WO2022116406A1

WO2022116406A1 - Open-type co-culture organ-on-a-chip and use thereof

Info

Publication number: WO2022116406A1
Application number: PCT/CN2021/080069
Authority: WO
Inventors: 肖荣荣; 刘建闯; 李珮文; 张晓会; 周宇
Original assignee: 北京大橡科技有限公司
Priority date: 2020-12-02
Filing date: 2021-03-10
Publication date: 2022-06-09

Abstract

Disclosed in the present application is an open-type co-culture organ-on-a-chip. The co-culture organ-on-a-chip comprises one or more culture units, wherein each culture unit comprises a central liquid storage hole, which is a stepped blind hole; one or more culture holes are provided in a bottom wall of each central liquid storage hole, and a co-culture hole is provided in a stepped surface of each central liquid storage hole and runs in an axial direction; and each central liquid storage hole comprises a column hole section protruding out of a surface of the body of the organ-on-a-chip. The co-culture organ-on-a-chip is open, the culture holes and a co-culture channel are located in a two-dimensional plane, and openings face the same side, such that operation difficulty is significantly reduced. An arrangement layout of the central liquid storage holes in the co-culture organ-on-a-chip is designed in a standardized manner, the open-type operation channel is highly compatible with operation and detection apparatuses in the market, and the co-culture organ-on-a-chip is simpler, more convenient and more suitable for industrial popularization. Further disclosed in the present application is the use of a co-culture organ-on-a-chip in constructing a multicell co-culture organ model.

Description

Open co-culture organ chip and its application

This application is based on the Chinese patent application with the application number of 202011389825.9 and the application date of December 2, 2020, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is incorporated herein by reference.

This application is based on the Chinese patent application with the application number of 202022865811.1 and the filing date of December 2, 2020, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is incorporated herein by reference.

This application is based on the Chinese patent application with the application number of 202120355192.3 and the filing date of February 8, 2021, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is incorporated herein by reference.

technical field

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

Background technique

Cells or tissues in a complete body require interaction and communication between organs, such as the innervation of the nervous system, the "dialogue" dialogue of the immune system, and the nutritional support of interstitial cells. At present, although traditional well plate culture platforms such as 24, 96, and 384 well plates are widely used in biological research fields such as cell culture, there are still some technical bottlenecks that limit their application in organ co-culture. Ordinary culture plates cannot achieve co-culture between cells. Microfluidic organ-on-a-chip technology is an emerging technology, which can overcome this limitation by designing various channels and be applied to the co-culture of various organs. However, the reported organ-on-a-chip technology has some technical bottlenecks that limit its large-scale application. First of all, the stability and reproducibility of the product are poor, and the product structure is complex, such as a closed channel, which requires specialized technical personnel to operate, the stability and reproducibility are relatively poor, and subsequent analysis is difficult. Secondly, the product has low throughput, poor standardization, and poor equipment compatibility, making it difficult to popularize and apply.

In the process of implementing the embodiments of the present disclosure, it is found that there are at least the following problems in the related art: the existing co-culture organ chip is a closed structure, which leads to complicated operation, difficult subsequent detection and analysis, low throughput and poor standardization.

SUMMARY OF THE INVENTION

In order to provide a basic understanding of some aspects of the disclosed embodiments, a brief summary is given below. This summary is not intended to be an extensive review, nor to identify key/critical elements or delineate the scope of protection of these embodiments, but rather serves as a prelude to the detailed description that follows.

The embodiments of the present disclosure provide an open co-culture organ chip and its application, to solve the problems of the existing co-culture organ chip being a closed structure, resulting in complicated operation, difficult subsequent detection and analysis, low throughput and poor standardization.

In some embodiments, the open co-culture organ chip includes one or more culture units, each culture unit includes a central liquid storage hole, which is a stepped blind hole; a bottom wall of the central liquid storage hole is provided with a or a plurality of culture holes, a co-cultivation channel is axially extended on the stepped surface of the central liquid storage hole.

In some embodiments, the aforementioned co-culture organ chip is used to construct a multi-cell co-culture organ model.

The open co-culture organ chip provided by the embodiment of the present disclosure can achieve the following technical effects:

The co-culture organ chip provided in the embodiments of the present disclosure is open, and the openings of the culture hole and the co-cultivation channel face the same side, so that cell planting can be completed on the same side (eg, the upper side) of the organ chip, which greatly reduces the difficulty of operation. By standardizing the layout of the central liquid storage hole on the co-culture organ chip, the open operation channel has strong compatibility with market operation, testing equipment, and imaging equipment, and is convenient for subsequent on-machine testing. As well as cell recovery for RNA, protein extraction and other analysis, it is simpler and more suitable for industrialization. Moreover, the operation is simple, no professional technicians are required, the application scope of the organ chip is expanded, and the universality is improved. The open co-culture organ chip can realize the contact/non-contact co-culture of cells or tissue micro-organs, and can realize interaction without mutual contamination. It can be used for the in vitro construction and long-term culture of multi-cell and multi-organ models, and then it can be used to construct multi-cell co-culture models or multi-organ co-culture models in vitro.

The foregoing general description and the following description are exemplary and explanatory only and are not intended to limit the application.

Description of drawings

One or more embodiments are exemplified by the accompanying drawings, which are not intended to limit the embodiments, and elements with the same reference numerals in the drawings are shown as similar elements, The drawings do not constitute a limitation of scale, and in which:

1 is a schematic top view of a culture unit of an open co-culture organ chip provided by an embodiment of the present disclosure;

Fig. 2 is the sectional structure schematic diagram of A-A in Fig. 1;

Fig. 3 is the sectional structure schematic diagram of another kind of culture unit of A-A direction shown in Fig. 1;

4 is a schematic top-view structural diagram of a culture unit of another open type co-culture organ chip provided in an embodiment of the present disclosure;

Fig. 5 is the sectional structure schematic diagram of B-B in Fig. 4;

6 is a schematic top-view structural diagram of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

Fig. 7 is the sectional structure schematic diagram of the C-C direction in Fig. 6;

8 is a schematic top-view structural diagram of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

9 is a schematic top-view structural diagram of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

10 is a schematic top-view structural diagram of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

11 is a schematic top-view structural diagram of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

Fig. 12 is the sectional structure schematic diagram of the D-D direction in Fig. 11;

13 is a schematic cross-sectional structural diagram of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

14 is a schematic cross-sectional structural diagram of a culture unit of another open type co-culture organ chip provided in an embodiment of the present disclosure;

15 is a schematic exploded structure diagram of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

16 is a schematic diagram of an explosion structure of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

17 is a schematic cross-sectional structural diagram of a culture unit of another open type co-culture organ chip provided by an embodiment of the present disclosure;

18 is a schematic exploded structure diagram of another culture unit provided in an embodiment of the present disclosure, taken along the A-A direction in FIG. 1 ;

19 is a schematic structural diagram of a culture chamber module provided by an embodiment of the present disclosure;

20 is a schematic structural diagram of another culture chamber module provided by an embodiment of the present disclosure;

21 is a schematic cross-sectional structural diagram of a culture unit of another co-culture organ chip provided in an embodiment of the present disclosure;

22 is a schematic cross-sectional structural diagram of a culture unit of another co-culture organ chip provided in an embodiment of the present disclosure;

23 is a schematic cross-sectional structural diagram of a culture unit of another co-culture organ chip provided in an embodiment of the present disclosure;

24 is a schematic cross-sectional structural diagram of a culture unit of another co-culture organ chip provided in an embodiment of the present disclosure;

25 is a schematic cross-sectional structural diagram of a culture unit of another co-culture organ chip provided in an embodiment of the present disclosure;

26 is an exploded schematic diagram of a cross-sectional structure of a culture unit of another co-culture organ chip provided in an embodiment of the present disclosure;

27 is a cross-sectional structural schematic diagram of another culture unit provided in an embodiment of the present disclosure, taken along the D-D direction in FIG. 11 ;

28 is a cross-sectional structural schematic diagram of another culture unit provided in an embodiment of the present disclosure, taken along the D-D direction in FIG. 11;

29 is a schematic structural diagram of an open co-culture organ chip provided by an embodiment of the present disclosure;

30 is a graph showing the results of fluorescent staining of tumor cells in a tumor-liver co-model constructed in accordance with an embodiment of the present disclosure;

FIG. 31 is a graph showing the results of fluorescent staining of human primary liver cells in a tumor-liver co-model constructed according to an embodiment of the present disclosure;

32 is a graph showing the results of drug sensitivity detection of tumor cells in a tumor-liver co-model constructed in accordance with an embodiment of the present disclosure;

33 is a graph showing the results of hepatotoxicity detection of human primary liver cells in a tumor-liver co-model constructed according to an embodiment of the present disclosure;

Reference number:

10. Central liquid storage hole; 11. Step surface; 12. Large aperture section; 13. Small aperture section; 20. Culture hole; 30. Co-cultivation channel; co-cultivation storage channel; 32, co-cultivation 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 (chip platform); 101, enclosure; 102, liquid storage tank; 110, first liquid storage layer; 120, second liquid storage layer; 130, culture layer; 131, planting area; 140, bottom plate; 150, central liquid storage straight hole; 160, upper platform; 170, lower platform; 200, culture chamber module; 210, culture area; 211, separation edge; 220, wall; 300 310, an annular member; 320, a first annular member; 321, a communication hole; 330, a second annular member; 410, a side liquid storage column hole.

Detailed ways

In order to understand the features and technical contents of the embodiments of the present disclosure in more detail, the implementation of the embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings, which are for reference only and are not intended to limit the embodiments of the present disclosure. In the following technical description, for the convenience of explanation, numerous details are provided to provide a thorough understanding of the disclosed embodiments. However, one or more embodiments may be practiced without these details. In other instances, well-known structures and devices may be shown simplified in order to simplify the drawings.

The terms "first", "second" and the like in the description and claims of the embodiments of the present disclosure and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data so used may be interchanged under appropriate circumstances for the purposes of implementing the embodiments of the disclosure described herein. Furthermore, the terms "comprising" and "having", and any variations thereof, are intended to cover non-exclusive inclusion.

In the embodiments of the present disclosure, the orientations or positional relationships indicated by the terms "upper", "lower", "inner", "middle", "outer", "front", "rear", etc. are based on the orientations shown in the drawings or Positional relationship. These terms are primarily used to better describe the embodiments of the present disclosure and embodiments thereof, and are not intended to limit the fact that the indicated device, element, or component must have a particular orientation, or be constructed and operated in a particular orientation. In addition, some of the above-mentioned terms may be used to express other meanings besides orientation or positional relationship. For example, the term "on" may also be used to express a certain attachment or connection relationship in some cases. For those of ordinary skill in the art, the specific meanings of these terms in the embodiments of the present disclosure can be understood according to specific situations.

In addition, the terms "arranged", "connected" and "fixed" should be construed broadly. For example, "connection" may be a fixed connection, a detachable connection, or a unitary construction; it may be a mechanical connection, or an electrical connection; it may be a direct connection, or an indirect connection through an intermediary, or two devices, elements or Internal connectivity between components. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present disclosure can be understood according to specific situations.

Unless stated otherwise, the term "plurality" means two or more.

It should be noted that the embodiments in the embodiments of the present disclosure and the features in the embodiments may be combined with each other in the case of no conflict.

1-29, an embodiment of the present disclosure provides an open co-culture organ chip, including one or more culture units, each culture unit includes a central liquid storage hole 10, which is a stepped blind hole; One or more culture holes 20 are provided on the bottom wall of the liquid hole 10 , and one or more co-cultivation channels 30 are arranged on the stepped surface 11 of the central liquid storage hole 10 along the axial direction.

The co-culture organ chip of the embodiment of the present disclosure is open, and the openings of the culture hole 20 and the co-culture channel 30 face the same side, so that the cell planting can be completed on the same side (eg, the upper side) of the organ chip, which greatly reduces the difficulty of operation. . By standardizing the layout of the central liquid storage hole 10 on the co-culture organ chip, the open operation channel has strong compatibility with market operation and testing equipment, and is convenient for subsequent on-machine testing and cell testing. It is easier to recover and perform analysis such as RNA and protein extraction, and is more suitable for industrialization. Moreover, the operation is simple, no professional technicians are required, the application scope of the organ chip is expanded, and the universality is improved.

In the embodiment of the present disclosure, cells/organs planted in different culture holes 20 in the central reservoir hole 10 can be independently cultured, and different cells/organs planted in the culture holes 20 and the co-culture channel 30 respectively can be cultured in a non-contact co-culture. Therefore, the open co-culture organ chip can realize the non-contact co-culture of cells or tissue micro-organs, and can be used for the in vitro construction and long-term culture of multi-cell and multi-organ models, and then can be used for in vitro construction of multi-cell Co-culture model or multi-organ co-culture model.

In the embodiment of the present disclosure, the central liquid storage hole 10 of the culture unit is a stepped blind hole, which includes a large-diameter section 12 and a small-diameter section 13 in communication, and the large-diameter section 12 is located in the upper layer. Then, when the large-diameter section 12 of the central liquid storage hole 10 is filled with a culture medium or a drug diluent, it can provide the required nutrient solution or the drug to be tested for the cells/organs in the culture hole 20 and the co-cultivation channel 30 When only the small aperture section 12 of the central reservoir hole 10 is filled with culture medium or drug diluent, it only provides the required nutrient solution or the drug to be tested for the cells/organs in the culture hole 20, that is, the central reservoir The small-diameter section 12 of the liquid hole 10 is an exclusive liquid storage hole of the culture hole 20 . However, when the liquid level of the medium or drug diluent filled in the co-cultivation channel 30 does not exceed the stepped surface 11 of the central liquid storage hole 10, the upper layer of the co-cultivation channel 30 serves as the exclusive liquid storage hole of the lower layer of the culture hole section. .

A plurality of culture holes 20 at the bottom of the central reservoir hole 10 can be seeded with the same or different types of cells. If the same cells are seeded, they can be selectively seeded according to the amount of the sample. For example, only one area (one culture hole 20) can be seeded. ) is sufficient for use. The cells of the second organ are cultured in the co-cultivation channel 30, the upper layer of which is used as a dedicated liquid storage channel, and the bottom/bottom wall is used for planting and culturing the cells of the second organ. Cells of the same or different types can also be seeded in the co-cultivation channel 30, which can be determined according to actual needs.

In the embodiment of the present disclosure, the culture hole 20 and the co-culture channel 30 are through holes or blind holes. Wherein, as shown in the figure, when the culture hole 20 and the co-culture channel 30 are through holes, the co-culture organ chip further includes a bottom plate 140 disposed at the bottom of the culture hole and the co-culture channel. It is placed on the bottom plate 140 during use.

In the embodiment of the present disclosure, the shape of the central liquid storage hole 10 and the culture hole 20 is not limited, and may be geometric shapes such as a circular hole, an ellipse, a square, a rectangle, a fan, or a polygon (for example, a hexagon, an octagon, etc.). Under the condition that the design requirements are met, the shape of the central liquid storage hole 10 is based on the design basis of containing as much culture medium or drug diluent as possible, and the shape of the culture hole 20 is based on the design basis for facilitating growth.

Optionally, the diameter of the large-diameter section 12 of the central liquid storage hole 10 ranges from 15 to 20 mm. Optionally, the diameter of the large-diameter section 12 of the central liquid storage hole 10 is a circular hole with a diameter ranging from 15 to 18 mm. Optionally, the diameter of the large aperture section 12 of the central liquid storage hole 10 is 16.5 mm.

Optionally, the depth of the central liquid storage hole 10 is 15-20 mm. Optionally, the depth of the central liquid storage hole 10 is 17-18 mm. Optionally, the depth of the central reservoir hole 10 is 17.4 mm.

Optionally, as shown in FIG. 2 , the central liquid storage hole 10 is a column hole. There are pillars protruding from the surface of the organ chip body 100, and a hole structure is formed on the pillars.

Optionally, as shown in FIG. 3 , the central liquid storage hole 10 is a concave hole. It is opened in the organ chip body 100 .

Optionally, as shown in FIG. 2 and FIG. 5 , the radial width d of the stepped surface 11 of the central liquid storage hole 10 is 0.5-5 mm. That is, the radius of the small-diameter segment 13 of the central liquid storage hole 10 is smaller than the radius of the large-diameter segment 12 by 0.5-5 mm.

For the culture wells 20 , they are arranged at the bottom of the central liquid storage hole 10 , and the number and arrangement 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 culture wells 20 is 1, 2, 3, 4, 5 or more, and the specific number can be determined according to actual needs. The arrangement of the plurality of culture wells 20 may be an array arrangement, for example, a circular array or a rectangular array.

Optionally, the volume of the culture well 20 is 1-50 μL. The side length dimension of the culture well 20 is not limited, as long as the volume requirement is met. Of course, other values are also possible, as long as cells can be cultured.

As for the co-culture channel 30 , which is used for culturing the second organ, in the axial direction, it includes an upper-layer liquid storage channel 301 and a lower-layer culture channel 302 . While the open end of the co-cultivation channel 30 is located on the stepped surface 11 of the central liquid storage hole 10, the open end of the co-cultivation channel 30 is higher than the culture hole 20 for non-contact co-culture. Its structural form is not limited, as long as it is guaranteed to form non-contact with the culture hole 20 at the bottom of the central liquid storage hole 10 .

In some embodiments, as shown in FIG. 1 and FIG. 7 , the co-cultivation channel 30 includes a closed/non-closed annular slot along the circumference of the stepped surface 11 of the central liquid storage hole 10 . That is, the co-cultivation channel 30 is a connected integral structure. In this embodiment, when the co-cultivation channel 30 includes a closed annular slot, it is a blind hole. Ensure structural integrity.

Optionally, as shown in FIG. 1 and FIG. 4 , a non-closed annular slot is provided on the stepped surface 11 of the central liquid storage hole 10 along the axial direction to serve as a co-cultivation channel 30 .

Optionally, as shown in FIG. 6 and FIG. 7 , the stepped surface 11 of the central liquid storage hole 10 is provided with a closed annular slot extending in the axial direction as the co-cultivation channel 30 . In this embodiment, the annular slotted hole may be an annular slotted hole, which is a blind hole. Ensure the integrity of the organ chip body.

In some embodiments, as shown in conjunction with FIGS. 8-10 , the co-cultivation channel 30 includes a plurality of channels. That is, a plurality of channels are opened along the axial direction on the stepped wall surface of the central liquid storage hole 10 . In this embodiment, a plurality of holes are distributed along the circumferential direction of the stepped surface 11 of the central liquid storage hole 10 . The cross-section of the channel is not limited, and can be geometric shapes such as circle, ellipse, square, rectangle, fan ring or polygon (such as hexagon, octagon, etc.) Hold more culture medium or drug diluent as the design basis.

Optionally, as shown in FIG. 8 , on the stepped surface 11 of the central liquid storage hole 10 , a plurality of channels with fan-shaped cross-sections extending in the axial direction are provided as co-cultivation channels 30 .

Optionally, as shown in FIG. 9 , the stepped surface 11 of the central liquid storage hole 10 is axially extended with a plurality of channels with circular cross-sections as co-cultivation channels 30 .

Optionally, as shown in FIG. 10 , on the stepped surface 11 of the central liquid storage hole 10 , a plurality of channels with elliptical cross-sections extending in the axial direction are provided as co-cultivation channels 30 .

In some embodiments, the volume of the co-culture channel 30 is 20-500 μL. Ensure a certain amount of inoculation. Here, the volume of the co-cultivation channel 30 refers to the entire volume of a closed/non-closed annular slot or the total volume of a plurality of channels. Of course, the volume of the co-cultivation channel 30 is not limited to this numerical range, and if the structure allows, the volume of the co-cultivation channel 30 can be expanded to meet different inoculum requirements.

In some embodiments, as shown in FIG. 4 to FIG. 7 , the co-cultivation channel 30 includes a connected upper layer liquid storage section 301 and a lower layer culture section 302 ; the size of the lower layer culture hole section 302 is smaller than or equal to the size of the upper layer liquid storage hole section 301 size. When it is equal to, the co-cultivation channel 30 is a straight channel (shown in conjunction with FIG. 2 and FIG. 3 ), and the shape is simple. When less than 30, the co-cultivation channel 30 is a stepped channel, that is, its cross section is stepped (as shown in FIG. 5 and FIG. 7 ), which can avoid the loss of inoculated cells and improve the inoculation rate.

As shown in FIG. 4 and FIG. 5 , the co-cultivation channel 30 is provided with a non-closed annular slot extending axially on the stepped surface 11 ; the radial width of the lower culturing section 302 is smaller than the radial width of the upper liquid storage section 301 .

Optionally, one side wall of the non-closed annular slot is stepped. Optionally, the outer sidewall of the non-closed annular slot is stepped.

In some embodiments, as shown in FIG. 18 , the area containing the culture hole 20 on the bottom wall of the central liquid storage hole 10 is divided to form a culture chamber module 200; an installation position is set on the bottom wall of the central liquid storage hole 10, The culture chamber module 200 is detachably arranged at the installation position of the bottom wall of the central liquid storage hole 10 . Then the culture chamber module 200 can be disassembled from the co-culture organ chip, or assembled, and can be flexibly replaced. The co-culture organ chip body 100 after the culture chamber module 200 is removed can be regarded as a chip platform 100. Therefore, the culture chamber module 200 with the appropriate shape, number and size of the culture holes 20 can be selected to be assembled to the actual needs. On the chip platform 100, it can meet the requirements of accurate experimental design, be flexible, increase the application scenarios of organ chips, and increase the application upper limit of organ chips. In this embodiment, a central liquid storage hole 10 and a culture chamber module 200 detachably arranged therein constitute a culture unit. There are one or more culture units on the chip platform 100 . The co-culture organ chip in this embodiment can be defined as a detachable/assembleable co-culture organ chip.

In this embodiment, on the chip platform 100 , the central liquid storage hole 10 may adopt a through-hole structure, and the port at the bottom end of the central liquid storage hole 10 is the installation position. The culture chamber module 200 is detachably disposed on the port at the bottom end of the central liquid storage hole 10 .

In this embodiment, the culture chamber module 200 can be detachably assembled on the chip platform 100 by means of colloidal bonding, tenon-and-mortise structure fitting, or chemical bonding.

Optionally, the mortise and tenon structure is used for fitting. Specifically, a protruding tenon (tenon) is provided on the peripheral wall of the culture chamber module 200, a concave tenon (mortise) is provided on the inner peripheral wall of the installation position (or bottom port) of the central liquid storage hole, the tenon and snap into place to removably set the culture chamber module 200 on the mounting position (or bottom port).

Optionally, chemical bonding is used. Specifically, the culture cell module 200 is made of polydimethylsiloxane (PDMS) material. After being treated by oxygen plasma, it can be bonded to the smooth surface of the chip platform of glass or its own material. Chemical bonding occurs to form a stable co-culture chamber structure.

In the co-culture organ chip of this embodiment, the number and specifications of the culture chamber modules 200 are not limited, and appropriate culture chamber modules 200 are assembled on the chip platform 100 according to actual needs. Optionally, the plurality of culturing chamber modules 200 at least include the same number of culturing chamber modules 200 as the number of the central liquid storage holes 10 on the chip platform 100. Of course, among the multiple culturing chamber modules 200, the number of The number of culture wells can be the same or different, and the shapes can also be the same or different, and can be combined at will.

In this embodiment, culture cell modules 200 of one specification or multiple specifications are assembled on a plurality of central liquid storage holes 10 of a chip platform 100 respectively. The specifications of the culture chamber module 200 include one or more of the number of culture compartments, the size of the culture compartment, the shape of the culture compartment, the size of the culture compartment module 200 and the shape of the culture compartment module 200 . By assembling the culture chamber modules 200 of various specifications on one chip platform 100, an experiment to verify the differences of samples produced by different culture partitions under the same culture environment conditions can be performed.

In the embodiment of the present disclosure, the shape of the culture hole is not limited, and it may be one or more culture holes 20 (as shown in FIG. 1 ) provided on the culture chamber module 200 , or it may be a separation rib on the culture chamber module 200 . One or more culture areas 210 defined by 211 (shown in Figures 19 and 20).

In this embodiment, the structure of the culture hole 20 on the culture chamber module 200 may refer to the above-mentioned related content of the culture hole 20 .

In some embodiments, the stepped portion in which the co-cultivation channel 30 is arranged in the central liquid storage hole 10 is divided to form a culture channel module. After the culture channel module is divided, the central liquid storage hole 10 which is a stepped hole includes a central liquid storage straight hole 150, and a culture channel module. The culture channel module is detachably arranged in the central liquid storage straight hole 150, and the culture channel The first end of the channel module is flush with the bottom end of the central liquid storage straight hole 150 and is provided with an installation position; wherein, the end surface of the second end of the culture channel module (equivalent to the aforementioned stepped surface 11 ) is provided with an axially extending The co-cultivation channel 30, or the culture channel module cooperates with the central liquid storage straight hole 150 to form the co-cultivation channel 30. In this embodiment, the stepped portion in which the co-cultivation channel 30 is arranged in the stepped central liquid storage hole 10 is divided, so that the stepped portion forms an independent module, which can be disassembled or assembled. That is, in this embodiment, the structure of the co-cultivation channel 30 is also designed to be detachable to form an independent culture channel module. The culture channel module can be flexibly replaced. When in use, a culture channel module with a co-cultivation channel 30 having a suitable specification is selected for assembly. to the central liquid storage straight hole 150. In this embodiment, the molding process of the chip platform 100 is simplified. The chip platform 100 can be a plate body with one or more straight holes as the central liquid storage straight hole 150 . The structure is simple and the molding is easy. When in use, the culture channel module and the culture chamber module 200 can be assembled into the straight hole.

Optionally, the culture channel module is detachably disposed in the central liquid storage straight hole 150 by means of colloidal bonding, mortise and tenon structure fitting or chemical bonding.

Optionally, the mortise and tenon structure is used for fitting (not shown in the figure). A protruding tenon (tenon) is arranged on the peripheral wall of the culture channel module, and a concave mortise (mortise) is arranged on the inner peripheral wall of the central liquid storage straight hole. in the central reservoir straight hole.

Optionally, chemical bonding is used. Specifically, the culture channel module is made of polydimethylsiloxane (PDMS) material. After being treated by oxygen plasma, it can be formed with the smooth surface of the chip platform of glass or its own material. Chemically bonded to form a stable co-culture unit structure.

In some embodiments, as shown in FIG. 21 , the first culture channel module is in the shape of a column, and a through hole is provided on it in the axial direction (the through hole is the small diameter section 13 of the central liquid storage hole 10 ), The port at one end serves as an installation location, and a co-cultivation channel 30 extending in the axial direction is provided on the end wall of the second end. In this embodiment, the culture channel module is disassembled/assembled as an independent module, and the culture chamber module 200 is disassembled/assembled on the first end of the culture channel module 300 . The outer side wall of the first type of culture channel module is arranged in contact with the inner wall of the central liquid storage straight hole 150, and can be connected by colloidal bonding or tenon-and-mortise fitting.

In some embodiments, the second type of culture channel module includes an annular member 310 , and the annular member 310 is detachably disposed in the central straight liquid storage hole 150 in a manner of contacting with the inner wall of the central liquid storage straight hole 150 or at a set interval. Inside, a co-cultivation channel 30 is formed in cooperation with the central liquid storage hole 10 (eg, its inner wall); and its first end (ie, the end flush with the bottom end of the central liquid storage straight hole 150 ) is provided with a mounting position . In this embodiment, the shape of the outer side wall of the annular member 310 (opposite to the inner wall of the central liquid storage straight hole 150 ) may be determined according to the structure and shape of the co-cultivation channel 30 .

In this embodiment, when the annular member 310 and the inner wall of the central liquid storage straight hole 150 are arranged at a set interval, the shape of the annular member 310 and the central liquid storage straight hole 150 may be the same or different, which is not limited.

Optionally, as shown in FIG. 22 , the outer side wall of the first ring member 310 is linear, and the outer side wall and the inner wall of the central liquid storage straight hole 150 are detachably arranged in the central liquid storage in a manner of a set interval. Inside the straight hole 150. The co-cultivation channel 30 constructed in this embodiment is a straight slot hole, that is, the size of the lower culturing section 302 is equal to the size of the upper liquid storage section 301 . In addition, the co-cultivation channel 30 constructed in this embodiment is a non-closed annular slot, and the non-closed position may be the detachable connection between the annular member 310 and the central liquid storage straight hole 150 . In this embodiment, the set interval is the radial width of the co-cultivation channel 30, which is determined according to actual needs.

Optionally, the second type of ring member 310 has a stepped outer side wall; it is detachably disposed in the central liquid storage straight hole 150 in a manner that the outer wall of the bottom step is in contact with the inner wall of the central liquid storage straight hole 150 or at a set interval. Inside. In the second type of ring member 310 in this embodiment, the bottom step is the largest step in the size of the ring member.

In this embodiment, when the outer wall of the bottom step of the second type of annular member 310 is in contact with the inner wall of the central liquid storage straight hole 150 and is detachably disposed in the central liquid storage straight hole 150, the constructed co-cultivation channel 30 is a closed annular shape Slotted holes and blind holes. The co-cultivation channel 30 is a straight channel or a stepped channel according to the number of steps of the stepped outer sidewall.

In this embodiment, when the outer wall of the bottom step of the second type of ring member 310 and the inner wall of the central liquid storage straight hole 150 are detachably disposed in the central liquid storage straight hole 150 at a set interval, the constructed co-cultivation channel 30 is a stepped channel, that is, the size of the lower culturing section 302 of the co-cultivation channel 30 is smaller than the size of the upper liquid storage section 301, which can avoid the loss of inoculated cells and improve the inoculation rate. In this embodiment, the set interval is the radial width of the lower culturing section 302 of the co-cultivation channel 30, which is determined according to actual needs.

In some embodiments, the third culture channel module includes a first annular member 320 and a second annular member 330 . The first annular member 320 is detachably disposed in the central liquid storage straight hole 150 and its outer wall is in contact with the inner wall of the central liquid storage straight hole 150 . The second annular member 330 is sleeved inside the first annular member 320 , and is connected and cooperated with the first annular member 320 to form the co-cultivation channel 30 . Its first end (the end flush with the bottom end of the central liquid storage straight hole 150 ) is provided with an installation position. Wherein, the first ring member 320 is fixedly connected or detachably connected with the second ring member 330 . In this embodiment, when the second annular member 330 and the first annular member 320 are fixedly connected, the structure can be the same as the aforementioned first culture channel module (as shown in FIG. 8 ), which is an integral and independent module; When it is detachably connected, the culture channel module is further divided, which makes the structure of the co-culture organ chip more flexible and has more application scenarios.

In this embodiment, the structure of the second ring member 330 may be the same as that of the ring member 310 , and correspondingly defined as the first type of second ring member 330 (the outer side wall is linear) and the second type of second ring member 330 (outer side wall). The walls are stepped). It is not repeated here.

Optionally, the second annular member 330 is detachably sleeved inside the first annular member 320 in a manner of being in contact with the inner wall of the first annular member 320 or at a set interval. The outer side wall of the second ring member 330 is opposite to the inner side wall of the first ring member 320 to form a co-cultivation channel 30 .

Optionally, the inner side wall of the first ring member 320 is linear or stepped. Among them, as shown in FIG. 26 , the first annular member 320 whose inner sidewall is linear is referred to as the first type of first annular member. As shown in FIG. 23 to FIG. 25 , the first annular member 320 whose inner sidewall is in a stepped shape is referred to as the second type of first annular member.

Optionally, a third type of culture channel module includes a first type of first annular member 320 and a first type of second annular member 330, the first type of second annular member 330 and the inner wall of the first annular member 320 are arranged They are detachably arranged inside the first ring member 320 in a spaced manner. Refer to the structure shown in FIG. 2 . The formed co-cultivation channel 30 is a non-closed annular slot and is a through hole.

Optionally, another third type of culture channel module includes a first type of first ring member 320 and a second type of second ring member 330, and a second type of second ring member 330 and the first type of first ring member 320 The inner wall of the ring contacts or is detachably disposed inside the first ring member 320 in a set interval. The formed co-cultivation channel 30 is a non-closed annular slot and is a through hole.

Optionally, another third type of culture channel module, as shown in FIG. 23 , includes a second type of first ring member 320 and a first type of second ring member 330, the first type of second ring member 330 is combined with the first type of second ring member 330. The inner walls of the second type of first ring members 320 are detachably disposed inside the second type of first ring members 320 in a manner of setting intervals. The cross-sectional shape of the constructed co-cultivation channel 30 is stepped and is a through hole.

Alternatively, the first-type second annular member 330 is detachably disposed inside the second-type first annular member 320 in a manner of being in contact with the inner wall of the second-type first annular member 320 . The constructed co-cultivation channel 30 can be a straight slot hole or a stepped slot hole, and is a blind hole.

Optionally, another third type of culture channel module includes a second type of first annular member 320 and a second type of second annular member 330, the first type of second annular member 330 is connected to the inner wall of the first annular member 320. Contact (as shown in FIG. 24 ) or detachably arranged inside the first ring member 320 in a set interval. In this embodiment, the height of the bottom step of the second type of first ring member 320 and the height of the bottom step of the second type of second ring member 330 are the same or different; Well co-culture channel 30.

In a detachable/assembleable co-culture organ chip according to an embodiment of the present disclosure, both the culture channel module and the culture chamber module 200 can be disassembled/assembled. Therefore, after the size of the central liquid storage hole 150 of the chip platform 100 is determined , the size of the co-cultivation channel 30 (for example, the radial width of the co-cultivation channel 30 ) can be adjusted by adjusting the size of the annular member 310 or the second annular member 330 , so that the planting area of the co-cultivation channel 30 and the cultivation hole 20 can be adjusted In order to indirectly adjust the proportion of cells seeded in the culture channel module and the culture chamber module 200, it is more flexible and changeable. According to actual needs, it is sufficient to assemble different culture channel modules and culture chamber modules 200 on the chip platform 100 .

In some embodiments, the culture chamber module 200 is fixedly connected to the culture channel module to form a co-culture module; the co-culture module is detachably arranged in the central liquid storage straight hole 150 . That is, the culture chamber module 200 is fixedly connected in the installation position (or, the first end port) of the first end of the culture channel module, or both are integrally formed. In this embodiment, the co-cultivation module constitutes an integral independent module, which can be assembled into the central liquid storage straight hole 150 of the chip platform 100, which is convenient for disassembly and assembly.

Optionally, in the first type of co-cultivation module, the culture channel module includes a ring member 310, and the culture chamber module 200 is fixedly connected to the installation position of the end of the ring member 310 to form a co-cultivation module. Specifically, the annular member 310 is integrally formed with the culture chamber module 200 , that is, the co-cultivation module has an open culture dish structure, and one or more culture culture holes 20 are provided on the bottom wall. The first co-cultivation module of this embodiment forms an integral independent module. For example, the culture chamber module 200 shown in FIG. 22 is fixedly attached to the ring member 310 .

Optionally, in the first type of co-cultivation module, as shown in FIG. 24 , the culture channel module includes a first annular member 320 and a second annular member 330, and the culture chamber module 200 is fixedly connected to the second annular member 330 to form a co-cultivation module. Cultivation module. Specifically, the second annular member 330 is integrally formed with the culture chamber module 200 to form an open culture dish structure. In this embodiment, when the first annular member 320 and the second annular member 330 are fixedly connected, the co-cultivation module forms an integral independent module. Referring to FIG. 21 , the culture chamber module 200 is fixedly connected to the culture channel module 300 . When the first annular member 320 and the second annular member 330 are detachably connected, the co-cultivation module includes the detachably connected first annular member 320 and the culture dish structure, as shown in FIGS. 23 and 24 .

The area (or volume) of the co-cultivation channel 30 can be adjusted by increasing or decreasing the size of the first co-cultivation module or the size of the petri dish structure in the second co-cultivation module (ie, the area of the culture chamber module 200 ). , and then adjust the ratio of the cells seeded in the culture well 20 of the culture chamber module 200 to the co-culture channel 30 .

In some embodiments, as shown in FIG. 24 , FIG. 25 and FIG. 26 , when the culture channel module in the co-cultivation module includes the first annular member 320 , the second end of the first annular member 320 is extended to make the first annular member 320 extend. Pieces 320 form a reservoir structure. Then, the thickness of the chip platform 100 can be reduced. In this embodiment, the extended first annular member 320 , the second annular member 330 and the culture chamber module 200 constitute a complete culture unit, which realizes the overall detachable assembly of a culture unit and is more flexible to use.

In some embodiments, the culture channel module and the culture chamber module 200 are prepared using materials having one or more properties of biocompatibility, bionics, cell permeability and degradability. Biocompatible, biomimetic or cell-permeable modular materials not only satisfy specific structural functions, but also better ensure the normal growth state of cells in in vitro models and are similar to those in vivo, reducing external factors. Effects of biomimetic function in an in vitro model. The degradable module material improves the environmental friendliness of the module.

Optionally, the culture channel module and the culture chamber module 200 are prepared by using biocompatible materials through 3D printing technology.

Optionally, the culture channel module and the culture chamber module 200 are prepared by techniques such as laser engraving.

In some embodiments, the culture chamber module 200 is fabricated using a hydrogel material. Expanding the application scenario, you can do cell migration or transfer experiments. Optionally, the culture chamber module 200 is prepared by using a soft hydrogel material. For example, methacrylated gelatin (gelma), alginate or collagen.

In some embodiments, as shown in FIG. 23 , the co-cultivation channel 30 includes an upper-layer liquid storage section 301 and a lower-layer cultivation section 302 in communication; with the upper-layer liquid storage section 301 and the central liquid storage hole 10 (or the central liquid storage straight hole 150) between the side walls are removable. That is, the side wall of the co-cultivation channel 30 on the side of the culture chamber module 200 corresponding to the upper-layer liquid storage section 301 is detachable. This part of the side wall is similar to the wall 220 between the culture hole 20 and the co-culture channel 30 in the culture chamber module 200, which can avoid mixed cross-contamination of samples/culture solutions in the two module areas. In this embodiment, the enclosure wall 220 is designed to be detachable, so that the organ chip can flexibly switch between the advantages of improving the exchange efficiency of culture components and avoiding mixed cross-contamination of samples in different culture areas. For example, 200 plant organoids in the culture chamber module and 30 stromal cells are planted in the co-culture channel. When static/dynamic co-culture is performed, the wall is removed and the wall-free mode is adopted, which can better promote the elimination of metabolic waste in the culture chamber. and promote the influence of the small molecules produced by the stromal cells B in the co-culture channel 30 on the cells A in the culture well 20 of the culture chamber module 200, so as to better improve the exchange efficiency of culture components and achieve the advantages of the co-culture system. When the cells A in the culture wells are digested and collected, after removing the medium in the organ chip, the wall is assembled, for example, a mortise-and-mortise structure is used to fix the wall, so as to isolate the connection with the co-culture channel 30 on the outside, and avoid the co-culture channel. 30 The cross-contamination of stromal cells B to cells A in the culture chamber ensures the purity of the collected target cells.

Optionally, the ring member 310 or the second ring member 330 is divided into detachable upper and lower halves. In some embodiments, as shown in FIG. 11 and FIG. 12 , the co-culture organ chip further includes side liquid storage holes 40 and communication channels 50 , and a plurality of side storage holes 10 are provided around each central liquid storage hole 10 . The liquid hole 40; the communication channel 50 communicates with the central liquid storage hole 10 and the side liquid storage holes 40 around it, and/or, communicates the co-cultivation channel 30 with the lateral liquid storage holes 40 around the central liquid storage hole 10 where it is located . The co-culture chip in this embodiment is defined as a dynamic co-culture organ chip, which can realize dynamic culture or dynamic co-culture of the culture well 20 and/or the co-culture channel 30 , and can also be compatible with various fluid manipulation methods. Real-time dynamic updating of the culture environment in the culture well 20 and/or the co-culture channel 30 can be achieved.

The dynamic co-culture organ chip of this embodiment has three communication modes. The first is that the first communication channel 51 only communicates with the central liquid storage hole 10 and the paired side liquid storage holes 40 around it; the second is the second communication The channel 52 only communicates with the co-cultivation channel 30 and the paired side liquid storage holes 40 around the central liquid storage hole 10 where it is located; the third type is that the communication channel 50 communicates with the central liquid storage hole 10 and its surrounding paired sides at the same time. The liquid storage hole 40, and the pair of side liquid storage holes 40 that communicate with the co-cultivation channel 30 and the periphery of the central liquid storage hole 10 where the co-cultivation channel 30 is located. In the third communication mode, the communication channel 50 may be the first communication channel 51 and the second communication channel 52 set independently, or may be a communication channel 50 that integrates the first communication channel 51 and the second communication channel 52 together (as shown in Figure 12).

In this embodiment, there are two or more side liquid storage holes 40 around each central liquid storage hole 10 to realize dynamic culture. Optionally, the side liquid storage holes 40 are arranged around the central liquid storage hole 10 in pairs. That is, one or more pairs of side liquid storage holes 40 may be provided around each central liquid storage hole 10 , not limited to the pair of side liquid storage holes 40 shown in FIG. 11 . A communication channel (eg, a first communication channel 51 ) is provided between the central liquid storage hole 10 and each side liquid storage hole 40 to communicate with each other. Moreover, the number of communication channels between the central liquid storage hole 10 and each side liquid storage hole 40 is not limited to the one shown in FIG. 11 , and multiple communication channels may be provided to improve the dynamic culture effect.

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

In this embodiment, based on the paired arrangement of the side liquid storage holes 40 , the communication channels 50 are also arranged in pairs. Wherein, when the communication channel 50 communicates with the co-cultivation channel 30 and the paired side liquid storage holes 40 around the central liquid storage hole 10 where the co-cultivation channel 30 is located, a corresponding number of paired communication channels 50 are arranged according to the structure of the co-cultivation channel 30 to ensure that Microfluidic control can be realized in the co-cultivation channel 30 . For example, for FIG. 8 to FIG. 9 , when the co-cultivation channel 30 includes a plurality of channels, each channel is correspondingly provided with a pair/multiple pairs of side liquid storage holes, and each side liquid storage hole is respectively connected with the corresponding hole. That's it.

Optionally, as shown in FIG. 12 , the side liquid storage holes 40 are column holes. The same as the aforementioned central liquid storage hole 10 in the form of a column hole.

Optionally, the side liquid storage holes 40 are concave holes. Reference is made to the aforementioned central reservoir hole 10 in the form of a concave hole.

In this embodiment, the size design of the communication channel 50 is based on realizing the microfluidic control of the central liquid storage hole 10 and/or the medium in the co-culture. Its cross-sectional shape is also not limited, and may be circular, square or other geometric shapes.

In some embodiments, the cross-sectional area of the communication channel 50 ranges from 0.01 to 100 mm ² . Within this cross-sectional area range, the microfluidic dynamic culture can be better realized.

Optionally, the cross section of the communication channel 50 is square, the width is in the range of 0.1-10 mm, and the height is in the range of 0.1-10 mm.

Optionally, the cross section of the communication channel 50 is square, the width is in the range of 0.5-5 mm, and the height is in the range of 0.5-5 mm.

Optionally, the cross section of the communication channel 50 is square, the width is in the range of 2 mm, and the height is in the range of 2 mm.

In this embodiment, the shape of the side liquid storage holes 40 is not limited, and may be geometric shapes such as round holes, ellipses, squares, rectangles, sectors, or polygons (such as hexagons, octagons, etc.). In some cases, the shape of the side liquid storage hole 40 is designed to hold as much culture medium or drug diluent as possible.

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

In the embodiment of the present disclosure, in the aforementioned detachable/assembleable co-culture chip, when the culture channel module is separated from the chip platform 100, a communication hole is provided on the side wall of the corresponding position of the culture channel module, Used to communicate with the communication channel 50 . A communication hole 321 is provided on the first ring member 320 as shown in FIG. 27 .

In some embodiments, as shown in FIG. 27 , when the side liquid storage hole 40 is a column hole, the column hole part of the side liquid storage hole 40 may be divided to form an independent side liquid storage column hole 410 . The side liquid storage hole 410 is detachably disposed in the concave hole portion of the side liquid storage hole 40 . In this embodiment, the volume of the side liquid storage holes 40 can be increased, and the thickness of the chip platform 100 can also be reduced.

Optionally, the size of the side liquid storage column hole 410 may be equal to or larger than the size of the side liquid storage hole 40 . When it is larger than that, more culture medium or drug dilution can be contained.

In the embodiment of the present disclosure, for the detachable/assembleable co-culture organ chip, when designing the detachable connection, reference can be made to the aforementioned connection method of the culture chamber module 200 and the culture channel module 300, for example, colloidal bonding, tenon and mortise The manner of structural chimeric or chemical bonding will not be repeated here.

In the open co-culture organ chip of the embodiment of the present disclosure, as long as it has one or more culturing units, the specific molding method is not limited.

In some embodiments, the open co-culture organ chip is integrally injection molded. Alternatively, in a detachable/assembleable co-culture chip, the chip platform 100 can be an integral structure. Optionally, the chip platform 100 is a plate body, and a plurality of central liquid storage holes 10 or central liquid storage straight holes 150 are opened on the plate body. For example, one-piece injection molding, or laser etching on the plate body, etc. For example, polystyrene (PS) and polymethyl methacrylate (PMMA) are used for integral injection molding. These materials are low-cost, easy to injection mold, and have no toxicity to cells and no specificity. Sexual adsorption.

In some embodiments, the open co-culture organ-on-a-chip or platform on a chip is obtained through a layered fabrication and assembly configuration. After layered processing, it is assembled, broken into parts, and the molding process is simplified.

A specific structural form of an open co-culture organ chip is given below, but is not limited to this specific structural form.

In some embodiments, as shown in FIG. 13 to FIG. 17 , the open co-culture organ chip includes a first liquid storage layer 110 , a second liquid storage layer 120 and a culture layer 130 that are 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 second liquid storage holes around each second liquid storage hole. A plurality of co-cultivation storage channels 31; the culture layer 130 is provided with one or more planting areas 131 and one or more co-cultivation planting channels 32 surrounding each planting area 131, and each planting area 131 is provided with one or more co-cultivation planting channels 32. A plurality of culture wells 20 . The first liquid storage hole, the second liquid storage hole and the planting area 131 are coaxially arranged to form the central liquid storage hole 10 ;

In the embodiment of the present disclosure, the open co-culture organ chip includes a three-layer chip structure, and the three-layer chip structure can be stacked and connected in sequence. The layers of chips can be assembled together using a sealing process such as double-sided tape, ultrasound, thermal bonding, plasma, and hot pressing.

In the embodiment of the present disclosure, the material of the chip structure of each layer is PMMA, PS, or the like. The fabrication of each layer structure may adopt soft lithography, molding method, laser etching, machining, LIGA technology or one-time injection molding to obtain the chip structure of each layer.

In this embodiment, when the organ chip body 100 is processed in layers, it may be divided and processed according to the structural features of the central liquid storage hole 10 , the culture hole 20 and the co-culture channel 30 .

Optionally, the diameter of the first liquid storage hole is larger than the diameter of the second liquid storage hole, and after the two are stacked, a stepped hole is formed. In this embodiment, the first liquid storage hole is the large aperture section 12 of the central liquid storage hole 10 , and the second liquid storage hole is the small aperture section 13 of the central liquid storage hole 10 . It is only necessary to process a plurality of through holes in the first liquid storage layer 110 and the second liquid storage layer 120, and the forming/processing is simple.

Optionally, the size of the co-cultivation reservoir channel 31 is greater than or equal to the size of the co-cultivation planting channel 32 . In this embodiment, the co-cultivation liquid storage channel 31 is the same as the aforementioned upper-layer liquid storage section 301 , and the co-cultivation planting channel 32 is the same as the aforementioned lower-layer culture section 302 , which simplifies the molding process.

In some embodiments, as shown in FIG. 14 and FIG. 16 , for the dynamic co-culture organ chip, the first liquid storage layer 110 is further provided with a plurality of first side liquid storage holes 41 and a first communication channel 51 . The first side liquid storage holes 41 are arranged in pairs around the first liquid storage hole (eg, on both sides); the first communication channels 51 are respectively connected to the first liquid storage hole and the pair of first side storage holes on both sides of the first liquid storage hole. 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 communication channels 52, and the plurality of second side liquid storage holes 42 are arranged in pairs Around the second liquid storage hole (eg, on both sides); the second communication channels 52 respectively communicate with the second liquid storage hole and the pair of second side liquid storage holes 42 on both sides thereof.

This embodiment corresponds to the aforementioned three communication modes, wherein, for the third communication mode, the first communication channel 51 and the second communication channel 52 are independently arranged or can be buckled to form a communication channel 50 . The independent setting means that even after the first liquid storage layer 110 and the second liquid storage layer 120 are engaged, the first communication channel 51 and the second communication channel 52 remain independent, and no mixed flow occurs.

For the embodiment in which the culture hole 20 and/or the co-culture channel 30 are through holes, as shown in FIG. 17 , the open co-culture organ chip further includes a bottom plate 140 on which the culture layer 130 is stacked. Easy to inoculate and cultivate. The bottom plate 140 can be a glass or PS bottom plate.

In the embodiment of the present disclosure, the first liquid storage hole (large aperture section 12 ) on the first liquid storage layer 110 is a column hole; when the side liquid storage hole 40 is provided, the first side liquid storage hole 41 is also a column hole. Column hole (as shown in Figure 16). In this embodiment, the first liquid storage layer 110 has a plurality of column holes protruding from the surface thereof, so as to prevent fluids in different holes from interacting with each other and avoid contamination.

In some embodiments, as shown in FIG. 29 , the central liquid storage hole 10 (the first liquid storage hole) adopts a column hole; Enclosures 101 are provided on the four peripheral edges of the surface of the chip body 100 , that is, the enclosures 101 are enclosed on the four peripheral edges of the surface of the organ chip body 100 . Then, a liquid storage tank is formed between the enclosure 101 and the column hole, which reduces the evaporation of the medium during the cultivation process and improves the cultivation effect.

In the embodiment of the present disclosure, the first liquid storage hole (large aperture section 12 ) and the second liquid storage hole (small aperture section 13 ) are formed by a through-hole type central liquid storage hole. After the liquid storage layers 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 are surrounded by a central liquid storage hole 10 forming a stepped blind hole. At this time, the through-hole type central liquid storage hole 10 is formed. The area surrounded by the hole on the surface of the culture layer 130 is the culture layer planting area 131, and the size of the culture layer planting area 131 can be consistent with the size of the second liquid storage hole, or it can be smaller than the size of the second liquid storage hole, Without limitation, the culture hole 31 may be arranged in the culture layer planting area 131 .

In the embodiment of the present disclosure, another chip platform 100 can be obtained by layering and assembling. After layered processing, it is assembled and broken into pieces, which simplifies the molding process and facilitates the molding of the structure on the chip platform 100 .

In some embodiments, the chip platform 100 including the side liquid storage holes 40 and the communication channel 50 is designed as a two-layer structure. As shown in FIG. 28 , the chip body includes an upper platform 160 and a lower platform 170 . The upper platform 160 is provided with a plurality of first side liquid storage holes 41 and a first communication channel 51 , and a plurality of first side liquid storage holes 41 Pairs are arranged around the first liquid storage hole (eg, on both sides); the first communication channels 51 are respectively connected to the first liquid storage hole and the pair of first side liquid storage holes 41 on both sides of the first liquid storage hole; and/or, the lower layer The platform 170 is also provided with a plurality of second side liquid storage holes 42 and a plurality of second communication channels 52, and the plurality of second side liquid storage holes 42 are arranged in pairs around the second liquid storage holes (eg, two side); the second communication channel 52 is respectively connected to the second liquid storage hole and the pair of second side liquid storage holes 42 on both sides thereof. The first liquid storage hole and the second liquid storage hole are coaxially arranged to form a central liquid storage hole 10 or a central liquid storage straight hole 150, and the first side liquid storage hole 41 and the second side liquid storage hole 42 are coaxially arranged The side reservoir holes 40 are formed.

This embodiment corresponds to the aforementioned three communication modes, wherein, for the third communication mode, the first communication channel 51 and the second communication channel 52 are independently arranged or can be snapped together to form a communication channel 50 (as shown in FIG. 28 ). The independent setting means that even after the upper platform 160 and the lower platform 170 are engaged, the first communication channel 51 and the second communication channel 52 remain independent, and no mixed flow occurs.

In the embodiment of the present disclosure, the two-layer platforms of the chip platform 100 may be assembled together by using a sealing process such as double-sided tape, ultrasonic, thermal bonding, plasma, and thermal pressing.

In the embodiment of the present disclosure, as shown in FIG. 29 , enclosures 101 are provided on the four peripheral edges of the surface of the chip platform 100 , that is, the enclosures 101 are enclosed on the four peripheral edges of the surface of the chip platform 100 . Then, the enclosure 101 is surrounded to form a liquid holding tank 102, which reduces the evaporation of the medium during the culturing process and improves the culturing effect.

In the embodiment of the present disclosure, the material of the chip platform and each culture module (eg, the culture chamber module and the culture channel module) of the detachable/assembled co-culture organ chip is PMMA, PS, and the like. Among them, the culture chamber module can be prepared by using a hydrogel material, which can be disassembled/assembled more conveniently while realizing its cell culture function. The fabrication of each structure can adopt soft lithography, molding method, laser etching, machining, LIGA technology or one-time injection molding to obtain the chip structure of each layer.

In the embodiment of the present disclosure, the central liquid storage hole 10 or the central liquid storage straight hole 150 on the chip platform 100 may be a concave hole, as shown in FIGS. 21 to 23 , opened on the body of the chip platform 100 . It can also be a pillar hole, as shown in FIG. 18 , there are pillars protruding from the surface of the chip platform 100 , and a hole structure is formed on the pillars. Of course, a central liquid storage hole structure formed by extending upward from the second end of the first annular member 320 may also be used to reduce the thickness of the chip platform 100 .

In the embodiment of the present disclosure, the side liquid storage holes on the chip platform 100 may also be concave holes or column holes. Not limited, it can be determined according to actual needs.

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

In this embodiment, in the multi-cell co-culture organ model, the multi-cells may be one or more cells from the same organ, or may be multiple types of cells from different organs. Depending on the type of cells, the constructed organ model can be a single-organ model or a multi-organ model.

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

S11, respectively inoculating the first cells in the culture wells 20 of the co-culture organ chip, and inoculating the second cells in the co-cultivation channel 30 of the co-culture organ chip;

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

Wherein, in step S11 and step 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 identical, a single-organ model is constructed. When the first organ and the second organ are different, a multi-organ model is constructed.

In step S12, the medium added to the central liquid storage hole 10 and the co-cultivation channel 30 may be the same or different.

Optionally, when different, that is, the first medium is added to the small aperture section 13 of the central liquid storage hole 10, the second medium is added to the co-cultivation channel 30, and the first medium and the second medium are controlled. The liquid level does not exceed the stepped surface 11 of the central liquid storage hole 10 . Independent culture of the first and second cells is guaranteed.

Optionally, when it is the same, only the first medium needs to be filled into the central liquid storage hole 10, and the co-cultivation channel 30 is also filled with the first medium. Co-culture of the first cell and the second cell is achieved.

Optionally, when the co-culture organ chip adopts the aforementioned dynamic co-culture organ chip, the culture adopts dynamic culture. Dynamic culture parameters are not limited.

S21. Inoculate the first cell in the culture hole 20 of the co-culture organ chip, then add the first culture medium into the central liquid storage hole 10, and culture to construct the first organ model.

S22. After the first set time (for example, 72h) of constructing the first module, remove the first medium, then inoculate the second cells in the co-cultivation channel 30, and then add the first medium into the central liquid storage hole 10. culture medium, adding the first culture medium or the second culture medium into the co-cultivation channel 30, culturing, and constructing the first organ-second organ model.

Wherein, 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 identical, a single-organ model is constructed. When the first organ and the second organ are different, a multi-organ model is constructed.

In step S22, the medium added to the central liquid storage hole 10 and the co-cultivation channel 30 may be the same or different.

In step S22, the first set time is not limited, and can be determined according to factors such as the type of the first cells and the inoculation amount. Optionally, the first set time is 72h.

In some embodiments, the application also includes the application of the constructed multi-cell co-culture organ model to study the interaction of multiple cells; or, to conduct the co-evaluation study of organ damage and drug efficacy or the in vitro drug of organ metabolism of drugs The application of effectiveness evaluation research.

Specifically, after the organ model is constructed in step S12 or the first organ-second organ model is constructed in step S22, the step of detecting is also included.

Optionally, it also includes: S30, after the organ model/first-second organ module is continuously cultivated for a second set time, adding a mixed culture medium solution containing the drug to be studied into the central liquid storage hole 10, and then culturing again. / Dynamic culture to obtain the organ model to be tested. Various tests can be performed on the organ model to be tested.

In step S30, the second setting time is not limited, and may be determined according to factors such as the types of the first cells and the second cells and the inoculation amount. Optionally, the second set time is 24h.

In the embodiments of the present disclosure, the first cells include tumor cells; for example, HCT-116, NCI-H460, or MDA-MB-231. The second cell includes one or any combination of human primary liver cells, liver cell lines LO2 and HapRG, and the like.

Optionally, the constructed multi-cell co-culture organ model is subjected to the study of contact/non-contact immune response of various cells; or, the study of non-contact co-culture of cells and feeder cells is carried out.

The following takes the application of constructing a tumor-liver co-model for organ damage and drug efficacy research or in vitro drug efficacy research of organ metabolized drugs as an example to specifically describe the co-culture organ chip of the embodiment of the present disclosure.

Example 1

The tumor-liver co-model was constructed using the dynamic co-culture organ chip of the culture unit as shown in Figure 16, including the following steps:

S41, sterilizing the open dynamic co-culture organ chip, then adding a mixed cell suspension containing tumor cells and matrix materials into the culture well 20, culturing at 37° C. to form a gel; obtaining a gel-forming tumor organ chip; A first medium (eg, DMEM+serum or 1640 basal medium+serum) was added to the central reservoir hole 10 of the gel-forming organ microchip, and then cultured/dynamically cultured at 37°C to construct a tumor model. Wherein, the mixed cell suspension comprising tumor cells and matrix material is a single cell suspension comprising matrix material and model cells, and the pH value is 6.5-7.5. Among them, tumor cells refer to cell lines that respond to anticancer drugs, such as HCT116. The addition amount of the first medium can fill up the central liquid storage hole 10, and the side liquid storage holes 40 are also filled with the first medium; meet the training needs.

S42. The human primary liver cells are converted into a single cell suspension, centrifuged and resuspended, and a cell suspension of a specific density is prepared as required. In a 1.5ml EP tube, add a specific volume of collagen (pH adjusted with NaOH, hepes buffer solution) or other matrix materials such as matrigel in proportion to obtain a mixed cell suspension to ensure the concentration (collagen is 1-2mg/ml , Matrigel is 30%-80%) under the 3D material to form a good three-dimensional structure. After 72 hours of construction and culture in step S41, the first medium is removed, and the mixed cell suspension is added to the lower culture section 302 of the co-cultivation channel 30, mixed with a pipette gun, and then quickly transferred to the co-culture by pipetting. In the lower culture section 302 of the channel 30, after the cell planting is completed, it is placed at 37°C for 10 minutes to ensure that the matrix material can form a good gel. The tumor cell line medium is added to the small-diameter section 13 of the central liquid storage hole 10 (the second liquid storage hole of the second liquid storage layer 120 ), and the primary liver medium is added to the co-cultivation channel 30 . Continue dynamic culture at 37℃ for 24h; construct and obtain tumor-liver co-model.

S43. Perform drug stimulation on the tumor-liver co-model: after the liver-tumor co-model is constructed and the co-cultivation continues for 24 hours, the central reservoir hole 10 is filled with the preparation medium containing the specific type and concentration of drugs to be screened (with 10 μM antitumor drug sunitinib as an example). The dynamic culture was continued at 37°C for 120h.

S431. Detection of drug susceptibility results: after the drug stimulation in step S43 for 120h, the culture medium with the drug is removed, and Cell titer glo is added to the small aperture section 13 (or the second liquid storage hole) of the central liquid storage hole 10 for 3D The ATP of the cultured cells was evaluated. This step is applicable to, but not limited to, the above characterization methods, other characterization reagents or methods are also compatible, such as cell titer blue, high-content imaging technology to image the number of live and dead cells.

S432. Hepatotoxicity detection: after the drug stimulation in step S43 for 120 hours, the medium with the drug is removed, and the evaluation reagent is added to the co-cultivation channel 30. In this part, a commercial kit can be used to respond to the liver function after drug treatment. Perform albumin, α-GST or metabolic enzymes for characterization. The characterization method can be Elasa kit or extract mRNA to quantify the expression of major metabolic enzymes. In addition, existing ATP and metabolic capacity detection methods can be used to characterize the activity of hepatocytes after drug action, and mitochondrial membrane potential kits or mitochondrial reactive oxygen species kits can also be used to characterize cell mitochondrial function. Therefore, single-parameter as well as multi-parameter can be used to characterize the hepatotoxic effects of drugs.

In this Example 1, the tumor cells and human primary liver cells in the tumor-liver co-model constructed in step S42 were respectively characterized by F-actin and nuclear staining, and the fluorescent staining results of the tumor cells as shown in Figure 30 were obtained. Figure 31 shows the characterization diagram of the fluorescent staining results of human primary liver cells. It can be seen that tumor cells and human primary liver cells express more tight junction proteins and are highly biomimetic.

In this Example 1, as shown in the drug susceptibility test result of step S431 shown in FIG. 32 , it can be seen that after adding the drug (sunitinib), the tumor inhibition rate is only 30%, indicating that the drug has almost no resistance to the drug at this concentration. tumor effect.

In this Example 1, as shown in the graph of the liver toxicity test results in step S432 shown in FIG. 33 , it can be seen that after adding the drug (sunitinib), the inhibition rate is close to 100%, indicating that at this concentration, the drug has a strong hepatic damage effect.

The foregoing description and drawings sufficiently illustrate the embodiments of the present disclosure to enable those skilled in the art to practice them. Other embodiments may include structural and other changes. The examples represent only possible variations. Unless expressly required, individual components and functions are optional and the order of operations may vary. Portions and features of some embodiments may be included in or substituted for those of other embodiments. Embodiments of the present disclosure are not limited to the structures that have been described above and shown in the accompanying drawings, and various modifications and changes may be made without departing from the scope thereof. The scope of the present disclosure is limited only by the appended claims.

Claims

An open co-culture organ chip, characterized in that it comprises: one or more culturing units, and each of the culturing units includes:

The central liquid storage hole is a stepped blind hole; the bottom wall of the central liquid storage hole is provided with one or more culture holes, and the stepped surface of the central liquid storage hole is axially extended with a co-cultivation channel.
The co-culture organ chip according to claim 1, wherein the co-culture channel comprises a closed/non-closed annular slot along the circumference of the stepped surface of the liquid storage hole;

Alternatively, the co-cultivation channel includes a plurality of channels.
The co-culture organ chip according to claim 2, wherein the volume of the co-culture channel is 20-500 μL.
The co-culture organ chip according to claim 1, wherein the volume of the culture well is 1-50 μL.
The co-culture organ chip according to claim 1, wherein the area containing the culture hole on the bottom wall of the central liquid storage hole is divided to form a culture chamber module; on the bottom wall of the central liquid storage hole An installation location is provided, and the culture chamber module is detachably arranged at the installation location of the bottom wall of the central liquid storage hole.
The co-culture organ chip according to claim 5, characterized in that, the stepped portion provided with the co-culture channel in the central liquid storage hole is divided to form a culture channel module; then, the central liquid storage hole, include:

Central liquid storage straight hole;

The culture channel module is detachably arranged in the central liquid storage straight hole, the first end of which is flush with the bottom end of the central liquid storage straight hole and is provided with an installation position;

Wherein, the co-culture channel extending in the axial direction is provided on the end surface of the second end of the culture channel module, or the culture channel module cooperates with the central liquid storage straight hole to form the co-culture channel.
The co-culture organ chip according to claim 6, wherein,

The culture channel module includes:

an annular member, which is detachably arranged in the central liquid storage straight hole in a manner of contacting with the inner wall of the central liquid storage straight hole or at a set interval, and cooperates with the central liquid storage straight hole to form a co-cultivation channel; and the first end of the ring member is provided with an installation position;

Alternatively, the culture channel module includes:

a first annular member, which is detachably arranged in the central liquid storage straight hole, and the outer wall of the first annular member is in contact with the inner wall of the central liquid storage straight hole;

A second annular member is sleeved in the first annular member, and is connected and cooperated with the first annular member to form the co-cultivation channel; and a first end of the second annular member is provided with an installation position;

Wherein, the first ring member is fixedly connected or detachably connected with the second ring member.
The co-culture organ chip according to claim 7, wherein,

The culture channel module includes an annular member, the annular member is annular and its outer sidewall is linear or stepped;

Alternatively, the culture channel module includes a first annular member, and the inner sidewall of the first annular member is linear or stepped;

Alternatively, the culture channel module includes a second annular member, and the outer sidewall of the second annular member is linear or stepped.
The co-culture organ chip according to claim 7, wherein the culture channel module comprises a first annular member, and the second end of the first annular member is extended, so that the first annular member forms a liquid storage Pore structure.
The co-culture organ chip according to claim 6, wherein the culture chamber module is fixedly connected to the installation position of the culture channel module to form a co-culture module; the co-culture module is detachably arranged on the into the central reservoir straight hole.
The co-culture organ chip according to any one of claims 5 to 10, wherein the culture chamber module is prepared from a hydrogel material;

Or, when the co-culture organ chip includes a culture channel module, the culture channel module and the culture chamber module adopt one of biocompatibility, biomimetic, cell penetrability and degradability or Materials with various properties are prepared.
The co-culture organ chip according to any one of claims 1 to 10, further comprising:

side liquid storage holes, each of the central liquid storage holes is provided with a plurality of the side liquid storage holes;

The communication channel communicates with the central liquid storage hole and the lateral liquid storage holes on both sides thereof, and/or communicates with the co-cultivation channel and the lateral liquid storage holes on both sides of the central liquid storage hole where the co-cultivation channel is located.
The co-culture organ chip according to claim 12, wherein the cross-sectional area of the communication channel ranges from 0.01 to 100 mm 2 .
The co-culture organ chip according to claim 12, wherein when the side liquid storage hole is a column hole, the column hole part of the side liquid storage hole is divided to form a side liquid storage column hole; The side liquid storage column hole is detachably arranged in the concave hole portion of the side liquid storage hole.
The co-cultivation organ chip according to any one of claims 1 to 10, wherein the co-cultivation channel comprises a connected upper-layer liquid storage section and a lower-layer culture section; the size of the lower-layer culture hole section is smaller than or Equal to the size of the upper liquid storage hole section.
The co-culture organ chip according to claim 15, wherein the side wall between the upper liquid storage section and the central liquid storage hole is detachable.
The application of the co-culture organ chip according to any one of claims 1 to 16 for constructing a multi-cell co-culture organ model.
The application according to claim 17, wherein the constructing a multi-cell co-culture organ model comprises:

respectively inoculating the first cells in the culture wells of the co-culture organ chip, and inoculating the second cells in the co-cultivation channel of the co-cultivation organ chip;

respectively adding the first culture medium to the central liquid storage hole, adding the first culture medium or the second culture medium to the co-cultivation channel, culturing, and constructing an organ model;

Alternatively, the construction of a multi-cell co-culture organ model includes:

inoculating the first cell in the culture hole of the co-culture organ chip, then adding the first culture medium into the central liquid storage hole, culturing, and constructing the first organ model;

After the first set time of constructing the first organ module, the first medium is removed, and the second cells are seeded in the co-cultivation channel, and then the first medium is added to the central reservoir hole to add the co-culture. Adding the first culture medium or the second culture medium into the channel, culturing, and constructing 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 are the same or different ;

When adding the second medium into the co-cultivation channel, the liquid level of the first medium and the second medium does not exceed the stepped surface of the central liquid storage hole;

When the co-culture organ chip adopts the co-culture organ chip according to any one of claims 12 to 16, the culture adopts dynamic culture.
The application according to claim 17, further comprising: the application of the constructed multi-cell co-culture organ model to study the interaction of multiple cells;

Alternatively, conduct co-evaluation studies of organ damage and drug efficacy or the application of in vitro efficacy evaluation studies of organ-metabolized drugs.