CN116590144A

CN116590144A - Lung chip, lung model, construction method of lung model and compound detection method

Info

Publication number: CN116590144A
Application number: CN202211230645.5A
Authority: CN
Inventors: 顾忠泽; 陈早早; 张静; 欧阳珺; 朱建峰; 葛健军; 沙利烽
Original assignee: Jiangsu Aiweide Biotechnology Co ltd; Southeast University Suzhou Medical Device Research Institute
Current assignee: Jiangsu Aiweide Biotechnology Co ltd; Southeast University Suzhou Medical Device Research Institute
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2023-08-15

Abstract

The invention provides a lung chip, a lung model, a construction method thereof and a detection method of a compound, belonging to the technical field of tissue engineering. The lung chip includes: a sealing layer, a lower culture layer, a separation membrane layer, an upper culture layer and a connecting layer which are sequentially laminated; wherein, the connecting layer is provided with an inlet group and an outlet group; the upper culture layer is provided with an upper culture unit connected with the inlet group and the outlet group, the upper culture unit comprises a first upper culture chamber and a second upper culture chamber which are communicated, the first upper culture chamber is used for culturing a three-dimensional lung bronchus structure, and the second upper culture chamber is used for culturing a three-dimensional alveolus structure; the lower culture layer is provided with a lower culture unit connected with the inlet group and the outlet group, and the lower culture unit comprises a first lower culture chamber and a second lower culture chamber which are communicated with each other; the lower culture unit and the upper culture unit are arranged oppositely, and substance exchange occurs through the isolating membrane layer, so that the constructed model has higher simulation degree and has a function closer to that of a real lung organ of a human body.

Description

Lung chip, lung model, construction method of lung model and compound detection method

Technical Field

The invention belongs to the technical field of tissue engineering, and particularly relates to a lung chip, a lung model, a construction method of the lung model, and a detection method of a compound by using the lung chip.

Background

The lung is an important respiratory organ of a human body, and in recent years, environmental problems are serious, haze is frequent, and lung influence caused by environmental changes is attracting more and more attention; chronic respiratory diseases (e.g., asthma and chronic obstructive pulmonary disease) create a tremendous public health burden, while poor lifestyle causes an increase in the prevalence of lung cancer. In addition, viral-induced pulmonary diseases (e.g., influenza) are also of increasing interest, and research into pulmonary diseases is being raised to a new level. Therefore, establishing an in vitro lung organ tissue model, and being able to simulate lung infection and inflammatory response, is a vital task in the biomedical field.

The culture of lung epithelial cells is applied to the field of life sciences for a long time based on a 2D cell culture model, but the 2D cell culture model is too simplified in terms of cell-cell and cell-matrix interactions, it is difficult to simulate complex interactions between various cells inside a human body, and the response of two-dimensional cells to external stimuli is greatly different from the response of three dimensions in the human body, so that the application is greatly restricted.

The animal model is used as the most widely applied 3D model at present, but because of the large structural difference between the animal model and the lung of a human body, the established model is insensitive to the detection of the efficacy of the medicine, the phenomenon of off-target of the medicine exists at the same time, the occurrence and development processes of diseases cannot be intuitively monitored, the detection of the efficacy of the medicine is insensitive, the phenomenon of off-target of the medicine exists at the same time, the phenomenon of off-target of the medicine still brings great uncertainty and challenges to the development process of new medicine, and the phenomenon of effective animal experiments and ineffective human body inspection exists for a long time, so that the application limitation is very large.

Accordingly, there is an urgent need to provide a lung chip, a lung model, and a method for detecting compounds using the lung chip that can effectively simulate the three-dimensional tissue structure and microenvironment of human lung organs.

Disclosure of Invention

The invention aims to at least solve one of the technical problems in the prior art, and provides a lung chip, a lung model construction method thereof and a compound detection method by using the lung chip.

In one aspect of the present invention, there is provided a lung chip comprising: a sealing layer, a lower culture layer, a separation membrane layer, an upper culture layer and a connecting layer which are sequentially laminated; wherein, the liquid crystal display device comprises a liquid crystal display device,

the connecting layer is provided with an inlet group and an outlet group;

The upper culture layer is provided with an upper culture unit connected with the inlet group and the outlet group, the upper culture unit comprises a first upper culture chamber and a second upper culture chamber which are communicated, the first upper culture chamber is cultured with a three-dimensional lung bronchus structure, and the second upper culture chamber is cultured with a three-dimensional alveolus structure;

the lower culture layer is provided with a lower culture unit connected with the inlet group and the outlet group, and the lower culture unit comprises a first lower culture chamber and a second lower culture chamber which are communicated with each other;

the lower culture unit is arranged opposite to the upper culture unit, and substance exchange occurs through the isolating membrane layer.

Optionally, the first lower culture chamber and the second lower culture chamber are cultured with vascular structures, and the isolation membrane layer comprises a first isolation membrane and a second isolation membrane; wherein, the liquid crystal display device comprises a liquid crystal display device,

the first upper culture chamber, the first isolation film and the first lower culture chamber correspond to form a first culture unit;

the second upper culture chamber, the second isolation film, and the second lower culture chamber correspond to form a second culture unit.

Optionally, the three-dimensional lung bronchus structure and three-dimensional alveolus structure in the first upper culture chamber and/or the second upper culture chamber comprises epithelial cells, or epithelial cells seeded with the first immune cells;

The vascular structures in the first lower culture chamber and/or the second lower culture chamber comprise vascular endothelial cells, or vascular endothelial cells inoculated with first immune cells;

wherein the first immune cells are one or more of monocytes, macrophages, granulocytes, dendritic cells and mast cells.

Optionally, the lung chip further comprises a circuit board layer arranged on one side of the sealing layer away from the lower culture layer;

the circuit board layer is provided with a first measuring electrode group connected with the first culture unit, a second measuring electrode group connected with the second culture unit and a resistance measuring interface; the method comprises the steps of,

the first measuring electrode group and the second measuring electrode group are electrically connected with the resistance measuring interface through the circuit board layer so as to obtain the resistance values of the cell growing process in the first culture unit and the second culture unit.

Optionally, the circuit board layer is further provided with at least one observation window, and the observation window is opposite to the first culture unit and the second culture unit.

Optionally, the connecting layer is further provided with a first bubble removing part and a second bubble removing part, and,

The upper culture unit further comprises a cell flow channel group connected with the first upper culture chamber and the second upper culture chamber, and the cell flow channel group is also connected with the first bubble removing part;

the lower culture unit further comprises a blood vessel runner group connected with the first lower culture chamber and the second lower culture chamber, and the blood vessel runner group is also connected with the second bubble removing part; the method comprises the steps of,

the lung chip further comprises a bubble removal membrane layer arranged on one side, away from the upper culture layer, of the connecting layer, wherein the bubble removal membrane layer is arranged opposite to the first bubble removal part and the second bubble removal part, so that gas passes through the bubble removal membrane layer.

In another aspect of the present invention, there is provided a method for constructing a lung model, the method comprising:

performing surface modification on an upper culture unit in the lung chip, wherein the lung chip is the lung chip described above;

adding a lung bronchus cell suspension into a first upper culture chamber in the upper culture unit, adding an alveolus cell suspension into a second upper culture chamber, and forming a lung epithelial layer after co-culture;

and filling air into the upper culture unit, and culturing to obtain a three-dimensional lung bronchus structure with a three-dimensional structure and a lung model of alveolar tissue.

Optionally, the construction method further includes:

adding vascular cell suspension into the downward culture unit to form a vascular layer so as to obtain a vascular-lung model; and/or the number of the groups of groups,

adding immune cells to the upper culture unit or the lower culture unit to obtain a lung model with immune function; or alternatively, the process may be performed,

and adding a target reactant into the lung chip, and performing perfusion culture on a culture chamber in the lung chip to obtain a lung model with a disease analysis function.

In another aspect of the present invention, a lung model is provided, which is constructed by the aforementioned construction method.

In another aspect, the present invention provides a method for detecting a compound using the lung chip described above, comprising the following specific steps:

introducing each culture medium into each culture unit to culture or perfuse the lung bronchi structure, alveoli structure and/or vascular structure;

introducing a test compound into the upper culture unit or the lower culture unit;

and obtaining a regulating result of the compound to be tested on the lung bronchus structure, the alveolus structure and/or the vascular structure.

The invention (1) structurally provides a lung chip formed by tissue cascade of complex multi-layer structures, so that the simulation degree of a lung model constructed by the lung chip is higher, and the function is closer to that of a real lung organ of a human body; (2) Introducing resident immune cells (e.g., resident macrophages) and circulating immune cells (e.g., monocytes) to effect an inflammatory cascade in an in vitro three-dimensional lung tissue model; (3) Changes in three-dimensional lung tissue function in vitro, such as permeability, mucus secretion, and inflammation, can be detected in real time; (4) Can cause similar reaction to external effect (especially virus infection) and can be used for constructing various disease models; (5) A deep learning algorithm is provided for analyzing morphological changes of epithelium, macrophages and endothelium.

Drawings

FIG. 1 is an exploded view of a lung chip according to an embodiment of the invention;

FIG. 2 is a schematic diagram of a lung chip according to another embodiment of the invention;

FIG. 3 is a schematic diagram of a connection layer in a lung chip according to another embodiment of the invention;

FIG. 4 is a schematic diagram showing the structure of an upper culture layer in a lung chip according to another embodiment of the present invention;

FIG. 5 is a schematic diagram of the structure of the lower culture layer in the lung chip according to another embodiment of the present invention;

FIG. 6 is a schematic view showing a structure of a sealing layer in a lung chip according to another embodiment of the invention

FIG. 7 is a schematic diagram of a circuit board layer in a lung chip according to another embodiment of the invention;

FIG. 8 is a schematic diagram showing an exploded structure of a dual-chamber three-dimensional biochip according to another embodiment of the invention;

FIG. 9 is a schematic view of a connection layer according to another embodiment of the present invention;

FIG. 10 is a schematic diagram showing the structure of an upper culture layer according to another embodiment of the present invention;

FIG. 11 is a schematic view showing the structure of a lower culture layer according to another embodiment of the present invention;

FIG. 12 is a schematic diagram showing the overall assembly structure of a dual-chamber three-dimensional biochip according to another embodiment of the invention.

FIG. 13 shows the adhesion and penetration results of THP-1 cells into the endothelial layer after LPS stimulation of the lung chip in example 2 of the present invention;

FIG. 14 shows the result of the staining reaction of characteristic proteins after infection with spike protein in the lung chip constructed in example 3 of the present invention.

FIG. 15 is a schematic diagram of a lung model obtained by culture according to an embodiment of the present invention, and a schematic diagram of a detection result performed on the lung model.

FIG. 16 is a schematic illustration of the introduction of immune cells into a lung chip to mimic early inflammation in accordance with an embodiment of the present invention.

FIG. 17 is a comparative schematic of inflammatory response in accordance with an embodiment of the present invention.

Fig. 18 is a lung chip classification algorithm based on deep learning according to an embodiment of the invention.

Detailed Description

The present invention will be described in further detail below with reference to the drawings and detailed description for the purpose of better understanding of the technical solution of the present invention to those skilled in the art. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention belong to the protection scope of the present invention.

Unless specifically stated otherwise, technical or scientific terms used herein should be defined in the general sense as understood by one of ordinary skill in the art to which this invention belongs. The use of "including" or "comprising" and the like in the present invention is not intended to limit the shape, number, step, action, operation, component, original and/or group thereof referred to, nor exclude the presence or addition of one or more other different shapes, numbers, steps, actions, operations, components, original and/or group thereof. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or order of the indicated features.

In some descriptions of the present invention, unless specifically stated and limited otherwise, the terms "mounted," "connected," or "fixed" and the like are not limited to a physical or mechanical connection, but may include an electrical connection, whether direct or indirect through an intervening medium, that is internal to two elements or an interaction relationship between the two elements. And, the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate an azimuth or a positional relationship based on that shown in the drawings, are used only to indicate a relative positional relationship, which may be changed when the absolute position of the object to be described is changed, accordingly.

As shown in fig. 1 to 7, an aspect of the present invention proposes a lung chip, including: a sealing layer F, a lower culture layer E, an isolating film layer D, an upper culture layer C and a connecting layer B which are sequentially laminated; wherein, the connecting layer B is provided with an inlet group and an outlet group; an upper culture unit connected with the inlet group and the outlet group is arranged on the upper culture layer C; the upper culture unit comprises a first upper culture chamber C15 and a second upper culture chamber C16 which are communicated, wherein the first upper culture chamber C15 is used for culturing a three-dimensional lung bronchus structure, and the second upper culture chamber C16 is used for culturing a three-dimensional alveolus structure. The lower culture layer E is provided with a lower culture unit connected with the inlet group and the outlet group, and the lower culture unit comprises a first lower culture chamber E7 and a second lower culture chamber E8 which are communicated with each other; the lower culture unit is arranged opposite to the upper culture unit, and exchange of substances occurs through the isolating membrane layer D.

In this embodiment, the pulmonary chip may be made of methyl methacrylate (PMMA).

The embodiment forms a communicated double-chamber lung chip, the first upper culture chamber is equivalent to forming a lung bronchus cavity, the second upper culture chamber is equivalent to forming an alveolus cavity, the simulation of the alveolar structure and the lung bronchus structure of a human body can be realized, a microenvironment similar to the human body is constructed, and the functions are closer to those of a real lung organ of the human body.

Specifically, as shown in fig. 1, 2, 3 and 4, the isolating film layer D includes a first isolating film and a second isolating film; wherein, the first isolation film is sandwiched between the first lower culture chamber E7 and the first upper culture chamber C15, and the second isolation film is sandwiched between the second lower culture chamber E8 and the second upper culture chamber C16 for separating the upper and lower culture chambers of the upper and lower culture layers.

In this embodiment, the first upper culture chamber located on the upper layer, the first isolation film of the middle layer, and the first lower culture chamber located on the lower layer form a first culture unit, and the second upper culture chamber located on the upper layer, the second isolation film of the middle layer, and the second lower culture chamber located on the lower layer form a second culture unit.

The first isolation membrane and the second isolation membrane of this embodiment may be porous membranes, and may be used to exchange substances in the upper and lower culture chambers through the porous membranes while performing isolation.

It should be further noted that, in the first upper culture chamber and/or the second upper culture chamber of the present embodiment, the three-dimensional lung bronchi structure and the three-dimensional alveoli structure include epithelial cells, or epithelial cells inoculated with the first immune cells; the vascular structures in the first lower culture chamber and/or the second lower culture chamber comprise vascular endothelial cells, or vascular endothelial cells inoculated with first immune cells; wherein the first immune cell is one or more selected from monocyte, macrophage, granulocyte, dendritic cell and mast cell.

Further, as shown in fig. 1 to 3, the inlet group on the connection layer B includes a cell fluid inlet B1, a vascular fluid inlet B4, a first cell loading port B5 near the inlet and the outlet, a first vascular loading port B6 near the inlet and the outlet, a second cell loading port B7 far from the inlet and the outlet, and a second vascular loading port B8 far from the inlet and the outlet. And, the outlet group on the connection layer B includes a cellular fluid outlet B2, a vascular fluid outlet B3, and a bubble removing part is further provided on the connection layer B.

It should be noted that, in this embodiment, the number and structure of the bubble removing portions are not particularly limited, for example, as shown in fig. 1 to 5, a first bubble removing portion B10 and a second bubble removing portion B11 are provided on the connection layer B, and the two bubble removing portions include a flow passage and a through hole for removing bubbles. The first bubble removing part B10 corresponds to an upper culture unit on the upper culture layer C, and the second bubble removing part B11 corresponds to a lower culture unit on the lower culture layer E, so that bubbles are discharged after air is introduced into the upper culture unit, and the real microenvironment of the human lung is simulated.

Further, as shown in fig. 1 to 4, the upper culture unit on the upper culture layer C further comprises a cell flow channel group, specifically a cell fluid inflow channel C1, a cell fluid outflow channel C2, a first cell loading channel C5, a second cell loading channel C7, and a cell connecting flow channel C10. Wherein, cell fluid inflow channel C1 is connected with cell fluid entry B1, and cell fluid outflow channel C2 is connected with cell fluid export B2, and first cell loading channel C5 is connected with first cell loading mouth B5, and second cell loading channel C7 is connected with second cell loading mouth B7. Based on this, the cell fluid inflow channel C1, the cell connection flow channel C10, the second upper culture chamber C16, the first upper culture chamber C15, and the cell fluid outflow channel C2 are sequentially communicated in the fluid flow direction. The first upper culture chamber C15 and the second upper culture chamber C16 are communicated through the cell fluid outflow channel C2, so that the cell connection channel C10 is a connection channel of the cell fluid inflow channel C1, the cell fluid outflow channel C2 and the first bubble removing part B10, and the first bubble removing part B10 is close to the second upper culture chamber C16.

Further, as shown in fig. 1 to 3 and 5, the lower culture unit on the lower culture layer E further includes a vascular flow path group, specifically including a vascular fluid inflow path E1, a vascular fluid outflow path E2, a first vascular sample loading path E3, a second vascular sample loading path E4, and a vascular connecting flow path E6. Wherein, vascular fluid inflow passageway E1 is connected with vascular fluid entry B4, and vascular fluid outflow passageway E2 is connected with vascular fluid export B3, and first vascular sample application passageway E3 is connected with first vascular sample application mouth B6, and second vascular sample application passageway E4 is connected with second vascular sample application mouth B8. Based on this, the vascular fluid inflow channel E1, the vascular connecting flow channel E6, the second lower culture chamber E8, the first lower culture chamber E7, and the vascular fluid outflow channel E2 are sequentially communicated in the fluid flow direction. The first lower culture chamber E7 is communicated with the second lower culture chamber E8 through a vascular fluid outflow channel E2, so that the vascular connection channel E6 is a connection channel of the vascular fluid inflow channel E1, the vascular fluid outflow channel E2 and the second bubble removing portion B11, and the second bubble removing portion B11 is close to the second lower culture chamber E8.

It should be understood that when the culture solution is introduced into the lower culture unit through the vascular fluid inlet or the sample inlet on the connection layer, or when the gas is led out from the lower culture layer to the connection layer, the culture solution needs to pass through the upper culture layer, and thus, a plurality of through holes are further formed in the upper culture layer to drain the culture solution into the lower culture unit on the lower layer, and the gas is led out to the connection layer.

Illustratively, as shown in fig. 1 to 5, the culture layer C is further provided with a vascular fluid inflow through hole C4, a vascular fluid outflow through hole C3, a first vascular sample through hole C6, a second vascular sample through hole C8, a gas discharge through hole C11, a gas discharge through hole C12, a gas discharge through hole C13, and a gas discharge through hole C14. The vascular fluid inlet B4, the vascular fluid inflow through hole C4, and the vascular fluid inflow channel E1 are sequentially communicated in the fluid flow direction. The first blood vessel sample adding port B6, the first blood vessel sample adding through hole C6 and the first blood vessel sample adding channel E3 are communicated in sequence according to the fluid flowing direction. The second blood vessel sample adding port B8, the second blood vessel sample adding through hole C8 and the second blood vessel sample adding channel E4 are communicated in sequence according to the fluid flowing direction. The vascular fluid outflow channel E2, the vascular fluid outflow through hole C3, and the vascular fluid outlet B3 are sequentially communicated in the fluid flow direction. And, the vascular fluid inflow path E1, the vascular fluid outflow path E2, and the vascular connecting flow path E6 are connected to the second bubble removing portion B11 on the connecting layer B through the gas discharge through hole C11, the gas discharge through hole C12, the gas discharge through hole C13, and the gas discharge through hole C14.

Still further, based on being provided with first bubble removal portion and second bubble removal portion on the tie layer, the lung chip of this embodiment still includes bubble removal rete, and this bubble removal rete sets up in bubble removal portion one side of deviating from the upper culture layer, and the bubble in the culture unit flow path can be got rid of after this bubble removal rete combines with each culture unit on the lung chip to make gas pass through this rete and arrange outside, and liquid can not pass through, prevent that the bubble from flowing into the culture cavity and causing the influence to tissue culture.

Specifically, as shown in fig. 1 to 3, the bubble removal film layer a includes a first bubble removal film and a second bubble removal film, the first bubble removal film is disposed opposite to the first bubble removal portion B10 on the connection layer B, the second bubble removal film is disposed opposite to the second bubble removal portion B11 on the connection layer B, and each through hole and flow channel on the bubble removal portion is sealed with the bubble removal film to remove bubbles in the culture flow path.

In the present embodiment, the material of the bubble removing film is not particularly limited, and for example, the bubble removing film may be a porous polytetrafluoroethylene film, a polydimethylsiloxane film, or the like.

Further, as shown in fig. 1, 6 and 7, the device further comprises a circuit board layer G, wherein the circuit board layer G is disposed on a side of the sealing layer F away from the lower culture layer E, and a first measurement electrode set and a second measurement electrode set are disposed on the circuit board layer G. The first measuring electrode group is arranged in the first culture unit in a penetrating way so as to obtain the resistance value of each cell growing process in the first culture unit. The second measuring electrode group is arranged in the second culture unit in a penetrating way so as to obtain the resistance value of each cell growing process in the second culture unit.

Specifically, as shown in fig. 2 to 7, the first measurement electrode group includes a measurement electrode G1, a measurement electrode G2, a measurement electrode G3, a measurement electrode G4, and the second measurement electrode group includes a measurement electrode G3, a measurement electrode G4, a measurement electrode G5, and a measurement electrode G6. The measurement electrodes G3 and G4 of the six measurement electrodes are common electrodes of the two culture units, the measurement electrodes G1, G4, and G5 are disposed in the upper culture unit, and the measurement electrodes G2, G3, and G6 are disposed in the lower culture unit.

Further, through holes F4, F2, and F6 corresponding to the three measurement electrodes (G1, G4, and G5) are provided in the sealing layer F, and through holes E9, E10, and E11 corresponding to the three measurement electrodes (G1, G4, and G5) are provided in the lower culture layer E. The measuring electrodes G1 sequentially penetrate through the through holes F4 and E9, the measuring electrodes G4 sequentially penetrate through the through holes F2 and E10, and the measuring electrodes G5 sequentially penetrate through the through holes F6 and E11, so that the three measuring electrodes are inserted into the fluid in the upper culture unit. That is, the measuring electrodes G2, G3, G6 are in contact with the fluid in the upper culture unit through the through holes F2, F4, F6 on the sealing layer F and the through holes E9, E10, E11 on the lower culture layer E.

Further, as shown in fig. 2 to 8, through holes F1, F5, F3 corresponding to the three measurement electrodes (G2, G3, G6) are provided in the sealing layer F. Wherein, the measurement electrode G2 is inserted into the through hole F1, the measurement electrode G3 is inserted into the through hole F5, and the measurement electrode G6 is inserted into the through hole F3, so that the three measurement electrodes are inserted into the lower culture unit. That is, the measuring electrodes G2, G3, G6 are in contact with the fluid in the lower culture unit through the through holes F1, F3, F5 on the sealing layer F.

In this embodiment, the measuring electrode G1, the measuring electrode G2, the measuring electrode G3, and the measuring electrode G4 form four transmembrane resistance measuring electrodes of the first culturing unit, so as to obtain the resistance value of the growing process of the lung cells in the first culturing unit. In addition, four transmembrane resistance measuring electrodes of the second culture unit are formed by the measuring electrode G5, the measuring electrode G6, the measuring electrode G3 and the measuring electrode G4, so that the resistance value of the lung cell growing process in the second culture unit is obtained.

In the present embodiment, the material of the measuring electrode is not particularly limited, and may be platinum, gold, graphite, or the like.

As shown in fig. 1 to 7, the circuit board layer G is further provided with a resistance measurement interface G9, and corresponding mounting through holes corresponding to the resistance measurement interface G9 are respectively formed in the connection layer B, the upper culture layer C, the lower culture layer E and the sealing layer F, and the resistance measurement interface G9 sequentially passes through the fourth mounting through hole F7, the third mounting through hole E5, the second mounting through hole C9 and the first mounting through hole B9, respectively, the second mounting through hole C9, the third mounting through hole E5 and the fourth mounting through hole F7. That is, a transmembrane resistance measuring interface is formed on the lung chip of the embodiment, and an external transmembrane resistance measuring device is connected through a USB interface, so as to evaluate the cell growth status and the fusion degree thereof by measuring the change of the resistance in the cell growth process in each culture unit.

The circuit board layer of the embodiment is the integration of a transmembrane resistance measuring electrode, a measuring circuit and an interface, and can obtain the resistance value in the cell growth process in the lung chip.

Further, the lung chip can be connected with the electrode adapter and the resistance measuring instrument respectively, and the connecting port of the electrode adapter comprises a current channel 1, a current channel 2, a voltage channel 1 and a voltage channel 2. Based on the structure, the transmembrane resistance measuring method comprises the following steps:

1) Four electrodes G1, G2, G3 and G4 measure the transmembrane resistance of the first culture unit, and four electrodes G3, G4, G5 and G6 measure the transmembrane resistance of the second culture unit. G1, G2, G3, G4, G5, G6 are connected with electrode interfaces on the chip through circuits on the circuit board. The electrode interface is connected with the electrode adapter and the transmembrane resistance measuring instrument through connecting wires. G1, G4, G5 are located on the on-chip culture layer and are in contact with the solution, and G2, G3, G6 are located on the under-chip culture layer and are in contact with the solution.

2) And measuring the transmembrane resistance of the first culture unit, connecting G1 with a voltage channel 1 of the electrode adapter through a circuit on the circuit board and an electrode interface on the chip, connecting G3 with a voltage channel 2 of the electrode adapter through a circuit on the circuit board and an electrode interface on the chip, connecting G4 with a current channel 1 of the electrode adapter through a circuit on the circuit board and an electrode interface on the chip, and connecting G2 with a current channel 2 of the electrode adapter through a circuit on the circuit board and an electrode interface on the chip. The transmembrane resistance measuring instrument measures the voltage and current at both sides of the porous membrane through four electrodes and displays the transmembrane resistance value.

3) And measuring the transmembrane resistance of the second culture unit, connecting G5 with the voltage channel 1 of the electrode adapter through a circuit on the circuit board and an electrode interface on the chip, connecting G3 with the voltage channel 2 of the electrode adapter through a circuit on the circuit board and an electrode interface on the chip, connecting G4 with the current channel 1 of the electrode adapter through a circuit on the circuit board and an electrode interface on the chip, and connecting G6 with the current channel 2 of the electrode adapter through a circuit on the circuit board and an electrode interface on the chip. The transmembrane resistance measuring instrument measures the voltage and the current at two sides of the porous membrane through four electrodes and displays the transmembrane resistance value of the second culture unit.

The transmembrane resistance value of this example correlates with the barrier function of the tissue, and the more complete the barrier, the greater the transmembrane resistance value. The transmembrane resistance of normal tissues is measured, and the transmembrane resistance of the drug is measured, and whether the barrier of the tissues is destroyed can be observed through numerical comparison.

Furthermore, the circuit board layer is also provided with at least one observation window which is arranged opposite to the first culture unit and the second culture unit and is used for observing the growth condition of lung tissues in each culture chamber.

Specifically, as shown in fig. 1 to 7, two observation windows, namely a first observation window G7 and a second observation window G8, are provided on the circuit board layer G, wherein the first observation window G7 corresponds to the first culturing unit, so that the cell growth conditions in the first upper culturing chamber C15 and the first lower culturing chamber E7 are observed through the first observation window G7. The second observation window G8 corresponds to the second culturing unit to observe the growth of cells in the second upper culturing chamber C16 and the second lower culturing chamber E8 through the second observation window G8.

As shown in fig. 8-12, the present application additionally provides an embodiment of a dual-chamber three-dimensional biochip.

Specifically, the dual-chamber three-dimensional biochip may include a connection layer B ', an upper culture layer C', a lower culture layer E ', and a sealing layer F' in this order from the top down. The corresponding positions on each layer are respectively provided with a positioning hole, each layer can be aligned by a jig, and the upper culture layer C 'and the lower culture layer E' are fixed between the connecting layer B 'and the sealing layer F' by means of hot press bonding, laser bonding, liquid glue or solid glue bonding, screws and the like. Fluids may be introduced into the upper and lower culture layers C 'and E', for culturing cells, etc. The connection layer B' may include a connection male, on which docking holes B1, B2, B3, B4 are provided.

Specifically, the first channel C1' is provided in the upper culture layer C ', and the first channel C1' may include a first upper culture chamber C15' and a second upper culture chamber C16' which are communicated with each other. The lower culture layer E 'is provided with a second channel E2', a third channel E3 'and a fourth channel E4'. The first channel C1', the second channel E2', the third channel E3 'and the fourth channel E4' are approximately parallel two by two. The third channel E3' may include a first lower culture chamber E7' and a second lower culture chamber E8' communicating with each other. The butt joint holes on the connecting layer B ' can be correspondingly communicated with the holes of the channels of the upper culture layer C ' and the lower culture layer E '. The wells C62, C64, C65, C67 on the upper culture layer C ' are respectively communicated with the wells on the corresponding positions on the connecting layer B ', and the wells C62, C64, C65, C67 can be used for adding the sample to the corresponding positions in the upper culture layer C '. The holes E31, E32, E33, E34 on the lower culture layer E ' are respectively communicated with the holes on the corresponding positions on the connecting layer B ' through the upper culture layer C ', and the holes E31, E32, E33, E34 on the lower culture layer E ' can be used for adding samples to the corresponding positions on the lower culture layer E '.

Optionally, the connection layer B ' may further be provided with a bubble removing chamber A2', and the bubble removing chamber A2' may include a first bubble removing chamber a21 and a second bubble removing chamber a22. The bubble removal chamber cover A1' can seal the bubble removal chamber A2' to ensure that fluid enters and exits through the aperture at the bottom end of the bubble removal chamber A2 '. The three channels E3' are communicated, and two communicating ports for communicating the first bubble removing chamber A21 with the second channel E2' and the third channel E3' are arranged at the bottom end of the first bubble removing chamber A21 and are positioned at the diagonal positions of the first bubble removing chamber A21. The second bubble removal chamber a22 is respectively communicated with the first channel C1', the fourth channel E4', and two communication ports for communicating the second bubble removal chamber a22 with the first channel C1', the fourth channel E4' are arranged at the bottom end of the second bubble removal chamber a22 and are positioned at the diagonal positions of the second bubble removal chamber a22. By this arrangement, bubbles can rise to the surface of the bubble removal chamber during passage through the bubble removal chamber, and bubbles in the fluid exiting the bubble removal chamber can be significantly reduced.

The bubble removing chamber can remove bubbles in the flow path, and prevent the bubbles from flowing into the culture chamber to affect tissue culture. For ease of understanding, fig. 12 shows a perspective view of one embodiment of the chip.

According to the three-dimensional lung biological tissue chip, a micro-fluidic technology is adopted, and the important structural parts of a lung organ, namely a culture chamber arranged in the three-dimensional lung biological tissue chip, and structures such as a fluid inlet, an inlet flow channel, a fluid outlet, an outlet flow channel and the like which are communicated with the culture chamber are connected on the three-dimensional lung biological tissue chip, so that functional structural units of the three-dimensional lung biological tissue and a growing microenvironment can be effectively simulated, more accurate control can be realized in time and space dimensions, the labor cost is further saved, and high-throughput, large-scale and standardized detection is realized. In addition, the three-dimensional culture of various cells can be realized by simultaneously culturing the lung cells on the membrane, and the introduction of the biological material can better simulate the interaction between the cells and the matrix, so that the method is an ideal in-vitro research model for scientific research and clinical detection. The three-dimensional lung biological tissue chip formed by the invention can obtain a lung model with corresponding disease function after being subjected to certain physical, chemical or biological treatment so as to carry out deeper disease research or drug development research.

On the other hand, the invention provides a method for constructing a lung model based on the lung chip structure, which comprises the following specific steps:

s1, pretreatment: and sterilizing the lung chip and the flow path system obtained in the previous step. In a sterile environment, the coating solution is injected into the upper culture unit through the first cell loading port C5.

In the embodiment, the surface of the culture unit is modified by using the coating liquid, so that the adhesion probability of cells in the later stage is improved, and the success rate of cell culture is improved.

Optionally, the lung chip and flow system may be sterilized prior to cell seeding, for example, by irradiation, ethylene oxide, ultraviolet light, or alcohol.

Alternatively, collagen II (0.2 mg/ml), collagen I (0.1 mg/ml), and collagen IV (0.05 mg/ml) may be used as the coating solution for culturing three-dimensional pulmonary bronchi structures, and a mixture of collagen III (0.2 mg/ml), collagen I (0.1 mg/ml), and collagen IV (0.05 mg/ml) may be used as the coating solution for culturing three-dimensional pulmonary bronchi structures.

S2, co-culturing a three-dimensional lung bronchus structure and alveolar tissue: the cell suspension containing lung bronchus cells is added into a first upper culture chamber C15 through a first cell sample adding port C5 on the upper culture unit, alveolar cells are added into a second upper culture chamber C16 through a second cell sample adding port C7, the first upper culture chamber C15 and the second upper culture chamber C16 are communicated, and three-dimensional lung bronchus structures with three-dimensional structures and alveolar tissues with three-dimensional structures are respectively obtained through culture.

It is further noted that the cell sources of the present embodiment include lung immortalized cells, lung primary cells, lung organoid digestive cells, or lung cancer immortalized cells, lung cancer primary cells, lung cancer organoid digestive cells, or IPSC differentiated cells.

Further, based on the above-mentioned forming of the lung model with the dual chambers, on the basis of the lung chip structure described above, a lung model with a vascular function may also be formed, and the process of constructing the lung model may further include:

s3, blood vessel functional culture: and in a sterile environment, respectively injecting vascular cell suspension into two lower culture chambers in the lower culture layer through a first vascular sample adding port E3 and a second vascular sample adding port E4, and culturing to form a three-dimensional vascular layer, thereby obtaining the vascular-lung chip.

It should be noted that the vascular cells added in this embodiment may be one or more of vascular endothelial cells, vascular myofibroblasts, and vascular smooth muscle cells.

It should be understood that when forming the vascular-pulmonary chip, the third step may also be performed before the second step, with the vascular layer being formed and the pulmonary epithelial layer being formed.

Furthermore, immune function can be further increased on the vascularization lung chip, and the construction process of the lung model can further comprise the following steps on the basis of the above process:

S4, inoculating immune cells: within 1-7 days before the end of the third step, the first immune cells were inoculated on the surface of the epithelial cells in the upper culture unit, and a lung model with immune function was obtained. Or alternatively, the process may be performed,

s5, constructing a circulating immune cell: the lower culture unit is perfused with a medium containing at least one immune cell, for example: THP-1 cells are used to construct immune functions, and whether the immune functions are successfully constructed can be determined by observing adhesion and penetration of the THP-1 cells to vascular layers. That is, the present embodiment may form a lung model having an immune function by inoculating immune cells in an upper culture unit of an upper layer, or may also perfuse a culture medium of immune cells in a lower culture layer of a lower layer.

Furthermore, the disease analysis function can be added on the vascularization lung chip, and the construction process of the lung model can further comprise the following steps of:

s6, virus treatment: and processing the bronchus-alveolus model by using a target virus to obtain a target lung model, performing perfusion culture on the target lung model in the culture chamber by using a compound to be tested, and evaluating the influence result of the compound to be tested on the disease by analyzing the change of the compound to be tested, namely forming the lung model with a disease analysis function.

Specifically, the lung model production steps are as follows:

first, the lung chip and the flow system obtained above are sterilized by irradiation, ethylene oxide, ultraviolet rays or alcohol.

Secondly, injecting coating liquid into the upper culture unit and/or the lower culture unit respectively through an inlet port in a sterile environment; wherein the coating liquid is collagen solution, and is put into a culture box at the temperature of 35-37 ℃ for incubation, taken out after 1-2 hours, and washed for 1-2 hours by PBS circulation.

Specifically, static incubation can be performed for 1-2 hours, cells can be attached to the membrane, and then the culture medium is poured into the first culture unit and the second culture unit at a volume flow rate of 1 mu L/min, wherein the upper layer of the lung chip can be poured with DMEM/F12K culture medium, and the lower layer of the lung chip can be poured with a mixture of DMEM and RPMI-1640 in a volume ratio of 50:50 as the culture medium.

Third, two lower culture chambers in the lower culture layer are respectively injected with 1x10 through a first blood vessel sample adding port E3 and a second blood vessel sample adding port E4 in a sterile environment ⁴ ～1x10 ⁶ Individual cells/cm ² Is cultured to form a three-dimensional vascular layer. Thereafter, the chip was turned over and the lung chip was placed in 5% CO at 37 ℃C ₂ And standing the incubator with 95% humidity for 1-24 hours.

The blood vessel cells added in the lower culture layer in the step are one or more of blood vessel endothelial cells, blood vessel myofibroblasts and blood vessel smooth muscle cells.

It should be noted that, when only the lung chip structure having the dual chambers needs to be formed, this step may be omitted, that is, the lower culture layer may not be cultured, the blood vessel layer may not be formed, and only the upper culture layer may be cultured.

Fourth, in a sterile environment, the first upper culture chamber and the second upper culture chamber in the on-chip culture layer are respectively injected with a sample containing 1x10 through the sample injection port of the layer ⁴ ～1x10 ⁶ Individual cells/cm ² Is cultured to form the lung epithelial layer. Then put into 37 ℃ and 5 percent CO ₂ And standing the incubator with 95% humidity for 1-24 hours.

The cells added in the upper culture layer are lung bronchial epithelial cells and alveolar epithelial cells, and the cell sources comprise lung immortalized cells, lung primary cells, lung organoid digestive cells, lung cancer immortalized cells, lung cancer primary cells, lung cancer organoid digestive cells, or IPSC differentiated cells.

It should be further noted that, in some embodiments, the fourth step and the third step may be interchanged, that is, the fourth step is performed to inject the cell suspension into the upper culture layer to form the lung epithelial layer, and the third step is performed to inject the cell suspension into the lower culture layer to form the vascular layer.

Fifthly, connecting the lung chip with a pump driven culture system, injecting 10mL of corresponding cell culture medium into each liquid storage tube, starting the culture system, and starting continuous perfusion culture on an upper culture layer and a lower culture layer on the chip.

Sixthly, placing the culture system with the started perfusion culture and the lung chip into a system with 37 ℃ and 5 percent CO ₂ And (3) automatically filling and culturing for 2-4 days in a 95% humidity incubator, and then filling clean air into an upper culture layer of the chip, and continuously culturing for 3-21 days, wherein lung biological tissues with three-dimensional structures can be formed in the lung chip.

It should be appreciated that a lung chip having an alveolar-pulmonary bronchus dual-chamber structure can be formed based on the above process, and a dual-chamber lung chip having vascular function can also be formed. On this basis, a lung model having a different function can be formed by adding different external factors, for example, immune cells can be introduced into the culture unit, a lung model having an immune function can be obtained, and viruses or the like can be introduced into the culture unit, so that a lung model having a disease function can be obtained.

In some preferred embodiments, the method further comprises the steps of:

Seventh, culture knotAdding first immune cell suspension into upper and lower culture layers of lung chip 1-7 days before bundling, adding 37deg.C and 5% CO ₂ And standing the incubator with 95% humidity for 1-24 hours, starting a perfusion culture system, and continuing to culture until the end.

The first immune cells of the present embodiment include monocytes, macrophages, granulocytes, dendritic cells, mast cells, and the like. The ratio of the concentration of the cell suspension to the epithelial or endothelial cells is 1 (3-30).

Eighth, after culturing to form a lung three-dimensional tissue chip, in some embodiments, the lower culture layer of the chip is subjected to a culture containing 1X10 ³ ～1x10 ⁵ The culture medium of each immune cell is continuously perfused, and aggregation and membrane penetration of the immune cells to the vascular layer are observed.

In other preferred embodiments, the method further comprises the steps of forming a lung model with disease analysis functions:

seventh, the three-dimensional tissue lung chip formed by culturing can also be used for preparing disease models by means of chemical substance stimulation, virus infection and the like, and viruses comprise true viruses, false viruses, proteins containing specific binding sites and the like.

Specifically, a lung chip model that mimics a new coronavirus infection can be obtained, for example, by incubating the lung epithelial layer of the lung chip with SARS-CoV-2 Spike protein. The lung chip model simulating the prepared novel coronavirus infection can be applied to the scenes of detecting the action of virus inhibitors or detecting the action of protective devices and the like.

The lung chip of the embodiment can be used for drug screening detection, disease model construction and environment assessment, and can be used for replacing in vitro tests of human bodies and living animals.

In another aspect of the present invention, a lung model is provided, and the lung model is constructed by the aforementioned construction method, and the lung model may be a lung model with alveoli-bronchus double chambers, a lung model with vascular function, a lung model with immune function, or a lung model with disease analysis function.

In another aspect of the present invention, a method for detecting a compound using the lung chip described above is provided, comprising the following specific steps:

first, a lung cell culture medium is introduced into the upward culture unit to culture or perfuse the lung bronchial structures and alveolar tissue.

Secondly, introducing a compound to be tested into the upward culture unit;

thirdly, after incubation for a corresponding time, liquid is taken out through a fluid inlet or a fluid outlet of the upper culture layer for detection and analysis, so that the regulation result of the compound to be detected on the lung bronchus structure and the alveolus tissue is obtained.

In other preferred embodiments, since the lung chip may further include a vascular layer, the method for detecting a compound further includes:

First, a lung cell culture medium is added into the upward culture unit to culture or perfuse a lung bronchus structure and an alveolus structure, and a vascular cell culture medium is added into the downward culture unit to culture or perfuse a vascular structure.

Secondly, introducing a compound to be tested into the downward culture unit;

thirdly, after incubation for a corresponding time, liquid is taken out through a fluid inlet or a fluid outlet of the upper culture layer for detection and analysis, so that the regulation result of the compound to be detected on the lung bronchus structure, the alveolus structure and/or the vascular structure is obtained.

The three-dimensional lung tissue chip of the embodiment can perform physical action or drug stimulation, drug permeation, drug absorption and other tests to obtain the growth condition of cells through the measurement resistance of the circuit board layer, and of course, three-dimensional lung biological tissues in the chip can also be taken out to perform detection such as slicing, staining and the like to obtain the biological activity and other conditions of the cells, and the influence result of the compound to be tested on the cells is obtained according to the overall change of the growth condition of the cells.

The invention constructs a lung chip with alveolar and pulmonary broncho-luminal, allows multiple immune cells to integrate into the system, can observe amplified inflammatory signals through dynamic interactions between macrophages, epithelial cells, endothelial cells and circulating monocytes, and can study viral or bacterial infections among different individuals. The device can effectively simulate the microenvironment of the lung of a human body, and can be used for detecting and observing the microenvironment of the lung of the human body and constructing various lung disease models.

The lung chip, the lung model and its construction method, and the method of detecting compounds using the lung chip will be described in detail below:

example 1

This example illustrates an example of vascularized lung broncho-alveolar chip culture, specifically comprising the following processes:

firstly, sterilizing a lung chip and a flow path system by adopting gamma ray irradiation, injecting collagen coating liquid into an upper culture layer C of the chip through a cell fluid inflow channel C1 in a sterile environment, placing the chip into a 37 ℃ incubator for incubation for 1h, taking out the chip, and circularly cleaning the chip for 1h by using PBS.

Second, the first upper culture chamber C15 in the upper culture unit is filled with a liquid containing 1X10 through the first cell loading port B5 of the lung chip in a sterile culture environment ⁵ Individual cells/cm ² Is injected with 1x10 by a second vascular sample-adding port B8 into a second upper culture chamber C16 in the upper culture unit ⁵ Individual cells/cm ² Placing the suspension of the primary alveolar epithelial cells in a sterile incubator for standing for 4 hours to enable the lung epithelial cells in each culture cavity to be fully attached to the surface of the porous membrane.

Thirdly, after the cells are adhered, connecting the lung chip with a pump driven culture system, injecting 10ml of corresponding cell culture medium into each liquid storage tube, starting the culture system, and starting continuous perfusion culture on an upper culture layer C and a lower culture layer E of the lung chip.

The lung chip was flipped over after the fourth, 7 days and the injection of culture chambers in the sub-lung chip culture layer was totalized 2x10 through the fluid inlet in that layer ⁴ Individual cells/cm ² Is prepared from the mixed suspension of primary vascular endothelial cells and smooth muscle cells through loading in 5% CO at 37 deg.C ₂ The incubator with 95% humidity was left to stand for 4 hours.

Fifth, connecting the lung chip with the culture system, injecting 10ml of corresponding culture medium into each culture bottle, starting the culture system, and filling the culture systemThe culture system of the injection culture is put into 37 ℃ and 5% CO together with the lung chip ₂ An incubator with 95% humidity was automatically perfused for 2 days.

Sixthly, evacuating the culture medium of the upper culture layer corresponding to the culture bottle, starting the culture system, injecting flowing clean air into the culture chamber in the upper culture layer, continuously supplying the culture solution into the culture chamber in the lower culture layer, and continuously culturing for 14 days to obtain the lung bronchus-alveolus model.

Specifically, a culture environment with a gas-liquid interface can be established by gently sucking the medium away and pumping air into the apical chamber at a rate of 1. Mu.l/min after alveolar epithelial cells are confluent. The culture medium of the mixed suspension of lung bronchial epithelial cells, pulmonary alveolar epithelial cells, primary vascular endothelial cells and smooth muscle cells flows into the first lower culture chamber and the second lower culture chamber to provide nutrients for the mixed suspension of lung bronchial epithelial cells, pulmonary alveolar epithelial cells, primary vascular endothelial cells and smooth muscle cells.

The embodiment utilizes the lung chip with double culture chambers to simulate the vascularized lung structures connected with bronchi and alveoli of the lung, realizes dynamic culture through a lung chip system, provides gas-liquid culture conditions required by cell maturation and differentiation, effectively simulates the microenvironment for growth of lung tissues, prolongs the in-vitro culture time of the lung tissues, also integrates the functional structure of the lung tissues, ensures that the constructed in-vitro three-dimensional lung tissues have higher simulation degree, and can better simulate the response of real human lung organs to external stimulation.

In addition, the lung chip formed in this embodiment can perform physical action or drug stimulation, drug permeation, drug absorption and other tests, and the drug is added through a fluid inlet in the culture layer on the chip or liquefied drug is added into the culture chamber through a sample adding port, and after incubation for a corresponding time, detection and analysis such as slice staining can be performed by taking out the tissue through the culture medium. Wherein the physical action includes mechanical action, electrical action, optical action, radiation, etc. The medicine comprises medicines, chemicals, biological agents and the like for treating lung related diseases. Staining includes cell activity indicator staining, HE staining, immunofluorescence staining, and the like.

Example 2

This example illustrates the culture of an immunofunctional lung broncho-alveolar chip, specifically comprising the following steps:

First, ultraviolet rays are used to sterilize the lung chip and the flow path system.

Second, in a sterile culture environment, the first and second lower culture chambers are injected 1X10 through vascular fluid inflow channel E1 in lower culture layer E ⁴ Individual cells/cm ² HUVEC cell suspension of vascular endothelial cells, and placing into 5% CO at 37deg.C ₂ The incubator with 95% humidity was left to stand for 4 hours.

Taking out the lung chip from the incubator after the fourth and 7 days, turning over the lung chip, and injecting 1x10 into the first upper culture chamber C15 in the upper culture layer C through the first cell sample inlet B5 and the first cell sample channel C5 of the lung chip in a sterile environment ⁵ Individual cells/cm ² Is injected into a second upper culture chamber C16 in the upper culture layer C through a second cell sample adding port B7 and a second cell sample adding channel C7, wherein the upper culture chamber C contains 1x10 ⁵ Individual cells/cm ² The human lung adenocarcinoma cells NCI-H441 cell suspension is placed in a sterile incubator for standing for 4 hours to allow the cells in the culture chamber to be fully attached to the surface of the porous membrane. 15ml of fresh medium was injected into each flask, and the culture system was started to perform continuous perfusion culture.

Fifthly, placing the system started to perform perfusion culture and the lung chips into a 37 ℃ incubator, and automatically culturing for 2 days until human lung adenocarcinoma cells grow into compact tissues, thus obtaining the alveolar tissue structure.

Sixthly, evacuating a culture bottle for supplying a culture medium to the upper culture layer of the lung chip, starting a culture system, injecting flowing clean air into the first upper culture chamber and the second upper culture chamber of the lung chip, continuously supplying a culture solution into the culture chambers, and continuously culturing for 3 days to obtain the lung bronchus-alveolus chip model.

Seventh, after epithelial differentiation, macrophages were differentiated at 5X 10 ³ Individual cells/cm ² Inoculating on the surface of epithelial cells. The lower culture layer of the lung chip is subjected to continuous 24-hour perfusion culture, and a lung bronchus-alveolus chip model with immune function is obtained.

Of course, in other embodiments, the following procedure may be used to form a lung broncho-alveolar chip model with immune function, as follows:

the upper culture layer of the lung chip model was treated with 10. Mu.g/m Lipopolysaccharide (LPS) solution for 24 hours, and the lower culture layer of the lung chip was perfused with a culture medium containing THP-1 cells, and adhesion and penetration of THP-1 cells to the vascular layer was observed (see FIG. 13).

Example 3

This example illustrates the culture of a lung broncho-alveolar chip with viral function, and specifically includes the following steps:

first, the lung chip and the flow path system are sterilized by gamma radiation.

Second, in a sterile culture environment, a first upper culture chamber C15 in the upper culture layer C is filled with a liquid containing 2.5x10 through a first cell loading port B5 and a first cell loading channel C5 of the lung chip ⁵ Individual cells/cm ² The mixed cell suspension (ratio 10:1) of human lung bronchial epithelial cells BEAS-2B and macrophages is injected into the second cell sample inlet B7 and the second cell sample channel C7 to inject the mixture containing 2.5x10 into the second upper culture chamber C16 in the upper culture layer C ⁵ Individual cells/cm ² The mixed cell suspension of the human lung adenocarcinoma cells Calu-3 and macrophages (the ratio is 10:1) is placed in a sterile incubator to stand overnight, so that the cells in the culture chamber are fully attached to the surface of the porous membrane.

Third, the lung chip is turned over and the culture chambers in the layer are injected 2.5x10 through the fluid inlet in the culture layer under the lung chip ⁵ Individual cells/cm ² Is described. Placing in 37 ℃ and 5% CO ₂ The incubator with 95% humidity was left to stand for 24 hours.

Fourthly, after the cells are adhered, connecting the lung chip with a pump driven culture system, injecting 10mL of corresponding cell culture medium into each liquid storage tube, starting the culture system, and starting continuous perfusion culture on the on-chip culture layer and the under-chip culture layer.

Fifth, the system with perfusion culture started is put into 37 ℃ and 5% CO together with the lung chip ₂ An incubator with 95% humidity automatically cultures for 2 days until alveolar epithelial cells grow into dense epithelial tissue on the upper surface of the lung interstitium.

Sixthly, evacuating a culture bottle for supplying culture medium to the culture layer on the lung chip, starting a culture system, injecting flowing clean air into the upper culture layer, continuously supplying culture solution into a culture chamber, and continuously culturing for 3 days.

Seventh, after epithelial differentiation, macrophages were grown at 2.5X10 ⁴ Individual cells/cm ² Inoculating on the surface of epithelial cells. The lower culture layer of the lung chip is subjected to continuous 24-hour perfusion culture, and a lung bronchus-alveolus chip model with immune function is obtained.

Eighth, lung chips were treated with 250ng/mL SARS-CoV-2 (2019-nCOV) Spike protein at 4℃for 1 hour and washed twice with PBS (phosphate buffered saline) to obtain lung chips mimicking the infection with the novel coronavirus (staining reactions of the chips to the characteristic proteins after infection are shown in FIG. 14).

As an example of an embodiment of the present specification, the lung epithelial cell line BEAS-2B, NCI-H441, A549, calu-3, the human monocyte line THP-1, the human endothelial cell line HUVEC are available from the Global biological resource center (ATCC). BEAS-2B cells were cultured in DMEM supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% GlutaMAXTM supplement and 1% penicillin-streptomycin solution. Calu-3 cells were cultured in MEM, 10% heat-inactivated FBS and 1% penicillin-streptomycin solution were added. NCI-H441 and a549 cells were cultured in RPMI1640 supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin solution. HUVECs were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin solution. THP-1 cells were cultured in RPMI1640, 10% heat-inactivated FBS and 1% GlutaMAXTM supplement and 1% penicillin-streptomycin solution and 0.05mM 2-mercaptoethanol were added. After 24 hours of treatment with PMA in 50nM, macrophages were induced by THP-1. All cells were maintained under standard culture conditions (37 ℃,5% co2 in humidified air) and all cells were used within 7 passages after receiving.

In the embodiment of the present specification, the lung chip may be subjected to inflammatory stimulus treatment. Specifically, the inflammatory stimulus treatment may be that the first upper culture chamber and the second upper culture chamber may be treated with 10. Mu.g/mL LPS in a 50:50 mixture of DMEM and RPMI-1640 medium for 24 hours. Meanwhile, the first lower culture chamber and the second lower culture chamber may be perfused with a control medium in a 50:50 mixture of DMEM and RPMI-1640 medium or a mixture to which THP-1 cells are added as a medium. After the inflammatory stimulus treatment, the effluent from the upper culture chamber was collected and the chemokines and inflammatory regulatory factors (IL-6, TNF-. Alpha.and MCP-1) in the effluent were detected using ELISA kit.

In the examples herein, after obtaining a lung chip cultured with a lung model, the lung chip may be incubated with SARS-CoV-2 (2019-nCOV) Spike protein (250 ng/mL) for 1 hour at 4 ℃. After treatment, cells were washed twice with PBS and fixed overnight with 4% paraformaldehyde at 4 ℃. Immunocytochemistry studies were performed on non-permeabilized cells with primary antibodies to ACE2 (4. Mu.g/ml) and to Spike (4. Mu.g/ml) to detect protein co-localization. Finally, the appropriate fluorescent secondary antibodies were added for 2 hours at 1:200 dilution and nuclei were stained with DAPI. Images were taken with a microscope and processed by software.

In some embodiments, the cells may be transduced with SARS-CoV-2 pseudovirions and the lung chip incubated with 50 μl of medium containing pseudovirions (107 iu/ml). After overnight incubation, the cells were replaced with fresh medium. Transduction efficiency was measured by enhanced fluorescent protein expression of pseudoviruses. In the pseudo-viral particle diffusion test, after diffusion, the medium was continuously circulated in the lower layer of the upper culture layer for 30 minutes.

In some embodiments, the treatment of inflammatory responses may be performed using a lung chip. As one example, monocyte adhesion may employ the following steps: can be used for living dyeing of vascular endothelial layer at 37 ℃ by using Hoechst 3334230 minutes. THP-1 cells were labeled with CMTPX (1. Mu.M) for 1 hour before use and the lung chips were treated with LPS (10. Mu.g/ml) for 24 hours before use, and monocytes (5X 10) ⁵ Individual cells/mL) was perfused in the lung chip at a rate of 0.1mL/min for 30 minutes. Four or five random areas in the lung model can be time-lapse photographed using a microscope, photomicrographs obtained, and cytotechnologically quantified by counting the attached monocytes. For measurement of monocyte adhesion, alveolar/pulmonary bronchial epithelial cells can be seeded in the lower layer, while vascular endothelial cells are cultured in the upper layer to avoid the influence of gravity.

In some embodiments, multiple assays may be performed on alveolar/pulmonary bronchial epithelial cells, as well as vascular endothelial cells, to evaluate the multi-directional simulation of a lung model in a lung chip. Specifically, it is possible to perform cell viability, histological and immunofluorescent staining, scanning electron microscope images, barrier function analysis (transepithelial resistance measurement, permeability), inflammation classification detection, and the like.

Wherein the detection of cell viability can be performed by live/dead cell staining. Specifically, the lung chip may be gently washed 3 times with DPBS, then incubated with 60ul of dye at 37℃for 30 minutes, and observed under a microscope.

In some embodiments, the lung model may be isolated by cutting along the edge of the barrier film with a surgical knife, fixed in 10% neutral buffered formalin and paraffin embedded upon histological examination of the lung model. The 5 μm deparaffinized sections were transferred to a glass slide for staining. And obtaining a dyed bright field image for analyzing the morphological structure of the lung model.

Specifically, for immunofluorescent staining, the lung chips may be washed by first infusing PBS into the lung chips, then fixed with 4% neutral buffered formalin for 1 hour without running, and then washed by gently flowing PBS. The lung model cultured on the lung chip was permeabilized with 0.3% Triton X-100 in PBS for 20 min and immersed in a blocking buffer consisting of PBS containing 10% normal goat serum and 0.3% Triton X-100 for 1 hr at room temperature. They were then combined with anti-ZO-1 (1:100 dilution), anti-VE-cadherin (1:100 dilution), anti-ACE 2 (1:100 dilution) or Actin-Tracker Green (1:300 dilution). When staining cell adhesion molecules on endothelial cells, the cells are not permeabilized to reveal surface antigens. In all studies, antibodies diluted in blocking buffer were introduced into the channel and incubated for 1 hour at room temperature or overnight at 4 ℃. After washing with PBS for 5 min, the samples were washed with either secondary anti-goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:300 dilution) or Alexa Fluor 647 (1:300 dilution) for 1 hour at room temperature and with PBS. The lung chip was then disassembled and the first and second barrier films were removed and examined under a microscope. And determining whether the morphological structure of the lung model is complete or not by analyzing the fluorescence images of the upper layer and the lower layer of the isolating membrane.

In some embodiments, for micro-morphological observations, the isolation membrane of the lung chip was fixed in cold 4% paraformaldehyde for 2 hours for Scanning Electron Microscope (SEM) detection. Prior to observation, the isolation membrane was rinsed in PBS, then dehydrated through a 25% to 100% gradient of ethanol solution and incubated for up to 10 minutes in each interval. Air-drying the incubated sample, fixing with conductive adhesive and coating with a thin layer of gold under vacuum for scanning electron microscopy imaging using a scanning electron microscope.

Among other things, barrier function analysis may include transepithelial electrical resistance measurement and penetration ability detection.

In some embodiments, to determine transepithelial resistance, the resistance of the lung chip under different conditions may be measured using a trans-membrane resistance meter to study barrier function. Specifically, referring to fig. 7, the transmembrane resistance measurement method may include the steps of:

(1) Four electrodes G1, G2, G3 and G4 measure the transmembrane resistance of the first culture unit, and four electrodes G3, G4, G5 and G6 measure the transmembrane resistance of the second culture unit. G1, G2, G3, G4, G5, G6 are connected with electrode interfaces on the chip through circuits on the circuit board. The electrode interface is connected with the electrode adapter and the transmembrane resistance measuring instrument through connecting wires. G1, G4, G5 are located on the on-chip culture layer and are in contact with the solution, and G2, G3, G6 are located on the under-chip culture layer and are in contact with the solution. The connection ports of the electrode adapter include a current channel 1, a current channel 2, a voltage channel 1 and a voltage channel 2.

(2) And measuring the transmembrane resistance of a first culture unit (consisting of a first upper culture chamber, an isolation film layer and a first lower culture chamber), connecting G1 with a voltage channel 1 of an electrode adapter through a circuit on a circuit board and an electrode interface on a chip, connecting G3 with a voltage channel 2 of the electrode adapter through the circuit on the circuit board and the electrode interface on the chip, connecting G4 with a current channel 1 of the electrode adapter through the circuit on the circuit board and the electrode interface on the chip, and connecting G2 with a current channel 2 of the electrode adapter through the circuit on the circuit board and the electrode interface on the chip. The transmembrane resistance measuring instrument measures the voltage and current at both sides of the porous membrane through four electrodes and displays the transmembrane resistance value.

(3) And measuring the transmembrane resistance of a second culture unit (consisting of a second upper culture chamber, an isolation film layer and a second lower culture chamber), connecting G5 with a voltage channel 1 of an electrode adapter through a circuit on a circuit board and an electrode interface on a chip, connecting G3 with a voltage channel 2 of the electrode adapter through the circuit on the circuit board and the electrode interface on the chip, connecting G4 with a current channel 1 of the electrode adapter through the circuit on the circuit board and the electrode interface on the chip, and connecting G6 with a current channel 2 of the electrode adapter through the circuit on the circuit board and the electrode interface on the chip. The transmembrane resistance measuring instrument measures the voltage and the current at two sides of the porous membrane through four electrodes and displays the transmembrane resistance value of the second culture unit.

The transmembrane resistance is related to the barrier function of the tissue, and the more complete the barrier, the greater the transmembrane resistance. The transmembrane resistance of normal tissues is measured, and the transmembrane resistance of the drug is measured, and whether the barrier of the tissues is destroyed can be observed through numerical comparison.

In some embodiments, to check for permeability, the lung chip may be perfused with an acetyl azide solution (50 μm) and tested 4 hours of perfusion. That is, 30. Mu.L of dextran solution may be poured into the first upper culture chamber and the second upper culture chamber of the lung chip while 30. Mu.L of control medium is poured into the lower layer of the upper culture layer, and then the lung chip is incubated at 37℃for 4 hours. At the detection point, the lower layer solution was diluted and transferred into a 96-well plate. Each sample was repeated three times. The amount of dextran collected was then measured using a microplate reader at excitation and emission wavelengths of 355nm and 460nm (for acetyl azide). Standard curves were plotted for fluorescence concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/mL. Finally, the percentage permeability (Papp) value of the sample was calculated using the following equation.

Where J is the molecular flux, A is the total area of diffusion, and ΔC is the average gradient.

In some embodiments, a deep learning algorithm may be employed to classify inflammation. Specifically, bright field cell images of 200 different classes (LPS and Control) can be captured, respectively. The entire dataset was then split into training and test sets at a ratio of 7:3. The training set is used to fine tune the pre-trained deep learning network and the accuracy of the inflammation classification is assessed on the test set.

For example, the image may be pre-processed and patch extracted. Specifically, each bright field image can be converted into a gray scale image first, so as to avoid the influence of hue and saturation and minimize the influence of different imaging conditions. The image is then normalized. Next, patches are extracted from the gray scale image using 224 x 224 sliding windows with 25% overlap. After patch extraction, 32,000 image patches can be obtained. During the training phase, the probability of random horizontal, vertical rollover and random rotation can be increased.

By way of example, the VGG-19 Network can be used to obtain features of different scales by combining the convolution sets and then using the full connection layer as a classifier to distinguish between the different categories. In particular, a combination of convolution blocks and full connection blocks with different characteristic channels may be used to achieve an end-to-end accurate image classification method. For example, the convolution block consists of 16 convolution layers and 4 maximum pooling layers, with the convolution kernel set to 3*3. The full connection block may be composed of 3 full connection layers. The result of the network input 224 x 224 corresponds to the output of 1*2, and the output structure may be the probability of a certain class.

Alternatively, the VGG-19 model can be pre-trained on ImageNet, optimizing parameters in the convolution block for feature extraction. By pre-training the model on ImageNet, more useful advanced features can be attracted for image classification. The parameters in the convolution block are then frozen and the network is retrained on our data set to optimize the parameters in the fully connected layer.

And (3) experimental results show that:

fig. 15 is a schematic view of a lung model obtained by culture, and a schematic view of a result of detection performed on the lung model.

Wherein fig. 15-II shows a frozen section of a lung chip and its HE staining pattern (including lower and higher magnifications), wherein a cultured lung epithelial (epihetlium) structure is above the barrier film (basement membrane, green part) and a cultured vascular endothelial (endohetlium) structure is below the barrier film. FIG. 15-III shows microscopic images of vascular endothelial (endothesium) structure, barrier membrane (Base-2 b) and lung adenocarcinoma cells (NCI-H441) at day 5 of growth, shown by Scanning Electron Microscopy (SEM). Fig. 15-IV shows live/dead staining of lung epithelial (epi) and vascular endothelial (endothesium) structures within the lung chip cavity, showing high viability of cells in the lung chip (day 7).

Fig. 16 is a schematic of the introduction of immune cells in a lung chip to mimic early inflammation. Wherein, FIG. 16A-i is a schematic diagram of seeding macrophages on the epithelial layer, FIG. 16A-ii is a HE staining pattern of macrophages, and FIG. 16A-iii is a scanning electron microscope pattern of macrophages; FIG. 16B is a schematic representation of the monocyte attachment of vascular endothelium with or without LPS solution treatment (10. Mu.g/ml) during perfusion, wherein endothelial cells stained nuclei with NucBlue (blue), monocytes appear red, scale bar 200 μm; FIG. 16C is a schematic representation of a lung chip electron micrograph showing the attachment of monocytes to endothelium with or without LPS solution treatment; FIG. 16D is a quantitative comparison of monocyte adhesion to endothelium in a lung chip with or without LPS solution. Significance was calculated by one-way ANOVA (one-way ANOVA) by means of a graph-based post hoc tests. * P <0.05, p <0.001.N.s., no significance.

Figure 17 shows a comparative schematic of inflammatory response. Wherein in FIG. 17A, the expression of inflammatory factors (including TNF-. Alpha., IL6 and MCP 1) was increased by more than 8-fold for circulating monocytes over the system without circulating monocytes, indicating an enhanced inflammatory response of the perfusion system, as shown in FIG. 17A, respectively, for secretion of inflammatory cytokines in lung chips under different conditions, wherein (I) is TNF-a, (II) is MCP-1, and (III) is IL-6 (pg/ml). Meanwhile, the inflammatory response resulted in a significant decrease in transmembrane resistance with the circulation of monocytes, with FIG. 17B-I showing the transmembrane resistance measured in the bronchi-alveoli of the lung and FIG. 17B-II showing the permeability measured in the lung chip showing the transfer of small fluorescent molecules from the upper to the lower lumen over time; figures 17B-III show calculated lung bronchopermeabilities and alveolar spaces under different conditions, where "w/o M LPS" means no macrophages but treated with LPS solution. The data were compared to 6 samples from each of the three experiments and significance was calculated by one-way analysis of variance from Tukey post-hoc testing. * p <0.05, < p <0.001.N.s., no significance. This suggests that triple culture of macrophages, monocytes and lung bronchi/alveolar epithelial cells exacerbates inflammation. An increase in mucus secretion can also be observed in the alveolar spaces stimulated by LPS.

The lung chip system may contain multiple flow cells, different layers and multiple tissues, resulting in complex imaging and reduced image quality. To better quantify changes in lung epithelial cells and macrophages caused by inflammation, embodiments of the present application employ deep learning-based algorithms to classify the state of these macrophage-attached epithelial cell layers. VGG-19 network models can be used to predict whether cells are normal (unstimulated) epithelial cells (FIG. 18A, left panel). The VGG network model used by the system is shown (fig. 13A, right). In some examples, the network may output predictive labels (control and stimulus groups) with pre-processed images of 224x224 size as input. The first two convolution groups have 2 convolution layers, with feature maps of 64 and 128, respectively. The remaining three convolution groups contain 4 convolution layers, with feature maps of 256, 512, respectively. The first two fully-connected layers each had 4096 channels, and the third performed a 2-way experimental condition classification, thus containing 2 channels (one for each class). The output is in two states representing either a controlled (no stimulus) condition or an abnormal (stimulus) condition, as shown in fig. 18B. Both the fluorescence image and the bright field image were tested and better results were used. As the number of exercises increases, the accuracy of the model increases (fig. 18C). It can be seen that after multiple iterative training, the accuracy rate is rapidly improved to more than 0.9 (compared with the ground trunk), which indicates that the accuracy rate is more than 90%. Fig. 18D shows a schematic diagram of whether a separation membrane is contained between the upper and lower chambers of the lung chip. Figure 18E shows that deep learning forgets to predict the number of lung bronchi/alveoli tissues stimulated by different treatments of lung chips (including control, LPS and pseudovirion groups), and experiments were performed using four chips per group in six independent tests.

The invention provides a lung chip, a lung model and a construction method thereof, and a method for detecting a compound by using the lung chip, which have the following beneficial effects:

the first, the invention provides the gas-liquid culture condition needed by the maturation of the lung tissue cell on the lung chip structure, and can realize the serial connection or parallel connection of the tissues of different functional parts of the lung, so that the constructed model has higher simulation degree and the function is closer to the real lung organ of the human body.

Secondly, the lung chip model constructed by the invention can be added into immune cells for co-culture or immune cells are added into a flow path system to construct an immune circulation system, so that the interaction with the immune system in the process of drug action is increased.

Third, the lung chip constructed by the invention has similar functions as the real lung organs, can cause similar reactions to exogenous actions (especially viral infection), and can be used for constructing various disease models.

It is to be understood that the above embodiments are merely illustrative of the application of the principles of the present invention, but not in limitation thereof. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the invention, and are also considered to be within the scope of the invention.

Claims

1. A lung chip, comprising: a sealing layer, a lower culture layer, a separation membrane layer, an upper culture layer and a connecting layer which are sequentially laminated; wherein, the liquid crystal display device comprises a liquid crystal display device,

the connecting layer is provided with an inlet group and an outlet group;

2. The lung chip according to claim 1, wherein the first lower culture chamber and the second lower culture chamber are cultured with vascular structures, and the separation membrane layer comprises a first separation membrane and a second separation membrane; wherein, the liquid crystal display device comprises a liquid crystal display device,

3. The lung chip according to claim 1, wherein the three-dimensional lung bronchial structures and three-dimensional alveolar structures in the first upper culture chamber and/or the second upper culture chamber comprise epithelial cells or epithelial cells seeded with first immune cells;

4. The lung chip according to claim 3, further comprising a circuit board layer arranged on a side of said sealing layer facing away from said lower culture layer;

5. The lung chip according to claim 4, wherein at least one viewing window is further provided on the circuit board layer, the viewing window being arranged opposite to the first culture unit and the second culture unit.

6. The lung chip according to claim 5, wherein the connection layer is further provided with a first bubble removing portion and a second bubble removing portion, and,

7. A method for constructing a lung model, the method comprising:

surface modification of an upper culture unit in the lung chip, the lung chip being as claimed in any one of claims 1 to 6;

8. The construction method according to claim 7, characterized in that the construction method further comprises:

9. A lung model constructed by the construction method according to claim 7 or 8.

10. A method for detecting compounds using the lung chip according to any of claims 1 to 6, comprising the specific steps of: