Cell culture chip

Abstract

The utility model provides a cell culture chip which comprises a first substrate, a second substrate, a first cell culture chamber and a second cell culture chamber, a plurality of circulation micropores are formed in the second substrate; the third substrate is provided with a second cell culture chamber and a third cell culture chamber, a plurality of micro-channels are arranged between the second cell culture chamber and the third cell culture chamber, and the two ends of each micro-channel are communicated with the second cell culture chamber and the third cell culture chamber respectively; two ends of the circulating micropores are respectively communicated with the first cell culture chamber and the second cell culture chamber, so that cells in the second cell culture chamber can be respectively contacted and communicated with cells in the first cell culture chamber and the third cell culture chamber through the plurality of circulating micropores and the microchannels; and material exchange and cell co-culture in a cross-cell culture room are completed to promote the integrity of organoid functions. According to the utility model, the cell co-culture and functional integrity in the prior art are enhanced.

Description

Technical Field

[0001] The utility model relates to the technical field of organ chips, in particular to a cell culture chip. Background Art

[0002] Organ-on-a-chip technology is an emerging technology that constructs the core tissue structure of human organs in vitro, simulating the microenvironment, including the cells, molecules, and shear forces surrounding the organs, to achieve the key physiological functions of the organs. Unlike traditional two-dimensional cell culture, organ-on-a-chips culture human cells in three dimensions on microfluidics chips (microfluidics chip technology integrates the basic operating units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, into a micrometer-scale chip, automatically completing the entire analysis process). This achieves a high degree of similarity to the organ or tissue structure in the human body, while simulating the in vivo microenvironment as much as possible, thereby reproducing the various functions of human organs in vitro.

[0003] To establish a culture system more similar to the in vivo environment, enabling cell communication and promoting growth and proliferation, co-culture techniques have been developed based on cell culture techniques. These techniques include two construction modes: direct co-culture and indirect co-culture. Indirect co-culture involves seeding two or more cell types in separate cell culture areas, then placing these areas in the same environment, allowing the different cell types to share the same culture system without direct contact. This co-culture system plays a positive role in inducing cell differentiation, maintaining cell function and viability, regulating cell proliferation, and increasing the production of metabolites.

[0004] Most organ chips have two cell culture areas and microchannels on their surfaces. Two types of cells are implanted in the two cell culture areas respectively. The culture fluid of one cell enters the culture area of the other cell through the connecting channel to achieve material exchange between the two cells and promote each other's induced differentiation. However, this organ chip can only achieve material exchange between two different cells, while the internal environment of the human body allows material exchange between multiple cells, resulting in poor biomimetic properties of this organ chip and insufficient integrity of the organoids on the organ chip. Utility Model Content

[0005] The main purpose of the utility model is to provide a cell culture chip to solve the problem of insufficient integrity of cell structure and function on organ chips in the prior art.

[0006] To achieve the above object, the present utility model provides a cell culture chip, comprising: a first substrate, on which a first cell culture chamber is provided for culturing a first cell; a second substrate, on which a plurality of flow-through micropores are provided; a third substrate, on which a second cell culture chamber and a third cell culture chamber are provided, the second cell culture chamber is used for culturing a second cell, the third cell culture chamber is used for culturing a third cell, and there are a plurality of microchannels between the second cell culture chamber and the third cell culture chamber, and both ends of each microchannel are respectively communicated with the second cell culture chamber and the third cell culture chamber; both ends of the flow-through micropores are respectively communicated with the first cell culture chamber and the second cell culture chamber, so that the culture solution in the first cell culture chamber can enter the third cell culture chamber through the plurality of flow-through micropores and microchannels.

[0007] Further, an annular flow-through portion is provided on the second substrate, and a plurality of flow-through micropore groups are provided on the annular flow-through portion. The plurality of flow-through micropore groups are arranged at intervals along the circumferential direction of the annular flow-through portion. Each flow-through micropore group includes a plurality of flow-through micropores, and the plurality of flow-through micropores are arranged at intervals along the radial direction of the annular flow-through portion.

[0008] Further, the third cell culture chamber is circular, the second cell culture chamber is annular, the second cell culture chamber is arranged on the circumferential outer side of the third cell culture chamber, the shape of the second cell culture chamber is adapted to that of the annular flow-through portion, and the central axes of the third cell culture chamber, the second cell culture chamber and the annular flow-through portion are arranged to coincide.

[0009] Further, a first liquid injection port and a second liquid injection port are provided on the first substrate, a first communication port and a second communication port are provided on the second substrate, and a first liquid inlet and a second liquid inlet are provided on the third substrate. The first liquid inlet is communicated with the second cell culture chamber, the first communication port is communicated with the first liquid injection port, and the first communication port is communicated with the first liquid inlet, so that the second cell mixture flows through the first liquid injection port, the first communication port and the first liquid inlet in sequence and enters the second cell culture chamber; the second liquid inlet is communicated with the third cell culture chamber, the second communication port is communicated with the second liquid injection port, and the second communication port is communicated with the second liquid inlet, so that the third cell mixture flows through the second liquid injection port, the second communication port and the second liquid inlet in sequence and enters the third cell culture chamber; wherein, the second cell mixture includes a second cell and a culture solution, and the third cell mixture includes a third cell and a culture solution.

[0010] Further, a first liquid outlet and a second liquid outlet are provided on the first substrate, a third communication port and a fourth communication port are provided on the second substrate, and a third liquid outlet and a fourth liquid outlet are provided on the third substrate. The third liquid outlet is communicated with the second cell culture chamber, the third communication port is communicated with the first liquid outlet, and the third communication port is communicated with the third liquid outlet, so that the culture medium in the second cell culture chamber flows out successively through the third liquid outlet, the third communication port and the first liquid outlet; the fourth liquid outlet is communicated with the third cell culture chamber, the fourth communication port is communicated with the second liquid outlet, and the fourth communication port is communicated with the fourth liquid outlet, so that the culture medium in the third cell culture chamber flows out successively through the fourth liquid outlet, the fourth communication port and the second liquid outlet.

[0011] Further, a first liquid inlet channel and a first liquid outlet channel are provided on the first substrate. One end of the first liquid inlet channel and one end of the first liquid outlet channel are both communicated with the first cell culture chamber, and the other end of the first liquid inlet channel is used for introducing a first cell mixture; wherein, the first cell mixture includes first cells and a culture medium.

[0012] Further, the other end of the first liquid inlet channel forms a third liquid inlet port. The cell culture chip further includes a filter member, and the filter member is arranged in the first liquid inlet channel and between the third liquid inlet port and the first cell culture chamber to filter the incoming first cell mixture.

[0013] Further, a second liquid inlet channel and a third liquid inlet channel are provided on the third substrate. One end of the second liquid inlet channel is used to form a first liquid inlet port, the other end of the second liquid inlet channel is communicated with the second cell culture chamber, one end of the third liquid inlet channel is used to form a second liquid inlet port, and the other end of the third liquid inlet channel is communicated with the third cell culture chamber.

[0014] Further, a second liquid outlet channel and a third liquid outlet channel are provided on the third substrate. One end of the second liquid outlet channel is used to form a third liquid outlet port, the other end of the second liquid outlet channel is communicated with the second cell culture chamber, one end of the third liquid outlet channel is used to form a fourth liquid outlet port, and the other end of the third liquid outlet channel is communicated with the third cell culture chamber.

[0015] Further, the cell culture chip further includes a plurality of barrier members, and the plurality of barrier members are arranged at intervals in the circumferential direction of the third cell culture chamber between the third cell culture chamber and the second cell culture chamber, and a microchannel is formed between any two adjacent barrier members.

[0016] Applying the technical solution of the present utility model, the cell culture chip includes a first substrate, a second substrate, and a third substrate. A first cell culture chamber is provided on the first substrate, a plurality of flow-through micropores are provided on the second substrate, a second cell culture chamber and a third cell culture chamber are provided on the third substrate, and there are a plurality of microchannels between the second cell culture chamber and the third cell culture chamber. Since the two ends of the microchannels are respectively connected to the second cell culture chamber and the third cell culture chamber, and the two ends of the flow-through micropores are respectively connected to the first cell culture chamber and the second cell culture chamber, the first cell culture chamber, the second cell culture chamber, and the third cell culture chamber are interconnected with each other, enabling the first cell, the second cell, and the third cell to exchange substances with each other pairwise. For example, the culture solution in the first cell culture chamber can enter the third cell culture chamber through a plurality of flow-through micropores and microchannels, enabling the third cell in the third cell culture chamber to not only exchange substances with the second cell but also exchange substances with the first cell, improving the mimicry of the cell culture chip of the present application and solving the problem of insufficient integrity of the cell structure and function on the organ chip in the prior art, and improving the co-culture efficiency of the three types of cells on the cell culture chip of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings forming a part of this application are used to provide a further understanding of the present utility model. The schematic embodiments of the present utility model and their descriptions are used to explain the present utility model and do not constitute an improper limitation of the present utility model. In the drawings:

[0018] Figure 1 Shows an exploded schematic view of an embodiment of the cell culture chip according to the present utility model;

[0019] Figure 2 Shows Figure 1 A partial enlarged schematic view at A in;

[0020] Figure 3 Shows Figure 1 A partial enlarged schematic view at B in;

[0021] Figure 4 Shows a schematic view of the normalized fluorescence intensity in the third cell culture chamber changing with time.

[0022] Wherein, the above-mentioned accompanying drawings include the following reference numerals:

[0023] 10. First substrate; 11. First cell culture chamber; 12. First liquid injection port; 13. Second liquid injection port; 14. First liquid outlet; 15. Second liquid outlet; 16. First liquid inlet channel; 17. First liquid outlet channel; 20. Second substrate; 21. Second cell culture chamber; 22. Annular circulation part; 23. Circulation micropores; 24. First communication port; 25. Second communication port; 26. Third communication port; 27. Fourth communication port; 30. Third substrate; 31. Third cell culture chamber; 32. Microchannel; 33. First liquid inlet; 34. Second liquid inlet; 35. Third liquid outlet; 36. Fourth liquid outlet; 37. Second liquid inlet channel; 38. Third liquid inlet channel; 39. Second liquid outlet channel; 391. Third liquid outlet channel; 40. Filter element; 50. Barrier element; 61. First alignment mark; 62. Second alignment mark; 63. Third alignment mark. Detailed implementation manners

[0024] It should be noted that the terms used herein are only for describing the detailed implementation manners and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] Unless otherwise specifically stated, the relative arrangements, numerical expressions, and numerical values of the components and steps described in these embodiments do not limit the scope of the present application. At the same time, it should be understood that, for the convenience of description, the dimensions of the various parts shown in the drawings are not drawn in actual proportional relationships. Technologies, methods, and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the said technologies, methods, and devices should be regarded as part of the authorized specification. In all the examples shown and discussed here, any specific value should be construed as merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that like reference numerals and letters denote like items in the following drawings, and thus, once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0026] In the description of the present application, it should be understood that the orientation or positional relationship indicated by orientation words such as "front, rear, upper, lower, left, right", "lateral, vertical, perpendicular, horizontal" and "top, bottom", etc. is usually based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present application and simplifying the description. Without contrary instructions, these orientation words do not indicate or imply that the device or component referred to must have a specific orientation or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation on the protection scope of the present application; the orientation words "inside, outside" refer to the inside and outside relative to the contour of each component itself.

[0027] Please refer to Figures 1 to 4 , the present utility model provides a cell culture chip, comprising: a first substrate 10, on which a first cell culture chamber 11 is provided for culturing a first cell; a second substrate 20, on which a plurality of flow microholes 23 are provided; a third substrate 30, on which a second cell culture chamber 21 and a third cell culture chamber 31 are provided. The second cell culture chamber 21 is used for culturing a second cell, and the third cell culture chamber 31 is used for culturing a third cell. There are a plurality of microchannels 32 between the second cell culture chamber 21 and the third cell culture chamber 31, and both ends of each microchannel 32 are respectively communicated with the second cell culture chamber 21 and the third cell culture chamber 31; both ends of the flow microholes 23 are respectively communicated with the first cell culture chamber 11 and the second cell culture chamber 21, so that the culture solution in the first cell culture chamber 11 can enter the third cell culture chamber 31 through the plurality of flow microholes 23 and the microchannels 32.

[0028] The cell culture chip of the present utility model includes a first substrate 10, a second substrate 20 and a third substrate 30. A first cell culture chamber 11 is provided on the first substrate 10, a plurality of flow microholes 23 are provided on the second substrate 20, a second cell culture chamber 21 and a third cell culture chamber 31 are provided on the third substrate 30, and there are a plurality of microchannels 32 between the second cell culture chamber 21 and the third cell culture chamber 31. Since both ends of the microchannels 32 are respectively communicated with the second cell culture chamber 21 and the third cell culture chamber 31, and both ends of the flow microholes 23 are respectively communicated with the first cell culture chamber 11 and the second cell culture chamber 21, the first cell culture chamber 11, the second cell culture chamber 21 and the third cell culture chamber 31 are mutually communicated with each other, so that material exchange can occur between the first cell, the second cell and the third cell pairwise. For example, the culture medium in the first cell culture chamber 11 can enter the third cell culture chamber 31 through a plurality of flow microholes 23 and microchannels 32, so that the third cell in the third cell culture chamber 31 can not only exchange substances with the second cell, but also the third cell can exchange substances with the first cell, completing the material exchange across cell culture chambers, improving the mimicry of the cell culture chip of the present application, solving the problem of insufficient integrity of the cell structure and function on the organ chip in the prior art, and improving the co-culture of the three cells and the integrity of the organoid on the cell culture chip of the present application. [[ID=I]]

[0029] Specifically, the flow diameters of the flow microholes 23 and the microchannels 32 are relatively small, so the flow rate of the culture medium flowing out of the three cell culture chambers is small, and a large amount of culture medium remains in the three cell culture chambers, which will not affect the normal growth of the cells in the three cell culture chambers.

[0030] Specifically, a first alignment mark 61 is provided on the first substrate 10, a second alignment mark 62 is provided on the second substrate 20, and a third alignment mark 63 is provided on the third substrate 30. When assembling the three substrates, by aligning the first alignment mark 61, the second alignment mark 62 and the third alignment mark 63 in sequence, it is ensured that the first substrate 10, the second substrate 20 and the third substrate 30 are assembled in place, and further ensured that both ends of the flow microholes 23 are respectively communicated with the first cell culture chamber 11 and the second cell culture chamber 21. Among them, there are a plurality of first alignment marks 61, a plurality of second alignment marks 62, and a plurality of third alignment marks 63. The plurality of first alignment marks 61 and the plurality of second alignment marks 62 are provided in one-to-one correspondence, and the plurality of third alignment marks 63 and the plurality of second alignment marks 62 are provided in one-to-one correspondence. The first alignment mark 61, the second alignment mark 62 and the third alignment mark 63 are all in a cross shape.

[0031] In this embodiment, as Figure 2As shown in the figure, an annular flow-through portion 22 is provided on the second substrate 20. A plurality of flow-through micropore groups are provided on the annular flow-through portion 22. The plurality of flow-through micropore groups are arranged at intervals in the circumferential direction of the annular flow-through portion 22. Each flow-through micropore group includes a plurality of flow-through micropores 23. The plurality of flow-through micropores 23 are arranged at intervals in the radial direction of the annular flow-through portion 22.

[0032] Specifically, the arrangement of the plurality of flow-through micropores 23 increases the flow rate of the culture medium in the first cell culture chamber 11 and the culture medium in the second cell culture chamber 21, thereby increasing the rate of material exchange between the first cell and the second cell, and further increasing the rate of material exchange among the first cell, the second cell, and the third cell, which helps to promote the growth of the three types of cells on the cell culture chip of the present application.

[0033] In this embodiment, as Figure 3 shown, the third cell culture chamber 31 is circular, the second cell culture chamber 21 is annular, the second cell culture chamber 21 is arranged on the circumferential outer side of the third cell culture chamber 31, the shape of the second cell culture chamber 21 is adapted to the shape of the annular flow-through portion 22, and the central axes of the third cell culture chamber 31, the second cell culture chamber 21, and the annular flow-through portion 22 are arranged to coincide.

[0034] Specifically, the shape of the first cell culture chamber 11 is adapted to the shape of the annular flow-through portion 22, and the shape of the second cell culture chamber 21 is adapted to the shape of the annular flow-through portion 22. Such an arrangement can prevent the situation that some of the plurality of flow-through micropores 23 in the annular flow-through portion 22 are not fully utilized due to the annular flow-through portion 22 being too large, and can also prevent the situation that the flow rate of the culture medium in the first cell culture chamber 11 and the culture medium in the second cell culture chamber 21 is relatively slow due to the annular flow-through portion 22 being too small, further ensuring that the first cell and the second cell can fully perform material exchange.

[0035] Specifically, the central axes of the third cell culture chamber 31, the second cell culture chamber 21, and the annular flow-through portion 22 are arranged to coincide, which ensures that the first cell culture chamber 11 and the annular flow-through portion 22 are in full contact, and the second cell culture chamber 21 and the annular flow-through portion 22 are in full contact, ensuring that the first cell, the second cell, and the third cell can fully perform material exchange.

[0036] In this embodiment, as Figure 1As shown, a first liquid injection port 12 and a second liquid injection port 13 are provided on the first substrate 10, a first communication port 24 and a second communication port 25 are provided on the second substrate 20, and a first liquid inlet 33 and a second liquid inlet 34 are provided on the third substrate 30. The first liquid inlet 33 is communicated with the second cell culture chamber 21, the first communication port 24 is communicated with the first liquid injection port 12, and the first communication port 24 is communicated with the first liquid inlet 33, so that the second cell mixture flows through the first liquid injection port 12, the first communication port 24 and the first liquid inlet 33 in sequence and enters the second cell culture chamber 21; the second liquid inlet 34 is communicated with the third cell culture chamber 31, the second communication port 25 is communicated with the second liquid injection port 13, and the second communication port 25 is communicated with the second liquid inlet 34, so that the third cell mixture flows through the second liquid injection port 13, the second communication port 25 and the second liquid inlet 34 in sequence and enters the third cell culture chamber 31; wherein, the second cell mixture includes second cells and a culture medium, and the third cell mixture includes third cells and a culture medium.

[0037] Specifically, the first liquid injection port 12 and the first liquid inlet 33 are communicated through the first communication port 24, so that the second cell mixture can smoothly flow into the second cell culture chamber 21, avoiding interference of the first substrate 10 and the second substrate 20 with the inflow of the second cell culture medium into the second cell culture chamber 21; the second liquid inlet 34 and the second liquid injection port 13 are communicated through the second communication port 25, so that the third cell mixture can smoothly flow into the third cell culture chamber 31, avoiding interference of the first substrate 10 and the second substrate 20 with the inflow of the third cell culture medium into the third cell culture chamber 31.

[0038] Specifically, injection tubes are provided on both the first liquid injection port 12 and the second liquid injection port 13, and the other ends of the injection tubes are connected to an injection pump. The injection pump is used to pump the first cell mixture, the second cell mixture and the third cell mixture into the injection tubes with a certain pressure, and the fluid shear stress of the first cell mixture, the second cell mixture and the third cell mixture can be dynamically adjusted to be less than or equal to 25 dyn / cm 2 within the range.

[0039] Specifically, the first substrate 10, the second substrate 20 and the third substrate 30 are sequentially placed in the vertical direction; the culture medium contains beneficial molecules of cells, and at the same time, due to the small flow diameters of the flow-through micropores 23 and the microchannels 32, cells are not allowed to pass through, and only the culture medium is allowed to pass through.

[0040] In this embodiment, as Figure 1As shown, a first liquid outlet 14 and a second liquid outlet 15 are provided on the first substrate 10, a third communication port 26 and a fourth communication port 27 are provided on the second substrate 20, and a third liquid outlet 35 and a fourth liquid outlet 36 are provided on the third substrate 30. The third liquid outlet 35 is communicated with the second cell culture chamber 21, the third communication port 26 is communicated with the first liquid outlet 14, and the third communication port 26 is communicated with the third liquid outlet 35, so that the culture solution in the second cell culture chamber 21 flows out through the third liquid outlet 35, the third communication port 26 and the first liquid outlet 14 in sequence; the fourth liquid outlet 36 is communicated with the third cell culture chamber 31, the fourth communication port 27 is communicated with the second liquid outlet 15, and the fourth communication port 27 is communicated with the fourth liquid outlet 36, so that the culture solution in the third cell culture chamber 31 flows out through the fourth liquid outlet 36, the fourth communication port 27 and the second liquid outlet 15 in sequence.

[0041] Specifically, the third communication port 26 is used to communicate the third liquid outlet 35 and the first liquid outlet 14, ensuring the smooth outflow of the culture solution in the second cell culture chamber 21, avoiding the interference of the first substrate 10 and the second substrate 20 with the outflow of the culture solution in the second cell culture chamber 21, and thus ensuring the cultivation of the second cells in a flowing environment; the fourth communication port 27 is used to communicate the fourth liquid outlet 36 and the second liquid outlet 15, ensuring the smooth outflow of the culture solution in the third cell culture chamber 31, avoiding the interference of the first substrate 10 and the second substrate 20 with the outflow of the culture solution in the third cell culture chamber 31, and thus ensuring the cultivation of the third cells in a flowing environment.

[0042] Specifically, injection tubes are provided on both the first liquid outlet 14 and the second liquid outlet 15.

[0043] Specifically, in order to ensure the growth efficiency of the cells, the first cell, the second cell and the third cell all need to be cultivated in a flowing environment. For example, after the second cell mixture flows into the second cell culture chamber 21, the injection tubes on the first liquid injection port 12 and the first liquid outlet 14 are both closed (clamping the injection tubes with clips) until the cells in the second cell mixture adhere to the wall in the second cell culture chamber 21, so that the cells remain in the cell culture chamber. Then, the injection tubes on the first liquid injection port 12 and the first liquid outlet 14 are both opened, and the culture solution in the second cell mixture flows out of the second cell culture chamber 21.

[0044] In this embodiment, as Figure 1 shown, a first liquid inlet channel 16 and a first liquid outlet channel 17 are provided on the first substrate 10. One end of the first liquid inlet channel 16 and one end of the first liquid outlet channel 17 are both communicated with the first cell culture chamber 11, and the other end of the first liquid inlet channel 16 is used for introducing the first cell mixture; wherein, the first cell mixture includes the first cells and the culture solution.

[0045] Specifically, the other end of the first liquid inlet channel 16 and one end of the first liquid outlet channel 17 are both connected to the injection tube. The first liquid inlet channel 16 is used to connect the injection tube and the first cell culture chamber 11, ensuring that the first cell mixture smoothly enters the first cell culture chamber 11. The first liquid outlet channel 17 is used to connect the injection tube and the first cell culture chamber 11, ensuring that the culture solution in the first cell culture chamber 11 smoothly flows out, and ensuring that the first cells are cultured in a flowing environment.

[0046] In this embodiment, as Figure 1 shown, the other end of the first liquid inlet channel 16 forms a third liquid inlet. The cell culture chip further includes a filter member 40. The filter member 40 is disposed in the first liquid inlet channel 16, and the filter member 40 is disposed between the third liquid inlet and the first cell culture chamber 11 to filter the incoming first cell mixture.

[0047] Specifically, the filter member includes a plurality of filter columns. The plurality of filter columns are spaced apart along the extending direction of the first liquid inlet channel 16. A flow-through gap is formed between any two adjacent filter columns. The flow-through gap only allows cells and the culture solution to pass through, and does not allow impurities such as large-diameter dust and bubbles to pass through. Thus, the filter member can filter the first cell mixture and prevent impurities from entering the first cell culture chamber 11.

[0048] In this embodiment, as Figure 1 shown, a second liquid inlet channel 37 and a third liquid inlet channel 38 are provided on the third substrate 30. One end of the second liquid inlet channel 37 is used to form a first liquid inlet 33, the other end of the second liquid inlet channel 37 is connected to the second cell culture chamber 21, and one end of the third liquid inlet channel 38 is used to form a second liquid inlet 34, and the other end of the third liquid inlet channel 38 is connected to the third cell culture chamber 31.

[0049] Specifically, the second liquid inlet channel 37 is used to connect the first liquid inlet 33 and the second cell culture chamber 21, thereby ensuring the smooth injection of the second cell mixture. The third liquid inlet channel 38 is used to connect the second liquid inlet 34 and the third cell culture chamber 31, thereby ensuring the smooth injection of the third cell mixture.

[0050] Specifically, there are a plurality of filter members 40. The filter members 40 are disposed in the second liquid inlet channel 37 and the third liquid inlet channel 38. The filter members 40 are used to filter the second cell mixture and the third cell mixture to prevent impurities from entering the second cell culture chamber 21 and the third cell culture chamber 31.

[0051] In this embodiment, as Figure 1As shown in the figure, a second liquid outlet channel 39 and a third liquid outlet channel 391 are provided on the third substrate 30. One end of the second liquid outlet channel 39 is used to form a third liquid outlet 35, the other end of the second liquid outlet channel 39 is communicated with the second cell culture chamber 21, one end of the third liquid outlet channel 391 is used to form a fourth liquid outlet 36, and the other end of the third liquid outlet channel 391 is communicated with the third cell culture chamber 31.

[0052] Specifically, the second liquid outlet channel 39 is used to connect the third liquid outlet 35 and the second cell culture chamber 21 to ensure the smooth outflow of the culture solution in the second cell culture chamber 21 and ensure the cultivation of the second cells in a flowing environment; the third liquid outlet channel 391 is used to connect the third cell culture chamber 31 and the fourth liquid outlet 36 to ensure the smooth outflow of the culture solution in the third cell culture chamber 31 and ensure the cultivation of the third cells in a flowing environment.

[0053] In this embodiment, as Figure 1 shown, the cell culture chip further includes a plurality of barrier members 50. The plurality of barrier members 50 are arranged at intervals in the circumferential direction of the third cell culture chamber 31 between the third cell culture chamber 31 and the second cell culture chamber 21, and a microchannel 32 is formed between any two adjacent barrier members 50.

[0054] Specifically, the barrier member 50 is used to prevent the second cells from entering the third cell culture chamber 31 and at the same time prevent the third cells from entering the second cell culture chamber 21. A microchannel 32 is formed between any two adjacent barrier members 50 to enable the culture solution in the second cell culture chamber 21 and the third cell culture chamber 31 to flow out smoothly, thereby ensuring that the first cells, the second cells, and the third cells can perform pairwise material exchange.

[0055] Specifically, each barrier member 50 has four interconnected barrier edges. The first barrier edge is connected to the second cell culture chamber 21, the second barrier edge is connected to the third cell culture chamber 31, and the other two barrier edges are used to jointly form two microchannels 32 with the other two barrier edges of the adjacent barrier member 50. The connection between the other two barrier edges and the second barrier edge is arc-shaped, and such a setting helps the culture solution in the third cell culture chamber 31 to flow into the second cell culture chamber 21.

[0056] During specific implementation, the manufacturing method of the cell chip of the present invention is as follows:

[0057] 1. According to the structure of the first substrate 10, a silicon wafer mold with a height of 100 μm is fabricated through a photolithography process. The silicon wafer mold is placed flat in a container, and PDMS mixed at a ratio of 10:1 is poured in. Then, it is placed in a vacuum chamber for degassing, and then put into an oven for curing. After curing, the PDMS on the silicon wafer mold is cut into an appropriate size and peeled off. The first alignment mark 61 is etched on the first substrate 10, and the first substrate 10 is pasted with a cleaning tape for standby; among them, the PDMS mixed at a ratio of 10:1 means that the proportion of the PDMS body is 10 and the proportion of the PDMS curing agent is 1. PDMS (Polydimethylsiloxane) refers to polydimethylsiloxane, which is a polymer and is applied in various fields such as pharmaceuticals, daily chemical products, food, and construction.

[0058] 2. According to the structure of the third substrate 30, a silicon wafer mold with a 3-μm-high channel reverse pattern is fabricated using a positive photoresist through a double patterning process. Then, according to the structures of the second cell culture chamber 21 and the third cell culture chamber 31, a silicon wafer mold with a height of 100 μm is fabricated on the original 3-μm-high structure silicon wafer using a negative photoresist through a double patterning process and a mask alignment method. The third alignment mark 63 is etched on the third substrate 30 to facilitate the subsequent manufacturing process in the same way as the first step, and the third substrate 30 is formed.

[0059] 3. According to the structure of the annular flow portion 22, a 10-μm-high circular microcolumn is fabricated through an etching process. Then, 15:1 PDMS is poured into a container without a pattern, and after curing, it is cut into an appropriate size and peeled off. The blank PDMS and the circular microcolumn are attached with a silane reagent. Then, the blank PDMS is placed in a spin coater, and an appropriate amount of 10:1 PDMS is poured on it. After setting the parameters, spin coating is performed to form a 10-μm-thick film. The circular microcolumn is pressed on the 10-μm-thick film, and then a 2-kg weight is pressed on the circular microcolumn overnight. After heating for eight hours, the 10-μm-thick film is removed. At this time, the annular flow portion 22 is formed on the 10-μm-thick film. Then, the second alignment mark 62 is etched on the second substrate 20, and the second substrate 20 is formed.

[0060] 4. After preparing 3 substrates, the first substrate 10 and the third substrate 30 with pre-drilled communication ports are cross-bonded at a 90-degree angle. Then, the first liquid outlet 14, the second liquid outlet 15, the first liquid injection port 12, the second liquid injection port 13, the first liquid inlet channel 16, and the first liquid outlet channel 17 are drilled. After removing the third substrate 30, the first substrate 10 and the second substrate 20 are subjected to plasma treatment, aligned and bonded under a microscope, and then the third communication port 26, the fourth communication port 27, the first communication port 24, and the second communication port 25 are drilled. Then, the third substrate 30 is subjected to plasma treatment and aligned and bonded under a microscope to complete the fabrication of the cell culture chip of the present application. Among them, plasma treatment, also known as plasma processing, is a technology that uses the characteristics of plasma for various process treatments. Plasma is a conductive medium composed of positive and negative charge ions, molecules, atoms, and atomic groups generated by gas ionization. In plasma treatment, various effects of plasma, such as activation, cross-linking, etching, cleaning, and modification, can be applied to improve the physical and chemical properties of the material surface.

[0061] Specifically, the usage method of the cell chip of the present utility model is as follows:

[0062] 1. Before inoculating cells, the first substrate 10 and the third substrate 30 are embedded with a reagent including fibronectin and Matrigel. After treatment, the chip is placed in an incubator at 37°C for half an hour, and then rinsed with a 37°C culture medium to remove the air bubbles in the first substrate 10 and the third substrate 30.

[0063] 2. Inject the corresponding cell mixture into the first cell culture chamber 11, the second cell culture chamber 21, and the third cell culture chamber 31. The density of the cell mixture is about 10 8 cells / ml. After the cells cover 80% of the area of the cell culture chamber, the injection tubes on the first liquid injection port 12 and the first liquid outlet 14 are both closed (clamp the injection tubes), and then the cell culture chip is placed in an incubator and wait for the cells to adhere. Since the cell types are different, the waiting time for adhesion is different. Taking the completion of cell adhesion as the limit, the time is 2 - 3 h. Among them, the first cell, the second cell, and the third cell can be pericytes, vascular endothelial cells, and astrocytes, respectively.

[0064] 3. Perform apparent permeability and chip integrity tests on the cell culture chip of the present application. The specific process is as follows: Inject a fluorescent tracer at a fixed flow rate from the first liquid injection port 12. The fluorescent tracer can be sodium fluorescein, Dextran - 4000, Dextran - 40000, OA647. The injection time is 1 hour, and a fluorescence camera is used to photograph and record its diffusion time, and observe the fluorescence intensity in the third cell culture chamber 31 to determine whether the structure of the cell culture chip of the present application is intact. Among them, such asFigure 4 As shown, there is no problem with the structural design and fabrication of the cell culture chip.

[0065] From the above description, it can be seen that the above embodiments of the present utility model achieve the following technical effects:

[0066] The cell culture chip of the present utility model includes a first substrate 10, a second substrate 20, and a third substrate 30. A first cell culture chamber 11 is provided on the first substrate 10, a plurality of flow microholes 23 are provided on the second substrate 20, a second cell culture chamber 21 and a third cell culture chamber 31 are provided on the third substrate 30, and there are a plurality of microchannels 32 between the second cell culture chamber 21 and the third cell culture chamber 31. Since both ends of the microchannels 32 are respectively connected to the second cell culture chamber 21 and the third cell culture chamber 31, and both ends of the flow microholes 23 are respectively connected to the first cell culture chamber 11 and the second cell culture chamber 21, the first cell culture chamber 11, the second cell culture chamber 21, and the third cell culture chamber 31 are mutually connected to each other, enabling the first cell, the second cell, and the third cell to exchange substances with each other pairwise. For example, the culture solution in the first cell culture chamber 11 can enter the third cell culture chamber 31 through a plurality of flow microholes 23 and microchannels 32, enabling the third cell in the third cell culture chamber 31 to not only exchange substances with the second cell but also with the first cell, completing the substance exchange across cell culture chambers, improving the biomimicry of the cell culture chip of the present application, solving the problem of low co-culture efficiency of cells on the organ chip in the prior art, and improving the structural and functional integrity of the three types of cells on the cell culture chip of the present application.

[0067] For the sake of convenience in description, spatial relative terms such as "above...", "over...", "on the upper surface of...", "above" etc. can be used here to describe the spatial positional relationship between a device or feature shown in the figure and other devices or features. It should be understood that the spatial relative terms are intended to encompass different orientations in use or operation in addition to the orientation of the device described in the figure. For example, if the device in the drawing is inverted, the device described as "above other devices or structures" or "over other devices or structures" will then be positioned as "below other devices or structures" or "under other devices or structures". Thus, the exemplary term "above..." can include both the orientations of "above..." and "below...". The device can also be positioned in other different ways (rotated 90 degrees or in other orientations), and corresponding interpretations should be made for the spatial relative descriptions used here.

[0068] In addition, it should be noted that the use of terms such as "first" and "second" to limit components is only for the convenience of distinguishing the corresponding components. Without additional statements, these terms have no special meaning, and therefore should not be construed as a limitation on the scope of protection of this application.

[0069] The above are only the preferred embodiments of the present utility model and are not intended to limit the present utility model. For those skilled in the art, various modifications and variations can be made to the present utility model. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present utility model shall be included within the scope of protection of the present utility model.

Claims

1. A cell culture chip, characterized in that, Comprising: A first substrate (10) having a first cell culture chamber (11) provided thereon, and the first cell culture chamber (11) is used for culturing first cells; A second substrate (20) having a plurality of flow-through micropores (23) provided thereon; A third substrate (30) having a second cell culture chamber (21) and a third cell culture chamber (31) provided thereon, the second cell culture chamber (21) is used for culturing second cells, the third cell culture chamber (31) is used for culturing third cells, and there are a plurality of microchannels (32) between the second cell culture chamber (21) and the third cell culture chamber (31), and both ends of each of the microchannels (32) are respectively communicated with the second cell culture chamber (21) and the third cell culture chamber (31); Both ends of the flow-through micropores (23) are respectively communicated with the first cell culture chamber (11) and the second cell culture chamber (21), so that the culture solution in the first cell culture chamber (11) can enter the third cell culture chamber (31) through the plurality of flow-through micropores (23) and the microchannels (32).

2. The cell culture chip according to claim 1, wherein, An annular flow-through portion (22) is provided on the second substrate (20), and a plurality of flow-through micropore groups are provided on the annular flow-through portion (22), the plurality of flow-through micropore groups are arranged at intervals in the circumferential direction of the annular flow-through portion (22), each of the flow-through micropore groups includes a plurality of the flow-through micropores (23), and the plurality of flow-through micropores (23) are arranged at intervals in the radial direction of the annular flow-through portion (22).

3. The cell culture chip according to claim 2, characterized in that, The third cell culture chamber (31) is circular, the second cell culture chamber (21) is annular, the second cell culture chamber (21) is provided on the circumferential outer side of the third cell culture chamber (31), the shape of the second cell culture chamber (21) is adapted to the shape of the annular flow-through portion (22), and the central axes of the third cell culture chamber (31), the second cell culture chamber (21) and the annular flow-through portion (22) are arranged to coincide with each other.

4. The cell culture chip according to claim 1, characterized in that, A first liquid injection port (12) and a second liquid injection port (13) are provided on the first substrate (10), a first communication port (24) and a second communication port (25) are provided on the second substrate (20), and a first liquid inlet (33) and a second liquid inlet (34) are provided on the third substrate (30). The first liquid inlet (33) is communicated with the second cell culture chamber (21), the first communication port (24) is communicated with the first liquid injection port (12), and the first communication port (24) is communicated with the first liquid inlet (33), so that the second cell mixture sequentially flows through the first liquid injection port (12), the first communication port (24), and the first liquid inlet (33) and enters the second cell culture chamber (21); the second liquid inlet (34) is communicated with the third cell culture chamber (31), the second communication port (25) is communicated with the second liquid injection port (13), and the second communication port (25) is communicated with the second liquid inlet (34), so that the third cell mixture sequentially flows through the second liquid injection port (13), the second communication port (25), and the second liquid inlet (34) and enters the third cell culture chamber (31); wherein, the second cell mixture includes the second cells and the culture medium, and the third cell mixture includes the third cells and the culture medium.

5. The cell culture chip according to claim 1, characterized in that A first liquid outlet (14) and a second liquid outlet (15) are provided on the first substrate (10), a third communication port (26) and a fourth communication port (27) are provided on the second substrate (20), and a third liquid outlet (35) and a fourth liquid outlet (36) are provided on the third substrate (30). The third liquid outlet (35) is communicated with the second cell culture chamber (21), the third communication port (26) is communicated with the first liquid outlet (14), and the third communication port (26) is communicated with the third liquid outlet (35), so that the culture medium in the second cell culture chamber (21) sequentially flows out through the third liquid outlet (35), the third communication port (26), and the first liquid outlet (14); the fourth liquid outlet (36) is communicated with the third cell culture chamber (31), the fourth communication port (27) is communicated with the second liquid outlet (15), and the fourth communication port (27) is communicated with the fourth liquid outlet (36), so that the culture medium in the third cell culture chamber (31) sequentially flows out through the fourth liquid outlet (36), the fourth communication port (27), and the second liquid outlet (15).

6. The cell culture chip according to claim 1, wherein A first liquid inlet channel (16) and a first liquid outlet channel (17) are provided on the first substrate (10). One end of the first liquid inlet channel (16) and one end of the first liquid outlet channel (17) are both communicated with the first cell culture chamber (11), and the other end of the first liquid inlet channel (16) is used for introducing a first cell mixture; wherein, the first cell mixture includes the first cells and the culture medium.

7. The cell culture chip according to claim 6, characterized in that, The other end of the first liquid inlet channel (16) forms a third liquid inlet, and the cell culture chip further includes a filter member (40). The filter member (40) is disposed in the first liquid inlet channel (16), and the filter member (40) is disposed between the third liquid inlet and the first cell culture chamber (11) to filter the incoming first cell mixture.

8. The cell culture chip according to claim 4, wherein A second liquid inlet channel (37) and a third liquid inlet channel (38) are disposed on the third substrate (30). One end of the second liquid inlet channel (37) is used to form the first liquid inlet (33), the other end of the second liquid inlet channel (37) communicates with the second cell culture chamber (21), one end of the third liquid inlet channel (38) is used to form the second liquid inlet (34), and the other end of the third liquid inlet channel (38) communicates with the third cell culture chamber (31).

9. The cell culture chip according to claim 5, wherein A second liquid outlet channel (39) and a third liquid outlet channel (391) are disposed on the third substrate (30). One end of the second liquid outlet channel (39) is used to form the third liquid outlet (35), the other end of the second liquid outlet channel (39) communicates with the second cell culture chamber (21), one end of the third liquid outlet channel (391) is used to form the fourth liquid outlet (36), and the other end of the third liquid outlet channel (391) communicates with the third cell culture chamber (31).

10. The cell culture chip according to claim 1, wherein, The cell culture chip further includes a plurality of barrier members (50). The plurality of barrier members (50) are spaced apart along the circumferential direction of the third cell culture chamber (31) between the third cell culture chamber (31) and the second cell culture chamber (21), and a microchannel (32) is formed between any two adjacent barrier members (50).

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