Organ chip for multi-tissue co-culture and method for detecting modulation of multi-tissue function

Abstract

The application provides an organ chip for multi-tissue co-culture and a method for detecting the regulation of multi-tissue functions. The organ chip comprises a sealing layer, a culture layer and a connecting layer which are sequentially stacked, the connecting layer is provided with a connecting inlet group and a connecting outlet group, the culture layer is provided with a culture inlet group, a culture outlet group, an extravascular tissue flow channel group, a first group of culture chambers and a vascular culture chamber, the sealing layer is provided with an intravascular flow inlet, an intravascular flow outlet and an intravascular flow channel group, the connecting inlet group, the culture inlet group, the extravascular tissue flow channel group, the vascular culture chamber, the first group of culture chambers, the culture outlet group and the connecting outlet group form an extravascular tissue culture channel, and the connecting inlet group, the culture inlet group, the intravascular flow inlet, the intravascular flow channel group, an artificial blood vessel, the intravascular flow outlet, the culture outlet group and the connecting outlet group form an intravascular culture channel, which generalizes the multi-cell structure of a living organ and provides extracorporeal blood perfusion.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological tissue engineering and biological medicine technology, and particularly relates to an organ chip for multi-tissue co-culture and a method for detecting the function regulation of multi-tissue. BACKGROUND

[0002] Toxicity detection of different organs or entire systems of human body is an important part of pharmacokinetics and pharmacodynamics research. Traditional methods mainly use animal experiments and two-dimensional cell culture mode to detect it, although many achievements have been made, but due to the limitations of cycle, cost, precision, ethics and other factors, it is difficult to predict the response of human body to various drugs. For example, the current cell culture experiment using animal model has a long experimental period, high cost, and the difference between animal tissue organs and human tissue organs is large, so the animal model cannot effectively predict the drug response of human body, and cannot meet the needs of in vitro human toxicity and efficacy testing. Secondly, there is also two-dimensional cell culture, which is still a single cell study, lacks systematicness, and cannot comprehensively analyze the mechanism of action and the toxicity to other tissues.

[0003] And the human organ chip, by combining methods of cell biology, engineering and biomaterials and other disciplines, can reflect the main structure and functional characteristics of human tissue organs by simulating various living cells, tissue organ microenvironments in vitro, can accurately control multiple system parameters, and can reflect the real situation of human body compared with traditional toxicology animal experiments, and has more specificity in new drug screening. Therefore, using microfabrication technology to establish a more human environment biomimetic system has become a research hotspot of in vitro physiological model.

[0004] However, with the development of organ chip technology, its application still has certain limitations, for example, although three-dimensional cell culture can construct three-dimensional chip structure of organ tissue, providing a new model for drug research and development, it is still a single organ static study, and the single organ chip cannot comprehensively reflect the complexity, functional changes and integrity of organ function of the body, cannot generalize the multi-cell structure, tissue interface and related physical microenvironment of the key functional unit of the living organ, and cannot simulate the real environment of the human body through blood circulation and vascular filtration to realize nutrition and drug absorption, or the real environment of human tumor metastasis and invasion.

[0005] Therefore, in view of the above technical problems, the present application provides an organ chip for multi-tissue co-culture and a method for detecting the function regulation of multi-tissue. SUMMARY

[0006] The present application aims to at least solve one of the technical problems existing in the prior art, and provides an organ chip for multi-tissue co-culture and a method for detecting the function regulation of multi-tissue.

[0007] In one aspect of the present application, an organ-on-a-chip for multi-tissue co-culture is provided, comprising a sealing layer, a culture layer and a connection layer arranged in sequence, the connection layer being provided with a connection inlet group and a connection outlet group; the culture layer being provided with a culture inlet group, a culture outlet group, an extravascular tissue flow channel group, a first group of culture chambers for placing three-dimensional cells or tissues or an in-vitro simulated organ, and a vascular culture chamber for placing an artificial blood vessel; the sealing layer being provided with an intravascular flow inlet, an intravascular flow outlet and an intravascular flow channel group;

[0008] The connection inlet group, the culture inlet group, the extravascular tissue flow channel group, the extravascular passage formed by the vascular culture chamber after the artificial blood vessel is placed, the first group of culture chambers, the culture outlet group and the connection outlet group form an extravascular tissue culture passage;

[0009] The connection inlet group, the culture inlet group, the intravascular flow inlet, the intravascular flow channel group, the intravascular passage formed by the vascular culture chamber after the artificial blood vessel is placed, the intravascular flow outlet, the culture outlet group and the connection outlet group form an intravascular culture passage.

[0010] Preferably, the culture layer is further provided with an independent flow channel group and a second group of culture chambers for placing three-dimensional cells or tissues or an in-vitro simulated organ;

[0011] The connection inlet group, the culture inlet group, the independent flow channel group, the second group of culture chambers, the culture outlet group and the connection outlet group form an independent culture passage.

[0012] Preferably, the extravascular tissue flow channel group comprises an extravascular tissue inlet flow channel, an extravascular tissue outlet flow channel, a vascular culture chamber outlet flow channel, a micro-mixing flow channel and a first group of culture chamber inlet flow channels; wherein,

[0013] The culture inlet group, the extravascular tissue inlet flow channel, the vascular culture chamber, the vascular culture chamber outlet flow channel, the micro-mixing flow channel, the first group of culture chamber inlet flow channels, the first group of culture chambers, the extravascular tissue outlet flow channel and the culture outlet group are sequentially connected.

[0014] Preferably, the extravascular tissue inlet flow channel, the extravascular tissue outlet flow channel and the micro-mixing flow channel are arranged on the side of the culture layer facing the sealing layer, the vascular culture chamber outlet flow channel and the first group of culture chamber inlet flow channels are arranged on the side of the culture layer away from the sealing layer, and the vascular culture chamber and the first group of culture chambers are arranged on the culture layer; and,

[0015] The culture layer is further provided with a first fluid conversion hole and a second fluid conversion hole, the blood vessel culture chamber outlet flow channel and the micro-mixing flow channel are communicated through the first fluid conversion hole, and the micro-mixing flow channel and the first group of culture chamber inlet flow channels are communicated through the second fluid conversion hole.

[0016] Preferably, the intravascular flow channel group comprises an intravascular inlet flow channel and an intravascular outlet flow channel; wherein,

[0017] The intravascular flow inlet, the intravascular inlet flow channel, the artificial blood vessel, the intravascular outlet flow channel and the intravascular flow outlet are sequentially communicated.

[0018] Preferably, the independent flow channel group comprises an independent inlet flow channel, an independent outlet flow channel and a second group of culture chamber outlet flow channels.

[0019] The culture inlet group, the independent inlet flow channel, the second group of culture chambers, the second group of culture chamber outlet flow channels, the independent outlet flow channel and the culture outlet group are sequentially communicated.

[0020] Preferably, the independent inlet flow channel and the independent outlet flow channel are arranged on the side of the culture layer facing the sealing layer, the second group of culture chamber outlet flow channels are arranged on the side of the culture layer away from the sealing layer, and the second group of culture chambers are arranged on the culture layer; and,

[0021] The culture layer is further provided with a third fluid conversion hole, and the second group of culture chamber outlet flow channels and the independent outlet flow channel are communicated through the third fluid conversion hole.

[0022] Preferably, the first group of culture chambers and the second group of culture chambers are correspondingly arranged.

[0023] Preferably, the culture layer is further provided with a fixing groove at both ends of the blood vessel culture chamber, the fixing groove is provided with a fixing connection assembly inside, and the end of the artificial blood vessel is fixedly connected with the fixing connection assembly; and,

[0024] The fixing connection assembly is provided with a connection flow channel, and the intravascular inlet flow channel and the intravascular outlet flow channel are communicated with the artificial blood vessel through the connection flow channel.

[0025] In another aspect of the present application, a detection method for the regulation of multiple tissue functions is provided, which uses the organ chip as described above to detect the regulation of multiple tissue functions, and the detection method comprises:

[0026] Introducing each culture medium into each culture channel to culture three-dimensional cells or tissues or an in-vitro simulated organ and an artificial blood vessel;

[0027] Introducing a test substance into the intravascular culture channel.

[0028] carrying out at least one activity detection analysis on the three-dimensional cells or the tissue or the in-vitro simulated organ to obtain the regulation result of the test substance on multi-tissue function.

[0029] The organ chip of the present application comprises a single artificial blood vessel and a culture chamber composed of human organ-specific cells, the culture chamber recapitulates in vitro the multicellular structure, tissue interface and related physical microenvironment of the key functional unit of the living organ, and provides in-vitro blood vessel perfusion, so as to fully embody the role of endothelial cells in promoting drug absorption, distribution, metabolism, excretion (ADME) and toxicity in vitro. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 An exploded view of the organ chip of an embodiment of the present application;

[0031] Figure 2 A structural schematic view of the culture layer in the organ chip of an embodiment of the present application;

[0032] Figure 3 A structural schematic view of the sealing layer in the organ chip of an embodiment of the present application;

[0033] Figure 4 A structural schematic view of the connecting layer in the organ chip of an embodiment of the present application;

[0034] Figure 5 A structural schematic view of the artificial blood vessel fixation in the culture layer of an embodiment of the present application;

[0035] Figure 6 A structural schematic view of the fixation connecting assembly in the culture layer of an embodiment of the present application;

[0036] Figure 7 A structural schematic view of the culture system of another embodiment of the present application;

[0037] Figure 8 A vascular endothelial image of an organ tissue of another embodiment of the present application;

[0038] Figure 9 A myocardial cell microsphere image of an organ tissue of another embodiment of the present application;

[0039] Figure 10 A graph showing the beating frequency of myocardial cell microspheres of an organ tissue of another embodiment of the present application varying with the concentration of the culture medium;

[0040] Figure 11 An NCI-H23 tumor microsphere image of an organ tissue of another embodiment of the present application;

[0041] Figure 12A cell activity curve of an organ tissue NCI-H23 tumor microsphere of another embodiment of the present application changes with days of drug action;

[0042] Figure 13 An organ tissue NCI-H23 tumor microsphere image of another embodiment of the present application changes with days of drug action;

[0043] Figure 14 A cell activity curve of an organ tissue myocardial cell microsphere of another embodiment of the present application changes with days of drug action;

[0044] Figure 15 A cell activity curve of an organ tissue NCI-H23 tumor microsphere of another embodiment of the present application changes with days of drug action;

[0045] Figure 16 An organ tissue NCI-H23 tumor microsphere image of another embodiment of the present application changes with days of drug action. DETAILED DESCRIPTION

[0046] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in combination with the drawings and specific embodiments.

[0047] As Figures 1 to 6 shown, the present application proposes an organ chip for multi-tissue co-culture, which comprises a sealing layer C, a culture layer B and a connecting layer A arranged in sequence, the connecting layer A is provided with a connecting inlet group and a connecting outlet group; the culture layer B is provided with a culture inlet group, a culture outlet group, an extravascular tissue flow channel group, a first group of culture chambers for placing three-dimensional cells or tissues or an in-vitro simulated organ, and a vascular culture chamber for placing an artificial blood vessel; the sealing layer C is provided with an intravascular flow inlet, an intravascular flow outlet and an intravascular flow channel group. Among them, the connecting inlet group, the culture inlet group, the extravascular tissue flow channel group, the vascular culture chamber and the extravascular passage formed after the artificial blood vessel is placed, and the first group of culture chambers, the culture outlet group and the connecting outlet group form an extravascular tissue culture passage. The connecting inlet group, the culture inlet group, the intravascular flow inlet, the intravascular flow channel group, the intravascular passage formed after the artificial blood vessel is placed, the vascular culture chamber, the intravascular flow outlet, the culture outlet group and the connecting outlet group form an intravascular culture passage.

[0048] In combination with Figure 7As shown, the first medium 71 is introduced into the extravascular tissue culture channel, and the second medium 72 containing the to-be-tested substance is introduced into the intravascular culture channel. Due to the concentration difference, pressure difference, and the like, the to-be-tested substance is exchanged after being absorbed and filtered by the artificial blood vessel in the vascular culture chamber. The first medium 71 outside the artificial blood vessel transports the to-be-tested substance exuded from the second medium 72 in the artificial blood vessel to the first group of culture chambers to act on the three-dimensional cells or tissues or the in-vitro simulated organs in the first group of culture chambers, that is, the intravascular culture channel and the extravascular tissue culture channel are connected through the artificial blood vessel.

[0049] It should be noted that the to-be-tested substance is not specifically limited in the embodiment, for example, a drug acting on tissues.

[0050] The embodiment forms the extravascular channel and the intravascular channel by placing the artificial blood vessel in the vascular culture chamber. The to-be-tested substance is absorbed and filtered by the intravascular channel formed by the artificial blood vessel, and the substance exchange, for example, drug exchange, occurs between the vascular culture chamber and the extravascular channel. After the drug is fully mixed with the first medium, the drug acts on the three-dimensional cells or tissues or the in-vitro simulated organs in the first group of culture chambers, thereby realizing the process of simulating the drug transportation, absorption, and filtration in the human body and the drug acting on organs through blood vessels in vitro. Moreover, based on the communication between the artificial blood vessel and the connection inlet group, the to-be-tested substance can be directly added into the artificial blood vessel through the connection inlet group, thereby improving the convenience of the to-be-tested substance adding process.

[0051] Specifically, as shown in FIG. 1, the vascular culture chamber 1 is connected with the connection inlet group 2 through the artificial blood vessel 3. The connection inlet group 2 is connected with the first group of culture chambers 4, and the first group of culture chambers 4 is connected with the second group of culture chambers 5. Figures 1 to 7As shown, the connection inlet group of the embodiment includes the extravascular connection inlet A2, the intravascular connection inlet A3, and the independent connection inlet A5, the connection outlet group includes the extravascular connection outlet A1, the intravascular connection outlet A4, and the independent connection outlet A6; the culture inlet group includes the extravascular culture inlet B2, the intravascular culture inlet B3, and the independent culture inlet B5, and the culture outlet group includes the extravascular culture outlet B1, the intravascular culture outlet B4, and the independent culture outlet B6. Among them, the extravascular connection inlet A2 and the extravascular culture inlet B2 are in corresponding communication, and the extravascular connection outlet A1 and the extravascular culture outlet B1 are in corresponding communication, so as to introduce the first culture medium 71 from the extravascular connection inlet A2 into the extravascular culture inlet B2 and into the extravascular tissue culture channel, and then discharge the first culture medium 71 from the extravascular culture outlet B1 to the extravascular connection outlet A1 and return to the culture bottle corresponding to the first culture medium. Secondly, the intravascular connection inlet A3, the intravascular culture inlet B3, and the intravascular flow inlet C3 are in corresponding communication, and the intravascular flow outlet C4, the intravascular culture outlet B4, and the intravascular connection outlet A4 are in corresponding communication, so as to introduce the second culture medium 72 from the intravascular connection inlet A3 into the intravascular culture inlet B3 and then into the intravascular flow inlet C3, and then into the intravascular culture channel, and then discharge the second culture medium 72 from the intravascular flow outlet C4 to the intravascular culture outlet B4 and then to the intravascular connection outlet A4, and return to the culture bottle corresponding to the second culture medium, so as to form a system connection area.

[0052] It should be understood that the extravascular connection inlet, the intravascular connection inlet, the independent connection inlet, the extravascular connection outlet, the intravascular connection outlet, the independent connection outlet, the extravascular culture inlet, the intravascular culture inlet, the independent culture inlet, the extravascular culture outlet, the intravascular culture outlet, and the independent culture outlet of the embodiment can be through holes to form the corresponding communication relationship.

[0053] In some preferred embodiments, the extravascular culture inlet, the intravascular culture inlet, the independent culture inlet, the extravascular culture outlet, the intravascular culture outlet, and the independent culture outlet are set as through holes with a diameter of 1 mm.

[0054] In some preferred embodiments, the intravascular flow inlet and the intravascular flow outlet are circular grooves with a diameter of 1 mm and a depth of 0.5 mm.

[0055] Further, as Figure 1 and Figure 2As shown, the extravascular tissue flow channel group includes an extravascular tissue inlet flow channel B2-1, an extravascular tissue outlet flow channel B1-1, a vascular culture chamber outlet flow channel B2-2, a micro-mixed flow channel B10, and a first group of culture chamber inlet flow channels (this first group of culture chamber inlet flow channel is equivalent to the sub-flow channel B12-2 of the first group of culture chambers); wherein, the extravascular culture inlet B2, the extravascular tissue inlet flow channel B2-1, the vascular culture chamber B13 after the placement of the artificial blood vessel E, the vascular culture chamber outlet flow channel B2-2, the micro-mixed flow channel B10, the first group of culture chamber inlet flow channels, the first group of culture chambers B12, the extravascular tissue outlet flow channel B1-1, and the extravascular culture outlet B1 are sequentially connected.

[0056] It should be noted that this embodiment does not impose specific limitations on how each flow channel is set up, as long as the flow channels can be connected. For example, grooves can be set on the culture layer.

[0057] In some preferred embodiments, such as Figure 1 and Figure 2 As shown, the extravascular tissue inlet channel B2-1, the extravascular tissue outlet channel B1-1, and the micro-mixing channel B10 are all located on the side of culture layer B facing the sealing layer C (the lower surface of the culture layer). The vascular culture chamber outlet channel B2-2 and the first group of culture chamber inlet channels are located on the side of culture layer B away from the sealing layer C (the upper surface of the culture layer). The vascular culture chamber B13 and the first group of culture chambers B12 are perforated on culture layer B. In addition, the culture layer B is also provided with a first fluid conversion hole B9 and a second fluid conversion hole B11. The vascular culture chamber outlet channel B2-2 and the micro-mixing channel B10 switch the fluid direction through the first fluid conversion hole B9 to achieve communication between the channels. The micro-mixing channel B10 and the second connecting channel switch the fluid direction through the second fluid conversion hole B11 to achieve communication between the channels.

[0058] In some preferred embodiments, such as Figure 2 As shown, the first group of culture chambers B12 includes five sub-culture chambers, namely the first sub-culture chamber, the second sub-culture chamber, the third sub-culture chamber, the fourth sub-culture chamber, and the fifth sub-culture chamber. One end of the first sub-culture chamber is connected to the micro-mixed flow channel through sub-flow channel B12-2, and the other end is connected to the second sub-culture chamber through sub-flow channel B12-1. One end of the third sub-culture chamber is connected to the second sub-culture chamber through sub-flow channel B12-2, and the other end is connected to the fourth sub-culture chamber through sub-flow channel B12-1. One end of the fifth sub-culture chamber is connected to the fourth sub-culture chamber through sub-flow channel B12-2, and the other end is connected to the extravascular tissue outlet flow channel B1-1, serving as a drug testing area.

[0059] In some preferred embodiments, sub-channel B12-2 is disposed on the side of the culture layer away from the sealing layer, and sub-channel B12-1 is disposed on the side of the culture layer facing the sealing layer.

[0060] It should be noted that the size of each sub-flow channel, each flow channel in the extravascular tissue flow channel group, the first group of culture chambers, the blood vessel culture chamber and the fluid conversion hole is not specifically limited in this embodiment.

[0061] In some preferred embodiments, each sub-flow channel and each flow channel in the extravascular tissue flow channel group is provided as a groove with a width of 0.5 mm and a depth of 0.5 mm, and the specific length and shape can be set according to actual needs, and is connected with the upper surface of the sealing layer or the lower surface of the connecting layer to form a closed fluid passage.

[0062] In some preferred embodiments, each sub-culture chamber in the first group of culture chambers is a through hole with a diameter of 4 mm.

[0063] In some preferred embodiments, the blood vessel culture chamber is a rectangular groove penetrating the thickness of the culture layer, used to install an artificial blood vessel, and the culture layer is connected with the lower surface of the connecting layer and the upper surface of the sealing layer to form a closed blood vessel culture chamber as a blood vessel culture area, wherein the artificial blood vessel in the blood vessel culture chamber forms an intravascular passage, and the remaining space of the blood vessel culture chamber forms an extravascular passage, which can exchange substances with the intravascular passage formed by the artificial blood vessel.

[0064] In some preferred embodiments, the first fluid conversion hole and the second fluid conversion hole are through holes with a diameter of 2 mm, used to convert the direction of the fluid, introduce the fluid in the lower flow channel of the culture layer into the upper flow channel, or introduce the fluid in the upper flow channel of the culture layer into the lower flow channel, to realize the communication of the flow channels.

[0065] Further, as shown in Figures 1 to 3 , the intravascular flow channel group includes an intravascular inlet flow channel C3-1 and an intravascular outlet flow channel C4-1; wherein the intravascular inlet C3, the intravascular inlet flow channel C3-1, the intravascular outlet flow channel C4-1 and the intravascular outlet C4 are sequentially communicated.

[0066] In some preferred embodiments, as shown in Figure 1 and Figure 3 , the intravascular inlet flow channel C3-1 and the intravascular outlet flow channel C4-1 are both arranged on the side of the sealing layer C facing the culture layer B, i.e. the upper surface of the sealing layer, and in addition, since the artificial blood vessel is arranged in the blood vessel culture chamber on the culture layer, a first connecting hole C3-2 and a second connecting hole C4-2 are also arranged on the upper surface of the sealing layer C, the first connecting hole C3-2 is located at one end of the intravascular inlet flow channel C3-1 close to the artificial blood vessel E, and the second connecting hole C4-2 is located at one end of the intravascular outlet flow channel C4-1 close to the artificial blood vessel E, and the artificial blood vessel is communicated with the intravascular inlet flow channel and the intravascular outlet flow channel through the above two connecting holes.

[0067] It should be noted that the size of each flow channel and the connecting hole is not specifically limited in the embodiment.

[0068] In some preferred embodiments, the intravascular inlet flow channel and the intravascular outlet flow channel are both grooves with a width of 0.5 mm and a depth of 0.5 mm, and the specific length and shape thereof can be set according to actual needs, and the upper surface of the sealing layer or the lower surface of the connecting layer is connected to form a closed fluid channel.

[0069] In some preferred embodiments, the first connecting hole and the second connecting hole are both circular grooves with a diameter of 1 mm and a depth of 0.5 mm.

[0070] Further, in order to fix the artificial blood vessel in the blood vessel culture chamber, the culture layer is further provided with a fixing groove at both ends of the blood vessel culture chamber, the fixing groove contains a fixing connecting assembly, the end of the artificial blood vessel is fixedly connected with the fixing connecting assembly, and the fixing connecting assembly is provided with a connecting flow channel, and the intravascular inlet flow channel and the intravascular outlet flow channel are both communicated with the artificial blood vessel through the connecting flow channel.

[0071] It should be noted that the fixing of the fixing connecting assembly and the artificial blood vessel and the communication of the artificial blood vessel and other flow channels are not specifically limited in the embodiment.

[0072] Exemplarily, as shown in Figs. 1 and 2, the fixing connecting assembly includes two fixing blocks and two connecting pipes, which are a first fixing block D1, a second fixing block D3, a first connecting pipe D2 and a second connecting pipe D4. Figure 5 and Figure 6 The first fixing block D1 and the second fixing block D3 are accommodated in the corresponding fixing grooves, and the first fixing block D1 and the second fixing block D3 are respectively provided with the first connecting pipe D2 and the second connecting pipe D4 on the side facing the artificial blood vessel. The first ends of the two connecting pipes are respectively inserted into the two ends of the artificial blood vessel E, the second end of the first connecting pipe D2 is fixed in the first fixing block D1, the first fixing block D1 is further provided with a first connecting flow channel, the first connecting flow channel is communicated with the first connecting pipe, so as to realize the communication of the intravascular inlet flow channel and the artificial blood vessel through the first connecting flow channel and the first connecting pipe. In addition, the second end of the second connecting pipe D4 is fixed in the second fixing block D3, the second fixing block D3 is further provided with a second connecting flow channel, the second connecting flow channel is communicated with the second connecting pipe, so as to realize the communication of the intravascular outlet flow channel and the artificial blood vessel through the second connecting flow channel and the second connecting pipe.

[0073] In some preferred embodiments, the first connecting pipe and the second connecting pipe can adopt a hard pipe with an outer diameter of 2 mm, an inner diameter of 1 mm and a length of 10 mm, which can be processed or injection molded from a high molecular material, or can be processed from a material without biological toxicity such as glass and stainless steel.

[0074] Further, in order to truly reflect the effect of the test substance on the tissue organs in the first group of culture chambers, a control experiment group should be set under the same external conditions, that is, the application also provides an independent culture area, which is specifically as follows: as shown in Figures 1 to 4 , the third culture layer B is also provided with an independent flow channel group and a second group of culture chambers for placing three-dimensional cells or tissues or in-vitro simulated organs, and the connection inlet group, the culture inlet group, the independent flow channel group, the second group of culture chambers, the culture outlet group and the connection outlet group form an independent culture channel of a control area.

[0075] It should be noted that the embodiment does not make specific limitations on the three-dimensional cells or tissues or in-vitro simulated organs placed in the first group of culture chambers and the second group of culture chambers, for example, they can be myocardial cell microspheres, tumor microspheres or a combination of the two.

[0076] Exemplarily, as shown in Figures 1 to 4 , the independent flow channel group includes an independent inlet flow channel B5-1, an independent outlet flow channel B6-1 and a second group of culture chamber outlet flow channels, so that the third culture medium is introduced into the independent culture inlet B5 through the independent connection inlet A5, and further flows into the independent inlet flow channel B5-1, the second group of culture chambers B7, the second group of culture chamber outlet flow channels (which are equivalent to the sub-flow channels B7-2 of the second group of culture chambers), the independent outlet flow channel B6-1, and then is introduced out of the independent culture outlet B6 through the independent connection outlet A6.

[0077] In combination with Figure 7 , the third culture medium 73 is introduced into the independent culture channel, and the third culture medium 73 flows into the second group of culture chambers through the independent inlet flow channel, so as to act on the three-dimensional cells or tissues or in-vitro simulated organs in the second group of culture chambers.

[0078] In some preferred embodiments, as shown in Figure 1 and Figure 2 , the independent inlet flow channel B5-1 and the independent outlet flow channel B6-1 are arranged on the side of the culture layer B facing the sealing layer C (the lower surface of the culture layer), the second group of culture chamber outlet flow channels are arranged on the side of the culture layer B away from the sealing layer C (the upper surface of the culture layer), the second group of culture chambers B7 are arranged through the culture layer B, the culture layer B is also provided with a third fluid conversion hole B8, and the second group of culture chamber outlet flow channels and the independent outlet flow channel B6-1 convert the fluid direction through the third fluid conversion hole B8, so as to be connected.

[0079] It should be noted that, in order to truly reflect the effect of the test substance on the three-dimensional cells or tissues or in-vitro simulated organs, the first group of culture chambers and the second group of culture chambers are correspondingly arranged on the culture layer, including the correspondence of position and quantity, the two groups of culture chambers are symmetrically arranged on both sides of the culture layer along the width direction thereof, and the two groups of culture chambers each include five sub-culture chambers.

[0080] Specifically, as shown in Figure 2 The second group of culture chambers includes five sub-chambers, i.e., a sixth sub-chamber, a seventh sub-chamber, an eighth sub-chamber, a ninth sub-chamber, and a tenth sub-chamber. One end of the sixth sub-chamber is in communication with the independent inlet flow channel, and the other end is in communication with the seventh sub-chamber through the sub-flow channel B7-2. One end of the eighth sub-chamber is in communication with the seventh sub-chamber through the sub-flow channel B7-1, and the other end is in communication with the ninth sub-chamber through the sub-flow channel B7-2. One end of the tenth sub-chamber is in communication with the ninth sub-chamber through the sub-flow channel B7-1, and the other end is in communication with the independent outlet flow channel through the sub-flow channel B7-2, serving as an independent culture area.

[0081] In some preferred embodiments, as shown in Figure 2 The sub-flow channel B7-2 is arranged on the side of the culture layer away from the sealing layer, and the sub-flow channel B7-1 is arranged on the side of the culture layer facing the sealing layer.

[0082] It should be noted that the size of the sub-flow channel, each flow channel in the independent flow channel group, the second group of culture chambers, and the third fluid switching hole is not specifically limited in this embodiment.

[0083] In some preferred embodiments, each sub-flow channel and each flow channel in the independent flow channel group is a groove with a width of 0.5 mm and a depth of 0.5 mm, and the specific length and shape can be set according to actual needs, and the upper surface of the sealing layer or the lower surface of the connecting layer is connected to form a closed fluid passage.

[0084] In some preferred embodiments, each sub-chamber in the second group of culture chambers is arranged as a through hole with a diameter of 4 mm.

[0085] It should be noted that the materials of the sealing layer, the culture layer, the connecting layer, and the fixing block in this embodiment can be processed or injection molded from PMMA, PC, COC, PE, PP, and other high-molecular materials without biological toxicity, or can be processed from glass, stainless steel, and other materials without biological toxicity. Of course, other materials can also be used by those skilled in the art.

[0086] It should be further noted that the specific connection of the sealing layer, the culture layer, the connecting layer, and the fixing block is not limited.

[0087] For example, the fixed block and the connecting tube are pre-assembled and sealed by a non-biological toxic adhesive. Next, the upper surface of the sealing layer C and the lower surface of the culture layer B and the lower surface of the fixed block are connected by double-sided adhesive, biological adhesive, ultrasonic bonding, laser bonding or other methods to form a closed flow channel and a semi-closed culture chamber. Further, the lower surface of the connecting layer A and the upper surface of the culture layer B are connected by double-sided adhesive or other fast-acting adhesives after the artificial blood vessels and the cell microspheres of the 3D tissue organ are loaded into the organ chip to form a closed flow channel and a culture chamber. Of course, in addition to the above, those skilled in the art can also adopt other connection methods to connect the sealing layer C, the culture layer B and the connecting layer A according to actual needs, and the present embodiment does not specifically limit this.

[0088] The chip culture layer of the present embodiment mainly has five regions. The first region is a system connection region formed by the culture inlet group and the culture outlet group to realize the connection of the culture layer with the connecting layer and the sealing layer. The second region is an independent culture region formed by the second group of culture chambers and the independent flow channel group. The third region is a blood vessel culture region formed by the blood vessel culture chamber and the artificial blood vessels placed therein to realize drug exchange based on concentration difference. The fourth region is a micro-mixing region formed by the micro-mixing flow channel to mix the test substance with the culture medium. The fifth region is a drug test region formed by the first group of culture chambers. The five regions and the stacked sealing layer, connecting layer form three independently controlled circulating culture channels, i.e., a culture channel for control test, a blood vessel internal culture channel containing the test substance and a tissue culture channel outside the blood vessel.

[0089] The organ chip of the present application realizes the simultaneous performance of multiple circulating culture channels and the connection with multiple culture media of the culture system. These culture chambers recapitulate the multicellular structure, tissue interface and related physical microenvironment of the key functional units of the living organ in vitro, and simultaneously provide in vitro blood vessel perfusion. The endothelial cells can fully reflect the role of promoting drug absorption, distribution, metabolism, excretion (ADME) and toxicity in vitro, and the effect of the test substance (e.g., drug) on the tissue organ in the culture chamber can be reflected.

[0090] In another aspect of the present application, a method for detecting the regulation of multiple tissue functions is provided. The organ chip described above is used to detect the regulation of multiple tissue functions. The detection method comprises the following steps in the sterile environment of the organ chip:

[0091] First, introduce each culture medium into each culture channel to culture three-dimensional cells or tissues or in vitro simulated organs, and artificial blood vessels.

[0092] Specifically, as shown in Figure 7 the first culture medium 71 is introduced into the extracorporeal tissue culture channel to culture the three-dimensional cells or tissues or in vitro simulated organs in the first group of culture chambers.

[0093] In some preferred embodiments, as shown in Figure 7 In order to truly reflect the effect of the test substance on the three-dimensional cells or tissues or in-vitro simulated organs in the first group of culture chambers, a control experiment group should be set up under the same external conditions, i.e. a third culture medium 73 is introduced into the independent culture channel to culture the three-dimensional cells or tissues or in-vitro simulated organs in the second group of culture chambers.

[0094] Second, introduce the test substance into the intravascular culture channel.

[0095] Specifically, as shown in Figure 7 The second culture medium 72 containing the test substance is introduced into the intravascular tissue culture channel, and the test substance in this embodiment is not specifically limited and can be a drug acting on tissues.

[0096] Of course, in order to determine that the three-dimensional cells or tissues or in-vitro simulated organs have good functional characteristics, in some preferred embodiments, the second culture medium 72 can be a culture medium without the test substance, and the second culture medium 72 is replaced with a culture medium containing the test substance after a period of culture.

[0097] It should be noted that the test substance in this embodiment is exchanged in the vascular culture chamber, and the second culture medium 72 in the artificial blood vessel leaks out the test substance in the vascular culture chamber, and the first culture medium 71 outside the artificial blood vessel is mixed with the test substance and transported to the first group of culture chambers to act on the three-dimensional cells or tissues or in-vitro simulated organs in the first group of culture chambers.

[0098] Third, at least one activity detection analysis is performed on the three-dimensional cells or tissues or in-vitro simulated organs to obtain the regulation result of the test substance on the multi-tissue function.

[0099] In some preferred embodiments, the first activity analysis is performed on the artificial blood vessels and tissue organs cultured by the second culture medium 72 without the test substance to ensure that the blood vessels and myocardial microspheres have good functional characteristics. Then, the second activity analysis is performed on the tissue organs cultured by the second culture medium 72 with the test substance to obtain the influence result of the test substance on the tissue organs.

[0100] It should be understood that before introducing each culture medium into each culture channel, the organ chip needs to be assembled with the culture system, sterilized, and the like, for example, the assembled organ chip components, culture layer, connection layer and culture system are sterilized with ethylene oxide for standby.

[0101] Further, it is also needed to culture the artificial blood vessels and the tissue organs in advance, culture the artificial blood vessels to show functional features, and make cell microspheres of the tissue organs and culture the cell microspheres to show functional features. Then, the artificial blood vessels are put into the blood vessel culture chamber and inserted into the hard connecting tube on the connecting block to realize sealed connection, and then one cell microsphere is put into each sub-culture chamber of the second group of culture chambers B7 and the first group of culture chambers B12 respectively.

[0102] Further, after the assembly of the artificial blood vessels and the cell microspheres of the tissue organs is completed, the connecting layer and the culture layer are quickly and sealedly connected by using the non-biotoxic double-sided adhesive, and then the organ chip is connected with the culture system.

[0103] The organ chip of the present application realizes the co-culture of the tissue cell microspheres and other multi-cell tissues through the artificial blood vessels and the culture chamber, can simulate the microenvironment in vivo in vitro, and can realize the drug testing on the artificial organ tissue or tumor tissue through the filtration, absorption, shielding and the like of the artificial blood vessels.

[0104] The present application proposes a kind of organ chip for multi-tissue co-culture and the detection method for the function regulation of multiple tissues, with the following beneficial effects:

[0105] Firstly, the organ chip of the present application can be applied to the three-dimensional culture of multi-tissue organs and organoids.

[0106] Secondly, the culture chamber of the organ chip of the present application re-summarizes the multi-cell structure, tissue interface and related physical microenvironment of the key functional unit of the living organ in vitro, and at the same time provides in vitro blood vessel perfusion, which can simulate the real environment of the human body through blood circulation and vascular filtration and other functional circulations to realize nutrition and drug absorption, or tumor metastasis and invasion of the human body.

[0107] Thirdly, the organ chip of the present application can fully reflect the role of endothelial cells in promoting drug absorption, distribution, metabolism, excretion (ADME) and toxicity in vitro.

[0108] The principle and application of the organ chip for multi-tissue co-culture will be described in the following specific examples:

[0109] Example 1

[0110] This example describes the system principle of the perfusion culture of the organ chip, as follows:

[0111] As shown in Figures 1 to 7 , the organ chip and the culture system form three independently controlled circulating culture channels, as follows:

[0112] The first culture channel: the first culture medium 71 is driven by the pump Ml, enters the extravascular connection inlet A2 through the pipeline, goes down through the extravascular culture inlet B2, enters the extravascular tissue inlet flow channel B2-1, enters the extravascular channel after the artificial blood vessel in the blood vessel culture chamber B13, enters the first fluid conversion hole B9 through the blood vessel culture chamber outlet flow channel B2-2, goes down into the micro-mixing flow channel B10 through B9, after flowing through the micro-mixing flow channel B10 channel, enters the first group of culture chamber inlet flow channels upward through the second fluid conversion hole B11, flows through the five sub-culture chambers of the first group of culture chambers B12 in turn, enters the extravascular tissue outlet flow channel B1-1, goes up through the extravascular culture outlet B1, and returns to the culture bottle corresponding to the first culture medium through the extravascular connection outlet A1 and the pipeline, to form a circulating perfusion culture channel of the extravascular and drug test area culture chamber.

[0113] The second culture channel: the second culture medium 72 enters the intravascular connection inlet A3 through the pipeline after being driven by the pump M2, goes down through the intravascular culture inlet B3, the intravascular flow inlet C3, and the intravascular inlet flow channel C3-1, enters the artificial blood vessel E upward through the first connection hole C3-2 and the connecting pipe D2, enters the intravascular outlet flow channel C4-1 downward through the connecting pipe D4 and the second connection hole C4-2, and reaches the intravascular flow outlet C4, goes up through the intravascular culture outlet B4 and the intravascular connection outlet A4, and returns to the culture bottle corresponding to the second culture medium through the pipeline, to form a circulating channel of the second culture medium 72 in the intravascular.

[0114] The third culture channel: the third culture medium 73 is driven by the pump M3, enters the independent connection inlet A5 through the pipeline, goes down through the independent culture inlet B5, enters the independent inlet flow channel B5-1, flows through the five sub-culture chambers of the second group of culture chambers B7 in turn, enters the third fluid conversion hole B8 hole downward, enters the independent outlet flow channel B6-1 channel, goes up through the independent culture outlet B6, and returns to the culture bottle corresponding to the third culture medium through the independent connection outlet A6 and the pipeline, to form a circulating culture channel of the independent culture control area.

[0115] Based on the aforementioned culture channels, the second culture medium 72 in the intravascular culture channel containing the drug and the first culture medium 71 in the extravascular tissue culture channel undergo drug exchange in the vascular culture chamber B13 due to concentration and pressure differences, after absorption and filtration by the artificial blood vessel. The first culture medium 71 in the extravascular tissue culture channel carries the drug that has seeped out of the second culture medium 72 in the intravascular culture channel into the micro-mixed flow channel B10. After thorough mixing in the micro-mixed flow channel B10, the drug acts on the three-dimensional cells, tissues, or in vitro simulated organs in the first group culture chamber B12. This process simulates the process by which drugs are transported, absorbed, and filtered through blood vessels in the human body and then act on organs. In addition, the five sub-culture chambers of the second group culture chamber B7 in the independent culture pathway and the five sub-culture chambers of the first group culture chamber B12 contain the same three-dimensional cells, tissues, or in vitro simulated organs, serving as a control for the drug's effect on organs under the same external conditions, reflecting the effect of the drug on the three-dimensional cells, tissues, or in vitro simulated organs in the first group culture chamber B12.

[0116] Example 2

[0117] This example provides a method for detecting the regulatory effects of multiple tissue functions, using the organ-on-a-chip described above. During culture, the chip needs to be connected to the culture system, such as... Figure 7 As shown, the culture system includes a first culture medium 71, a second culture medium 72, and a third culture medium 73, pumps M1, M2, and M3, in... Figure 7 In this process, the first culture medium 71 is connected to the extravascular tissue culture channel via pump M1, the second culture medium 72 is connected to the intravascular culture channel via pump M2, and the third culture medium 73 is connected to the independent culture channel via pump M3. The specific culture method is as follows:

[0118] The assembled chip was connected to the culture system, and the chip and culture system were sterilized with ethylene oxide.

[0119] Artificial blood vessels were cultured using Huvec endothelial cells until they exhibited functional characteristics.

[0120] Induced human pluripotent stem cells were used to differentiate into human cardiomyocytes. Cardiomyocyte microspheres were then created using these microspheres and cultured until they exhibited functional characteristics, with the myocardium exhibiting a regular beat of 18 times per minute.

[0121] Under aseptic conditions, the sterilized chip, cultured artificial blood vessel, and cardiomyocyte microspheres were taken. The cultured artificial blood vessel was placed into the blood vessel culture chamber and inserted into the fixing block and connecting tube to achieve a sealed connection. The cultured cardiomyocyte microspheres were then placed into the first and second culture chambers in sequence.

[0122] The connection layer and the culture layer are sealed and connected by using a double-sided adhesive tape without biological toxicity;

[0123] In a sterile environment, the chip and the culture system are connected, and 15 mL of corresponding culture medium is respectively put into each culture bottle, the culture system is started, and when each culture medium fills the corresponding culture chamber and forms a flow perfusion loop, the chip is put into a 37°C sterile incubator together with the culture system;

[0124] After 24 h of continuous perfusion culture, the chip is taken out together with the culture system, and a high-content image analysis system is used to analyze the activity of the artificial blood vessels and the myocardial cell microspheres in the chip, so as to ensure that the artificial blood vessels and the myocardial cell microspheres have good functional characteristics, the vascular endothelium is arranged closely, has a certain directionality after continuous perfusion culture, and the peristaltic frequency of the myocardial microspheres is 18 times per minute. The blood vessel images and the myocardial microsphere images collected by the high-content system are shown in Figure 8 、 Figure 9 ;

[0125] In a sterile environment, the second culture medium corresponding to the artificial blood vessel is replaced with a culture medium containing adrenaline, and the adrenaline concentration is 10 -10 mol / L, 10 -9 mol / L, and 2x10 -9 mol / L, respectively. The chip is put into a 37°C sterile incubator together with the culture system for perfusion culture for 1 h, and an organ tissue is obtained.

[0126] The obtained organ tissue is further analyzed by using a high-content system. As shown in Figure 10 , the control group is the original second culture medium, that is, the control group without replacement of adrenaline. It is found through comparison that the beating frequency of the myocardial cell microspheres is obviously increased with the increase of the concentration of adrenaline, and the increase of the beating frequency of the myocardial cell microspheres is consistent with the influence law of adrenaline on heart tissue, which indicates that adrenaline can be absorbed, filtered, shielded and affected on the myocardial microsphere tissue through the artificial blood vessel.

[0127] Example 3

[0128] The present example provides a detection method for the regulation effect of multiple tissues, which uses the organ chip as recorded in the foregoing. When culturing, the chip needs to be connected to a culture system, as shown in Figure 7 , the culture system includes a first culture medium 71, a second culture medium 72, and a third culture medium 73, a pump M1, a pump M2, and a pump M3. In Figure 7 , the first culture medium 71 is connected to the extravascular tissue culture channel via the pump M1, the second culture medium 72 is connected to the intravascular culture channel via the pump M2, and the third culture medium 73 is connected to the independent culture channel via the pump M3. The specific culture method is as follows:

[0129] The assembled chip is connected to the culture system, and the chip and the culture system are sterilized by using ethylene oxide;

[0130] The artificial blood vessel is cultured by using Huvec endothelial cells, and the culture is continued until the artificial blood vessel exhibits functional characteristics;

[0131] The 3D tumor microspheres are prepared by using human lung cancer cells NCI-H23, and the culture is continued until the NCI-H23 tumor microspheres exhibit functional characteristics;

[0132] In a sterile culture environment, the sterilized chip, the cultured artificial blood vessel and the NCI-H23 tumor microspheres are taken, the cultured artificial blood vessel is placed into the blood vessel culture chamber, the artificial blood vessel is inserted into the fixing block and the connecting pipe to realize sealed connection, and the cultured tumor microspheres are sequentially placed into the first group of culture chambers and the second group of culture chambers;

[0133] The connecting layer and the culture layer are sealed and connected by using a double-sided adhesive tape without biological toxicity;

[0134] In a sterile environment, the chip and the culture system are connected, 15 mL of corresponding culture medium is respectively placed in each culture bottle, the culture system is started, and after the culture medium fills the corresponding culture chamber and forms a flow perfusion loop, the chip is taken together with the culture system into a 37°C sterile incubator;

[0135] After 24 hours of continuous perfusion culture, the chip is taken together with the culture system, the activity of the artificial blood vessel and the tumor microspheres in the chip is analyzed by using a high-content image analysis system, the artificial blood vessel and the NCI-H23 tumor microspheres have good functional characteristics, the blood vessel endothelium is arranged closely, has a certain directionality after continuous perfusion culture, the tumor microspheres have a deep color in the middle, a light color at the edge and a clear outline, and the tumor microsphere image collected by the high-content system is shown in Figure 11 ;

[0136] In a sterile environment, the second culture medium corresponding to the artificial blood vessel is replaced with a culture medium containing doxorubicin (Dox), the chip is taken together with the culture system into a 37°C sterile incubator, and continuous culture perfusion culture is continued for 10 days to obtain an organ tissue.

[0137] Further, the obtained organ tissue is further analyzed by using a high-content system, as shown in Figure 12 , and the activity of the NCI-H23 tumor microspheres in the first group of culture chambers B12 of the drug test area (corresponding to Dox-1 in Figure 12 ) and the NCI-H23 tumor microspheres in the five sub-culture chambers of the second group of culture chambers B7 of the independent control area (corresponding to Dox-2 in Figure 12 ) is analyzed on the 1st, 3rd, 5th, 7th and 10th day of the above continuous culture perfusion culture, and the analysis data and the collected images are shown inFigure 12 、 Figure 13 Through the above experimental data analysis, it is found that after 10 days of drug action, the activity of the five NCI-H23 tumor microspheres in the first group of culture chamber B12 drug test area is reduced to 20% of the activity of the NCI-H23 tumor microspheres in the second group of culture chamber B7 control group, which conforms to the effect of doxorubicin on human lung cancer cells NCI-H23, and indicates that doxorubicin can be absorbed, filtered, shielded and affected by artificial blood vessels to produce toxic effects on NCI-H23 tumor microsphere tissues.

[0138] It should be noted that the same method as described above can also be used to test docetaxel with a concentration of 40 umol / L, and the same experimental data are obtained as Figure 13 , which indicates that docetaxel (corresponding to DMSO in Figure 13 ) in the chip of the present application can also produce toxic effects on NCI-H23 lung cancer cells.

[0139] Example 4

[0140] The present example provides a method for detecting the regulation of multiple tissue functions. The organ chip described above is used for culture, and the chip is connected to a culture system, as shown in Figure 7 , which includes a first culture medium 71, a second culture medium 72, and a third culture medium 73, a pump M1, a pump M2, and a pump M3. In Figure 7 , the first culture medium 71 is connected to the extravascular tissue culture channel via the pump M1, the second culture medium 72 is connected to the intravascular culture channel via the pump M2, and the third culture medium 73 is connected to the independent culture channel via the pump M3. The specific culture method is as follows:

[0141] The above assembled chip is connected to the culture system, and the chip and the culture system are sterilized by using ethylene oxide;

[0142] Huvec endothelial cells are used to culture artificial blood vessels, and the culture is carried out until the artificial blood vessels exhibit functional characteristics;

[0143] Human multi-functional stem cells are used to differentiate into human myocardial cells, and myocardial cell microspheres are made of myocardial cells, and the culture is carried out until the myocardial cell microspheres exhibit functional characteristics, and the myocardium has a regular beat of 18 times per minute;

[0144] Human lung cancer cells NCI-H23 are used to make 3D tumor microspheres, and the culture is carried out until the NCI-H23 tumor myocardial cell microspheres exhibit functional characteristics;

[0145] Under aseptic conditions, sterilized chips, cultured artificial blood vessels, cardiomyocyte microspheres, and NCI-H23 tumor microspheres were taken. The cultured artificial blood vessels were placed into the vascular culture chamber and inserted into the fixation block and connecting tube to achieve a sealed connection. Then, two cultured cardiomyocyte microspheres and three NCI-H23 tumor microspheres were sequentially placed into the first culture chamber B12 and the second culture chamber B7. The connecting layer and the culture layer were sealed together using non-biotoxic double-sided tape.

[0146] In a sterile environment, connect the chip and the culture system, and put 15 mL of the corresponding culture medium into each culture bottle. Start the culture system. When each culture medium fills the corresponding culture chamber and forms a flow perfusion loop, put the chip together with the culture system into a 37°C sterile incubator.

[0147] After 24 hours of continuous perfusion culture, the chip and culture system were removed together. The activity of the artificial blood vessels, cardiomyocyte microspheres, and NCI-H23 tumor microspheres within the chip was analyzed using a high-content image analysis system to ensure that the artificial blood vessels, cardiomyocyte microspheres, and NCI-H23 tumor microspheres possess good functional characteristics. In a sterile environment, the second culture medium corresponding to the artificial blood vessels was replaced with a culture medium containing 5-fluorouracil (5-FU). The chip and culture system were then placed in a 37°C sterile incubator for continuous perfusion culture for 10 days to obtain organ tissues.

[0148] Further analysis of cardiomyocyte microspheres was conducted using a high-content system. During the aforementioned continuous perfusion culture process, cardiomyocyte microspheres (corresponding to...) were analyzed every 24 hours in two sub-culture chambers of culture chamber B12 in the drug testing area. Figure 14 Cardiac cell microspheres (corresponding to 5-FU-1) and two sub-culture chambers of culture chamber B7 in the second group of independent control area. Figure 14 The amplitude of the jumping frequency was analyzed using 5-FU-2. The graph of the analysis data is shown in the figure. Figure 14 The study found that 5-FU had no significant effect on the beating frequency of cardiomyocyte microspheres in the two sub-culture chambers of the drug test area in culture chamber B12 of the first group. It did have a damaging effect on the beating of cardiomyocyte microspheres, but this effect was not significant. In the second group, 5-FU significantly affected the beating frequency of cardiomyocyte microspheres in the control group (culture chamber B7) at both 24 and 48 hours, reducing the amplitude of the beating. The cardiomyocytes stopped beating on the seventh day and could not recover after washing. This experimental phenomenon is consistent with the effects of 5-FU on human myocardium, indicating that 5-FU can affect cardiomyocyte microsphere tissue after absorption, filtration, and shielding by artificial blood vessels.

[0149] Furthermore, the obtained organ tissues were further analyzed using a high-content system, such as... Figure 15The activity of the NCI-H23 tumor microspheres in the three sub-culture chambers of the first group of culture chambers B12 in the drug test area (corresponding to 5-FU-1) and the three sub-culture chambers of the second group of culture chambers B7 in the independent control area (corresponding to 5-FU-2) was analyzed on the 1st, 3rd, 5th, 7th and 10th days of the above continuous perfusion culture, and the analysis data and collected images are shown in Figure 15 , Figure 15 , Figure 15 , Figure 16 Through the above experimental data analysis, it is found that after 10 days of drug action, compared with the activity of the NCI-H23 tumor microspheres in the second group of culture chambers B7 in the control group, the activity of the three NCI-H23 tumor microspheres in the first group of culture chambers B12 in the drug test area is reduced by about 25%, which conforms to the effect of 5-FU on human lung cancer cells NCI-H23, and indicates that 5-FU can be absorbed, filtered and shielded by artificial blood vessels to produce toxic effects on NCI-H23 tumor microspheres.

[0150] In summary, the tested drug can be absorbed, filtered and shielded by artificial blood vessels to produce effects on myocardial cell microspheres and tumor microspheres. Compared with the control group without the action of artificial blood vessels, the experimental results are closer to the results found in actual clinical research, which indicates that the detection method for the regulation of multiple tissue functions of the present application can more truly reflect the effect of the tested drug in the human body.

[0151] The detection method for the regulation of multiple tissue functions of the present application provides a brand-new model for drug screening and disease research. The culture chamber re-summarizes the multicellular structure, tissue interface and related physical microenvironment of the key functional unit of the living organ in vitro, and provides in-vitro blood vessel perfusion, which can fully reflect the role of endothelium in promoting drug absorption, distribution, metabolism, excretion (ADME) and toxicity in vitro, so as to realize the real process of in-vitro simulation of drug, nutrition, etc. in the body through blood vessels to achieve transportation and absorption, which can replace animal experiments as an important platform for drug screening and drug toxicity experiments.

[0152] It can be understood that the above embodiments are only exemplary embodiments for illustrating the principles of the present application, and the present application is not limited thereto. Various modifications and improvements can be made by those skilled in the art without departing from the spirit and essence of the present application, and these modifications and improvements are also considered as the protection scope of the present application.

Claims

1. An organ-chip for multi-tissue co-culture, characterized by, The culture layer is provided with a culture inlet group, a culture outlet group, an extravascular tissue flow channel group, a first group of culture chambers for placing three-dimensional cells or tissues or an in-vitro simulated organ, and a blood vessel culture chamber; the sealing layer is provided with an intravascular flow inlet, an intravascular flow outlet, and an intravascular flow channel group; The connection inlet group, the culture inlet group, the extravascular tissue flow channel group, the blood vessel culture chamber, an extravascular passage formed after the blood vessel culture chamber is placed, the first group of culture chambers, the culture outlet group, and the connection outlet group form an extravascular tissue culture passage; The connection inlet group, the culture inlet group, the intravascular flow inlet, the intravascular flow channel group, an intravascular passage formed after the blood vessel culture chamber is placed, the intravascular flow outlet, the culture outlet group, and the connection outlet group form an intravascular culture passage; The extravascular tissue flow channel group comprises an extravascular tissue inlet flow channel, an extravascular tissue outlet flow channel, a blood vessel culture chamber outlet flow channel, a micro-mixing flow channel, and a first group of culture chamber inlet flow channels; wherein, The culture inlet group, the extravascular tissue inlet flow channel, the blood vessel culture chamber, the blood vessel culture chamber outlet flow channel, the micro-mixing flow channel, the first group of culture chamber inlet flow channels, the first group of culture chambers, the extravascular tissue outlet flow channel, and the culture outlet group are sequentially connected; The intravascular flow channel group comprises an intravascular inlet flow channel and an intravascular outlet flow channel; wherein, The intravascular flow inlet, the intravascular inlet flow channel, the artificial blood vessel, the intravascular outlet flow channel, and the intravascular flow outlet are sequentially connected.

2. The organ-on-chip according to claim 1, characterized in that The culture layer is further provided with an independent flow channel group and a second group of culture chambers for placing three-dimensional cells or tissues or an in-vitro simulated organ; The connection inlet group, the culture inlet group, the independent flow channel group, the second group of culture chambers, the culture outlet group, and the connection outlet group form an independent culture passage.

3. The organ-on-chip of claim 1, wherein The extravascular tissue inlet flow channel, the extravascular tissue outlet flow channel, and the micro-mixing flow channel are arranged on a side of the culture layer facing the sealing layer, the blood vessel culture chamber outlet flow channel and the first group of culture chamber inlet flow channels are arranged on a side of the culture layer away from the sealing layer, and the blood vessel culture chamber and the first group of culture chambers are arranged through the culture layer; and, The culture layer is further provided with a first fluid conversion hole and a second fluid conversion hole, the blood vessel culture chamber outlet flow channel and the micro-mixing flow channel are connected through the first fluid conversion hole, and the micro-mixing flow channel and the first group of culture chamber inlet flow channels are connected through the second fluid conversion hole.

4. The organ-chip of claim 2, wherein, The independent flow channel group comprises an independent inlet flow channel, an independent outlet flow channel, and a second group of culture chamber outlet flow channels; The culture inlet group, the independent inlet flow channel, the second group of culture chambers, the second group of culture chamber outlet flow channels, the independent outlet flow channel, and the culture outlet group are sequentially connected.

5. The organ-chip according to claim 4, characterized in that The independent inlet flow channel and the independent outlet flow channel are arranged on the side of the culture layer facing the sealing layer, the second group of culture chamber outlet flow channels are arranged on the side of the culture layer away from the sealing layer, and the second group of culture chambers are arranged through the culture layer; Furthermore, The culture layer is further provided with a third fluid conversion hole, and the second group of culture chamber outlet flow channels and the independent outlet flow channel are communicated through the third fluid conversion hole.

6. The organ-on-chip according to claim 5, characterized in that The first group of culture chambers and the second group of culture chambers are arranged correspondingly.

7. The organ-chip according to any one of claims 2 to 3, characterized in that, The culture layer is further provided with a fixing groove at both ends of the blood vessel culture chamber, the fixing groove contains a fixing connection assembly, and the end of the artificial blood vessel is fixedly connected with the fixing connection assembly; and The fixing connection assembly is provided with a connection flow channel, and the intravascular inlet flow channel and the intravascular outlet flow channel are communicated with the artificial blood vessel through the connection flow channel.

8. A method for detecting the modulation of multiple tissue functions, characterized by, The organ chip of any one of claims 1 to 7 is used to detect the regulation of multi-tissue function, and the detection method comprises: introducing each culture medium into each culture channel to culture three-dimensional cells or tissues or an in-vitro simulated organ, and an artificial blood vessel; introducing a test substance into the intravascular culture channel; performing at least one activity detection analysis on the three-dimensional cells or the tissues or the in-vitro simulated organ to obtain the regulation result of the test substance on the multi-tissue function.

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