CN116333881A

CN116333881A - Three-channel organ chip and application and use method thereof

Info

Publication number: CN116333881A
Application number: CN202310250066.5A
Authority: CN
Inventors: 邱京晶; 孟家旭; 徐祎春; 韩峻松; 苏军; 丁岩汀
Original assignee: SHANGHAI BIOCHIP CO Ltd
Current assignee: SHANGHAI BIOCHIP CO Ltd
Priority date: 2023-03-15
Filing date: 2023-03-15
Publication date: 2023-06-27

Abstract

The invention provides a three-channel organ chip, which comprises a cover body and a base body, wherein the cover body is provided with a gas channel, and the base body is provided with a plurality of cell culture holes and a plurality of culture liquid channels; the gas channel is in fluid communication with the cell culture well, and the fluid within the gas channel and/or the culture fluid channel remains flowing under the force of an external force. According to the three-channel organ chip, the flowable gas channel and the culture liquid channel are introduced, so that a flowing gas-liquid environment is formed in the chip, gas and liquid shearing force can be provided for cells cultured on the chip, the complexity of the cells capable of receiving external stimulus factors is increased, and the tumor in-vivo environment is fully simulated. In addition, by arranging a plurality of groups of culture units, different culture items can be simultaneously carried out on one chip, so that the detection flux is improved, multi-component lung cancer related cells are co-cultured, and culture liquid perfusion of different components is simultaneously carried out, so that the method is more suitable for establishing an accurate and efficient in-vitro drug screening platform.

Description

Three-channel organ chip and application and use method thereof

Technical Field

The invention relates to the technical field of 3D tumor cell culture chips, in particular to a three-channel organ chip and application and use methods thereof.

Background

Among various malignant tumors, lung cancer always occupies the first place of morbidity and mortality, and is a large killer seriously harming human health. The patients are often treated by surgical operation and traditional chemotherapy in clinic; in recent years, new drugs are continuously emerging, the application of tumor immunotherapy greatly widens the application range of patients, but the application range of surgery and chemotherapy is limited, and the existing targeted drugs cannot cover all patients, so that the five-year survival rate of clinical lung cancer patients is always less than 20%. Therefore, the preparation method has very practical clinical significance and economic value in the field of development of new anti-lung cancer drugs.

2D cell culture is an important experimental means for researching the occurrence and development rules of diseases and early research of medicines, but the single and planar cell culture mode leads the cells to lose the original interaction with other cells and extracellular matrixes in a body environment, and after the cells lose the extracellular information transmission modes, the original state and the gene expression mode of the cells are also greatly changed, so that positive data obtained in a cell experiment can not always obtain ideal results after in vivo experimental verification; however, although the experimental results of animals are more reliable, the experimental cost of experimental animals, especially non-human primates, is often very high, in recent years, the requirements of multi-national government authorities on animal welfare and ethics are put forward, the development of animal experiments is greatly hindered, and more importantly, the physiological environment of mice and even primates' monkeys is not completely similar to human body, and the in vitro and in vivo experimental results are greatly different, so that it is very necessary to develop an in vitro experimental platform capable of better simulating the human body environment.

The 3D culture technology is a complex in-vitro cell culture means integrating multiple types of cells and extracellular matrixes, and can greatly enrich the types of external stimulus factors acceptable by cells in the in-vitro culture process, so that the in-vivo environment of a human body can be reflected more truly. On the basis, a working platform formed by mounting a 3D culture system on an automatically controllable microfluidic chip is called an organ chip, is hopeful to replace the traditional cell culture, and becomes a main cell experiment form in the subsequent drug research and development process.

The existing lung organ chip often has the problems that the flux is low, the in-vivo environment cannot be well simulated, and the like, and the purpose of the application is to provide the high-flux lung cancer organ chip which is provided with a 3D tumor microsphere culture system and can fully simulate the in-vivo environment of tumors based on a microfluidic system pair.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, a main object of the present invention is to provide a three-way organ chip, which can be loaded with a 3D tumor microsphere culture system and fully simulate the in vivo environment of a tumor on the basis of the three-way organ chip. Other objects of the invention are also: 1) By arranging a plurality of groups of detection channels, the detection flux is improved, different culture projects can be simultaneously carried out on one chip, and for example, the inhibition effect detection of different drugs on tumor cells can be realized on one chip; 2) And by combining a microfluidic technology, the cell culture is automatically controlled so as to improve the experimental efficiency.

To achieve the above and other related objects, the present invention is achieved by comprising the following technical solutions.

The first aspect of the invention provides a three-way organ chip, which comprises a cover body and a base body, wherein the cover body comprises a gas channel, and the base body is provided with a plurality of cell culture holes and a plurality of culture liquid channels; the gas channel is in fluid communication with the cell culture well, preferably the gas channel is in direct fluid communication with the cell culture well, and the fluid within the gas channel and the culture fluid channel is maintained in flow under the force of an external force. By introducing the gas channel and the culture liquid channel into the chip, the gas channel and the culture liquid channel can form a flowing environment through an external driving pump, and multi-component lung cancer related cell co-culture can be realized, so that the in-vivo tumor environment can be fully simulated.

In some preferred embodiments, the cell culture well is provided with a porous partition membrane at the connection point with the culture liquid channel, the porous partition membrane plays a role of interval, so as to limit the cells in the cell culture well to enter the culture liquid channel, and the existence of the porous membrane forms a liquid exchange plane, so that liquid, small molecules and some biological macromolecules can permeate.

In some preferred embodiments, the upper membrane surface of the porous spacer membrane is provided with a low cell adhesion layer, and the material of the low cell adhesion layer is a simulated extracellular matrix component; preferably, the material of the cell blocking layer is matrigel. For reducing the cytophilicity of the attachment surface. The cell-blocking layer is formed by dripping matrigel into the bottom of the hole with a micropipette under the operating environment of 4 ℃, transferring to the environment of 37 ℃ and standing for 10 minutes.

In some preferred embodiments, the plurality of cell culture wells are arranged in a plurality of rows, and a culture fluid channel is arranged below each row of cell culture wells; the cell culture holes are not communicated, and the culture liquid channels are not communicated. That is, each row of cell culture holes and the corresponding culture liquid channels form a relatively independent and closed culture unit, so that the aim of simultaneously detecting the growth inhibition capability of different drugs on the same chip can be fulfilled.

In some preferred embodiments, the cover is provided with at least two vent holes, and the preferred vent holes protrude out of the cover, i.e. the protruding cover forms a section of external connection pipe, each vent hole is an inlet and outlet for fluid in the gas channel, and each vent hole is used for externally connecting a vent pipe, and the vent pipe is communicated with the driving pump. The cover body is provided with a plurality of culture solution infusion ports, each culture solution channel at least corresponds to two culture solution infusion ports, and two ends of each culture solution channel are respectively in fluid communication with the two culture solution infusion ports. Preferably, the culture solution channel comprises a long channel and an uplink channel arranged at two ends of the long channel; the long channel is positioned below the cell culture hole; one end of the ascending channel is communicated with the long channel, the other end of the ascending channel protrudes out of the substrate and forms an outer interface of the culture liquid channel, and the outer interface of the culture liquid channel is used for externally connecting a liquid pipeline. More specifically, each culture liquid channel corresponds to two culture liquid infusion ports on the cover body and a culture liquid channel external interface on the base body, and the culture liquid channel external interface on the base body is communicated with the liquid through pipeline and the driving pump, so that automatic control of fluid is realized. Preferably, a closed-loop unidirectional flow mode is realized through the connection mode of each vent hole, the culture solution infusion port, the external port of the culture solution channel, the external driving pump and the fluid pipeline. It is known to a person skilled in the art how a unidirectional flow pattern is formed, for example by arranging an inlet and an outlet in sequence along a certain direction, the inlet and the outlet being respectively externally connected to a driving pump.

The second aspect of the invention provides a three-way organ-chip system comprising the three-way organ-chip described above and a drive pump.

The third aspect of the invention provides a method for using the three-way organ chip or system in researching the killing effect of candidate anti-tumor drugs on lung cancer cells, comprising the following steps:

1) Obtaining lung tumor microspheres; preferably, the lung tumor microspheres are obtained by mixed culture of human lung cancer cells and human respiratory epithelial cells; more preferably, the tumor microspheres seeded in the chip should have a diameter greater than 300. Mu.M;

2) Preparing endothelial cell suspension; preferably, the cell suspension comprises human vascular endothelial cells;

3) Injecting the cell suspension in the step 2) into the culture solution channel, and sealing and standing for culture;

4) After the cells in the culture solution channel cling to the wall and grow on the inner surface of the culture solution channel, injecting the lung tumor microspheres and the cell culture solution in the step 1) into the cell culture holes;

5) After the three-channel organ chip is closed, the fluid in the culture liquid channel and the gas channel is circulated for culture respectively through an external driving pump.

Preferably, after the cells in the culture solution channel in step 4) adhere to the wall and begin to proliferate, a layer of matrigel is paved and solidified at the bottom of the cell culture hole 21, and then the cultured tumor microspheres and the culture solution are connected into the culture hole together. It is worth noting that matrigel is preferably added in this step, too early to facilitate long-term storage.

It should be noted that, in the three-channel organ chip of the present invention, the three channels respectively refer to a gas channel 11 formed by air flowing in the chip, a cancer cell channel formed by arranging the cell culture holes 21, and a culture solution channel 22 (containing endothelial cells), and at least 3 different types of cells can be co-cultured in the chip.

As described above, the three-channel organ chip of the present invention has mainly the following advantageous effects:

1) The three-channel organ chip introduces a gas channel and a culture liquid channel which can flow fluid, can provide gas and liquid shearing force for cells cultured on the chip, and increases the complexity of external stimulus factors acceptable by the cells, thereby fully simulating the in-vivo environment of tumors.

2) The three-channel organ chip can realize the capability of simultaneously detecting different drugs to inhibit the growth of tumor cells on the same chip by arranging a plurality of relatively independent and closed culture units.

3) The three-channel organ chip realizes automatic control of cell culture by externally connecting a driving pump so as to achieve the aim of improving the working efficiency.

4) In addition to tumor cells, epithelial cells and vascular endothelial cells, the system may also incorporate immune cells, fibroblasts, etc. to increase on-chip cell complexity.

Drawings

Fig. 1 is a schematic diagram showing the overall structure of a three-channel chip according to the present invention.

Fig. 2 shows a schematic side view of a three-channel chip of the present invention.

Fig. 3 shows a schematic side view of a three-channel chip of the present invention.

FIG. 4 is a schematic top view of a substrate of a three-channel chip of the present invention

The reference numerals in fig. 1 to 4 are as follows:

1. cover body

11. Gas channel

111. Cavity bottom

12. Vent hole

13. Infusion port of culture solution

2. Matrix body

21. Cell culture well

22. Culture liquid channel

221. Long channel

222. Uplink channel

223. Outer joint of culture liquid channel

23. Porous separator

Detailed Description

Further advantages and effects of the present invention will become apparent to those skilled in the art from the description given herein below, by way of specific examples.

Please refer to fig. 1 to 4. It should be understood that the structures, proportions, sizes, etc. shown in the drawings are for illustration purposes only and should not be construed as limiting the invention to the extent that it can be practiced, since modifications, changes in the proportions, or otherwise, used in the art, do not materially affect the advantages of the invention and which are otherwise, used in the practice of the invention, which fall within the spirit and scope of the invention. Also, the terms such as "upper," "lower," "left," "right," "middle," and "a" and the like recited in the present specification are merely for descriptive purposes and are not intended to limit the scope of the invention, but are intended to provide relative positional changes or modifications without materially altering the technical context in which the invention may be practiced.

Example 1

As shown in fig. 1 to 4, the present invention provides a three-channel organ chip comprising a lid 1 and a base 2, the lid 1 being provided with a gas channel 11 and a vent hole 12, the base 2 being provided with a plurality of cell culture holes 21 and a plurality of culture liquid channels 22. In addition, the gas channel 11 is in fluid direct communication with the cell culture well 21, and the fluid in the gas channel 11 and the culture liquid channel 22 is kept flowing by the pushing of an external force. The culture liquid channel 22 is a main place for culture liquid to flow and vascular endothelial cells to survive, the cell culture holes 21 are used for inoculating 3D tumor cell microspheres and taking out the culture liquid for detection after the experiment is finished, and the vent holes 12 can be used for introducing air and allowing the air to leave, so that a flowing air layer is formed inside the chip. According to the method, the gas channel 11 and the culture liquid channel 22 are introduced into the chip, the gas channel 11 and the culture liquid channel 22 can form flowing gas and liquid environments through the external driving pump, multi-component lung cancer related cells can be co-cultured, and culture liquid perfusion of different components can be performed simultaneously, so that the in-vivo environment of tumors can be fully simulated. In general, through holes for ventilation and liquid ventilation are formed in the cover body to be respectively communicated with the gas channel 11 and the culture liquid channel 22, and a driving force is provided by an external pump to form a closed-loop unidirectional flow system inside the chip.

In one embodiment, the three-way organ-chip material is a transparent material; specifically, the cover body 1 and the base body 2 are made of high polymer materials; more specifically, the material of the cover 1 and/or the base 2 is selected from polystyrene or polyethylene.

In one embodiment, as shown in fig. 2, the cover 1 is provided with at least two ventilation holes 12, each ventilation hole 12 is in fluid communication with the gas channel 11, and each ventilation hole 12 is provided with a section of external pipe (the external pipe protrudes out of the cover 1) or directly externally connected with a ventilation pipeline. Preferably, the external connection pipe at the vent hole 12 is integrally formed with the cover body 1, and when external force driving is required to be provided, the external connection pipe is externally connected with a vent pipe and connected with a driving pump to form a flow system, preferably a unidirectional flow system. In addition, the cover 1 is provided with a plurality of culture solution infusion ports 13, each culture solution channel 22 at least corresponds to two culture solution infusion ports 13, and two ends of each culture solution channel 22 are respectively in fluid communication with two culture solution infusion ports 13. Specifically, the culture liquid channel includes a long channel 221, and an uplink channel 222 disposed at two ends of the long channel 211, the long channel 211 is located below the cell culture hole 21, one end of the uplink channel 222 is communicated with the long channel 221, the other end of the uplink channel protrudes out of the substrate 2 and forms a culture liquid channel external interface 223, and the culture liquid channel external interface 223 is used for externally connecting a liquid pipeline. Specifically, the outer interface 223 of the culture liquid channel on each substrate 2 is used for externally connecting a liquid pipeline, that is, the liquid pipeline is communicated with the outer interface 223 of the culture liquid channel after passing through the culture liquid infusion port 13, and when external force driving needs to be provided, the liquid pipeline is connected with a driving pump to form a flow system, preferably a unidirectional flow system.

In a specific embodiment, as shown in fig. 2 and fig. 3, the cover 1 is detachably connected to the base 2, and the cover 1 is detachably connected to the base 2 by sliding connection, plugging connection, threaded connection or snap connection. For example, the cover 1 includes a top cover and a peripheral connection portion detachably connected to the base 2, and a cavity portion is formed in a state where the cover 1 is connected to the base 2, and the cavity portion forms the gas passage 11. More specifically, in the state where the cover 1 is connected to the base 2, the vertical distance from the cavity bottom 111 on the side of the cavity portion far from the base 2 to the plane where the top of the cell culture hole 21 is located is 9-12mm, optionally 9-10mm,10-11mm, and 11-12mm. The specific distance between the cavity bottom 111 and the cell culture well 21 is maintained so that the gas passage 11 is formed under the cover.

In some preferred embodiments, a porous partition membrane 23 is provided at the communication between the cell culture well 21 and the culture liquid channel 22, and the cell culture well 21 and the culture liquid channel 22 are partitioned by the porous partition membrane 23. Preferably, the pore diameter on the porous diaphragm is 0.4-5 mu M, and the pore diameter in the range can enable liquid, small molecules and biological macromolecules to freely permeate, but has a certain limitation on cells. Under the condition that the two conditions that the membrane material can support the growth of cells and the pore size can not lead the cells to leak into the culture solution channel 22 from the cell culture pores 21 are satisfied, an experimenter can customize the pore size and the membrane material according to the self requirements. In general, the material of the porous spacer film 23 is a polymer material having a plurality of pores; specifically, the material of the porous spacer film 23 is at least one selected from polystyrene, polyethylene terephthalate and polybutylene terephthalate.

As shown in fig. 2, the porous separation membrane 23 is arranged at the communication position of the cell culture hole 21 and the culture solution channel 22, so that the liquid in the culture solution channel can be diffused into the cell culture hole to realize liquid exchange, tumor cells (normal epithelial cells) and vascular endothelial cells are separated and grown in two relatively independent and mutually connected spaces, and circulating immune cells can be additionally reintroduced into the system, so that the interaction of cells of micro-environments and other components of tumors in a human body and tumor cells can be better simulated.

In some preferred embodiments, the membrane surface of porous spacer membrane 23 is provided with a low cell adhesion layer; specifically, the material of the low cell adhesion layer is an extracellular matrix component; more specifically, the cell blocking layer is made of matrigel. Specifically, taking a PET film as an example, a layer of matrigel imitating extracellular matrix components is coated on the PET film, so that the tumor microspheres can maintain the original shape after being inoculated into the cell culture wells 21.

In some preferred embodiments, as shown in FIG. 4, a plurality of cell culture wells 21 are arranged in a plurality of rows, and a culture medium channel 22 is provided at the bottom of each row of cell culture wells 21. Specifically, the cell culture wells 21 are not connected, and the culture medium passages 22 are not connected. More specifically, the bottom of the cell culture well may be provided with a porous spacer film for forming a spacer, or the cell culture well 21 may be a through hole, and the bottom of the cell culture well 21 is provided with at least one layer of the porous spacer film 23. Specifically, each row of the culture medium channels 22, through which the cell culture holes, the porous partition membrane 23 positioned below the cell culture holes and one culture medium channel positioned below the cell culture holes are arranged, form a relatively closed culture unit, and a plurality of culture units can be arranged to improve the flux and realize the simultaneous cell culture of a plurality of culture liquids with different components.

Example 2

As shown in fig. 2, 3 and 4, the cover 1 is provided with 2 vent holes 12 protruding from the cover 1 and 8 culture solution infusion ports 13, and each vent hole 12 and culture solution infusion port 13 is provided with a silica gel plug. The substrate 2 is provided with 24 cell culture wells 21 and 4 culture liquid passages 22. Each culture medium channel 22 comprises a long channel 221 with the same width as the cross section diameter of the cell culture hole 21, an uplink channel 222 at two ends and a culture medium channel external interface 223. Tumor microspheres (including tumor cells and epithelial cells) and immune cells can be inoculated in the cell culture holes 21, and vascular endothelial cells and fibroblasts can be inoculated in the culture fluid channels 22, so that a multi-component 3D cell culture system is formed. The relative independence of the culture medium channel 22 and the cell culture well 21 ensures that the chip is simultaneously perfused with at most four different components of culture medium. More specifically, the cover body 1 is detachably connected with the base body 2, the cover body 1 includes a peripheral connection portion and a cavity portion, the peripheral connection portion is detachably connected with the base body 2, and in a connection state of the cover body 1 and the base body 2, the cavity portion forms the air channel 11, which is an air layer flowing unidirectionally.

Specifically, after the 2 vent holes of the chip are communicated with the gas pipeline, the gas can flow in/out so as to form a flowing air layer inside the chip; the 8 culture liquid infusion ports 13 are positions where a liquid passing pipeline is externally connected with the outer interface 223 of the culture liquid channel, and the liquid passing pipeline is communicated with the driving pump to form a flowing liquid environment inside the chip. When the chip is not in use, the vent hole 12 and the culture solution infusion port 13 on the cover body 1 are both provided with silica gel plugs to isolate the external environment, and when the chip is in use, the silica gel plugs are opened and stored so as to be reloaded back to the chip after the experiment is finished, thereby realizing the recycling.

More specifically, the size of the chip is similar to that of a conventional 24-well cell culture plate, more specifically, the substrate 2 is provided with 24 cell culture wells 21 and 4 culture solution channels 22, the 24 cell culture wells 21 are arranged on the substrate 2 according to a 6x 4 arrangement mode, the upper openings of the cell culture wells 21 are used for inoculating a 3D cell culture system and taking out cells/culture solution for detection after the experiment is finished, and each 1 culture solution channel 22 is positioned under 6 cell culture wells 21 and has the same width as the diameter of the cell culture well 21. Each culture liquid channel 22 has a long channel 221 parallel to the long axis of the chip, an upward channel 222 and a culture liquid channel external interface 223 on both sides, and the culture liquid channel external interface 223 can be connected with a liquid passage, so that the culture liquid flows in from one side and then flows out. The cell culture hole 21 is a through hole, the lower opening is provided with a porous partition membrane, or the bottom wall of the cell culture hole 21 is directly prepared into the porous partition membrane, and the porous partition membrane enables a liquid exchange plane to be formed between the cell culture hole 21 and the culture liquid channel 22, so that liquid can be freely exchanged between the cell culture hole 21 and the culture liquid channel 22.

More specifically, as shown in fig. 4, each row is 6 cell culture wells 21, each column is 4 cell culture wells 21, the cell culture wells 21 are right circular in cross section, and the diameters are: 7-9 mm, the depth is: the distance between the centers of the cross sections of two holes in each row is 10-12 mm: 15-18 mm, the distance between the centers of the cross sections of two holes in each row is as follows: 20-24 mm; the bottom of the cell culture hole 21 is a plane, the manufacturing material is porous high polymer material so that liquid can freely flow between the cell culture hole 21 and the culture solution channel 22, the top of the cell culture hole 21 is opened upwards so that a tumor 3D culture system can be inoculated and the culture solution can be taken out for detection, and the liquid level in the cell culture hole should not exceed 1/2 of the height of the hole. The thickness of the bottom plate at the lowest side of the base body 2 is as follows: 1 mm-5 mm.

More specifically, the cross section of the culture fluid channel 22 is rectangular, the length is the same as the diameter of the cell culture hole, and the width is: 3-5 mm, the whole channel length is: 110mm. Wherein the width of the channel part completely under the 6 cell culture holes 21 is uniform and is 10mm; the two ends extend to the short axis of the chip by about 10mm, the width gradually contracts until the two ends of the channel vertically extend upwards to form an uplink channel 222, the cross section is circular, and the diameter is: 3-5 mm, continuing to extend to form a circular opening; specifically, one end of the up channel 222 is communicated with the long channel 221, and the other end of the up channel protrudes out of the substrate 2 to form a culture fluid channel outer interface 223, and the length of the culture fluid channel outer interface 223 is as follows: 3-5 mm for connecting the liquid pipeline so as to form a flowing liquid environment inside the chip. The surface of the top of the culture liquid channel 22 connected with the cell culture holes is a porous diaphragm, so that the liquid exchange is facilitated. When the chip works, the flow direction of the liquid in the culture liquid channel is unidirectional.

More specifically, the length, width and height dimensions of the chip are: 127mm x 85mm x 27mm except for the porous diaphragm 23, the whole chip is made of Polystyrene (PS) or Polyethylene (PE) material, so that the cell culture is suitable, and the finished product is of a full transparent appearance, and is convenient for microscopic observation. The length, width and height dimensions of the detachable cover body 1 are as follows: 127mm x 85mm x 27mm after the cover body 1 is buckled, the vertical height from the inner surface of the cover body 1 to the surface of the culture layer is as follows: 9-12 mm; the cover body 1 is provided with 2 right circular vent holes 12 protruding out of the cover body 1, and the diameter is as follows: the center of the cross section of the vent hole 12 is positioned on the center axis of the cover body 1, 5-8 mm, and the distance between the centers of the two vent holes 12 is as follows: 40-45 mm, the distance between each circle center and the short axis of the cover body is as follows: 41-43.5 mm, the distance from the long axis of the cover body is as follows: 42.5mm, vent hole 12 upwards has the external connection mouth fixed with lid 1, length is: 8-12 mm, the vent hole 12 can be connected with a vent pipeline; in addition, the short shaft two sides of the cover body 1 are respectively provided with 4 culture solution infusion ports 13, the culture solution infusion ports 13 do not need to be provided with external connecting pipes, and the culture solution infusion ports 13 are used for enabling the liquid through pipeline to pass through. In order to prevent pollution, all holes on the chip are plugged by a silica gel plug when not in use, and are opened when in use.

More specifically, the culture liquid infusion port 13 on the chip cover 1 and the uplink pipeline 222 on the substrate 2 are in one-to-one correspondence with the culture liquid channel external interface 223, but the aperture of the culture liquid infusion port 13 on the cover 1 is slightly larger than that of the culture liquid channel external interface 223 of the substrate 2, and the whole hole can be exactly blocked when the liquid passing pipeline passes through, so that a relatively closed space is formed inside the chip.

Example 3

The corresponding system of the three-channel organ chip of the embodiment comprises the three-channel organ chip and a driving pump, wherein the driving pump is used for providing power for the fluid in the gas channel and the culture liquid channel, can realize automatic control of cell culture on the chip, and is not limited here, and the model of the driving pump is selected or customized according to the needs.

Example 4

The three-way organ-chip of example 1 or example 2 or the system of example 3 was used as follows:

before the chip is formally used, firstly, human lung cancer cells and human normal respiratory tract epithelial cells are mixed and inoculated according to the proportion of 2:1, and are cultured for 3-7 days to form tumor microspheres; simultaneously, culturing human vascular endothelial cells and preparing cell suspension for standby; next, a silica gel cover of a side culture solution infusion port 13 on the cover body 1 of the chip is opened (the silica gel cover is specially preserved after being removed), endothelial cell suspension is slowly injected into the culture solution channel 22 at an outer interface 223 of the culture solution channel of the short shaft of the side substrate 2 until the channel is full of cell suspension, then the silica gel cover is covered for sealing and static culture, the silica gel cover is opened for replacing culture solution every two days, after endothelial cells are attached to the wall and proliferated until the inner wall of the channel is full, all the silica gel covers are opened (the silica gel cover is continuously preserved after being removed) and replaced with new culture solution again, and meanwhile, tumor microspheres with the diameter of more than 300 mu M are taken out from the original pore plate and transferred into the cell culture holes 21 together with the culture solution; then the liquid and air pipelines which are communicated with the culture liquid channel 22 and the air holes 12 are connected, the cover body 1 is installed, and the pipelines are connected with the driving pump; finally, the pump is turned on, so that the fluid in the chip flows.

In particular, the method comprises the steps of,

1) When not in use, the cover body 1 and the base body 2 of the chip are buckled together, all holes on the cover body 1 are plugged by silica gel plugs, and the inside of the chip is a clean and sterile environment.

2) When the chip is used, firstly, a silica gel plug of a culture solution infusion port 13 on one side of the cover body 1 is opened, a 5ml syringe with a needle is used for slowly and dropwise adding a prepared endothelial cell suspension into the culture solution channel 22 from an outer interface 223 of the culture solution channel on one side of the base body 2 until the inside of the channel is full of culture solution (the height of the culture solution is controlled below an ascending channel 222, namely, the ascending channel 222 is free of culture solution), then the cover body 1 is buckled for static culture, and the culture solution is observed and replaced every 2-3 days; after endothelial cells are substantially full, the cover 1 is opened and all silica gel plugs are removed (silica gel plugs are preserved), and a layer of matrigel is spread on the bottom of the cell culture well 21 and cured. And then the cultured tumor microspheres and the culture solution are connected into the culture holes together, then the liquid in the culture solution channel 22 is completely removed, all liquid and air channels are connected, the whole chip is assembled, and finally an external driving pump is connected, so that the liquid and the gas flow in the chip.

Specifically, when the chip is in actual use, a plurality of culture units can be used at different time, and culture liquids with different components can be poured. The used culture broth was collected separately for subsequent detection.

3) The chip can be observed and photographed by a front microscope in the culture process.

4) After the chip is cultured, the detection means after the chip is used include the following steps: a) After the culture is finished, the chip is placed under a microscope for observation and the diameter of the tumor microspheres is measured; b) After the culture is completed, the original culture solution in the cell culture well 21 is removed, and the ATP content in the individual tumor microspheres is detected to determine the cell viability. c) Other methods: after the completion of the culture, the culture solution is directly taken out from the cell culture well 21, and specific indicators (cytokines, other exocrine proteins, etc., and the detection indicators are not specifically required) are detected according to the experimental requirements. If other detection modes exist, the detection method can be automatically added according to the actual requirement.

5) After the experiment is finished, the culture solution can be directly removed, cells adhered to the chip can be removed by trypsin digestion, and finally the whole chip can be thoroughly sterilized and reused by means of electron beam irradiation (such as gamma rays) (the number of times of reuse is recommended to be controlled to be 3 times or less).

In addition, when the chip is subjected to experiments, if immune cells are required to be added, the chip can be added to the chip for co-culture together with the culture solution while the endothelial cells grow fully and the tumor microspheres are inoculated, immune cells can be added to the cell culture hole 21 and the culture solution channel 22, and after the culture is finished, the immune cells can be subjected to independent related index detection.

Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. The three-channel organ chip is characterized by comprising a cover body (1) and a base body (2), wherein the cover body (1) comprises a gas channel (11), and the base body (2) is provided with a plurality of cell culture holes (21) and a plurality of culture liquid channels (22);

the gas channel (11) is in fluid communication with the cell culture well (21), and the fluid in the gas channel (11) and the culture liquid channel (22) is kept flowing under the pushing of external force.

2. The three-way organ-chip according to claim 1, wherein a porous spacer film (23) is provided at a communication place of the cell culture well (21) and the culture liquid channel (22);

and/or, the cover body (1) is provided with at least two vent holes (12), each vent hole (12) is used as an inlet and an outlet for fluid of the gas channel (11), and each vent hole (12) is used for arranging or externally connecting a vent pipeline.

3. The three-way organ-chip according to claim 2, wherein the plurality of cell culture wells (21) are arranged in a plurality of rows, and a culture liquid channel (22) is provided under each row of the cell culture wells (21);

and/or, each of the cell culture wells (21) is not in communication with each other; and/or, each culture liquid channel (22) is not communicated with each other;

and/or, a plurality of culture solution infusion ports (13) are arranged on the cover body (1), each culture solution channel (22) at least corresponds to two culture solution infusion ports (13), and each culture solution infusion port (13) is arranged as an inlet and outlet for fluid of the culture solution channel (22);

and/or the cell culture hole (21) is a through hole, and at least one layer of porous diaphragm (23) is arranged at the bottom of the cell culture hole (21).

4. A three-way organ-chip according to claim 3, wherein the material of said porous spacer film (23) is a polymeric polymer material capable of forming a porous structure; preferably, the material of the porous diaphragm (23) is selected from one of polystyrene, polyethylene terephthalate or polybutylene terephthalate;

and/or the pore diameter of the porous separation membrane is 0.4-5 mu M;

and/or, the culture solution channel (22) comprises a long channel (221) and an uplink channel (222) arranged at two ends of the long channel (211); the long channel (211) is located below the cell culture well (21); one end of the ascending channel (222) is communicated with the long channel (221), the other end of the ascending channel protrudes out of the substrate (2) and forms a culture liquid channel outer interface (223), the culture liquid channel outer interface (223) is in fluid communication with the culture liquid infusion port (13), and the culture liquid channel outer interface (223) is used for externally connecting a liquid pipeline.

5. The three-way organ-chip according to claim 4, wherein the membrane surface of the porous spacer membrane (23) is provided with a low cell adhesion layer; preferably, the material of the low cell adhesion layer is a simulated extracellular matrix component; more preferably, the material of the low cell adhesion layer is matrigel.

6. The three-way organ-chip according to claim 2, wherein the cover (1) is detachably connected to the base body (2); preferably, the detachable connection is a sliding connection, a plug connection, a threaded connection or a snap connection.

7. The three-way organ-chip according to claim 6, wherein the cover (1) comprises a top cover and a peripheral connection part, which is detachably connected to the base (2); a cavity part is formed in a state that the cover body (1) is connected with the base body (2), and the cavity part forms the gas channel (11).

8. The three-channel organ-in-a-chip according to claim 7, wherein the peripheral connection portion is located at the outer periphery of the lid (1);

and/or, in the state that the cover body (1) is connected with the base body (2), the vertical distance from the cavity bottom (111) at one side of the cavity part far away from the base body (2) to the plane of the top of the cell culture hole (21) is 9-12mm.

9. The three-way organ-chip of any one of claims 1-8, wherein the material of the three-way organ-chip is a transparent material; preferably, the material of the cover body (1) and/or the base body (2) is a high-molecular polymer material; more preferably, the material of the cover (1) and/or the base (2) is selected from polystyrene or polyethylene.

10. A three-way organ-chip system comprising the three-way organ chip of any one of claims 1-9 or a drive pump for powering a fluid within a gas channel and a culture fluid channel.

11. Use of the three-way organ-chip of any one of claims 1-9 and/or the three-way organ-chip system of claim 10 for studying the killing effect of candidate anti-tumor drugs on lung cancer cells.

12. A method of using the three-way organ-chip of any one of claims 1-9 or the three-way organ-chip system of claim 10 in studying the killing effect of candidate anti-tumor drugs on lung cancer cells, comprising the steps of:

1) Culturing lung tumor microspheres; preferably, the lung tumor microspheres are obtained by mixed culture of human lung cancer cells and human respiratory epithelial cells; more preferably, the tumor microspheres inoculated onto the chip for 3D culture have a diameter of greater than 300 μm;

4) After the cells in the culture solution channel cling to the wall and proliferate until cling to the inner surface of the channel, injecting the lung tumor microspheres in the step 1) together with the cell culture solution into the cell culture hole;

5) After the three-channel organ chip is closed, the liquid and the ventilation pipelines are connected, and the fluid in the liquid culture channel and the gas channel flows through the external driving pump to culture.