Open co-culture organ-chip and applications thereof

Abstract

The application relates to the technical field of biological tissue engineering, and discloses an open co-culture organ chip, which comprises one or more culture units, each culture unit comprises a center liquid storage hole in the form of a stepped blind hole; the bottom wall of the center liquid storage hole is provided with one or more culture holes, and the stepped surface of the center liquid storage hole is provided with a co-culture hole extending in the axial direction; and the center liquid storage hole comprises a column hole section protruding from the surface of the organ chip body. The co-culture organ chip is in an open form, the culture holes and the co-culture channel are located in a two-dimensional plane, and the openings are oriented to the same side, so that the operation difficulty is greatly reduced. Through standardized design of the arrangement and layout of the center liquid storage holes on the co-culture organ chip, the open operation channel is compatible with market operation and detection equipment, is more convenient, and is more suitable for industrial promotion. The application further discloses an application of the co-culture organ chip in construction of a multi-cell co-culture organ model.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological tissue engineering, for example to an open co-culture organ chip and application thereof. BACKGROUND

[0002] Cells or tissues need the interaction and communication between organs in the complete body, such as the domination of the nervous system, the "dialogue" of the immune system, and the nutritional support of interstitial cells. At present, although traditional well plates such as 24, 96, 384 well plates are widely used in the field of biological research such as cell culture, there are still some technical bottlenecks restricting their application in organ co-culture. Ordinary culture well plates cannot realize the co-culture between cells. Microfluidic organ chip technology is a new technology, which can overcome this limitation by designing various channels and be applied to the co-culture of various organs. However, the organ chip technology reported at present still has some technical bottlenecks restricting its large-scale application. Firstly, the stability and reproducibility of the product are poor, the product structure is complex, such as a closed channel, which needs to be operated by special technical personnel, and the stability and reproducibility are poor, and the subsequent analysis is difficult. Secondly, the product has low throughput and poor standardization, and the equipment compatibility is poor, which is difficult to popularize and apply.

[0003] In the implementation process of the embodiments of the present disclosure, it is found that at least the following problems exist in the related art: the existing co-culture organ chip is a closed structure, which leads to complex operation, difficult subsequent detection and analysis, low throughput and poor standardization. SUMMARY

[0004] The following presents a simplified summary of some aspects of the disclosed embodiments in order to provide a basic understanding of such embodiments. This summary is not an extensive overview of the embodiments described in detail in the following detailed description, and is not intended to identify key / critical elements of the embodiments or to delineate the scope of the embodiments. Its sole purpose is to present some aspects of the disclosed embodiments in a simplified form as a prelude to the more detailed description that is presented later.

[0005] The embodiments of the present disclosure provide an open co-culture organ chip and application thereof, to solve the problem that the existing co-culture organ chip is a closed structure, which leads to complex operation, difficult subsequent detection and analysis, low throughput and poor standardization.

[0006] In some embodiments, the open co-culture organ chip comprises one or more culture units, each culture unit comprising a center liquid storage hole in the form of a stepped blind hole; the center liquid storage hole is provided with one or more culture holes on the bottom wall, and a co-culture channel is provided on the stepped surface of the center liquid storage hole in the axial direction; the center liquid storage hole comprises a column hole segment protruding from the surface of the organ chip body.

[0007] In some embodiments, the co-culture organ chip is used for the application of constructing a multi-cell co-culture organ model.

[0008] The open co-culture organ chip provided by the embodiments of the present disclosure can achieve the following technical effects:

[0009] The co-culture organ chip provided by the embodiments of the present disclosure is open, the openings of the culture wells and the co-culture channels are on the same side, cell planting can be completed on the same side (for example, the upper side) of the organ chip, and the operation difficulty is greatly reduced. By standardizing the arrangement and layout of the central liquid storage holes on the co-culture organ chip, the open operation channel has strong compatibility with market operation, detection equipment and imaging equipment, is convenient for subsequent machine detection and cell recovery for RNA and protein extraction analysis, is simpler and more suitable for industrialization promotion. Moreover, the operation is simple and does not require professional technicians, thereby expanding the application range of the organ chip and improving the universality. The open co-culture organ chip can realize contact / non-contact co-culture of cells or tissue micro-organ, and can realize interaction on the premise of not polluting each other. The open co-culture organ chip can be used for in-vitro construction and long-term culture of multi-cell and multi-organ models, and can be used for in-vitro construction of a multi-cell co-culture model or a multi-organ co-culture model.

[0010] The foregoing general description and the following description are only exemplary and explanatory, and are not used to limit the present application. BRIEF DESCRIPTION OF DRAWINGS

[0011] One or more embodiments are exemplarily illustrated by corresponding drawings, which do not constitute limitation on the embodiments, elements with the same reference numerals in the drawings are shown as similar elements, the drawings do not constitute proportional limitation, and wherein:

[0012] Figure 1 is a top view structural schematic diagram of one culture unit of an open co-culture organ chip provided by the embodiments of the present disclosure;

[0013] Figure 2 is a sectional view structural schematic diagram of the A-A direction in Figure 1

[0014] Figure 3 is a top view structural schematic diagram of one culture unit of another open co-culture organ chip provided by the embodiments of the present disclosure;

[0015] Figure 4 is a sectional view structural schematic diagram of the B-B direction in Figure 5

[0016] Figure 5 is a top view structural schematic diagram of one culture unit of another open co-culture organ chip provided by the embodiments of the present disclosure;

[0017] Figure 6 is a sectional view structural schematic diagram of the C-C direction in Figure 5 ​​A cross-sectional structural schematic view in the direction of C-C;

[0018] Figure 7 A top view structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0019] Figure 8 A top view structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0020] Figure 9 A top view structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0021] Figure 10 A top view structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0022] Figure 11 A top view structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B; Figure 10 A cross-sectional structural schematic view in the direction of D-D;

[0023] Figure 12 A cross-sectional structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0024] Figure 13 An exploded structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0025] Figure 14 A cross-sectional structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0026] Figure 15 An exploded structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0027] Figure 16 A cross-sectional structural schematic view of another culture unit of the open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6B;

[0028] Figure 17 A structural schematic view of an open co-culture organ chip provided by the embodiments of the present disclosure is shown in FIG. 6A;

[0029] Figure 18 A fluorescence staining result characterization diagram of tumor cells in a tumor-liver co-model constructed by the embodiments of the present disclosure is shown in FIG. 7A;

[0030] Figure 19 A fluorescence staining result characterization diagram of human primary liver cells in a tumor-liver co-model constructed by the embodiments of the present disclosure is shown in FIG. 7B.

[0031] Figure 20 is a drug sensitivity detection result graph of tumor cells in a tumor-liver co-model constructed by the embodiment of the present disclosure;

[0032] Figure 21 is a hepatotoxicity detection result graph of human primary liver cells in a tumor-liver co-model constructed by the embodiment of the present disclosure.

[0033] Reference signs:

[0034] 10, central liquid storage hole; 11, stepped surface; 12, large pore diameter section; 13, small pore diameter section; 14, column hole section; 20, culture hole; 30, co-culture channel; 301, upper liquid storage section; 302, lower culture section; 31, co-culture liquid storage channel; 32, co-culture planting channel; 40, side liquid storage hole; 41, first side liquid storage hole; 42, second side liquid storage hole; 50, communication channel; 51, first communication channel; 52, second communication channel; 100, organ chip body; 101, enclosure; 102, liquid containing groove; 110, first liquid storage layer; 120, second liquid storage layer; 130, culture layer; 131, planting area; 140, bottom plate. DETAILED DESCRIPTION

[0035] In order to enable a more detailed understanding of the features and technical content of the embodiments of the present disclosure, the implementation of the embodiments of the present disclosure will be described in detail below, and the attached drawings are only used for reference and do not limit the embodiments of the present disclosure. In the following technical description, in order to facilitate explanation, through multiple details, a sufficient understanding of the disclosed embodiments is provided. However, one or more embodiments can still be implemented without these details. In other cases, in order to simplify the drawings, well-known structures and devices can be simplified.

[0036] The terms "first", "second", and the like in the specification and claims of the embodiments of the present disclosure and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily have to describe a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the embodiments of the present disclosure described herein can be implemented. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion.

[0037] In the embodiments of the present disclosure, the terms "upper", "lower", "inner", "middle", "outer", "front", "back" and the like indicate the orientation or positional relationship shown in the drawings. These terms are mainly used to better describe the embodiments of the present disclosure and its embodiments, and are not used to limit the indicated devices, elements or components to have a specific orientation, or to be constructed and operated in a specific orientation. In addition, in addition to indicating the orientation or positional relationship, the above-mentioned part of the terms can also be used to indicate other meanings, for example, the term "upper" can also be used to indicate a certain dependent relationship or connection relationship in some cases. For those skilled in the art, the specific meaning of these terms in the embodiments of the present disclosure can be understood according to the specific circumstances.

[0038] In addition, the terms "set", "connected", "fixed" should be broadly understood. For example, "connected" can be fixedly connected, detachably connected, or integrally configured; can be mechanically connected, or electrically connected; can be directly connected, or indirectly connected through an intermediate medium, or internal communication between two devices, elements or components. For those skilled in the art, the specific meaning of the above terms in the embodiments of the present disclosure can be understood according to the specific circumstances.

[0039] Unless otherwise specified, the term "a plurality of" means two or more.

[0040] It should be noted that the embodiments in the embodiments of the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0041] In combination with Figures 1-17 As shown, the present disclosure provides an open co-culture organ chip, which comprises one or more culture units. Each culture unit comprises a center liquid storage hole 10 in the form of a stepped blind hole; one or more culture holes 20 are arranged on the bottom wall of the center liquid storage hole 10, and one or more co-culture channels 30 are arranged on the stepped surface 11 of the center liquid storage hole 10 in the axial direction. Wherein, the center liquid storage hole 10 comprises a column hole segment 14 protruding from the surface of the organ chip body 100.

[0042] The co-culture organ chip of the embodiment of the present disclosure is open type, the openings of the culture wells 20 and the co-culture channels 30 are on the same side, cell planting can be completed on the same side (for example, the upper side) of the organ chip, which greatly reduces the operation difficulty; moreover, the central liquid storage holes 10 include the column hole sections 14, which can ensure the independence of the multiple central liquid storage holes 10 and avoid the mutual influence of the fluids in different holes and contamination. Through the standardized design of the arrangement and layout of the central liquid storage holes 10 on the co-culture organ chip, the open operation channel has strong compatibility with the market operation and detection equipment, is convenient for subsequent machine detection and cell recovery for RNA and protein extraction analysis, is simpler and more suitable for industrialization promotion. Moreover, the operation is simple and does not require professional technicians, which expands the application range of the organ chip and improves the universality.

[0043] In the embodiment of the present disclosure, the cells / organs planted in the central liquid storage holes 10 in different culture wells 20 can be independently cultured, and different cells / organs planted in the culture wells 20 and the co-culture channels 30 can be non-contact co-cultured, so that the open co-culture organ chip can realize non-contact co-culture of cells or tissue micro-organ and can be used for in-vitro construction and long-term culture of multi-cell and multi-organ models, and further can be used for in-vitro construction of multi-cell co-culture models or multi-organ co-culture models.

[0044] In the embodiment of the present disclosure, the central liquid storage holes 10 of the culture unit are ladder type blind holes, which include the large-diameter sections 12 and the small-diameter sections 13 in communication, and the large-diameter sections 12 are located on the upper layer. When the large-diameter sections 12 of the central liquid storage holes 10 are filled with culture medium or drug diluent, the cells / organs in the culture wells 20 and the co-culture channels 30 can be provided with the required nutrient solution or the drug to be tested; when only the small-diameter sections 12 of the central liquid storage holes 10 are filled with culture medium or drug diluent, only the cells / organs in the culture wells 20 can be provided with the required nutrient solution or the drug to be tested, that is, the small-diameter sections 12 of the central liquid storage holes 10 are exclusive liquid storage holes of the culture wells 20. When the liquid level of the culture medium or the drug diluent filled in the co-culture channels 30 does not exceed the ladder face 11 of the central liquid storage holes 10, the upper layer of the co-culture channels 30 serves as the exclusive liquid storage hole of the lower layer culture well section.

[0045] The multiple culture wells 20 at the bottom of the central liquid storage holes 10 can be inoculated with the same or different types of cells, and if the same cells are inoculated, selective planting can be performed according to the amount of the sample, for example, only one region (one culture well 20) can be planted to meet the use. The second organ cells are cultured in the co-culture channels 30, the upper layer thereof serves as an exclusive liquid storage channel, and the bottom / bottom wall is used for planting the second organ cells. The same or different types of cells can also be inoculated in the co-culture channels 30, which can be determined according to the actual needs.

[0046] In the embodiments of the present disclosure, the culture wells 20 and the co-culture channels 30 are through holes or blind holes. When the culture wells 20 and the co-culture channels 30 are through holes, the co-culture organ chip further comprises a bottom plate 140 arranged at the bottom of the culture wells and the co-culture channels. In use, the bottom plate 140 is placed on the bottom plate 140.

[0047] In the embodiments of the present disclosure, the shapes of the central liquid storage hole 10 and the culture well 20 are not limited and can be circular holes, elliptical holes, square holes, rectangular holes, fan-shaped holes or polygonal holes (for example, hexagonal holes, octagonal holes, etc.). In the case of meeting the design requirements, the shape of the central liquid storage hole 10 is designed to hold as much culture medium or drug diluent as possible, and the shape of the culture well 20 is designed to facilitate growth.

[0048] Optionally, the diameter of the large-diameter section 12 of the central liquid storage hole 10 is in the range of 15-20 mm. Optionally, the large-diameter section 12 of the central liquid storage hole 10 is a circular hole with a diameter in the range of 15-18 mm. Optionally, the diameter of the large-diameter section 12 of the central liquid storage hole 10 is 16.5 mm.

[0049] Optionally, the depth of the central liquid storage hole 10 is in the range of 15-20 mm. Optionally, the depth of the central liquid storage hole 10 is in the range of 17-18 mm. Optionally, the depth of the central liquid storage hole 10 is 17.4 mm.

[0050] Optionally, as shown in Figure 2 , the central liquid storage hole 10 is a column hole. The column protrudes from the surface of the organ chip body 100, and the hole structure is formed on the column.

[0051] Optionally, in combination with Figure 2 and Figure 4 , the radial width d of the stepped surface 11 of the central liquid storage hole 10 is in the range of 0.5-5 mm. That is, the radius of the small-diameter section 13 of the central liquid storage hole 10 is smaller than the radius of the large-diameter section 12 by 0.5-5 mm.

[0052] As for the culture wells 20 arranged at the bottom of the central liquid storage hole 10, the number and arrangement manner are not limited and can be determined according to the area and shape of the bottom of the central liquid storage hole 10. For example, the number of the culture wells 20 is one, two, three, four, five or more, and the specific number can be determined according to actual needs. The arrangement manner of the plurality of culture wells 20 can be array arrangement, for example, circular array or rectangular array, etc.

[0053] Optionally, the volume of the culture well 20 is in the range of 1-50 μL. The side length of the culture well 20 is not limited and can be any value as long as the volume requirement is met. Of course, other values can also be used as long as cells can be cultured.

[0054] For the co-culture channel 30, which is used for the culture of the second organ, in the axial direction, it includes the upper layer liquid storage channel 301 and the lower layer culture channel 302 which are communicated. While the open end of the co-culture channel 30 is located on the stepped surface 11 of the central liquid storage hole 10, the open end of the co-culture channel 30 is higher than the culture hole 20, which is used for non-contact co-culture. Its structure is not limited, as long as it can form a non-contact with the culture hole 20 at the bottom of the central liquid storage hole 10.

[0055] In some embodiments, in combination with Figure 1 and Figure 6 As shown, the co-culture channel 30 includes a closed / non-closed annular groove hole along the circumference of the stepped surface 11 of the central liquid storage hole 10. That is, the co-culture channel 30 is a whole structure. In this embodiment, when the co-culture channel 30 includes a closed annular groove hole, it is a blind hole. It ensures the integrity of the structure.

[0056] Alternatively, as shown in Figure 1 and Figure 3 The stepped surface 11 of the central liquid storage hole 10 is provided with a non-closed annular groove hole extending in the axial direction, which is used as a co-culture channel 30.

[0057] Alternatively, in combination with Figure 5 and Figure 6 The stepped surface 11 of the central liquid storage hole 10 is provided with a closed annular groove hole extending in the axial direction, which is used as a co-culture channel 30. In this embodiment, the annular groove hole can be a circular annular groove hole, which is a blind hole. It ensures the integrity of the organ chip body.

[0058] In some embodiments, in combination with Figures 7-9 As shown, the co-culture channel 30 includes a plurality of holes. That is, a plurality of holes are opened in the axial direction on the stepped wall surface of the central liquid storage hole 10. In this embodiment, the plurality of holes are distributed along the circumference of the stepped surface 11 of the central liquid storage hole 10. The cross section of the hole is not limited, which can be circular, elliptical, square, rectangular, sector ring or polygonal (such as hexagonal, octagonal, etc.) geometric shape. In the case of meeting the design requirements, the shape of the hole is designed to hold more culture medium or drug diluent as the design basis.

[0059] Alternatively, as shown in Figure 7 The stepped surface 11 of the central liquid storage hole 10 is provided with a plurality of holes with a sector ring cross section extending in the axial direction, which is used as a co-culture channel 30.

[0060] Alternatively, as shown in Figure 8 The stepped surface 11 of the central liquid storage hole 10 is provided with a plurality of holes with a circular cross section extending in the axial direction, which is used as a co-culture channel 30.

[0061] Alternatively, as shown in Figure 9As shown, the stepped surface 11 of the central liquid storage hole 10 is provided with a plurality of elliptical cross-section hole channels extending axially as the co-culture channel 30.

[0062] In some embodiments, the volume of the co-culture channel 30 is 20-500 μL. A certain inoculation amount is ensured. Here, the volume of the co-culture channel 30 refers to the overall volume of a closed / non-closed annular groove hole or the total volume of a plurality of hole channels. Of course, the volume of the co-culture channel 30 is not limited to this numerical range, and the volume of the co-culture channel 30 can be expanded to meet different inoculation amount requirements as the structure permits.

[0063] In some embodiments, in combination with Figures 3-6 As shown, the co-culture channel 30 includes an upper layer liquid storage section 301 and a lower layer culture section 302 in communication; the size of the lower layer culture hole section 302 is less than or equal to the size of the upper layer liquid storage hole section 301. When less than, the loss of inoculated cells can be avoided, and the inoculation rate is improved.

[0064] As shown in Figure 3 and Figure 4 As shown, the co-culture channel 30 is a non-closed annular groove hole provided on the stepped surface 11 and extending axially; the radial width of the lower layer culture section 302 is less than the radial width of the upper layer liquid storage section 301.

[0065] Optionally, one side wall of the non-closed annular groove hole is stepped. Optionally, the outer side wall of the non-closed annular groove hole is stepped.

[0066] In some embodiments, in combination with Figure 10 and Figure 11 As shown, the co-culture organ chip further includes a side liquid storage hole 40 and a communication channel 50, the side liquid storage hole 40 is provided in pairs around the liquid storage hole; the communication channel 50 connects the central liquid storage hole 10 and the paired side liquid storage holes 40 around it, and / or connects the co-culture channel 30 and the paired side liquid storage holes 40 around the central liquid storage hole 10 where the co-culture channel 30 is located. The co-culture chip of this embodiment is defined as a dynamic co-culture organ chip, which can realize dynamic culture or dynamic co-culture of the culture hole 20 and / or the co-culture channel 30, and can also be compatible with multiple fluid manipulation methods. Real-time dynamic update of the culture environment in the culture hole 20 and / or the co-culture channel 30 can be realized.

[0067] The dynamic co-culture organ chip of the embodiment has three communication modes. The first mode is that the first communication channel 51 only communicates the central liquid storage hole 10 and the pair of side liquid storage holes 40 around it. The second mode is that the second communication channel 52 only communicates the co-culture channel 30 and the pair of side liquid storage holes 40 around the central liquid storage hole 10 where the co-culture channel 30 is located. The third mode is that the communication channel 50 simultaneously communicates the central liquid storage hole 10 and the pair of side liquid storage holes 40 around it, and the co-culture channel 30 and the pair of side liquid storage holes 40 around the central liquid storage hole 10 where the co-culture channel 30 is located. In the third mode, the communication channel 50 can be the first communication channel 51 and the second communication channel 52 which are independently arranged, or the first communication channel 51 and the second communication channel 52 which are integrated into one communication channel 50 (as shown in FIG. 8). Figure 11

[0068] In the embodiment, a plurality of pairs of side liquid storage holes 40 can be arranged around one central liquid storage hole 10, and are not limited to one pair of side liquid storage holes 40 as shown in FIG. 7. A communication channel (such as the first communication channel 51) is arranged between the central liquid storage hole 10 and each side liquid storage hole 40 to communicate them. Moreover, the number of communication channels between the central liquid storage hole 10 and each side liquid storage hole 40 is not limited to one as shown in FIG. 7, and a plurality of communication channels can be arranged to improve the dynamic culture effect. Figure 10 Figure 10

[0069] Optionally, the side liquid storage holes 40 are arranged in pairs on opposite sides of the liquid storage hole to improve the balance of fluid flow and improve the dynamic culture effect.

[0070] In the embodiment, the communication channels 50 are arranged in pairs based on the pair arrangement of the side liquid storage holes 40. When the communication channel 50 communicates the co-culture channel 30 and the pair of side liquid storage holes 40 around the central liquid storage hole 10 where the co-culture channel 30 is located, a corresponding number of pairs of communication channels 50 are arranged according to the structure of the co-culture channel 30 to ensure that microfluid control can be achieved in the co-culture channel 30. For example, in the case of FIG. 6, the co-culture channel 30 includes a plurality of channels, and each channel is correspondingly provided with a pair / multiple pairs of side liquid storage holes, and each side liquid storage hole is respectively communicated with the corresponding channel. Figures 7-8

[0071] Optionally, as shown in FIG. 9, the side liquid storage hole 40 is a column hole. The same as the aforementioned central liquid storage hole 10 which is a column hole. Figure 11

[0072] In the embodiment, the size of the communication channel 50 is designed according to the microfluid control of the central liquid storage hole 10 and the culture medium in the co-culture. The cross-sectional shape is not limited and can be circular, square or other geometric shapes.

[0073] ​​​​​In some embodiments, the cross-sectional area of the communication channel 50 ranges from 0.01 mm2to 100 mm2. 2 Within the cross-sectional area range, microfluidic dynamic culture can be better achieved.

[0074] Optionally, the cross-section of the communication channel 50 is square, with a width ranging from 0.1 mm to 10 mm and a height ranging from 0.1 mm to 10 mm.

[0075] Optionally, the cross-section of the communication channel 50 is square, with a width ranging from 0.5 mm to 5 mm and a height ranging from 0.5 mm to 5 mm.

[0076] Optionally, the cross-section of the communication channel 50 is square, with a width of 2 mm and a height of 2 mm.

[0077] In this embodiment, the shape of the side liquid storage hole 40 is not limited and can be a circular hole, an elliptical hole, a square hole, a rectangular hole, a fan-shaped hole, or a polygonal hole (such as a hexagonal hole, an octagonal hole, etc.), and the shape of the side liquid storage hole 40 is designed to hold as much culture medium or drug diluent as possible under the condition of meeting the design requirements.

[0078] Optionally, the side liquid storage hole 40 adopts the hole size of a standard 48-well plate or a standard 96-well plate, which is convenient for subsequent machine detection and cell recovery for RNA and protein extraction analysis, and is simpler and more suitable for industrialization.

[0079] In the open co-culture organ chip of the embodiments of the present disclosure, only one or more of the aforementioned culture units are required, and the specific forming method is not limited.

[0080] In some embodiments, the open co-culture organ chip is integrally injection molded. For example, polystyrene (PS) and polymethyl methacrylate (PMMA) are integrally injection molded, which has low cost, is easy to injection mold, has no toxicity to cells, and has no specific adsorption.

[0081] In some embodiments, the open co-culture organ chip is obtained by layered processing and assembly. After layered processing and assembly, the whole is divided into parts, and the molding process is simplified.

[0082] The specific structure of an open co-culture organ chip is given below, but is not limited to this specific structure.

[0083] In some embodiments, the open co-culture organ chip is integrally injection molded. Figures 12-16As shown, the open co-culture organ chip includes a first liquid storage layer 110, a second liquid storage layer 120 and a culture layer 130 stacked in sequence. The first liquid storage layer 110 is provided with one or more first liquid storage holes; the second liquid storage layer 120 is provided with one or more second liquid storage holes and one or more co-culture liquid storage channels 31 arranged around each second liquid storage hole; the culture layer 130 is provided with one or more planting areas 131 and one or more co-culture planting channels 32 arranged around each planting area 131, and each planting area 131 is provided with one or more culture holes 20. The first liquid storage hole, the second liquid storage hole and the planting area 131 are coaxially arranged to form a central liquid storage hole 10; the co-culture liquid storage channel 31 and the co-culture planting channel 32 are arranged in one-to-one correspondence and in communication to form a co-culture channel 30.

[0084] In the embodiments of the present disclosure, the open co-culture organ chip includes a three-layer chip structure, and the three-layer chip structure is stacked and connected in sequence. The layers of chips can be bonded and assembled together by using sealing processes such as double-sided tape, ultrasonic, thermal bonding, plasma, hot pressing and the like.

[0085] In the embodiments of the present disclosure, the material of each layer of chip structure is PMMA, PS or the like. The manufacturing of each layer of structure can adopt soft lithography, molding method, laser etching, machining, LIGA or one-time injection molding to obtain each layer of chip structure.

[0086] In the embodiments, when the organ chip body 100 is layered and processed, the layered and processed organ chip body 100 can be segmented and processed according to the structural characteristics of the central liquid storage hole 10, the culture hole 20 and the co-culture channel 30.

[0087] Optionally, the diameter of the first liquid storage hole is greater than the diameter of the second liquid storage hole, and after the two are stacked, a stepped hole is formed. In the embodiments, the first liquid storage hole is the large-diameter section 12 of the central liquid storage hole 10, and the second liquid storage hole is the small-diameter section 13 of the central liquid storage hole 10. A plurality of through holes are processed in the first liquid storage layer 110 and the second liquid storage layer 120, respectively, which is simple in forming / processing.

[0088] Optionally, the size of the co-culture liquid storage channel 31 is greater than or equal to the size of the co-culture planting channel 32. In the embodiments, the co-culture liquid storage channel 31 is the same as the upper liquid storage section 301 described above, and the co-culture planting channel 32 is the same as the lower culture section 302 described above, which simplifies the molding process.

[0089] In some embodiments, as shown in FIG. 6, the co-culture organ chip includes a first liquid storage layer 110, a second liquid storage layer 120 and a culture layer 130 stacked in sequence. The first liquid storage layer 110 is provided with one or more first liquid storage holes; the second liquid storage layer 120 is provided with one or more second liquid storage holes and one or more co-culture liquid storage channels 31 arranged around each second liquid storage hole; the culture layer 130 is provided with one or more planting areas 131 and one or more co-culture planting channels 32 arranged around each planting area 131, and each planting area 131 is provided with one or more culture holes 20. The first liquid storage hole, the second liquid storage hole and the planting area 131 are coaxially arranged to form a central liquid storage hole 10; the co-culture liquid storage channel 31 and the co-culture planting channel 32 are arranged in one-to-one correspondence and in communication to form a co-culture channel 30. Figure 14 and Figure 15As shown, for a dynamic co-culture organ-on-a-chip, the first liquid storage layer 110 is further provided with a plurality of first side liquid storage holes 41 and a first connecting channel 51. The plurality of first side liquid storage holes 41 are arranged in pairs around the first liquid storage hole (e.g., on both sides); the first connecting channel 51 connects the first liquid storage hole to a pair of first side liquid storage holes 41 on both sides respectively; and / or, the second liquid storage layer 120 is further provided with a plurality of second side liquid storage holes 42 and a plurality of second connecting channels 52. The plurality of second side liquid storage holes 42 are arranged in pairs around the second liquid storage hole (e.g., on both sides); the second connecting channel 52 connects the second liquid storage hole to a pair of second side liquid storage holes 42 on both sides respectively.

[0090] This embodiment corresponds to the three communication methods described above. For the third communication method, the first communication channel 51 and the second communication channel 52 are independently configured or can be interlocked to form a single communication channel 50. "Independently configured" means that even after the first liquid storage layer 110 and the second liquid storage layer 120 are interlocked, the first communication channel 51 and the second communication channel 52 remain independent and do not mix.

[0091] For embodiments where the culture well 20 and / or co-culture channel 30 are through holes, combined with Figure 16 As shown, the open-cell co-culture organ-on-a-chip also includes a substrate 140, with a culture layer 130 stacked on top of the substrate 140. This facilitates inoculation and culture. The substrate 140 can be made of glass or PS.

[0092] In this embodiment of the disclosure, the first liquid storage hole (large diameter section 12) on the first liquid storage layer 110 is a cylindrical hole; when a side liquid storage hole 40 is provided, the first side liquid storage hole 41 is also a cylindrical hole (e.g. Figure 15 (As shown). In this embodiment, the first liquid storage layer 110 has multiple columnar holes protruding from its surface to prevent the fluids in different holes from affecting each other and to avoid contamination.

[0093] In some embodiments, such as Figure 17 As shown, the central liquid storage hole 10 (first liquid storage hole) is a cylindrical hole (cylindrical hole segment 14); or, when both the central liquid storage hole 10 and the side liquid storage holes 40 are cylindrical holes, a barrier 101 is provided on the four periphery of the surface of the organ-on-a-chip body 100, that is, the barrier 101 surrounds the four periphery of the surface of the organ-on-a-chip body 100. The barrier 101 and the cylindrical hole form a liquid storage tank, reducing the evaporation of the culture medium during the culture process and improving the culture effect.

[0094] In the embodiments of the present disclosure, the first liquid storage hole (large aperture section 12) and the second liquid storage hole (small aperture section 13) constitute a through-hole type central liquid storage hole. After the first liquid storage layer 110 and the second liquid storage layer 120 are sequentially stacked on the culture layer 130, the through-hole type central liquid storage hole and the surface of the culture layer 130 form a stepped blind hole type central liquid storage hole 10. At this time, the area of the through-hole type central liquid storage hole around the surface of the culture layer 130 is the culture layer planting area 131. The size of the culture layer planting area 131 can be consistent with the size of the second liquid storage hole, or can be smaller than the size of the second liquid storage hole, which is not limited. The culture hole 31 is arranged in the culture layer planting area 131.

[0095] The embodiments of the present disclosure provide an application of the co-culture organ chip for constructing a multi-cell co-culture organ model.

[0096] In the embodiments, in the multi-cell co-culture organ model, the multi-cell can be one or more cells from the same organ, or can be multiple cells from different organs. According to the types of cells, the constructed organ model is a single organ model or a multi-organ model.

[0097] In some embodiments, constructing a multi-cell co-culture organ model includes the following steps:

[0098] S11, respectively inoculating the first cell into the culture hole 20 of the co-culture organ chip and inoculating the second cell into the co-culture channel 30 of the co-culture organ chip;

[0099] S12, respectively adding the first culture medium into the central liquid storage hole 10 and adding the first culture medium or the second culture medium into the co-culture channel 30, and culturing to construct an organ model.

[0100] In steps S11 and S12, the first cell includes one or more cells of the first organ, and the second cell includes one or more cells of the second organ; the first organ and the second organ are the same or different. When the first organ and the second organ are the same, a single organ model is constructed. When the first organ and the second organ are different, a multi-organ model is constructed.

[0101] In step S12, the culture medium added into the central liquid storage hole 10 and the co-culture channel 30 can be the same or different.

[0102] Optionally, when they are different, that is, the first culture medium is added into the small aperture section 13 of the central liquid storage hole 10, and the second culture medium is added into the co-culture channel 30, the liquid levels of the first culture medium and the second culture medium are controlled to not exceed the stepped surface 11 of the central liquid storage hole 10. The independent culture of the first cell and the second cell is ensured.

[0103] Optionally, when the same, only need to fill the first medium into the central liquid storage hole 10, then the co-culture channel 30 is also filled with the first medium. Realize the co-culture of the first cell and the second cell.

[0104] Optionally, when the co-culture organ chip adopts the aforementioned dynamic co-culture organ chip, the culture adopts dynamic culture. The dynamic culture parameters are not limited.

[0105] In some embodiments, the multi-cell co-culture organ model is constructed, including the following steps:

[0106] S21, inoculate the first cell into the culture hole 20 of the co-culture organ chip, then add the first medium into the central liquid storage hole 10, culture, and construct the first organ model.

[0107] S22, after the first module is constructed for a first set time (for example, 72h), remove the first medium, then inoculate the second cell into the co-culture channel 30, then add the first medium into the central liquid storage hole 10, add the first medium or the second medium into the co-culture channel 30, culture, and construct the first organ-second organ model.

[0108] Among them, in steps S21 and S22, the first cell includes one or more cells of the first organ, and the second cell includes one or more cells of the second organ; the first organ and the second organ are the same or different. When the first organ and the second organ are the same, a single organ model is constructed. When the first organ and the second organ are different, a multi-organ model is constructed.

[0109] In step S22, the medium added into the central liquid storage hole 10 and the co-culture channel 30 can be the same or different.

[0110] Optionally, when different, that is, the first medium is added into the small hole diameter section 13 of the central liquid storage hole 10, and the second medium is added into the co-culture channel 30, the liquid level of the first medium and the second medium is controlled to not exceed the stepped surface 11 of the central liquid storage hole 10. Ensure independent culture of the first cell and the second cell.

[0111] Optionally, when the same, only need to fill the first medium into the central liquid storage hole 10, then the co-culture channel 30 is also filled with the first medium. Realize the co-culture of the first cell and the second cell.

[0112] Optionally, when the co-culture organ chip adopts the aforementioned dynamic co-culture organ chip, the culture adopts dynamic culture. The dynamic culture parameters are not limited.

[0113] In step S22, the first set time is not limited, which can be determined according to the type and inoculation amount of the first cell and other factors. Optionally, the first set time is 72h.

[0114] In some embodiments, the application further comprises an application of the constructed multi-cell co-cultured organ model for studying the interaction of multiple cells; or an application of the organ damage and drug efficacy co-evaluation study or the in-vitro drug efficacy evaluation study of organ metabolized drugs.

[0115] Specifically, after the organ model is constructed in step S12 or the first- second organ model is constructed in step S22, a detection step is further included.

[0116] Optionally, the method further comprises: S30, after the organ model / first-second organ model is cultured for a second set time, a mixed culture medium solution containing a drug to be studied is added to the central liquid storage hole 10, and then the organ model to be detected is obtained by culturing / dynamic culturing. The organ model to be detected is detected.

[0117] In step S30, the second set time is not limited and can be determined according to the types and inoculation amounts of the first cells and the second cells and the like. Optionally, the second set time is 24 h.

[0118] In the embodiments of the present disclosure, the first cells include tumor cells, such as HCT-116, NCI-H460, or MDA-MB-231, and the like. The second cells include one or a combination of any of human primary liver cells, hepatocyte cell line L02, and HapRG, and the like.

[0119] Optionally, the constructed multi-cell co-cultured organ model is used for studying the contact / non-contact immune response of multiple cells; or the non-contact co-culture of cells and feeder cells is studied.

[0120] The following takes the application of constructing a tumor-liver co-model for organ damage and drug efficacy study or in-vitro drug efficacy study of organ metabolized drugs as an example to specifically describe the co-cultured organ chip in the embodiments of the present disclosure.

[0121] Embodiment 1

[0122] The dynamic co-cultured organ chip using the culture unit as shown in Figure 15 is used to construct a tumor-liver co-model, including the following steps:

[0123] S41, sterilize the open dynamic co-culture organ chip, then add a mixed cell suspension containing tumor cells and matrix material into the culture well 20, culture at 37°C, gel; obtain a gelled tumor organ chip; then add a first culture medium (such as DMEM + serum or 1640 basic culture medium + serum) into the central reservoir well 10 of the gelled organ microchip, and continue to culture / dynamically culture at 37°C to construct a tumor model. The mixed cell suspension containing tumor cells and matrix material is a single cell suspension including matrix material and model cells, and the pH value is 6.5-7.5. The tumor cells refer to cell lines that respond to anticancer drugs, such as HCT116. The first culture medium can fill the central reservoir well 10, and the side reservoir well 40 is also filled with the first culture medium; or the first culture medium can partially fill the central reservoir well 10, and the filling amount is determined according to the culture requirement.

[0124] S42, prepare a single cell suspension of human primary liver cells, centrifuge and resuspend, and configure a cell suspension with a specific density as needed. In a 1.5ml EP tube, add a specific volume of collagen (which has been adjusted to pH with NaOH and hepes buffer solution) or other matrix materials such as Matrigel to obtain a mixed cell suspension, and ensure that the 3D material forms a good three-dimensional structure at this concentration (collagen is 1-2mg / ml, and Matrigel is 30%-80%). After culturing for 72h in step S41, remove the first culture medium, then add the mixed cell suspension into the lower culture section 302 of the co-culture channel 30, mix and homogenize with a pipette gun, quickly transfer and inoculate into the lower culture section 302 of the co-culture channel 30 with a pipette, and place it at 37°C for 10 minutes after cell planting to ensure that the matrix material can gel well. Add a tumor cell line culture medium into the small pore section 13 (second reservoir hole of the second reservoir layer 120) of the central reservoir well 10, and add a primary liver culture medium into the co-culture channel 30. Continue to dynamically culture at 37°C for another 24h; construct a tumor-liver co-model.

[0125] S43, drug stimulation of the tumor-liver co-model: after the liver-tumor co-model is constructed and continues to co-culture for 24h, fill the central reservoir well 10 with a formulated culture medium containing a specific type and concentration of drug to be screened (for example, 10μM of the antitumor drug sunitinib). Continue to dynamically culture at 37°C for another 120h.

[0126] S431, Drug Sensitivity Detection: After 120 hours of drug stimulation in step S43, remove the drug-containing culture medium. Add Cell titer glo to the small diameter section 13 (or the second reservoir) of the central reservoir 10 to evaluate the ATP content of the 3D cultured cells. This step is applicable to, but not limited to, the characterization methods described above. Other characterization reagents or methods are also compatible, such as Cell titer blue and high-content imaging techniques for imaging characterization of the number of live and dead cells.

[0127] S432, Hepatotoxicity Assay: After 120 hours of drug stimulation (step S43), the drug-containing culture medium is removed, and evaluation reagents are added to the co-culture channel 30. This section uses commercially available kits to characterize the liver function response after drug treatment using albumin, α-GST, or metabolic enzymes. Characterization methods can include the Elasa kit or quantification of the expression levels of major metabolic enzymes by extracting mRNA. In addition, existing ATP and metabolic capacity assays can be used to characterize hepatocyte activity after drug treatment, and mitochondrial membrane potential kits or mitochondrial reactive oxygen species kits can be used to characterize mitochondrial function. Therefore, single-parameter and multi-parameter methods can be used to characterize the hepatotoxicity results of the drug.

[0128] In this Example 1, the tumor cells and human primary liver cells in the tumor-liver co-model constructed in step S42 were characterized by F-actin and nuclear staining, respectively, to obtain the following results: Figure 18 The fluorescence staining results of the tumor cells shown are characterization diagrams and Figure 19 The fluorescence staining results of human primary liver cells shown in the image indicate that tumor cells and human primary liver cells express a high level of tight junction protein, demonstrating a high degree of biomimicry.

[0129] In this embodiment 1, as Figure 20 The drug sensitivity test results of step S431 shown in the figure can be seen that after adding the drug (sunitinib), the tumor inhibition rate was only 30%, indicating that at this concentration, the drug had almost no anti-tumor effect.

[0130] In this embodiment 1, as Figure 21 The hepatotoxicity test results for step S432 shown in the figure indicate that the inhibition rate was close to 100% after the addition of the drug (sunitinib), indicating that the drug has a strong hepatotoxic effect at this concentration.

[0131] The above description and drawings suffice to fully enable one skilled in the art to practice the embodiments of the present disclosure. Other embodiments can include structural and other changes. The embodiments are merely representative of possible variations. Individual components and functions are optional unless explicitly required, and the order of operations can be varied. Portions and features of some embodiments can be included in, or substituted for, portions and features of other embodiments. The embodiments of the present disclosure are not limited to the structures described above and shown in the drawings, and can be varied in a variety of ways. The scope of the present disclosure is limited only by the claims that follow.

Claims

1. An open-cell co-culture organ-on-a-chip, characterized in that, include: Multiple culture units, each of the culture units comprising: The central liquid storage hole is a stepped blind hole; one or more culture holes are provided on the bottom wall of the central liquid storage hole, and a co-culture channel is provided along the axial direction on the stepped surface of the central liquid storage hole. Side liquid storage holes are arranged in pairs around the central liquid storage hole; and A connecting channel connects the central liquid storage hole with the side liquid storage holes on both sides, and connects the co-culture channel with the side liquid storage holes on both sides of the central liquid storage hole; wherein, on the axial projection plane through the central liquid storage hole, the projection of the connecting channel falls into the open end side of the co-culture channel, and the remaining part falls into the large-diameter section of the central liquid storage hole. The central liquid storage hole includes a columnar section protruding from the surface of the organ-on-a-chip body; the side liquid storage holes are columnar holes. The co-culture channel includes an upper liquid storage section and a lower culture section connected in the axial direction. The open end of the co-culture channel is located on the stepped surface of the central liquid storage hole, so that the open end of the co-culture channel is higher than the culture hole. The co-cultivation channel includes a closed / open annular groove along the circumferential direction of the stepped surface of the central liquid reservoir, and is a connected integral structure.

2. The co-cultured organ-on-a-chip according to claim 1, characterized in that, The volume of the co-culture channel is 20–500 μL.

3. The co-cultured organ-on-a-chip according to claim 2, characterized in that, The volume of a co-cultivation channel refers to the overall volume of a closed / open annular slot or the total volume of multiple channels.

4. The co-cultured organ-on-a-chip according to claim 1, characterized in that, The volume of the culture well is 1–50 μL.

5. The co-cultured organ-on-a-chip according to any one of claims 1 to 4, characterized in that, The cross-sectional area of ​​the connecting channel ranges from 0.01 to 100 mm². 2 .

6. The co-cultured organ-on-a-chip according to claim 5, characterized in that, The cross-section of the connecting channel is square, with a width ranging from 0.1 to 10 mm and a height ranging from 0.1 to 10 mm.

7. The co-cultured organ-on-a-chip according to claim 5, characterized in that, The cross-section of the connecting channel is square, with a width ranging from 0.5 to 5 mm and a height ranging from 0.5 to 5 mm.

8. The co-cultured organ-on-a-chip according to any one of claims 1 to 4, characterized in that, The size of the lower culture section is less than or equal to the size of the upper liquid storage section.

9. The application of co-cultured organ-on-a-chip as described in any one of claims 1 to 8 for constructing multi-cell co-cultured organ models.

10. The application according to claim 9, characterized in that, The construction of the multi-cell co-culture organ model includes: The first cells were seeded into the culture wells of the co-culture organ chip, and the second cells were seeded into the co-culture channels of the co-culture organ chip. Add the first culture medium to the central reservoir well, and add either the first culture medium or the second culture medium to the co-culture channel, culture, and construct an organ model; Alternatively, the construction of the multi-cell co-culture organ model includes: The first cells were seeded into the culture wells of the co-cultured organ chip, and then the first culture medium was added into the central reservoir well. The cells were cultured to construct the first organ model. After constructing the first organ module for a first set time, the first culture medium is removed, and then the second cells are seeded into the co-culture channel. Then, the first culture medium is added to the central reservoir, and the first culture medium or the second culture medium is added to the co-culture channel. The cells are cultured to construct the first-second organ model. Wherein, the first cell includes one or more cells of a first organ, and the second cell includes one or more cells of a second organ; the first organ and the second organ may be the same or different; When the second culture medium is added into the co-culture channel, the liquid levels of both the first and second culture media do not exceed the stepped surface of the central reservoir. When the co-cultured organ-on-a-chip is the co-cultured organ-on-a-chip as described in claim 5 or 6, the culture is performed using dynamic culture.

11. The application according to claim 10, characterized in that, The first cell includes tumor cells; the second cell includes one or any combination of human primary liver cells, hepatocyte line LO2, and HapRG.

12. The application according to any one of claims 9 to 11, characterized in that, Also includes: The application of the constructed multi-cell co-cultured organ model in the study of interactions among various cells; Alternatively, it can be used for co-evaluation studies of organ damage and drug efficacy, or for in vitro efficacy evaluation studies of drugs that metabolize organs.

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