CN110669670B - Human body micro-ecosystem chip and use method thereof - Google Patents

Human body micro-ecosystem chip and use method thereof Download PDF

Info

Publication number
CN110669670B
CN110669670B CN201911110425.7A CN201911110425A CN110669670B CN 110669670 B CN110669670 B CN 110669670B CN 201911110425 A CN201911110425 A CN 201911110425A CN 110669670 B CN110669670 B CN 110669670B
Authority
CN
China
Prior art keywords
cell culture
cavity
culture cavity
chip
waste liquid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201911110425.7A
Other languages
Chinese (zh)
Other versions
CN110669670A (en
Inventor
孙长凯
欧阳琦镇
孙品
郝慧芳
姜迪
关水
刘海龙
滕岩
朱慧超
张航与
张建伟
张驰
余隽
田杨军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dajiniao Health Intelligence Medical Big Data Dalian Co ltd
Dalian University of Technology
Original Assignee
Dajiniao Health Intelligence Medical Big Data Dalian Co ltd
Dalian University of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dajiniao Health Intelligence Medical Big Data Dalian Co ltd, Dalian University of Technology filed Critical Dajiniao Health Intelligence Medical Big Data Dalian Co ltd
Priority to CN201911110425.7A priority Critical patent/CN110669670B/en
Publication of CN110669670A publication Critical patent/CN110669670A/en
Application granted granted Critical
Publication of CN110669670B publication Critical patent/CN110669670B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A human body micro-ecosystem chip belongs to the field of medicine research and human body related monitoring application, and realizes that a lower PDMS A plate constructs an organ chip integrating an intestinal-liver-heart/brain-female genital tract system by using a sandwich structure of a PDMS A plate-porous membrane-PDMS B plate to simulate real human body blood circulation; culturing human microorganisms in the upper PDMS B plate; the porous membrane filters the metabolites of the human microorganisms from the culture medium containing the human microorganisms, from the PDMS B plate into the PDMS A plate. Micro valves are arranged among all the cell culture chambers in the PDMS A plate in a micro-flow mode, and the flowing of a culture medium is adjusted by controlling the micro valves. The invention can be used for accurately, simply and efficiently researching, evaluating and controlling the interaction between the microbial metabolites of the human body and multiple organs of the individual; by culturing corresponding human body cells in the culture cavity and controlling the flow of the culture medium, the bionic of cerebrospinal fluid circulation and direct nerve connection existing in a real human body is realized, and a research foundation is laid for medicine research and human body related monitoring application.

Description

Human body micro-ecosystem chip and use method thereof
Technical Field
The invention belongs to the field of medicine research and human body related monitoring application, and relates to a human body micro-ecosystem chip integrated with a multi-organ system and a using method thereof, which can be used for accurately, simply and efficiently researching, evaluating and controlling the interaction between a human body microbial metabolite and the individual multi-organ.
Background
People live in a huge and complicated ecosystem network woven from a wide variety of microorganisms. With the recent introduction of the concept of the "gut-brain" axis, the role of intestinal microorganisms in human metabolism has been receiving more and more attention from researchers. The intestinal microorganisms are mainly located in the gastrointestinal tract of a human body, and the secreted metabolites of the intestinal microorganisms are absorbed by intestinal epithelial cells of the human body and enter the hepatic portal vein, so that the intestinal microorganisms enter the blood circulation of the human body and influence the metabolism of multiple organs of the human body. Short-chain fatty acids and lipopolysaccharides in human microbial metabolites are most concerned by researchers, and are closely linked with the generation and prevention of human-related diseases. Short chain fatty acids are organic fatty acids containing 1-6 carbon atoms and have been reported in the literature to reduce temporal lobe tissue hyperexcitability by treating a temporal lobe tissue section taken from the brain of a young male suffering from epilepsy. Among them, acetate, propionate and butyrate are involved in the related signal pathways of human body, and are more commonly used in the information exchange research between the intestinal microorganisms and the central nervous system. Lipopolysaccharide is a biological macromolecule composed of lipid and polysaccharide, has a complex structure, and is a component of the outer wall of the cell wall of gram-negative bacteria. Lipopolysaccharide has biological toxicity, generally, lipopolysaccharide can only be when the bacteria death or bacteria outer wall is damaged by external force, and will come off from the cell wall, and acts on animal cells to exert its toxic effect, and in research in intravenous injection of 1ng/mL lipopolysaccharide can cause mammalian shock response, so lipopolysaccharide is also called as bacterial endotoxin. The existing research on the correlation between human microbial metabolites and human metabolism is mostly carried out by using animal models, and the research results are extremely limited in practical application due to species differences between human and animals. With the continuous and deep research of 'organ chips' in recent years, the research of integrating multiple 'organ' systems to simulate real human physiological circulation is technically possible. In addition, the organ chip developed based on the current mature microfluidic technology naturally becomes the best research and evaluation platform for the interaction research of human body micro-ecology and human body multiple organs due to the advantages of low cost, small actual dosage and the like. At present, organ chips such as lung, intestine, liver, heart and the like are formed in domestic and foreign researches, and are close to corresponding real human organs in key functions. The single 'organ' chip is too simple to meet the requirement of researching the human physiological circulation, and the multiple 'organ' system integrated chip is an excellent research scheme for real human physiological circulation, has important clinical application value, but is still in the infancy in practice. Based on the background, the human microecological system chip integrated by the multi-organ system, which can be used for accurately, simply and efficiently researching, evaluating and controlling the interaction between the microbial metabolites of the human body and the multiple organs of the individual, is established, and has very high application value. Based on the chip, the bionics of blood circulation, cerebrospinal fluid circulation and direct nerve connection existing in a real human body can be realized by culturing corresponding human body cells in corresponding culture cavities and controlling the flow of a culture medium, and a research foundation is laid for medicine research and human body related monitoring application.
Disclosure of Invention
Aiming at the problems in the construction of the multi-organ integrated chip, the invention provides a multi-organ system integrated human body micro-ecosystem chip based on a micro-nanofluidic technology, which can be used for accurately, simply and efficiently researching, evaluating and controlling the interaction between the human body microorganism metabolite and the individual multi-organ. Specifically, a sandwich structure of a PDMS A plate-porous membrane-PDMS B plate is used for realizing that the lower PDMS A plate constructs an integrated organ chip for the intestine-liver-heart/brain-female genital tract system to simulate real human blood circulation; culturing human microorganisms in the upper PDMS B plate; the porous membrane filters the metabolites of human microorganisms from the medium containing them and allows them to pass from the PDMS B plate into the PDMS A plate. Corresponding micro valves are arranged in the micro-fluidic pipelines between the cell culture chambers in the PDMS A plate, and the flow of the culture medium can be adjusted by controlling the corresponding micro valves. In addition, the human body micro-ecological chip realizes the bionics of cerebrospinal fluid circulation and direct nerve connection existing in a real human body by culturing corresponding human body cells in corresponding culture cavities and controlling the flow of a culture medium, and lays a research foundation for medicine research and human body related monitoring application.
In order to achieve the purpose, the invention adopts the technical scheme that:
a human body micro-ecosystem chip integrated by a multi-organ system can be used for accurately, simply and efficiently researching, evaluating and controlling the interaction between human body microorganism metabolites and the individual multi-organ. The human body micro-ecosystem chip realizes the bionics of blood circulation, cerebrospinal fluid circulation and direct nerve connection existing in a real human body by culturing corresponding human body cells in a corresponding culture cavity and controlling the flow of a culture medium, and lays a research foundation for medicine research and human body related monitoring application. The human body micro-ecological chip comprises two layers of Polydimethylsiloxane (PDMS) plates and a layer of porous membrane, wherein the lower PDMS plate is marked as a PDMS A plate, and the upper PDMS plate is marked as a PDMS B plate. The sandwich structure of the PDMS A plate-porous membrane-PDMS B plate is formed by the two PDMS plates and the porous membrane, so that the interaction between the microbial metabolites of the human body and the individual multiple organs under the condition of being close to the real physiological circulation of the human body can be conveniently researched.
The lower PDMS A plate comprises 1-1 of hepatocyte culture medium input interface, 4 of culture medium input interface and culture cavity connecting channels 1-2, 1-3 of hepatocyte culture cavity, 4 of waste liquid output interface and culture cavity connecting channels 1-4, 1 of hepatocyte culture cavity waste liquid output interface 1-5, 1 of intestinal epithelial cell culture cavity and hepatocyte culture cavity connecting channel 1-6, 1 of intestinal epithelial cell culture medium input interface 1-7, 1 of intestinal epithelial cell culture cavity 1-8, 1 of intestinal epithelial cell culture cavity waste liquid output interface 1-9, 1 of intestinal epithelial cell culture cavity and female genital tract cell culture cavity connecting channel 1-10, 1 of female genital tract cell culture medium input interface 1-11, 1 of female genital tract cell culture cavity 1-12, 1-13 parts of female genital tract cell culture cavity waste liquid output interface, 1 part of connecting channel between heart or nerve cell culture cavity and female genital tract cell culture cavity, 1-15 parts of input interface of heart or nerve cell culture medium, 1-16 parts of heart or nerve cell culture cavity, 1-17 parts of waste liquid output interface between heart or nerve cell culture cavity and liver cell culture cavity, 1-18 parts of connecting channel between heart or nerve cell culture cavity and liver cell culture cavity, 1-19 parts of micro valve, and 1-20 parts of connecting channel between heart or nerve cell culture cavity and intestinal epithelial cell culture cavity.
The porous membrane is a polycarbonate membrane, so that most of microorganisms in the microorganism culture cavities 2-11 on the PDMS B plate are prevented from entering the intestinal epithelial cell culture cavities 1-8 by a culture medium, normal growth of cells in the PDMS A plate is guaranteed, microbial metabolites of a human body are in contact with multiple organs of the individual, and a research platform of the microbial metabolites of the human body in the fields of medical research and related monitoring and application of the human body is accurately and efficiently built.
The PDMS B plate comprises 1 hepatocyte culture medium inlet 2-1, 1 hepatocyte culture chamber waste liquid outlet 2-2, 1 bacterial liquid and microorganism culture medium inlet 2-3, 1 intestinal epithelial cell culture medium inlet 2-4, 1 intestinal epithelial cell culture chamber waste liquid outlet 2-5, 1 microorganism culture chamber waste liquid outlet 2-6, 1 female genital tract cell culture medium inlet 2-7, 1 female genital tract cell culture chamber waste liquid outlet 2-8, 1 cardiac or nerve cell culture medium inlet 2-9, 1 cardiac or nerve cell culture chamber waste liquid outlet 2-10, and 1 microorganism culture chamber 2-11.
The intestinal-liver-heart/brain-female genital tract chip is composed of an intestinal chip, a liver chip, a heart/brain chip and a female genital tract chip which are independent.
The intestinal chip consists of an intestinal epithelial cell culture cavity 1-8 on a PDMS A plate, a porous membrane and a PDMS B plate microorganism culture cavity 2-11. Wherein the intestinal epithelial cell culture chamber 1-8 on the PDMS A plate is in the shape of an ellipse which is composed of a semicircle at the left end and the right end and a rectangle in the middle. The left lower end of the device is connected with 1 intestinal epithelial cell culture medium input interface 1-7 through a culture medium input interface and a culture cavity connecting channel 1-2, and the right lower end of the device is connected with 1 intestinal epithelial cell culture cavity waste liquid output interface 1-9 through a waste liquid output interface and a culture cavity connecting channel 1-4. The intestinal epithelial cell culture medium input interface 1-7 is communicated with the intestinal epithelial cell culture medium input port 2-4 on the PDMS B plate, and is externally connected with an injection pump to ensure that the intestinal epithelial cells smoothly enter the intestinal epithelial cell culture cavity 1-8. The waste liquid output interface 1-9 of the intestinal epithelial cell culture cavity is communicated with the waste liquid output port 2-5 of the intestinal epithelial cell culture cavity on the PDMS B plate, so that the redundant waste liquid of the intestinal epithelial cell culture cavity can smoothly flow out of the intestinal epithelial cell culture cavity 1-8. The upper left end of the intestinal epithelial cell culture cavity 1-8 is provided with 1 intestinal epithelial cell culture cavity and hepatocyte culture cavity connecting channel 1-6 for connecting the intestinal chip and the hepatocyte, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The middle part of the upper end of the intestinal epithelial cell culture cavity 1-8 is provided with 1 heart or nerve cell culture cavity and intestinal epithelial cell culture cavity connecting channel 1-20 for connecting the intestinal chip and the heart/brain chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The upper right end of the intestinal epithelial cell culture cavity 1-8 is provided with 1 intestinal epithelial cell culture cavity and female genital tract cell culture cavity connecting channel 1-10 for connecting the intestinal chip and the female genital tract chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The shape of the microorganism culture cavity 2-11 on the PDMS B plate is an ellipse square formed by semicircles at two ends and a rectangle in the middle, and the size of the ellipse square is consistent with that of the intestinal epithelial cell culture cavity 1-8. The left end of the microorganism culture cavity 2-11 is connected with 1 bacterial liquid and microorganism culture medium input port 2-3 through a micro-fluidic pipeline, and is used for injecting the liquid and the microorganism culture medium through an injection pump. The right end of the microorganism culture cavity 2-11 is connected with a microorganism culture cavity waste liquid outlet 2-6 through a micro-fluidic pipeline for discharging redundant microorganism culture waste liquid. And an external conduit is connected behind the biological culture medium, the biological culture medium is opened only in the early stage of research, and is in a closed state in the rest time, so that the microbial culture waste liquid can smoothly enter the PDMS A plate.
The liver chip consists of liver cell culture cavities 1-3 on a PDMS A plate and an attached microfluidic pipeline. The shape of the liver cell culture cavity 1-3 is an ellipse square consisting of upper and lower semi-circles at two ends and a middle rectangle, the size of the liver cell culture cavity is consistent with that of the intestinal epithelial cell culture cavity 1-8, and the direction of the liver cell culture cavity is vertical to that of the intestinal epithelial cell culture cavity. The left upper end of the hepatocyte culture chamber 1-3 is connected with 1 hepatocyte culture medium input interface 1-1 through a culture medium input interface and culture chamber connecting channel 1-2, and the left lower end is connected with 1 hepatocyte culture chamber waste liquid output interface 1-5 through a waste liquid output interface and culture chamber connecting channel 1-4. The hepatocyte culture medium input interface 1-1 is communicated with the hepatocyte culture medium input port 2-1 on the PDMS B plate, and is externally connected with an injection pump to ensure that hepatocytes smoothly enter the hepatocyte culture chamber 1-3. The waste liquid output interface 1-5 of the hepatocyte culture chamber is communicated with the waste liquid output port 2-2 of the hepatocyte culture chamber on the PDMS B plate, so that the redundant waste liquid of the hepatocyte culture chamber can smoothly flow out of the hepatocyte culture chamber 1-3. The upper right end of the hepatocyte culture chamber 1-3 is provided with 1 connecting channel 1-18 for connecting the hepatocyte or nerve cell culture chamber and the hepatocyte culture chamber for connecting the hepatocyte chip and the cardiac/brain chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The right lower end of the liver cell culture cavity 1-3 is provided with 1 intestinal epithelial cell culture cavity and liver cell culture cavity connecting channel 1-6 for connecting the liver chip and the intestinal chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow.
The heart/brain chip is composed of heart or nerve cell culture cavities 1-16 on a PDMS A plate and a micro-fluidic pipeline attached to the heart or nerve cell culture cavities. The heart or nerve cell culture chamber 1-16 is an ellipse formed by a semicircle at the left and right ends and a rectangle in the middle, the size of the ellipse is consistent with that of the intestinal epithelial cell culture chamber 1-8, and the ellipse is parallel to the intestinal epithelial cell culture chamber in direction. The upper right end of the heart or nerve cell culture cavity 1-16 is connected with 1 heart or nerve cell culture medium input interface 1-15 through a culture medium input interface and a culture cavity connecting channel 1-2, and the upper left end is connected with 1 heart or nerve cell culture cavity waste liquid output interface 1-17 through a waste liquid output interface and a culture cavity connecting channel 1-4. The heart or nerve cell culture medium input interface 1-15 is communicated with the heart or nerve cell culture medium input port 2-9 on the PDMS B plate, and is externally connected with an injection pump to ensure that the heart or nerve cell smoothly enters the heart or nerve cell culture cavity 1-16. The waste liquid output interfaces 1-17 of the heart or nerve cell culture cavity are communicated with the waste liquid output ports 2-10 of the heart or nerve cell culture cavity on the PDMS B plate, so that the redundant waste liquid of the liver cell culture cavity can smoothly flow out of the heart or nerve cell culture cavity 1-16. The left lower end of the heart or nerve cell culture cavity 1-16 is provided with 1 heart or nerve cell culture cavity and liver cell culture cavity connecting channel 1-18 for connecting the heart/brain chip and the intestine chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The middle part of the lower end of the heart or nerve cell culture cavity 1-16 is provided with 1 heart or nerve cell culture cavity and intestinal epithelial cell culture cavity connecting channel 1-20 for connecting the heart/brain chip and the intestinal chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The right lower end of the heart or nerve cell culture cavity 1-16 is provided with 1 heart or nerve cell culture cavity and female genital tract cell culture cavity connecting channel 1-14 for connecting the heart/brain chip and the female genital tract chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow.
The female genital tract chip consists of female genital tract cell culture cavities 1-12 on a PDMS A plate and a micro-fluidic pipeline attached to the female genital tract cell culture cavities. The shape of the female genital tract cell culture cavity 1-12 is an ellipse which is composed of semicircles at the upper and lower ends and a rectangle at the middle, the size of the oval is consistent with that of the intestinal epithelial cell culture cavity 1-8, and the oval is vertical to the direction. The right lower end of the female genital tract cell culture cavity 1-12 is connected with 1 female genital tract cell culture medium input interface 1-11 through a culture medium input interface and a culture cavity connecting channel 1-2, and the right upper end is connected with 1 female genital tract cell culture cavity waste liquid output interface 1-13 through a waste liquid output interface and a culture cavity connecting channel 1-4. The female genital tract cell culture medium input interface 1-11 is communicated with the female genital tract cell culture medium input interface 2-7 on the PDMS B plate, and is externally connected with an injection pump to ensure that female genital tract cells smoothly enter the female genital tract cell culture cavity 1-12. The female genital tract cell culture cavity waste liquid output interface 1-13 is communicated with a female genital tract cell culture cavity waste liquid output port 2-8 on the PDMS B plate, so that the redundant female genital tract cell culture cavity waste liquid can smoothly flow out of the female genital tract cell culture cavity 1-12. The left upper end of the female genital tract cell culture cavity 1-12 is provided with 1 heart or nerve cell culture cavity and female genital tract cell culture cavity connecting channel 1-14 for connecting the heart/brain chip and the female genital tract chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the flowing fluid. The left lower end of the female genital tract cell culture cavity 1-12 is provided with 1 intestinal epithelial cell culture cavity and female genital tract cell culture cavity connecting channel 1-10 for connecting the female genital tract chip and the intestinal chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow.
Furthermore, the human body micro-ecological chip can realize three culture modes of high oxygen, low oxygen and no oxygen for microbial culture by controlling the oxygen content in the microbial culture medium.
Further, the ellipse sizes of the liver cell culture cavity 1-3 and the female genital tract cell culture cavity 1-12 are as follows: the diameter of the upper and lower end semi-circle is 200-.
Further, the ellipse sizes of the heart or nerve cell culture cavities 1-16 and the intestinal epithelial cell culture cavities 1-8 are as follows: the diameters of the left and right semi-circles are 200-.
Furthermore, all the connecting channels are micro-nano flow control channels with the length of 200-.
Furthermore, the sizes and shapes of all the culture medium input ports and the waste liquid output ports are circles with the diameters of 200-400 μm.
The porous membrane 3 is a polycarbonate membrane, the pore diameter is 0.01-0.5 μm, and the density is 1 × 103-1 × 106/cm 2.
Furthermore, the cells cultured in the cell culture chamber are not limited to the intestinal epithelial cells, liver cells, cardiac muscle cells, nerve cells and female genital tract cells mentioned above, and can be connected for many times to meet the bionic requirement on more complex human physiological circulation.
Furthermore, the method can promote the connection between nerve cells and intestinal epithelial cells and female genital tract cells, thereby realizing the physiological bionics of directly connecting the nerves with the intestines and the female genital tract.
A use method of a human body micro-ecosystem chip comprises the following contents:
the human body micro-ecological chip cultures corresponding nerve cells of different species in the cell culture cavities 1-3, 1-8, 1-12 and 1-16, realizes the bionics of the physiological circulation of 'intestine-liver-heart/brain-female genital tract-intestine' by controlling the corresponding micro-valve, the working state of the injection pump and the on-off of the corresponding waste liquid output port, so as to simulate the blood circulation of a real human body, and the specific operation flow is as follows: after all injection pumps, culture medium input ports and waste liquid output ports in the control micro-ecological chip are in a closed state, micro valves 1-19 on connecting channels 1-14 of an intestinal epithelial cell culture cavity and a liver cell culture cavity, connecting channels 1-18 of a heart or nerve cell culture cavity and the liver cell culture cavity and connecting channels 1-14 of the heart or nerve cell culture cavity and a female genital tract cell culture cavity are opened, and a waste liquid output port 2-8 of the female genital tract cell culture cavity is opened. Injecting a cell liquid culture medium into an intestinal epithelial cell culture medium input port 2-4 by using an injection pump, injecting a microorganism liquid culture medium into a bacterial liquid and microorganism culture medium input port 2-3, wherein the culture medium carries human microorganism metabolites to sequentially pass through an intestinal chip, a liver chip and a heart/brain chip, and finally is discharged from a female genital tract cell culture cavity waste liquid output port 2-8 to form the intestinal-liver-heart/brain chip. On the basis, when waste liquid discharged from a waste liquid outlet 2-8 of a female genital tract cell culture cavity is collected, the injection of the culture medium into an intestinal epithelial cell culture cavity inlet 2-4 and a bacterial liquid and microorganism culture medium inlet 2-3 is stopped, the waste liquid outlet 2-8 of the female genital tract cell culture cavity is closed, a micro valve 1-19 on a connecting channel 1-10 of the intestinal epithelial cell culture cavity waste liquid outlet 2-5 and the intestinal epithelial cell culture cavity and the female genital tract cell culture cavity is opened, a micro valve 1-19 on a connecting channel 1-6 of the intestinal epithelial cell culture cavity and a liver cell culture cavity is closed, then a cell liquid culture medium is injected into the liver cell culture medium inlet 2-1 by using an injection pump, and the injected liquid culture medium is finally discharged from the waste liquid outlet 2-5 of the intestinal epithelial cell culture cavity, the chip is formed by the chip of heart/brain-female genital tract-intestine. The bionics of the physiological circulation of the intestines-liver-heart/brain-female genital tract-intestines is realized by controlling the working state of the corresponding micro valve and the injection pump and the on-off of the corresponding waste liquid outlet. In addition, corresponding nerve cells of different species can be cultured in the cell culture cavities 1-3, 1-8, 1-12 and 1-16, and the simulation of the cerebrospinal fluid circulation of the real human body can be realized according to the same operation mode.
The cells cultured in the cell culture chamber of the human micro-ecological chip are not limited to the intestinal epithelial cells, liver cells, cardiac muscle cells, nerve cells, female genital tract cells mentioned above, and the chip can be connected many times to meet the demand of integrating more 'organs'. The specific operation flow is that after all injection pumps, culture medium input ports and waste liquid output ports in the micro-ecological chip are controlled to be in a closed state, micro valves 1-19 on three channels, namely culture cavity connecting channels 1-6, culture cavity connecting channels 1-18 and culture cavity connecting channels 1-14 are opened, and waste liquid output ports 2-8 of the female genital tract cell culture cavity are opened. And (3) injecting a culture medium into the intestinal epithelial cell culture medium inlet 2-4 by using a syringe pump, and enabling the culture medium to flow out from the female reproductive tract cell culture cavity waste liquid outlet 2-8. The flowing culture medium flows in from the cell culture medium inlet 2-4 of the human body micro-ecological chip with the same second block structure and the same on-off state of the culture medium inlet and the waste liquid outlet, and flows out from the waste liquid outlet 2-8 of the cell culture cavity of the second human body micro-ecological chip, and the circulation is carried out, so that the function of connecting the same chip for multiple times is realized. Wherein, the liver cell culture cavity 1-3 and the heart or nerve cell culture cavity 1-16 in each human body micro-ecological chip can culture human body cells except liver cells, cardiac muscle cells and nerve cells, which is convenient for meeting the bionic requirement of more complex human body physiological circulation.
The human body micro-ecological chip can realize physiological bionics of a nerve direct connection intestine and a female genital tract, and is used for researching neuron loop researches between a human brain and human body intestinal tracts and between the human brain and the female genital tract. The specific operation flow comprises the steps of culturing intestinal epithelial cells in the intestinal epithelial cell culture cavity 1-8, culturing female genital tract cells in the female genital tract cell culture cavity 1-12, and culturing nerve cells in the heart or nerve cell culture cavity 1-16. After all injection pumps, culture medium input ports and waste liquid output ports in the micro-ecological chip are controlled to be in a closed state, micro valves 1-19 on three channels, namely a culture cavity connecting channel 1-20, a culture cavity connecting channel 1-10 and a culture cavity connecting channel 1-14, are opened. After the waste liquid outlet 2-5 of the intestinal epithelial cell culture cavity is opened, a syringe pump is used for injecting a culture medium into the heart or nerve cell culture medium inlet 2-9, and the injected culture medium flows out from the waste liquid outlet 2-5 of the intestinal epithelial cell culture cavity. The process promotes the connection between nerve cells and intestinal epithelial cells and female genital tract cells, thereby realizing the physiological bionics of directly connecting the nerves with the intestines and the female genital tract.
The invention has the advantages that the micro-nano flow control technology system is utilized to integrate the intestinal chip, the liver chip, the heart/brain chip and the female genital tract chip, on the basis, the physiological circulation process of a real human body is simulated through the structures such as the micro-fluidic pipeline, the micro-valve and the like, the human body micro-ecological system close to the real human body physiological state is constructed, an effective solution is provided for the problem that the difference exists between a research model and the human body in the research of the human body metabolism action of the human body microbial metabolite, the efficiency of the research process and the reliability of the research result are greatly improved, and a good research foundation is laid for the research on the human body microbial metabolite and the human body metabolism.
Drawings
FIG. 1 is a diagram of the whole chip structure of the human body micro-ecosystem.
Fig. 2 is a schematic diagram of a microfluidic channel in a PDMS a plate.
Fig. 3 is a schematic diagram of a micro-nanofluidic channel in a PDMS B plate.
FIG. 4 is a functional diagram of a chip of the micro-ecosystem of a human body.
FIG. 5 is a schematic diagram of the chip for realizing blood circulation bionic operation;
FIG. 6 is a schematic diagram of the chip for realizing blood circulation bionic operation;
FIG. 7 is an operational diagram of a chip implementing a multiple connection integration function;
FIG. 8 is a schematic diagram of the bionic operation of the chip for realizing the neural direct connection;
in the figure: 1PDMS A plate; 2PDMS B plate; 3 a porous membrane;
1-1 hepatic cell culture medium input interface; 1-2 a connecting channel of a culture medium input interface and a culture cavity; 1-3 liver cell culture chamber; 1-4 connecting channel of waste liquid output interface and culture cavity; 1-5 hepatic cell culture cavity waste liquid output interface; 1-6 connecting channels of the intestinal epithelial cell culture cavity and the hepatocyte culture cavity; 1-7 intestinal epithelial cell culture medium input interface; 1-8 intestinal epithelial cell culture chambers; 1-9 intestinal epithelial cell culture cavity waste liquid output interface; 1-10 connecting channels of the intestinal epithelial cell culture cavity and the genital tract cell culture cavity; 1-11 genital tract cell culture medium input interface; 1-12 genital tract cell culture cavities; 1-13 genital tract cell culture cavity waste liquid output interface; 1-14 connecting channels of the heart or nerve cell culture cavity and the reproductive tract cell culture cavity; 1-15 heart or nerve cell culture medium input interface; 1-16 heart or nerve cell culture chambers; 1-17 heart or nerve cell culture cavity waste liquid output interface; 1-18 connecting channels of the heart or nerve cell culture cavity and the liver cell culture cavity; 1-19 micro valves; 1-20 connecting channels of heart or nerve cell culture cavity and intestinal epithelial cell culture cavity;
2-1 hepatic cell culture medium input port; 2-2, a waste liquid outlet of the hepatocyte culture cavity; 2-3 inputting ports of bacterial liquid and microbial culture medium; 2-4 intestinal epithelial cell culture medium input ports; 2-5 intestinal epithelial cell culture cavity waste liquid outlet; 2-6 a waste liquid outlet of the microbial culture cavity; 2-7 genital tract cell culture medium input ports; 2-8 genital tract cell culture cavity waste liquid outlet; 2-9 heart or nerve cell culture medium input ports; 2-10 outlet of waste liquid from heart or nerve cell culture cavity; 2-11 microorganism culture chambers;
Detailed Description
The invention is further described below with reference to the accompanying drawings.
A micro-ecological system chip for accurately, simply and efficiently researching, evaluating and controlling the interaction between microbial metabolites of a human body and multiple organs of an individual is integrated with a multi-organ system.
The integrated human body micro-ecological chip of the multiple "organ" system shown in fig. 1 is composed of two layers of PDMS (polydimethylsiloxane) plates, the lower PDMS plate is referred to as PDMS a plate 1, and the upper PDMS plate is referred to as PDMS B plate 2. The two PDMS plates and a porous membrane 3 form a sandwich structure of PDMS A plate-porous membrane-PDMSB plate.
The lower PDMS A plate is shown in figure 2 and comprises 1-1 of hepatocyte culture medium input interface, 4 of culture medium input interface and culture cavity connecting channel 1-2, 1-3 of hepatocyte culture cavity, 4 of waste liquid output interface and culture cavity connecting channel 1-4, 1 of hepatocyte culture cavity waste liquid output interface 1-5, 1 of intestinal epithelial cell culture cavity and hepatocyte culture cavity connecting channel 1-6, 1 of intestinal epithelial cell culture medium input interface 1-7, 1 of intestinal epithelial cell culture cavity 1-8, 1 of intestinal epithelial cell culture cavity waste liquid output interface 1-9, 1 of intestinal epithelial cell culture cavity and female genital tract cell culture cavity connecting channel 1-10, 1 of female genital tract cell culture medium input interface 1-11, 1 of female genital tract cell culture cavity 1-12, 1-13 parts of female genital tract cell culture cavity waste liquid output interface, 1 part of connecting channel between heart or nerve cell culture cavity and female genital tract cell culture cavity, 1-15 parts of input interface of heart or nerve cell culture medium, 1-16 parts of heart or nerve cell culture cavity, 1-17 parts of waste liquid output interface between heart or nerve cell culture cavity and liver cell culture cavity, 1-18 parts of connecting channel between heart or nerve cell culture cavity and liver cell culture cavity, 1-19 parts of micro valve, and 1-20 parts of connecting channel between heart or nerve cell culture cavity and intestinal epithelial cell culture cavity. The hepatocyte culture chamber 1-3 at the left end of the PDMS A plate and the female genital tract cell culture chamber 1-12 at the right end of the PDMS A plate have the same shape and size, are parallel to each other in the direction, and are all elliptic squares consisting of upper and lower semi-circles at the two ends and a middle rectangle. The semi-circle diameters of the upper and lower ends of the ellipse are 300 μm, the middle rectangle is 400 μm long, 300 μm wide and 250 μm high. The heart or nerve cell culture cavity 1-16 at the upper end of the PDMS A plate and the intestinal epithelial cell culture cavity 1-8 at the lower end of the PDMS A plate have the same shape and size, are parallel to each other in the direction, are vertical to the hepatocyte culture cavity 1-3, and are both ellipses consisting of semicircles at the left end and the right end and a rectangle in the middle. The diameter of the semi-circle at the left end and the right end of the ellipse is 300 μm, the length of the middle rectangle is 450 μm, the width is 300 μm, and the height is 250 μm.
The porous membrane 3 is a polycarbonate membrane having a pore size of about 5 μm and a density of 1X 105Per cm2The method ensures that most microorganisms in the microorganism culture cavities 2-11 on the PDMS B plate do not enter the intestinal epithelial cell culture cavities 1-8 by a culture medium, thereby ensuring the normal growth of cells in the PDMS A plate, enabling the human microorganism metabolites to be in contact with multiple organs of the individual, and accurately and efficiently building a research platform of the human microorganism metabolites in the fields of medical research and human body related monitoring application.
The PDMS B plate 2 is shown in figure 3 and comprises 1 input port 2-1 of a hepatocyte culture medium, 1 output port 2-2 of waste liquid of a hepatocyte culture chamber, 1 input port 2-3 of a bacterial liquid and microbial culture medium, 1 input port 2-4 of an intestinal epithelial cell culture medium, 1 output port 2-5 of waste liquid of the intestinal epithelial cell culture chamber, 1 output port 2-6 of waste liquid of the microbial culture chamber, 1 input port 2-7 of a female genital tract cell culture medium, 1 output port 2-8 of waste liquid of the female genital tract cell culture chamber, 1 input port 2-9 of a heart or nerve cell culture medium, 1 output port 2-10 of waste liquid of the heart or nerve cell culture chamber and 1 output port 2-11 of waste liquid of the microorganism culture chamber. Wherein the shape and the size of the microorganism culture cavity 2-11 which is positioned right above the intestinal epithelial cell culture cavity 1-8 are completely consistent with the shape and the size of the intestinal epithelial cell culture cavity 1-8. A porous membrane is arranged between the microorganism culture cavity 2-11 and the intestinal epithelial cell culture cavity 1-8 to isolate the microorganism from the cells, thereby not only ensuring the normal growth of the cells, but also ensuring the normal contact of the human microorganism metabolites with the cells.
The intestinal chip consists of an intestinal epithelial cell culture cavity 1-8 on a PDMS A plate, a porous membrane and a PDMS B plate microorganism culture cavity 2-11. Wherein the left lower end of an intestinal epithelial cell culture cavity 1-8 at the lower end of the PDMS A plate is connected with 1 intestinal epithelial cell culture medium input interface 1-7 through a culture medium input interface and a culture cavity connecting channel 1-2, and the right lower end of the PDMS A plate is connected with 1 intestinal epithelial cell culture cavity waste liquid output interface 1-9 through a waste liquid output interface and a culture cavity connecting channel 1-4. The intestinal epithelial cell culture medium input interface 1-7 is communicated with the intestinal epithelial cell culture medium input port 2-4 on the PDMS B plate, and is externally connected with an injection pump to ensure that the intestinal epithelial cells smoothly enter the intestinal epithelial cell culture cavity 1-8. The waste liquid output interface 1-9 of the intestinal epithelial cell culture cavity is communicated with the waste liquid output port 2-5 of the intestinal epithelial cell culture cavity on the PDMS B plate, so that the redundant waste liquid of the intestinal epithelial cell culture cavity can smoothly flow out of the intestinal epithelial cell culture cavity 1-8. The upper left end of the intestinal epithelial cell culture cavity 1-8 is provided with 1 intestinal epithelial cell culture cavity and hepatocyte culture cavity connecting channel 1-6 for connecting the intestinal chip and the hepatocyte, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The middle part of the upper end of the intestinal epithelial cell culture cavity 1-8 is provided with 1 heart or nerve cell culture cavity and intestinal epithelial cell culture cavity connecting channel 1-20 for connecting the intestinal chip and the heart/brain chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The upper right end of the intestinal epithelial cell culture cavity 1-8 is provided with 1 intestinal epithelial cell culture cavity and female genital tract cell culture cavity connecting channel 1-10 for connecting the intestinal chip and the female genital tract chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The shape of the microorganism culture cavity 2-11 on the PDMS B plate is an ellipse square formed by semicircles at two ends and a rectangle in the middle, and the size of the ellipse square is consistent with that of the intestinal epithelial cell culture cavity 1-8. The left end of the microorganism culture cavity 2-11 is connected with 1 bacterial liquid and microorganism culture medium input port 2-3 through a micro-fluidic pipeline, and is used for injecting the liquid and the microorganism culture medium through an injection pump. Wherein, by controlling the oxygen content in the microorganism culture medium, three culture modes of high oxygen, low oxygen and no oxygen for microorganism culture can be realized. The right end of the microorganism culture cavity 2-11 is connected with a microorganism culture cavity waste liquid outlet 2-6 through a micro-fluidic pipeline for discharging redundant microorganism culture waste liquid. And an external conduit is connected behind the biological culture medium, the biological culture medium is opened only in the early stage of research, and is in a closed state in the rest time, so that the microbial culture waste liquid can smoothly enter the PDMS A plate.
The liver chip consists of liver cell culture cavities 1-3 on a PDMS A plate and an attached microfluidic pipeline. The left upper end of the hepatocyte culture chamber 1-3 is connected with 1 hepatocyte culture medium input interface 1-1 through a culture medium input interface and culture chamber connecting channel 1-2, and the left lower end is connected with 1 hepatocyte culture chamber waste liquid output interface 1-5 through a waste liquid output interface and culture chamber connecting channel 1-4. The hepatocyte culture medium input interface 1-1 is communicated with the hepatocyte culture medium input port 2-1 on the PDMS B plate, and is externally connected with an injection pump to ensure that hepatocytes smoothly enter the hepatocyte culture chamber 1-3. The waste liquid output interface 1-5 of the hepatocyte culture chamber is communicated with the waste liquid output port 2-2 of the hepatocyte culture chamber on the PDMS B plate, so that the redundant waste liquid of the hepatocyte culture chamber can smoothly flow out of the hepatocyte culture chamber 1-3. The upper right end of the hepatocyte culture chamber 1-3 is provided with 1 connecting channel 1-18 for connecting the hepatocyte or nerve cell culture chamber and the hepatocyte culture chamber for connecting the hepatocyte chip and the cardiac/brain chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The right lower end of the liver cell culture cavity 1-3 is provided with 1 intestinal epithelial cell culture cavity and liver cell culture cavity connecting channel 1-6 for connecting the liver chip and the intestinal chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow.
The heart/brain chip consists of heart or nerve cell culture cavities 1-16 on a PDMS A plate and a micro-fluidic pipeline attached to the heart or nerve cell culture cavities. The upper right end of the heart or nerve cell culture cavity 1-16 is connected with 1 heart or nerve cell culture medium input interface 1-15 through a culture medium input interface and a culture cavity connecting channel 1-2, and the upper left end is connected with 1 heart or nerve cell culture cavity waste liquid output interface 1-17 through a waste liquid output interface and a culture cavity connecting channel 1-4. The heart or nerve cell culture medium input interface 1-15 is communicated with the heart or nerve cell culture medium input port 2-9 on the PDMS B plate, and is externally connected with an injection pump to ensure that the heart or nerve cell smoothly enters the heart or nerve cell culture cavity 1-16. The waste liquid output interface 1-17 of the heart or nerve cell culture cavity is communicated with the waste liquid output port 2-10 of the heart or nerve cell culture cavity on the PDMS B plate, so that the redundant waste liquid of the liver cell culture cavity can smoothly flow out of the heart or nerve cell culture cavity 1-16. The left lower end of the heart or nerve cell culture cavity 1-16 is provided with 1 heart or nerve cell culture cavity and liver cell culture cavity connecting channel 1-18 for connecting the heart/brain chip and the intestine chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The middle part of the lower end of the heart or nerve cell culture cavity 1-16 is provided with 1 heart or nerve cell culture cavity and intestinal epithelial cell culture cavity connecting channel 1-20 for connecting the heart/brain chip and the intestinal chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow. The right lower end of the heart or nerve cell culture cavity 1-16 is provided with 1 heart or nerve cell culture cavity and female genital tract cell culture cavity connecting channel 1-14 for connecting the heart/brain chip and the female genital tract chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow.
The female genital tract chip consists of female genital tract cell culture cavities 1-12 on a PDMS A plate and a micro-fluidic pipeline attached to the female genital tract cell culture cavities. The right lower end of the female genital tract cell culture cavity 1-12 is connected with 1 female genital tract cell culture medium input interface 1-11 through a culture medium input interface and a culture cavity connecting channel 1-2, and the right upper end is connected with 1 female genital tract cell culture cavity waste liquid output interface 1-13 through a waste liquid output interface and a culture cavity connecting channel 1-4. The female genital tract cell culture medium input interface 1-11 is communicated with the female genital tract cell culture medium input interface 2-7 on the PDMS B plate, and is externally connected with an injection pump to ensure that female genital tract cells smoothly enter the female genital tract cell culture cavity 1-12. The female genital tract cell culture cavity waste liquid output interface 1-13 is communicated with a female genital tract cell culture cavity waste liquid output port 2-8 on the PDMS B plate, so that the redundant female genital tract cell culture cavity waste liquid can smoothly flow out of the female genital tract cell culture cavity 1-12. The left upper end of the female genital tract cell culture cavity 1-12 is provided with 1 heart or nerve cell culture cavity and female genital tract cell culture cavity connecting channel 1-14 for connecting the heart/brain chip and the female genital tract chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the flowing fluid. The left lower end of the female genital tract cell culture cavity 1-12 is provided with 1 intestinal epithelial cell culture cavity and female genital tract cell culture cavity connecting channel 1-10 for connecting the female genital tract chip and the intestinal chip, and the middle of the channel is provided with 1 microvalve 1-19 for controlling the on-off of the fluid flow.
All the connecting channels mentioned above are micro-nano flow control channels, with the length of 200-. All mentioned medium input ports and waste liquid output ports are circles with a diameter of 200-.
After the human body micro-ecosystem chip is successfully manufactured through photoetching, reverse molding and the like, the micro-ecosystem chip can be used for microbial culture only by carrying out sterilization treatment, and the aim is to ensure the accuracy of research. The specific operation flow comprises the steps of firstly wiping the surface of the micro-nano fluidic control chip by using 75% alcohol, cleaning the surface, then putting the micro-nano fluidic control chip into a super-clean workbench, and irradiating for 2 hours by using an ultraviolet sterilizing lamp. After sterilization, the bubble removal operation is required, and the specific flow is to close the waste liquid outlet 2-2 of the hepatocyte culture chamber, the waste liquid outlet 2-5 of the intestinal epithelial cell culture chamber, the waste liquid outlet 2-8 of the female genital tract cell culture chamber and the waste liquid outlet 2-10 of the cardiac or nerve cell culture chamber shown in the figure 3, and open the waste liquid outlet 2-6 of the microbial culture chamber. 75% alcohol is sequentially introduced from a liver cell culture medium inlet 2-1, a bacterial liquid and microorganism culture medium inlet 2-3, an intestinal epithelial cell culture medium inlet 2-4, a female genital tract cell culture medium inlet 2-7 and a heart or nerve cell culture medium inlet 2-9 by using a syringe pump to prevent bubbles, deionized water and PBS0.01M from being generated inside the chip, and the pH value is 7.4. After the whole micro-fluidic pipeline is filled with the introduced liquid, the injection pump is closed after a certain volume of PBS buffer solution is collected from a waste liquid outlet 2-6 of the microbial culture cavity, and the bubble removal operation is finished. After sterilization and bubble removal, the inoculation operation of related human body cells and microorganisms is carried out, and the specific flow is that after all culture medium input ports and output ports shown in figure 3 are opened, 200 mug/mL collagen I solution is introduced from a liver cell culture medium input port 2-1, an intestinal epithelial cell culture medium input port 2-4, a female genital tract cell culture medium input port 2-7 and a heart or nerve cell culture medium input port 2-9 by using an injection pump, the collagen solution is filled in all cell culture chambers for a period of time as far as possible, and then the human body microecological chip is placed in a thermostat at 37 ℃ for incubation for 2h to enhance the adhesion between the collagen and the PDMS plate, further enhance the adhesion between the cells and the cell culture chambers, and then the human body cells can be inoculated. The specific operation of the human body cells and the microorganisms is to ensure that all input ports and output ports are in an open state, human body liver tissue cells are slowly injected into a liver cell culture medium input port 2-1 by using an injection pump, human body intestinal epithelial cells are slowly injected into an intestinal epithelial cell culture medium input port 2-4, female genital tract tissue cells are slowly injected into a female genital tract cell culture medium input port 2-7, myocardial tissue cells or related nerve tissue cells are slowly injected into a heart or nerve cell culture medium input port 2-9, and then the human body microecological chip is placed into a thermostat to be incubated for 1 hour at 37 ℃ to complete cell inoculation work. And then, carrying out inoculation operation of related microorganisms, specifically, slowly injecting bacterial liquid of corresponding microorganisms into the bacterial liquid and microorganism culture medium input ports 2-3 by using an injection pump, and then incubating for 1h at 37 ℃ in a thermostat, thereby completing all inoculation operations.
The human body micro-ecological chip realizes the bionics of the physiological circulation of 'intestine-liver-heart/brain-female genital tract-intestine' shown in figure 4 mainly by controlling the working state of the corresponding micro-valve, the injection pump and the on-off of the corresponding waste liquid output port, thereby simulating the blood circulation of a real human body. The specific operation flow is shown in fig. 5, after all injection pumps, culture medium inlets and waste liquid outlets in the control micro-ecological chip are in a closed state, the connecting channels 1-6 of the intestinal epithelial cell culture cavity and the liver cell culture cavity, the connecting channels 1-18 of the heart or nerve cell culture cavity and the liver cell culture cavity and the micro valves 1-19 on the connecting channels 1-14 of the heart or nerve cell culture cavity and the female genital tract cell culture cavity are opened, and the waste liquid outlets 2-8 of the female genital tract cell culture cavity are opened. Injecting a cell liquid culture medium into an intestinal epithelial cell culture medium input port 2-4 by using an injection pump, injecting a microorganism liquid culture medium into a bacterial liquid and microorganism culture medium input port 2-3, wherein the culture medium carries human microorganism metabolites to sequentially pass through an intestinal chip, a liver chip and a heart/brain chip, and finally is discharged from a female genital tract cell culture cavity waste liquid output port 2-8 to form the intestinal-liver-heart/brain chip. On the basis, when the waste liquid discharged from the waste liquid outlet 2-8 of the female genital tract cell culture cavity is collected, as shown in figure 6, the injection of the culture medium into the waste liquid outlet 2-4 of the intestinal epithelial cell culture cavity and the inlet 2-3 of the bacterial liquid and microorganism culture medium is stopped, the waste liquid outlet 2-8 of the female genital tract cell culture cavity is closed, the micro valves 1-19 on the waste liquid outlet 2-5 of the intestinal epithelial cell culture cavity and the connecting channel 1-10 of the intestinal epithelial cell culture cavity and the female genital tract cell culture cavity are opened, the micro valves 1-19 on the connecting channel 1-6 of the intestinal epithelial cell culture cavity and the liver cell culture cavity are closed, and then the injection pump is used for injecting the cell liquid culture medium into the inlet 2-1 of the liver cell culture cavity. The injected liquid culture medium is finally discharged from a waste liquid outlet 2-5 of the intestinal epithelial cell culture cavity to form the chip of 'heart/brain-female genital tract-intestine'. The bionics of the physiological circulation of the intestines-liver-heart/brain-female genital tract-intestines is realized by controlling the working state of the corresponding micro valve and the injection pump and the on-off of the corresponding waste liquid outlet. In addition, corresponding nerve cells of different species are cultured in the cell culture cavities 1-3, 1-8, 1-12 and 1-16, and the simulation of the cerebrospinal fluid circulation of the real human body is realized according to the same operation mode.
The cells cultured in the cell culture chamber of the human micro-ecological chip are not limited to the intestinal epithelial cells, liver cells, cardiac muscle cells, nerve cells, female genital tract cells mentioned above, and the chip can be connected many times to meet the demand of integrating more 'organs'. The specific operation flow is shown in fig. 7, after all injection pumps, culture medium input ports and waste liquid output ports in the micro-ecological chip are controlled to be in a closed state, micro valves 1-19 on three channels, namely culture cavity connecting channels 1-6, culture cavity connecting channels 1-18 and culture cavity connecting channels 1-14 are opened, and waste liquid output ports 2-8 of the female genital tract cell culture cavity are opened. And (3) injecting a culture medium into the intestinal epithelial cell culture medium inlet 2-4 by using a syringe pump, and enabling the culture medium to flow out from the female reproductive tract cell culture cavity waste liquid outlet 2-8. The flowing culture medium flows in from the cell culture medium input port 2-4 of the human body micro-ecological chip with the same structure and the same on-off state of the culture medium input port and the waste liquid output port, and flows out from the waste liquid output port 2-8 of the cell culture cavity of the second human body micro-ecological chip, and the circulation is carried out, so that the function of connecting the same chip for multiple times is realized. Wherein, the liver cell culture cavity 1-3 and the heart or nerve cell culture cavity 1-16 in each human body micro-ecological chip can culture human body cells except liver cells, cardiac muscle cells and nerve cells, which is convenient for meeting the bionic requirement of more complex human body physiological circulation.
The human body micro-ecological chip can realize physiological bionics of a nerve direct connection intestine and a female genital tract, and is used for researching neuron loop researches between a human brain and human body intestinal tracts and between the human brain and the female genital tract. As shown in FIG. 8, the specific operation flow is to culture intestinal epithelial cells in the intestinal epithelial cell culture chambers 1-8, female genital tract cells in the female genital tract cell culture chambers 1-12, and nerve cells in the heart or nerve cell culture chambers 1-16. After all injection pumps, culture medium input ports and waste liquid output ports in the micro-ecological chip are controlled to be in a closed state, micro valves 1-19 on three channels, namely a culture cavity connecting channel 1-20, a culture cavity connecting channel 1-10 and a culture cavity connecting channel 1-14, are opened. After the waste liquid outlet 2-5 of the intestinal epithelial cell culture cavity is opened, a culture medium is injected into the heart or nerve cell culture medium inlet 2-9 by using an injection pump, and the injected culture medium flows out from the waste liquid outlet 2-5 of the intestinal epithelial cell culture cavity. The process promotes the connection between nerve cells and intestinal epithelial cells and female genital tract cells, thereby realizing the physiological bionics of directly connecting the nerves with the intestines and the female genital tract.

Claims (10)

1. A human body micro-ecosystem chip is characterized in that the human body micro-ecosystem chip comprises two layers of polydimethylsiloxane plates and a porous membrane (3) between the two layers, a PDMS A plate (1) is positioned below the porous membrane, and a PDMSB plate (2) is positioned above the porous membrane, and is used for researching the interaction between human body microorganism metabolites and individual multiple organs under the condition of approaching to the real human body physiological circulation;
the lower PDMS A plate (1) comprises a hepatocyte culture cavity (1-3) positioned at the left end, an intestinal epithelial cell culture cavity (1-8) positioned at the lower end, a female genital tract cell culture cavity (1-12) positioned at the right end and a heart or nerve cell culture cavity (1-16) positioned at the upper end of the plate, wherein two ends of the four cell culture cavities are respectively communicated with a cell culture medium input interface and a cell culture medium output interface through connecting channels; the upper end of the liver cell culture cavity (1-3) is communicated with the left end of the heart or nerve cell culture cavity (1-16) through a connecting channel (1-18), the lower end of the liver cell culture cavity is communicated with the left end of the intestinal epithelial cell culture cavity (1-8) through a connecting channel (1-6), the upper end of the female genital tract cell culture cavity (1-12) is communicated with the right end of the heart or nerve cell culture cavity (1-16) through a connecting channel, the lower end of the female genital tract cell culture cavity is communicated with the right end of the intestinal epithelial cell culture cavity (1-8) through a connecting channel (1-10), and the middle part of the heart or nerve cell culture cavity (1-16) is communicated with the middle part of the intestinal epithelial cell culture cavity (1-8) through a connecting channel (1-20); micro valves (1-19) are arranged in the middle of the five connecting channels and are used for controlling the on-off of the flow of the fluid; the shape and the size of the liver cell culture cavity (1-3) are consistent with those of the female genital tract cell culture cavity (1-12), the directions are parallel to each other, the shape and the size of the heart or nerve cell culture cavity (1-16) are consistent with those of the intestinal epithelial cell culture cavity (1-8), the directions are parallel to each other, the liver cell culture cavity (1-3) is vertical to the intestinal epithelial cell culture cavity (1-8), and the female genital tract cell culture cavity (1-12) is vertical to the heart or nerve cell culture cavity (1-16); the four culture chambers are all of an oval structure and comprise two semi-circles at the end parts and a middle rectangle;
the PDMS B plate (2) comprises four cell culture medium input ports and four cell culture cavity waste liquid output ports, corresponds to and is communicated with cell culture medium input interfaces and cell culture medium output interfaces at two ends of four cell culture cavities on the PDMS A plate (1) respectively, and is externally connected with an injection pump; the device also comprises 1 microorganism culture cavity (2-11), the left end of the microorganism culture cavity (2-11) is communicated with the bacteria liquid and microorganism culture medium input ports (2-3) through a connecting channel and is used for inputting the bacteria liquid and the microorganism culture medium by an injection pump, and the right end of the microorganism culture cavity is communicated with the microorganism culture cavity waste liquid output port (2-6) through the connecting channel and is used for discharging redundant microorganism culture waste liquid; an external conduit is connected behind the biological culture medium, the biological culture medium is opened only in the early stage of research, and is in a closed state in the rest time, so that the microbial culture waste liquid can smoothly enter the PDMS A plate (1); the microorganism culture cavity (2-11) is positioned right above the intestinal epithelial cell culture cavity (1-8), the shapes and the sizes of the two cavities are completely consistent, a layer of porous membrane (3) is arranged between the microorganism culture cavity (2-11) and the intestinal epithelial cell culture cavity (1-8) to isolate microorganisms from cells, so that the metabolites of human microorganisms are normally contacted with the cells while the normal growth of the cells is ensured;
the sandwich structure of the PDMS A plate-porous membrane-PDMS B plate can respectively form an intestine chip, a liver chip, a heart/brain chip and a female genital tract chip, and the bionics of physiological circulation of intestine-liver-heart/brain-female genital tract-intestine is realized by controlling the working state of a corresponding micro valve, an injection pump and the on-off of a corresponding waste liquid output port; the method comprises the following specific steps:
the intestinal chip comprises an intestinal epithelial cell culture cavity (1-8) on a PDMS A plate, a porous membrane (3), a PDMS B plate microorganism culture cavity (2-11) and corresponding microfluidic pipelines, input interfaces and output interfaces thereof, and a microorganism culture cavity (2-11) on a PDMS B plate and corresponding microfluidic pipelines, input interfaces and output interfaces thereof; wherein, the intestinal epithelial cell culture medium input interface (1-7) is communicated with the intestinal epithelial cell culture medium input port (2-4) on the PDMS B plate, and is externally connected with an injection pump to ensure that the intestinal epithelial cells smoothly enter the intestinal epithelial cell culture cavity (1-8); the waste liquid output interface (1-9) of the intestinal epithelial cell culture cavity is communicated with a waste liquid output port (2-5) of the intestinal epithelial cell culture cavity on the PDMS B plate, so that the redundant waste liquid of the intestinal epithelial cell culture cavity can smoothly flow out of the intestinal epithelial cell culture cavity (1-8);
the liver chip comprises a liver cell culture cavity (1-3) on a PDMS A plate, a micro-fluidic pipeline, an input interface and an output interface which correspond to the liver cell culture cavity, a liver cell culture medium input port (2-1) on a PDMS B plate, and a waste liquid output port (2-2) of the liver cell culture cavity; the hepatocyte culture medium input interface (1-1) is communicated with the hepatocyte culture medium input port (2-1) on the PDMS B plate, and is externally connected with an injection pump to ensure that hepatocytes smoothly enter the hepatocyte culture chamber (1-3); the waste liquid output interface (1-5) of the hepatocyte culture cavity is communicated with a waste liquid output port (2-2) of the hepatocyte culture cavity on the PDMS B plate, so that the redundant waste liquid of the hepatocyte culture cavity can smoothly flow out of the hepatocyte culture cavity (1-3);
the heart/brain chip comprises heart or nerve cell culture cavities (1-16) on a PDMS A plate and micro-fluidic pipelines, input and output interfaces attached to the heart or nerve cell culture cavities, heart or nerve cell culture medium input ports (2-9) on a PDMS B plate, and waste liquid output ports (2-10) of the heart or nerve cell culture cavities; the heart or nerve cell culture medium input interface (1-15) is communicated with the heart or nerve cell culture medium input port (2-9) on the PDMS B plate, and is externally connected with an injection pump to ensure that the heart or nerve cells smoothly enter the heart or nerve cell culture cavity (1-16); the waste liquid output interface (1-17) of the heart or nerve cell culture cavity is communicated with a waste liquid output port (2-10) of the heart or nerve cell culture cavity on the PDMS B plate, so that the redundant waste liquid of the liver cell culture cavity can smoothly flow out of the heart or nerve cell culture cavity (1-16);
the female genital tract chip controls a female genital tract cell culture cavity (1-12) on the PDMS A plate and a micro-fluidic pipeline, an input interface and an output interface which are attached to the female genital tract cell culture cavity, a female genital tract cell culture medium input port (2-7) on the PDMS B plate and a female genital tract cell culture cavity waste liquid output port (2-8); the female genital tract cell culture medium input interface (1-11) is communicated with a female genital tract cell culture medium input port (2-7) on the PDMS B plate, and is externally connected with an injection pump to ensure that female genital tract cells smoothly enter the female genital tract cell culture cavity (1-12); the female genital tract cell culture cavity waste liquid output interface (1-13) is communicated with a female genital tract cell culture cavity waste liquid output port (2-8) on the PDMS B plate, so that the redundant female genital tract cell culture cavity waste liquid can smoothly flow out of the female genital tract cell culture cavity (1-12).
2. The micro-ecosystem chip for human body according to claim 1, wherein the micro-ecosystem chip for human body can realize three culture modes of high oxygen, low oxygen and no oxygen by controlling the oxygen content in the microorganism culture medium.
3. The micro ecosystem chip for human body according to claim 1, wherein the oval dimensions of the liver cell culture chamber (1-3) and the female genital tract cell culture chamber (1-12) are: the diameter of the upper and lower end semi-circle is 200-.
4. The micro ecosystem chip for human body according to claim 1, wherein the ellipse of the heart or nerve cell culture chamber (1-16) and the intestinal epithelial cell culture chamber (1-8) has the following dimensions: the diameters of the left and right semi-circles are 200-.
5. The micro-ecosystem chip for human body according to claim 1, wherein all the connection channels are micro-nano fluidic channels with a length of 200-600 μm, a width of 50-150 μm and a height of 100-300 μm.
6. The micro-ecosystem chip for human body according to claim 1, wherein the sizes and shapes of all the culture medium input ports and the waste liquid output ports are circles with a diameter of 200- "400 μm".
7. The personal micro-ecosystem chip according to claim 1, wherein the porous membrane (3) is a polycarbonate membrane with a pore size of 0.01 to 0.5 μm and a density of 1 x 103-1×106Per cm2。
8. The use method of the human microecological system chip according to any one of claims 1 to 7, wherein the human microecological chip is used for culturing corresponding nerve cells of different species in the cell culture chamber (1-3), the cell culture chamber (1-8), the cell culture chamber (1-12) and the cell culture chamber (1-16), and by controlling the working state of the corresponding microvalve, the injection pump and the on-off of the corresponding waste liquid outlet, the bionic of the physiological circulation of the 'intestine-liver-heart/brain-female genital tract-intestine' is realized, so as to simulate the blood circulation of a real human body, and the specific operation flow is as follows: after all injection pumps, culture medium input ports and waste liquid output ports in the micro-ecological chip are controlled to be in a closed state, micro valves (1-19) on connecting channels (1-14) of an intestinal epithelial cell culture cavity and a liver cell culture cavity, connecting channels (1-18) of a heart or nerve cell culture cavity and the liver cell culture cavity and connecting channels (1-14) of the heart or nerve cell culture cavity and a female genital tract cell culture cavity are opened, and waste liquid output ports (2-8) of the female genital tract cell culture cavity are opened; injecting a cell liquid culture medium into an intestinal epithelial cell culture medium input port (2-4) by using an injection pump, injecting a microorganism liquid culture medium into a bacterial liquid and microorganism culture medium input port (2-3), wherein the culture medium carries human microorganism metabolites to sequentially pass through an intestinal chip, a liver chip and a heart/brain chip, and finally is discharged from a female genital tract cell culture cavity waste liquid output port (2-8) to form an intestinal-liver-heart/brain chip; on the basis, when waste liquid discharged from a waste liquid outlet (2-8) of a female genital tract cell culture cavity is collected, the injection of the culture medium into an intestinal epithelial cell culture cavity inlet (2-4) and a bacteria liquid and microorganism culture medium inlet (2-3) is stopped, the waste liquid outlet (2-8) of the female genital tract cell culture cavity is closed, a micro valve (1-19) on a connecting channel (1-10) of the intestinal epithelial cell culture cavity waste liquid outlet (2-5) and the intestinal epithelial cell culture cavity and the female genital tract cell culture cavity is opened, a micro valve (1-19) on a connecting channel (1-6) of the intestinal epithelial cell culture cavity and a hepatocyte culture cavity is closed, then a cell liquid culture medium is injected into a hepatocyte culture medium inlet (2-1) by using an injection pump, and the injected liquid culture medium is finally discharged from the waste liquid outlet (2-5) of the intestinal epithelial cell culture cavity, forming a chip of 'heart/brain-female genital tract-intestine'; bionic of physiological circulation of intestine-liver-heart/brain-female genital tract-intestine is realized by controlling the working state of the corresponding micro valve and the injection pump and the on-off of the corresponding waste liquid outlet; in addition, corresponding nerve cells of different species can be cultured in the cell culture cavities (1-3), (1-8), (1-12) and (1-16), and the simulation of the cerebrospinal fluid circulation of the real human body can be realized according to the same operation mode.
9. The use method of the human body micro-ecosystem chip of any one of claims 1 to 7, characterized in that the chip of the human body micro-ecosystem chip can be connected for many times to meet the demand of integrating more 'organs'; the specific operation flow is that after all injection pumps, culture medium input ports and waste liquid output ports in the micro-ecological chip are controlled to be in a closed state, micro valves (1-19) on three channels, namely a culture cavity connecting channel (1-6), a culture cavity connecting channel (1-18) and a culture cavity connecting channel (1-14), are opened, and a waste liquid output port (2-8) of a female genital tract cell culture cavity is opened; injecting a culture medium into the intestinal epithelial cell culture medium inlet (2-4) by using a syringe pump, wherein the culture medium flows out from the female genital tract cell culture cavity waste liquid outlet (2-8); the flowing culture medium flows into the cell culture medium inlet (2-4) of the human body micro-ecological chip with the same second block structure and the same on-off state of the culture medium inlet and the waste liquid outlet, and flows out of the cell culture cavity waste liquid outlet (2-8) of the second human body micro-ecological chip, and the function of connecting the same chip for multiple times is realized by the circulation; wherein, the liver cell culture cavity (1-3) and the heart or nerve cell culture cavity (1-16) in each human body micro-ecological chip can culture human body cells except liver cells, cardiac muscle cells and nerve cells, and meet the bionic demand of more complex human body physiological circulation.
10. The use method of the human microecological system chip according to any one of claims 1 to 7, wherein the human microecological chip can realize physiological bionics in which nerves directly connect intestines and female genital tracts, so as to study the neuron circuit between the human brain and the human intestinal tract and female genital tract; the specific operation process comprises the steps of culturing intestinal epithelial cells in an intestinal epithelial cell culture cavity (1-8), culturing female genital tract cells in a female genital tract cell culture cavity (1-12), and culturing nerve cells in a heart or nerve cell culture cavity (1-16); after all injection pumps, culture medium input ports and waste liquid output ports in the micro-ecological chip are controlled to be in a closed state, micro valves (1-19) on three channels, namely a culture cavity connecting channel (1-20), a culture cavity connecting channel (1-10) and a culture cavity connecting channel (1-14) are opened; after opening the waste liquid outlet (2-5) of the intestinal epithelial cell culture cavity, injecting a culture medium into the heart or nerve cell culture medium inlet (2-9) by using a syringe pump, and enabling the injected culture medium to flow out of the waste liquid outlet (2-5) of the intestinal epithelial cell culture cavity; the process promotes the connection between nerve cells and intestinal epithelial cells and female genital tract cells, thereby realizing the physiological bionics of directly connecting the nerves with the intestines and the female genital tract.
CN201911110425.7A 2019-11-14 2019-11-14 Human body micro-ecosystem chip and use method thereof Active CN110669670B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911110425.7A CN110669670B (en) 2019-11-14 2019-11-14 Human body micro-ecosystem chip and use method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911110425.7A CN110669670B (en) 2019-11-14 2019-11-14 Human body micro-ecosystem chip and use method thereof

Publications (2)

Publication Number Publication Date
CN110669670A CN110669670A (en) 2020-01-10
CN110669670B true CN110669670B (en) 2022-06-03

Family

ID=69087289

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911110425.7A Active CN110669670B (en) 2019-11-14 2019-11-14 Human body micro-ecosystem chip and use method thereof

Country Status (1)

Country Link
CN (1) CN110669670B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106811413A (en) * 2015-11-30 2017-06-09 中国科学院大连化学物理研究所 Multiple organ chip based on microflow control technique and preparation method thereof
CN109576155A (en) * 2018-12-18 2019-04-05 大连理工大学 General nervous system chip
CN109837237A (en) * 2017-11-28 2019-06-04 中国科学院大连化学物理研究所 A kind of micro-fluidic intestines chip and its application based on nitrocellulose basilar memebrane

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2712918T3 (en) * 2012-09-28 2015-02-16 Tissuse Gmbh Multi-organ-chip with improved lifetime and homeostasis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106811413A (en) * 2015-11-30 2017-06-09 中国科学院大连化学物理研究所 Multiple organ chip based on microflow control technique and preparation method thereof
CN109837237A (en) * 2017-11-28 2019-06-04 中国科学院大连化学物理研究所 A kind of micro-fluidic intestines chip and its application based on nitrocellulose basilar memebrane
CN109576155A (en) * 2018-12-18 2019-04-05 大连理工大学 General nervous system chip

Also Published As

Publication number Publication date
CN110669670A (en) 2020-01-10

Similar Documents

Publication Publication Date Title
WO2022134294A1 (en) Detachable and reusable hydrophobic or super-hydrophobic microfluidic organ-on-a-chip
CN107312713A (en) A kind of micro-fluidic chip and its application
CN106581761A (en) Artificial liver tissue and preparation method thereof
CN106811413A (en) Multiple organ chip based on microflow control technique and preparation method thereof
CN207576440U (en) A kind of micro-fluidic chip of pneumatic operated valve control
CN111218404A (en) Bionic multi-organ chip and preparation method and application thereof
CN107312711B (en) Self-circulation tissue/organ chip device and manufacturing method thereof
CN108913592A (en) A kind of organ storage in vitro and culture apparatus and its method
RU171690U1 (en) Microfluidic Chip for the Creation of Cellular Models of Mammalian Organs
CN108485975A (en) Bionical chip kidney
US20160130543A1 (en) Modular Microtube Network for Vascularized Organ-On-A-Chip Models
CN107570246A (en) A kind of nematode chip and its microfluidic methods for selection of antibacterial
CN110669670B (en) Human body micro-ecosystem chip and use method thereof
CN207760352U (en) Media to cell culture modules, culture solution module, cell cultivation chip and cell culture cabinet
CN107955787B (en) Bionic intestine model construction method based on microfluidic technology
CN220166205U (en) Organ-like co-culture chip
US20210108178A1 (en) Systems and methods for multilane vasculature
CN208472114U (en) Bionical chip kidney
CN201643113U (en) Control system for two-way perfusion type bioreactor
CN107418891A (en) A kind of perfusion type bladder bacterial biof iotalm dynamic is incubated simulation system
CN220317828U (en) Parallel organoid chip system for evaluating placenta-peripheral organ adaptation of pregnant women
CN220413410U (en) Series-connected organoid chip system for evaluating preeclampsia multi-organ damage
CN219490026U (en) Three-dimensional dynamic culture colorectal tissue-like tumor chip
CN114437932B (en) Microfluidic chip, system and using method of multi-placenta vascular anastomosis model
CN212128201U (en) Cell co-culture system for simulating hepatic sinus

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant