0.8mm.

[0060] 2) The metabolic chambers 300 are parallel, and the intervals between the metabolic chambers 300 are the same. Preferably, the inner width of the metabolic chamber 300 is 0.5-1.5 mm, for example, the first metabolic chamber is 0.5 mm wide, the second metabolic chamber is 1.5 mm wide, and the third metabolic chamber is 0.5 mm wide. Preferably, the chamber depth of the metabolic chamber 300 is 80-150 μm, for example, 80-100 μm or 100-120 μm or 120-150 μm. 3) The angle between the upper fluid channel 100 and the metabolic chamber 300 is 30-120°, for example, 30-60° or 60-90° or 90-120°. Preferably, the upper fluid channel 100 shown in Figure 1 is arranged perpendicular to the metabolic chamber 300.

[0061] 4) The porous film layer 2 is made of at least one porous film. The pore size of the porous film layer 2 is 0.4-30 μm, for example, 0.4-1 μm or 1-30 μm. That is, the porous film layer 2 does not allow cells to penetrate, but must allow cellular metabolic secretions to pass through. Preferably, the material of the porous film layer 2 is an organic polymer material. Preferably, the thickness of the porous film layer 2 is 0.4-1.0 mm, for example, 0.1-0.5 mm or 0.5-1.0 mm. The material of the porous film layer 2 is polyethylene terephthalate or polycarbonate.

[0062] 5) The thickness of the chip lower layer 3 is smaller than the thickness of the chip upper layer 1. The thickness of the chip upper layer 1 is 2 to 3 mm, and the thickness of the chip lower layer 3 is 1 to 1.5 mm.

[0063] 6) The materials of the chip upper layer 1 and the chip lower layer 3 are both organic polymer materials. Preferably, the materials of the chip upper layer 1 and the chip lower layer 2 are both polydimethylsiloxane or COC plastic. Such materials have good biocompatibility.

[0064] To better understand this example, a method for using the porous membrane-based multi-stage drug metabolism organ chip is provided, comprising the following steps:

[0065] For example, three upper fluid channels 100 and two metabolic chambers 300 are used, namely, the first upper fluid channel, the second upper fluid channel, the third upper fluid channel, the first metabolic chamber, and the second metabolic chamber. The first metabolic chamber can communicate with the first and second upper fluid channels through a porous film layer, and the second metabolic chamber can communicate with the second and third upper fluid channels through a porous film layer.

[0066] 1) The chip upper layer 1, the porous film layer 2 and the chip lower layer 3 are assembled into a target chip in sequence, and the chip is sterilized and disinfected.

[0067] 2) Inject the same type of cells or different types of cells into the three upper fluid channels 100 in sequence.

[0068] 3) After the cells are stabilized in the upper fluid channel 100 and grow stably, the test drug is injected into the first upper fluid channel.

[0069] 4) The drug interacts with the cells in the first upper fluid channel to produce metabolic secretion 1, which penetrates into the first metabolic chamber of the lower fluid channel layer through the porous membrane. The pore size of the membrane restricts the cells to the first upper fluid channel layer.

[0070] 5) Secretion 1 will penetrate into the first metabolic chamber of the lower fluid channel layer. When secretion 1 is full in the first metabolic chamber, it will pass through the membrane again under the action of pressure difference and continue to interact with the cells of the second upper fluid channel to produce metabolic secretion 2. Similarly, secretion 2 will continue to penetrate into the next fluid channel layer and reach the second metabolic chamber of the next level.

[0071] 6) Similarly, after the secretion 2 in the second metabolic chamber is full, it will pass through the membrane again under the action of pressure difference and continue to interact with the cells in the third upper fluid channel to produce metabolic secretion 3.

[0072] As described above, secretion 1, secretion 2 or secretion 3 can be sampled and tested in their respective upper fluid flow channels to analyze the results.

[0073] More specifically, the above steps may also include: 1) cell collection: the desired test cells are digested with trypsin to convert the cells from an adherent state to a floating state, and the trypsin is terminated with a solution containing 20% ​​high sugar. The cell mixture is then transferred and centrifuged, and the supernatant is extracted and added with the culture medium to prepare a cell suspension with an appropriate cell concentration.

[0074] 2) Injecting collagen solution into the upper fluid channel, the solution infiltrates the porous film layer in the modified chip, and then placing the modified chip in an incubator at 37°C containing 5% CO2 to enhance cell adhesion on its surface.

[0075] 3) After removal from the incubator, perfuse the chip with phosphate-buffered saline (PBS) to wash away excess collagen solution. After washing, perfuse the cell suspension into the upper fluid channel. Then, inject the desired test drug into the perfusion port of the upper fluid channel layer. Finally, place the chip in a 37°C incubator with 5% CO2 for incubation.

[0076] Example 1

[0077] Referring to Figures 1 to 7 , in this example, N is 4, meaning that the lower surface of the chip's upper layer 1 is provided with a first fluid channel 101, a second fluid channel 102, a third fluid channel 103, and a fourth fluid channel 104, all parallel to each other and of decreasing length. The upper surface of the chip's lower layer 3 is provided with a first metabolic chamber 301, a second metabolic chamber 302, and a third metabolic chamber 303. The projection of the first metabolic chamber 301 on the chip's upper layer 1 overlaps with the first and second fluid channels 101 and 102, the projection of the second metabolic chamber 302 on the chip's upper layer 1 overlaps with the second and third fluid channels 102 and 103, and the projection of the third metabolic chamber 303 on the chip's upper layer 1 overlaps with the third and fourth fluid channels 103 and 104.

[0078] More specifically, the first fluid channel 101 is provided with a first channel groove 1011 and a left perfusion hole 1012 and a right perfusion hole 1013 located at both ends of the first channel groove 1011. The second fluid channel 102 is provided with a second channel groove 1021 and two left perfusion holes 1022 and two right perfusion holes 1023 located at both ends of the second channel groove 1021. The third fluid channel 103 is provided with a third channel groove 1031 and three left perfusion holes 1032 and three right perfusion holes 1033 located at both ends of the third channel groove 1031. The fourth fluid channel 104 is provided with a fourth channel groove 1041 and four left perfusion holes 1042 and four right perfusion holes 1043 located at both ends of the fourth channel groove 1041. Taking the first fluid channel 101 as an example, fluid enters the first channel groove 1011 from the right perfusion hole 1013 and can be used from the left perfusion hole 1012 or permeate through the porous film layer 2 to the chip lower layer 3.

[0079] 5 and 6 , the upper layer 1 of the chip is provided with a plurality of perfusion through-holes 200, which correspond to the first left perfusion hole 1012, the first right perfusion hole 1013, the second left perfusion hole 1022, the second right perfusion hole 1023, the third left perfusion hole 1032, the third right perfusion hole 1033, the fourth left perfusion hole 1042 and the fourth right perfusion hole 1043 of each upper fluid channel, that is, each perfusion through-hole 200 is coaxially aligned with each perfusion hole. When in use, different types of cell suspensions or test drugs are injected through each perfusion through-hole 200.

[0080] Regarding the metabolic chambers: Referring to Figure 7 , the first metabolic chamber 301 comprises a first middle chamber 3011 and a first transfer port 3012 located at one end of the first middle chamber 3011. The transfer port 3012 overlaps with the projection of the second left perfusion hole 1022 on the chip's upper layer 1, and the projections of the middle chamber 3011 and the first channel groove 1011 on the chip's upper layer 1 intersect. The second metabolic chamber 302 comprises a second middle chamber 3021 and a second transfer port 3022 located at one end of the second middle chamber 3021. The second transfer port 3022 overlaps with the projection of the third left perfusion hole 1032 on the chip's upper layer 1, and the projections of the second middle chamber 3021 and the second channel groove 1021 on the chip's upper layer 1 intersect. The third metabolic chamber 303 is equipped with a third middle chamber 3031 and a third transfer port 3032 located at one end of the third middle chamber 3031. The third transfer port 3032 overlaps with the projection of the fourth left perfusion hole 1042 on the chip's upper layer 1, while the projections of the third middle chamber 3031 and the third channel groove 1031 on the chip's upper layer 1 intersect. Each metabolic chamber is arranged perpendicular to each upper fluid channel and has the same structure. Furthermore, each transfer port is circular, with a diameter of 1 to 1.5 mm.

[0081] More specifically, referring to Figures 1 and 6, the second left perfusion hole 1022 of the second fluid channel 102 is aligned coaxially with the first transfer port 3012 of the first metabolic chamber 301, the third left perfusion hole 1032 of the third fluid channel 103 is aligned coaxially with the second transfer port 3022 of the second metabolic chamber 302, and the fourth left perfusion hole 1042 of the fourth fluid channel 104 is aligned coaxially with the third transfer port 3032 of the third metabolic chamber 303.

[0082] More specifically, referring to Figure 2, the area of ​​the porous film layer 2 is consistent with the size of the chip's upper layer 1 and lower layer 3. The porous film layer 2 is made of polyethylene terephthalate (PET), with a pore size of approximately 10 μm and a thickness of approximately 0.2 mm. This prevents cell passage but allows the passage of cellular metabolic products (secretions). Research has shown that the optimal thickness of the porous film layer 2 is between 0.1 mm and 0.30 mm, allowing secretions to pass back and forth across the porous film layer 2 and extending its service life.

[0083] The operation process of the chip is described using the specific example of N being 3 (liver and kidney structure):

[0084] (1) Suspensions of intestinal cells, hepatocytes, and kidney cells are injected into the first right perfusion hole 1013 of the first fluid channel 101, the second right perfusion hole 1023 of the second fluid channel 102, the third right perfusion hole 1033 of the third fluid channel 103, and the fourth right perfusion hole 1043 of the fourth fluid channel 104, respectively;

[0085] 2) After the intestinal cells in the first fluid channel 101 absorb the drug, secretions 1 are produced. Under the action of the external pressure difference, the secretions 1 are transferred through the porous membrane to the first metabolic chamber 301 in the lower fluid channel layer;

[0086] 3) Secretion 1 accumulates in the first metabolic chamber 301 and enters the second fluid channel 102 through the porous membrane under pressure to interact with liver cells to produce secretion 2, which then enters the second metabolic chamber 302 through the porous membrane;

[0087] 4) Similarly, if the secretion 2 accumulates in the second metabolic chamber 302, it will react with the renal cells in the third fluid channel 103 through the porous membrane under pressure to produce secretion 3, and then the secretion 3 will enter the third metabolic chamber 303 through the porous membrane;

[0088] 5) Finally, secretion 3 interacts with the liver endothelial cells in the fourth fluid channel 104 through the porous membrane to produce secretion 4.

[0089] During the secretion transport process, secretions can be extracted from each perfusion hole at any time to analyze the effect of metabolism at that level.

[0090] It is worth noting that the pressure difference can be achieved by external ventilation or an external pump, or by varying the volume of cell culture fluid. When varying the volume of culture fluid is used, the operation is simple and does not require the introduction of additional substances. Specifically, the principle to be followed is that the volume of culture fluid at the first right perfusion hole 1013 is greater than the volume of culture fluid at the first left perfusion hole 1012, the volume of culture fluid at the second left perfusion hole 1022 is greater than the volume of culture fluid at the second right perfusion hole 1023, the volume of culture fluid at the third left perfusion hole 1032 is greater than the volume of culture fluid at the third right perfusion hole 1033, the volume of culture fluid at the fourth left perfusion hole 1042 is greater than the volume of culture fluid at the fourth right perfusion hole 1043, the volume of culture fluid at the first left perfusion hole 1012 is greater than the volume of culture fluid at the second left perfusion hole 1022, the volume of culture fluid at the second left perfusion hole 1022 is greater than the volume of culture fluid at the third left perfusion hole 1032, and the volume of culture fluid at the third left perfusion hole 1032 is greater than the volume of culture fluid at the fourth left perfusion hole 1042. Furthermore, the use of pressure difference to promote the movement of secretions can ensure that the liquid level is maintained within a certain period of time (12 to 24 hours). Before the leveling, the secretions can fully interact with cells at all levels, and there is no need to frequently change the culture medium.

[0091] As described above, the chip of this application can extract secretion products from each upper fluid channel layer at any time during the metabolic process and analyze the effects of drugs on cells, thereby achieving the purpose of drug testing. It can also achieve the co-culture of multiple cells and simulate the effects of drugs interacting with different cells in the body, thereby studying the effects of drugs on multi-level cellular metabolism.

[0092] Example 2

[0093] The difference from Example 1 may also be that:

[0094] 1) Three identical cells, two identical cells, or the same cells can be injected into different upper fluid channels, which can be flexibly applied.

[0095] 2) Different cells can be injected into the same upper fluid channel to achieve co-culture to form a single organ simulation, that is, single organ simulation can be performed in multiple different upper fluid channels, thereby achieving simulation between multiple single organs in multiple different upper fluid channels.

[0096] 3) The chip of the present application can be used for testing a variety of drugs and can be applied flexibly.

[0097] 4) The number of chips N in the present application can be further increased, for example, 10, 20, or even 1000, so as to realize large-scale operations.

[0098] 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 skilled in 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 one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A multi-stage metabolic organ chip based on a porous membrane, characterized in that, The organ-on-a-chip sequentially includes an upper chip layer (1), a porous thin film layer (2), and a lower chip layer (3) from top to bottom; The lower surface of the upper chip layer (1) is provided with N upper fluid channels (100), and the upper fluid channels (100) do not cross each other; The upper surface of the lower chip layer (3) is provided with N - 1 metabolic chambers (300). The projections of the metabolic chambers (300) on the upper chip layer (1) are respectively located between two adjacent upper fluid channels (100) and overlap with the two adjacent upper fluid channels (100). Each metabolic chamber (300) communicates with its two adjacent upper fluid channels (100) through the porous thin film layer (2); Among them, the porous thin film layer (2) is made of a material that cells cannot penetrate but cell metabolic secretions can penetrate; N is an integer greater than or equal to 2.

2. The multi-stage metabolic organ chip based on a porous membrane according to claim 1, wherein, Perfusion through holes (200) penetrating the upper chip layer (1) are respectively provided on the upper chip layer (1) corresponding to both ends of the upper fluid channel (100); One side of the perfusion through hole (200) is exposed outside the organ-on-a-chip, and the other side communicates with the upper fluid channel (100).

3. The multi-stage metabolic organ chip based on a porous membrane according to claim 2, characterized in that, A liquid storage tube (400) is provided outside each perfusion through hole (200).

4. The multi-stage metabolic organ chip based on a porous membrane according to claim 3, wherein, The liquid storage tube (400) is made of glass or a polymer material.

5. The multi-stage metabolic organ chip based on a porous membrane according to claim 2, wherein One end of the metabolic chamber (300) is provided with a transfer port; the transfer port overlaps with the projection of the perfusion through hole (200) corresponding to one end of the upper fluid channel (100) on the upper chip layer (1).

6. The multi-stage metabolic organ chip based on a porous membrane according to claim 1, wherein It includes at least one of the following technical features: a1) Each of the upper fluid channels (100) is parallel; and / or, the intervals between the upper fluid channels (100) are the same; a2) Each of the metabolic chambers (300) is parallel; and / or, the intervals between the metabolic chambers (300) are the same; a3) The included angle between the upper fluid channel (100) and the metabolic chamber (300) is 30 - 120°; a4) In the use state, the upper chip layer (1), the porous thin film layer (2), and the lower chip layer (3) are sequentially bonded and sealed; a6) The porous thin film layer (2) is made of at least one porous thin film; and / or, the pore diameter of the porous thin film layer (2) is 0.4 - 30 μm; and / or, the material of the porous thin film layer (2) is an organic polymer material; and / or, the porous thin film layer is modified by collagen infiltration; a7) The thickness of the lower chip layer (3) is less than the thickness of the upper chip layer (1); a8) The porous thin film layer (2) covers each of the upper fluid channels (100) and each of the metabolic chambers (300); a9) The materials of the upper chip layer (1) and the lower chip layer (3) are both organic polymer materials.

7. The multi-stage metabolic organ chip based on a porous membrane according to claim 6, wherein, It includes at least one of the following technical features: a11) The inner width of the channel of the upper fluid channel (100) is 0.4 - 0.8 mm, and the inner widths of the channels of the upper fluid channels (100) are the same or different; a21) The inner width of the chamber of the metabolic chamber (300) is 0.5 - 1.5 mm, and the inner widths of the chambers of the metabolic chambers (300) are the same or different; a31) The upper fluid channel (100) is perpendicular to the metabolic chamber (300); a61) The thickness of the porous film layer (2) is 0.1 - 0.3 mm; and / or, the material of the porous film layer (2) is selected from one of polyethylene terephthalate, polyurethane, polytetrafluoroethylene, polyvinylidene fluoride or polycarbonate; a71) The thickness of the upper layer (1) of the chip is 2 - 3 mm; and / or, the thickness of the lower layer (3) of the chip is 1 - 1.5 mm; a91) The materials of the upper layer (1) and the lower layer (2) of the chip are both polydimethylsiloxane or COC plastic or polymethyl methacrylate.

8. The multi-stage metabolic organ chip based on a porous membrane according to any one of claims 1 to 7, characterized in that, On the lower surface of the upper layer (1) of the chip, there are a first fluid channel (101), a second fluid channel (102), a third fluid channel (103) and a fourth fluid channel (104) that are parallel to each other and decrease in length in sequence; On the upper surface of the lower layer (3) of the chip, there are a first metabolic chamber (301), a second metabolic chamber (302) and a third metabolic chamber (303); Wherein, the projection of the first metabolic chamber (301) on the upper layer (1) of the chip overlaps with the first fluid channel (101) and the second fluid channel (102), the projection of the second metabolic chamber (302) on the upper layer (1) of the chip overlaps with the second fluid channel (102) and the third fluid channel (103), and the projection of the third metabolic chamber (303) on the upper layer (1) of the chip overlaps with the third fluid channel (103) and the fourth fluid channel (104).

9. The multi-stage metabolic organ chip based on a porous membrane according to claim 8, wherein, The first fluid channel (101) is provided with a first channel groove (1011) and a left first perfusion hole (1012) and a right first perfusion hole (1013) located at both ends of the first channel groove (1011); The second fluid channel (102) is provided with a second channel groove (1021) and a left second perfusion hole (1022) and a right second perfusion hole (1023) located at both ends of the second channel groove (1021); the first metabolic chamber (301) is provided with a first middle chamber (3011) and a first transfer port (3012) located at one end of the first middle chamber (3011), the first transfer port (3012) overlaps with the projection of the left second perfusion hole (1022) on the upper layer (1) of the chip, and the first middle chamber (3011) and the first channel groove (1011) cross each other in the projection on the upper layer (1) of the chip; Wherein, the structures of the third fluid channel (103) and the fourth fluid channel (104) are the same as those of the first channel (101) and the second channel (102); The structures and arrangement manners of the second metabolic chamber (302) and the third metabolic chamber (303) are the same as those of the first metabolic chamber (301); and / or, the inner width of the first fluid channel (101) and the fourth fluid channel (104) is 0.4 - 0.6 mm; the inner width of the second fluid channel (102) and the third fluid channel (103) is 0.6 - 0.8 mm.

10. Use of the multi-level metabolic organ chip based on a porous membrane according to any one of claims 1 to 9 in disease simulation, drug development, and nutrition research.

11. The application according to claim 10, characterized in that, Use of the multi-level metabolic organ chip based on a porous membrane in studying multi-level drug metabolism in vitro.

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