CN115895895A - Blood vessel series-connected multi-organ chip model and application method thereof - Google Patents

Abstract

The invention relates to a multi-organ chip model with blood vessels connected in series and an application method thereof, and the model comprises a multi-organ interconnection chip substrate, organ culture chamber inserts and various organ blood vessel communicating pipelines, wherein a plurality of various organ culture chamber inserting holes are formed in the multi-organ interconnection chip substrate, the organ culture chamber inserts are arranged in the various organ culture chamber inserting holes, the organ culture chamber inserts are sequentially arranged to form a multi-organ simulation system, the multi-organ simulation system comprises at least two organ culture chamber inserts, and the organ culture chamber inserts are sequentially connected through various organ blood vessel communicating channels of the multi-organ interconnection chip substrate. The invention realizes the interaction and the connection among different organs by using the chip and the perfusion technology, is used for simulating various processes of absorption, distribution, metabolism, elimination or immunity of a medicament entering a human body in vitro, and can be used for application research such as organ interaction research, medicament evaluation and the like.

Description

A multi-organ chip model in series with blood vessels and its application method

technical field

The invention relates to the technical field of organ chip manufacturing, in particular to a multi-organ chip model in which blood vessels are connected in series and an application method thereof.

Background technique

Pharmacokinetic research involves a variety of organs. Taking oral drugs as an example, drugs in the human body involve many processes such as intestinal absorption, blood distribution, liver metabolism, and renal excretion, and sometimes cause damage to other organs such as skin inflammation and heart infarction. Adverse reactions. According to reports, the average development cycle of a new drug is about 13-15 years, costing about US$2 billion. However, behind the huge time, manpower and material costs, there are still extremely high risks—up to 90% of the drugs under research that have undergone animal experiments fail in clinical human trials due to poor efficacy or toxic side effects. The traditional physiological and pathological research technology platforms and means used for preclinical drug screening and development mainly include in vitro cell culture testing, animal experiments, etc., which still have many shortcomings for efficient drug screening.

Multi-organ chips integrate multiple organs on one chip to simulate the human body, and can be used to investigate the comprehensive effects of drugs on the entire human body. The co-cultivation of cells from different tissues and organs is achieved by means of integration and multi-modules, and the micro-tissues of different modules are interconnected by using microchannels or controllable fluids, simulating the interaction between different organs of the human body and the drug in vivo. Distribution. The development of multi-organ chips continues to develop in multiple directions of bionics, systematization, flux, and immunization.

The patent of this invention proposes a multi-organ chip with blood vessels connected in series, which allows multiple organs such as the intestine, liver, heart, kidney or skin to be integrated through the blood vessel structure, and can realize in vitro simulation of drug absorption, distribution, metabolism, The various processes of exclusion or immunization facilitate in vitro drug screening tests.

Contents of the invention

The technical problem to be solved by the present invention is to provide a multi-organ chip model with blood vessels connected in series and its application method, which uses chip and perfusion technology to realize the interaction and connection between different organs, and is used for in vitro simulation of drug absorption, distribution, Various processes of metabolism, elimination or immunity can be used in applied research such as organ interaction research and drug evaluation.

The technical solution adopted by the present invention to solve the technical problem is to provide a multi-organ chip model in which blood vessels are connected in series, the pluggable multi-organ interconnection culture chip includes a multi-organ interconnection An organ vascular communication pipeline, the substrate of the multi-organ interconnection chip is provided with several insertion holes for various organ culture chambers, and the insertion holes of the various organ culture chambers are all equipped with organ culture chamber inserts, and the described The organ culture chamber inserts are arranged in sequence to form a multi-organ simulation system. The multi-organ simulation system includes at least two organ culture chamber inserts, and the organ culture chamber inserts pass through the multiple organ vascular communication channels of the multi-organ interconnection chip substrate in sequence. to connect.

The edge of the substrate of the multi-organ interconnection chip is provided with an organ culture medium perfusion channel communicating with the insertion holes of various organ culture chambers.

The inlet and outlet ports of the organ culture medium perfusion channel are docked with external microfluidic pumping equipment to provide a stable liquid flow environment for organ culture.

In addition to cultivating cells, organoids or tissues of different organs inside the organ culture chamber insert, vascular cells or lymphatic vessel cells or other duct epithelial cells are simultaneously cultivated on the back of the organ culture chamber insert, so that different organs can pass through connected by blood vessels or lymphatic vessels.

The bottom of the organ culture chamber insert is provided with a porous membrane at the bottom of the chamber.

The porous membrane at the bottom of the small chamber allows the penetration of various biological or chemical substances such as culture medium, cytokines, cell metabolites, and drugs, so as to realize the mutual connection between various organs.

A method for applying a multi-organ chip model in series with blood vessels, comprising the following specific steps:

Step 1: Chip pretreatment: Collagen coating is performed on the bottom of the organ culture insert or on the surface of the inner membrane. The collagen can be Matrigel, type I collagen, rat tail collagen and other natural or polymer gels, so that the cells can grow during the growth process. Closely adhere to the surface of the membrane;

Step 2: Vascular formation method: add 20-50 μL of PBS solution containing 5-20% Matrigel to the blood vessel side of the porous membrane at the bottom of the organ culture chamber, and incubate at room temperature for 1 hour to complete the collagen coating on the blood vessel side. Further, adjust the concentration of vascular endothelial cells to 0.1-1×10 ⁶ cells/mL and inoculate them on the blood vessel side of the porous membrane at the bottom of the small chamber insert, and culture them upside down in a 37°C incubator until the cells adhere to the wall;

Step 3: Organ culture: According to the desired organ, select the corresponding culture method to form the components of the multi-organ chip;

Step 4: Inject drugs for experiments: You can choose to inject oral drugs or inject intravenous drugs. When administering drugs, the vascular channel of the multi-organ co-culture chip containing blood vessels is continuously circulatory or non-circulatory perfusion through the chip inlet.

The multi-organ chip of the present invention can simulate animal organs and plant organs. For animal organs, including but not limited to liver, lung, intestine, heart, kidney, fat, eye, ear, nose, islet, bone, brain, skin, blood vessel, uterus, periodontium, spleen, placenta, muscle, larynx, bone marrow and other organs. Wherein, the cells cultured in the cell culture room can be a single cell or a plurality of cells, and can be primary cells, stem cells or cell lines.

Preferably, the first organ culture chamber insert is used for culturing small intestine or intestinal organoids, the second culture organ culture chamber insert is used for culturing liver organoids, the third culture organ culture chamber insert is used for culturing heart organs, and the fourth The small chamber insert is used to cultivate skin organs or kidney organs. This is to select or arrange organs according to the absorption, distribution, digestion, immunity or excretion of drugs after they enter the human body. The multi-organ co-culture chip can select organs or arrange them in order. Organ arrangements can also be arranged in any manner.

In addition to cultivating cells, organoids or tissues of different organs in the insert of the multi-organ culture chamber, vascular cells or lymphatic vessel cells or other duct epithelial cells are simultaneously cultivated on the back of the insert, so that different organs can pass through blood vessels or lymph Tubes are connected to better simulate the connection between organs.

Intestinal organ culture method: adjust intestinal cells to 0.1-5× ¹⁰⁶ cells/mL cell suspension, add to the non-vascular side of the organ culture chamber insert, put it into a conventional cell well plate for cell-attached culture, and then transfer to a place containing Intestinal multi-organ co-culture chip for perfusion co-culture;

Hepatic organ culture method: mix hepatocytes or liver cancer cells or primary liver cells with other non-parenchymal cells in the liver, such as stellate cells, endothelial cells, macrophages, etc. Spheroids of uniform size are formed by various methods such as adsorption culture and microwell array culture, which are inoculated in the non-vascular side of the organ culture chamber insert at a certain concentration, and then perfused and co-cultured.

Cardiac organ culture method: The cardiomyocytes formed by stem cell iPSC differentiation have been formed into cardiomyocyte spheroids in various ways such as 3D printing, spin drop culture, low adsorption culture, microwell array culture, etc., and are cultured in a well plate until they can spontaneously develop After beating and contracting regularly, transfer to the non-vascular side of the organ culture chamber for perfusion co-culture.

Different organs were cultured with the corresponding medium, and the medium on the non-vascular side was replaced every 2 days.

When the invention provides a test mode of oral medicine, the test experiment of oral pharmacokinetics can be carried out in vitro.

When administering drugs, the vascular channel of the multi-organ co-culture chip containing blood vessels is continuously perfused with circulation or non-circulation through the chip inlet. The drug is added to the multi-organ chip through the non-vascular side of the intestinal organ, absorbed through the intestinal organ epithelium, and enters the vascular channel to achieve drug delivery. Different organs can absorb the drugs circulating in the blood vessels through the vascular endothelial barrier, and carry out absorption or metabolism or excretion or immunity.

The administration method can be a single administration, that is, a given drug is administered at a certain concentration within a period of time, or intermittent multiple administration or other administration methods can be performed according to the characteristics of different oral drugs.

When the invention provides an intravenous administration test mode, the intravenous drug test experiment can be carried out in vitro.

When administering, the drug is added to the vascular perfusion medium according to a specific concentration, and enters the multi-organ chip system through the chip inlet for continuous circulation or non-circulation perfusion. The administration method can be a single administration, that is, a given drug is administered at a certain concentration within a period of time, or intermittent multiple administration or other administration methods can be performed according to the characteristics of different drugs.

Beneficial effects: the present invention relates to a multi-organ chip model with blood vessels connected in series and its application method, which has the following advantages:

(1) The multi-organ chip of the present invention interconnects and cultivates organs with different functions through blood vessels to realize complex multi-organ testing experiments in vitro, which is of great significance for improving the bionicity of the human body chip and expanding the application range of the human body chip;

(2) The multi-organ chip of the present invention can self-assemble different organs according to requirements for different matching and assembly, so as to realize different experimental purposes such as drug ADME test or drug efficacy evaluation.

Description of drawings

Fig. 1 is a schematic diagram of a multi-organ interconnection chip according to the present invention;

Fig. 2 is a bright-field view of the porous membrane at the bottom of the organ culture chamber of vascular endothelial cells of the present invention;

Fig. 3 is the intestinal organ transmembrane barrier resistance figure after application of Sorafenib medicine in the present invention;

Fig. 4 is a curve diagram of drug concentration and time in the liver organ using Sorafenib in the present invention.

Illustration: 1. Multi-organ interconnection chip substrate, 2. Insertion holes of various organ culture chambers, 3. Organ medium perfusion channel, 4. Vascular communication channel of various organs, 5. Organ culture chamber insert, 6. Bottom of the chamber Porous membrane.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The embodiment of the present invention relates to a multi-organ chip model with blood vessels in series and its application method, as shown in Figure 1-2, including a multi-organ interconnection chip substrate 1, an organ culture chamber insert 5, and a variety of organ vascular communication channels 4 , the multi-organ interconnection chip substrate 1 is provided with several various organ culture chamber insertion holes 2, and the organ culture chamber inserts 5 are installed in the various organ culture chamber insertion holes 2, and the organ culture The culture chamber inserts 5 are arranged in sequence to form a multi-organ simulation system. The multi-organ simulation system includes at least two organ culture chamber inserts 5, and the organ culture chamber inserts 5 sequentially pass through various organs of the multi-organ interconnection chip substrate 1. The blood vessel communication channel 4 is connected.

The edge of the multi-organ interconnection chip substrate 1 is provided with an organ culture medium perfusion channel 3 communicating with the insertion holes 2 of various organ culture chambers.

The inlet and outlet ports of the organ culture medium perfusion channel 3 are connected with external microfluidic pumping equipment to provide a stable liquid flow environment for organ culture.

In addition to cultivating cells, organoids or tissues of different organs inside the organ culture chamber insert 5, vascular cells or lymphatic vessel cells or other duct epithelial cells are simultaneously cultured on the back of the organ culture chamber insert 5, so that different Organs are connected by blood vessels or lymphatic vessels.

The bottom of the organ culture chamber insert 5 is provided with a porous filter membrane 6 at the bottom of the chamber.

The porous filter membrane 6 at the bottom of the small chamber allows the penetration of various biological or chemical substances such as culture medium liquid, cytokines, cell metabolites, and drugs, so as to realize the mutual connection between various organs.

Example 1

A design and production diagram of a multi-organ interconnection chip with blood vessels connected in series is shown in Figure 1. It consists of a multi-organ culture chip substrate, a cover plate attached to the substrate, a high light-transmitting film attached to the substrate, and an array The composition of the organ culture chamber insert: the matrix 1 of the multi-organ interconnection chip includes the insertion hole 2 of the multi-organ culture chamber, the perfusion channel 3 of the organ culture medium and the communication channel 4 of the blood vessels of various organs. Among them, the insertion holes 2 of various organ culture chambers can be inserted into the organ culture chamber inserts 5 according to requirements, so as to realize arbitrary interconnection of various organs. Various organ culture chamber inserts 5 can be connected to each other through various organ blood vessel communication channels 4 . The organ culture chamber insert is mainly composed of the side wall of the chamber insert and the porous filter membrane 6 at the bottom of the chamber.

Example 2

In this example, the four main organs for absorption, distribution, metabolism, excretion or immunity after oral medicine enter the human body are combined and arranged, and the four organs are the intestine, liver, heart, kidney or skin in order to form an ADME multi-organ combination. On a multi-organ interconnection chip, at least one set of such ADME multi-organ combinations is included.

The ADME multi-organ combination includes at least one intestinal organ as the main organ for drug absorption. Before seeding enterocytes, the inner side of the organ culture chamber insert, that is, the non-vascular side, was coated with type I collagen at a concentration of 1–100 μg/mL overnight at 4°C.

Invert the insert of the type I collagen-coated organ culture chamber to coat the blood vessel side, add 10-500 μL of PBS solution containing 1-20% Matrigel to the blood vessel side of the porous filter membrane 6 at the bottom of the chamber, and incubate at room temperature for no less than After 1 hour, the collagen coating on the vessel side was completed.

Adjust the concentration of vascular endothelial cells to 0.1-1× ¹⁰⁶ cells/mL and inoculate them on the blood vessel side of the porous membrane at the bottom of the small chamber insert, and culture them in a 37°C incubator until the cells adhere to the wall.

Adjust the intestinal cells to 0.1-5× ¹⁰⁶ cells/mL cell suspension, add it to the non-vascular side of the porous membrane 6 at the bottom of the organ culture chamber insert 5, put it into a conventional cell well plate for cell attachment culture, and then transfer to Perfusion co-culture in a multi-organ co-culture chip containing intestinal tract;

Liver organ culture method: mix hepatocytes or liver cancer cells or primary liver cells with other non-parenchymal cells in the liver, such as stellate cells, endothelial cells, macrophages, etc. in a certain proportion, and use spin drop culture, low adsorption culture, Spheroids of uniform size are formed by various methods such as microwell array culture, which are inoculated in the non-vascular side of the organ culture chamber insert at a certain concentration, and then perfused and co-cultured.

Cardiac organ culture method: The cardiomyocytes formed by stem cell IPSC differentiation have been 3D printed, spin-drop cultured, low-adsorption cultured, or microwell array cultured to form cardiomyocyte spheroids, and cultured in the orifice plate until they can spontaneously undergo regular beating and contraction Transfer to the non-vascular side of the organ culture chamber for perfusion co-culture.

Different organs were cultured with the corresponding medium, and the medium on the non-vascular side was replaced every 1 to 3 days.

Example 3

Connect the above-mentioned intestinal organ to the liver, heart, kidney or skin, and use a diaphragm pump to perfuse the endothelial cell culture medium at a flow rate of 0.1-1000 μL per minute. Prior to drug testing, continuous perfusion was performed for 4 to 72 hours, during which heartbeat tests were performed every 1 to 24 hours.

After 72 hours, 50-600 μM free fatty acid was added to the vascular endothelial cell culture medium, and high-fat induction was carried out for 48 hours, during which a heartbeat test was performed every 1-24 hours.

The chip was tested with an oral liver cancer treatment drug-Sorafenib SORA, and intestinal organ culture medium containing 1-30 μM SORA was prepared and added to the inner side of the intestinal organ culture chamber to simulate the process of oral drug absorption in the intestine. During this period, a heartbeat test is performed every 24 hours, and the specific data are shown in Figure 3.

After the first administration, the organ culture medium of different organs or blood vessels was collected at 1 hour, 4 hours, 24 hours, and 48 hours, and the drug concentration test was performed by HPLC-MS. The specific parameters are shown in Figure 4.

The above is a detailed introduction of a multi-organ chip model with blood vessels connected in series and its application method provided by this application. In this paper, specific examples are used to illustrate the principle and implementation of this application. The description of the above examples is only for To help understand the method and its core idea of this application; at the same time, for those of ordinary skill in the art, according to the idea of this application, there will be changes in the specific implementation and application scope. In summary, the content of this specification It should not be construed as a limitation of the application.

Claims (7)

1. A multi-organ chip model with blood vessels connected in series, characterized in that: it includes a multi-organ interconnection chip substrate (1), an organ culture chamber insert (5) and a variety of organ vascular communication channels (4), and the multi-organ The interconnected chip substrate (1) is provided with several various organ culture chamber insertion holes (2), and the organ culture chamber inserts (5) are installed in the various organ culture chamber insertion holes (2). The organ culture chamber inserts (5) are arranged in sequence to form a multi-organ simulation system, the multi-organ simulation system includes at least two organ culture chamber inserts (5), and the organ culture chamber inserts (5) pass through the multi-organ The various organ blood vessel communication channels (4) of the interconnection chip base body (1) are connected. 2. A multi-organ chip model with blood vessels connected in series according to claim 1, characterized in that: the edge of the multi-organ interconnection chip substrate (1) is provided with insertion holes (2) communicating with various organ culture chambers organ media perfusion channel (3). 3. A multi-organ chip model with blood vessels in series according to claim 2, characterized in that: the inlet and outlet ports of the organ culture medium perfusion channel (3) are docked with external microfluidic pumping equipment for organ culture Provides a stable fluid flow environment. 4. A multi-organ chip model with blood vessels in series according to claim 3, characterized in that: in addition to cultivating cells, organoids or tissues of different organs in the inside of the organ culture chamber insert (5), The back of the organ culture chamber insert (5) simultaneously cultures blood vessel cells or lymphatic vessel cells or other duct epithelial cells, so that different organs are connected through blood vessels or lymphatic vessels. 5 . The multi-organ chip model with blood vessels in series according to claim 4 , characterized in that: the bottom of the organ culture chamber insert ( 5 ) is provided with a porous membrane ( 6 ) at the bottom of the chamber. 6 . 6. The multi-organ chip model of a kind of blood vessel series according to claim 5, characterized in that: the porous filter membrane (6) at the bottom of the small chamber allows a variety of media liquid, cytokines, cell metabolites, drugs, etc. Through the passage of biological or chemical substances, the interconnection between various organs is realized. 7. An application method of a multi-organ chip model in series with blood vessels as claimed in claim 6, characterized in that: comprising the following specific steps: Step 1: Chip pretreatment: Collagen coating is performed on the bottom of the organ culture insert or on the surface of the inner membrane. The collagen can be Matrigel, type I collagen, rat tail collagen and other natural or polymer gels, so that the cells can grow during the growth process. Closely adhere to the surface of the membrane; Step 2: Vascular formation method: add 20-50 μL of PBS solution containing 5-20% Matrigel to the blood vessel side of the porous membrane at the bottom of the organ culture chamber, and incubate at room temperature for 1 hour to complete the collagen coating on the blood vessel side. Further, adjust the concentration of vascular endothelial cells to 0.1-1×10 ⁶ cells/mL and inoculate them on the blood vessel side of the porous membrane at the bottom of the small chamber insert, and culture them upside down in a 37°C incubator until the cells adhere to the wall; Step 3: Organ culture: According to the desired organ, select the corresponding culture method to form the components of the multi-organ chip; Step 4: Inject drugs for experiments: You can choose to inject oral drugs or inject intravenous drugs. When administering drugs, the vascular channel of the multi-organ co-culture chip containing blood vessels is continuously circulatory or non-circulatory perfusion through the chip inlet.

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