CN214193293U

CN214193293U - Open type co-culture organ chip

Info

Publication number: CN214193293U
Application number: CN202022865811.1U
Authority: CN
Inventors: 肖荣荣; 刘建闯; 李珮文; 张晓会; 周宇
Original assignee: Beijing Da Xiang Technology Co ltd
Current assignee: Beijing Da Xiang Technology Co ltd
Priority date: 2020-12-02
Filing date: 2020-12-02
Publication date: 2021-09-14
Anticipated expiration: 2030-12-02

Abstract

The application relates to the technical field of biological tissue engineering, and discloses an open type co-culture organ chip which comprises one or more culture units, wherein each culture unit comprises a central liquid storage hole and is a stepped blind hole; be provided with one or more culture hole on the diapire in center liquid storage hole follow axial extension on the ladder face in center liquid storage hole and be provided with the coculture passageway. The co-culture organ chip is in an open type, the openings of the culture holes and the co-culture channel face to the same side, cell planting can be completed on the same side of the organ chip, and operation difficulty is greatly reduced. Through carrying out standardized design to the overall arrangement of central stock solution hole on this coculture organ chip, then its open-type operation passageway, with market operation and check out test set's compatibility strong, more simple and convenient, more be fit for the industrialization and promote.

Description

Open type co-culture organ chip

Technical Field

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

Background

Cells or tissues require interaction and communication between organs within the intact body, such as innervation of the nervous system, "dialogues" of the immune system, nutritional support of mesenchymal cells. At present, although traditional well plate culture platforms such as 24, 96, 384 well plates are widely applied in biological research fields such as cell culture, there are some technical bottlenecks that limit the application of the traditional well plate culture platforms in organ co-culture. The common culture well plate can not realize the intercellular coculture. The microfluidic organ chip technology is a new technology, and can be applied to co-culture of various organs by designing various channels to overcome the limitation. However, there are some technical bottlenecks to limit the large-scale application of the organ chip technology reported at present. Firstly, the stability and reproducibility of the product are poor, the product structure is complex, if a closed channel is formed, the operation of a special technician is needed, the stability and reproducibility are poor, and the subsequent analysis difficulty is high. Secondly, the flux of the product is low, the standardization degree is poor, the compatibility of the equipment is poor, and the popularization and the application are difficult.

In the process of implementing the embodiments of the present disclosure, it is found that at least the following problems exist in the related art: the existing co-culture organ chip is of a closed structure, so that the operation is complex, the subsequent detection and analysis are difficult, the flux is low and the standardization degree is poor.

SUMMERY OF THE UTILITY MODEL

The following presents a simplified summary in order to provide a basic understanding of some aspects of the disclosed embodiments. This summary is not an extensive overview nor is intended to identify key/critical elements or to delineate the scope of such embodiments but rather as a prelude to the more detailed description that is presented later.

The embodiment of the disclosure provides an open type co-culture organ chip, which is used for solving the problems of complex operation, difficult subsequent detection and analysis, low flux and poor standardization degree caused by the closed structure of the existing co-culture organ chip.

In some embodiments, the open cocultivation organ chip comprises one or more culture units, each culture unit comprising a central reservoir, in the form of a stepped blind hole; be provided with one or more culture hole on the diapire in center liquid storage hole follow axial extension on the ladder face in center liquid storage hole and be provided with the coculture passageway.

In some embodiments, the use of the aforementioned co-cultured organ chip for constructing a multi-cell co-cultured organ model.

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

the co-culture organ chip provided by the embodiment of the disclosure is in an open type, the openings of the culture holes and the co-culture channel face to the same side, cell planting can be completed on the same side (e.g., upper side) of the organ chip, and the operation difficulty is greatly reduced. By carrying out standardized design on the arrangement layout of the central liquid storage holes on the co-culture organ chip, the open operation channel has strong compatibility with commercial operation, detection equipment and imaging equipment, is convenient for subsequent on-board detection and cell recovery for RNA, protein extraction and other analysis, is simpler and more suitable for industrial popularization. The method is simple to operate, does not need professional technicians, enlarges the application range of the organ chip and improves the universality. The open type co-culture organ chip can realize contact type/non-contact type co-culture of cells or tissue micro-organs and realize interaction on the premise of mutual non-pollution. The method can be used for in vitro construction and long-time culture of multi-cell and multi-organ models, and further can be used for in vitro construction of multi-cell co-culture models or multi-organ co-culture models.

The foregoing general description and the following description are exemplary and explanatory only and are not restrictive of the application.

Drawings

One or more embodiments are illustrated by way of example in the accompanying drawings, which correspond to the accompanying drawings and not in limitation thereof, in which elements having the same reference numeral designations are shown as like elements and not in limitation thereof, and wherein:

FIG. 1 is a schematic top view of a culture unit of an open type co-culture organ chip according to an embodiment of the present disclosure;

FIG. 2 is a schematic sectional view taken along line A-A in FIG. 1;

FIG. 3 is a schematic sectional view of another culture unit in the direction A-A shown in FIG. 1;

FIG. 4 is a schematic top view of a culture unit of another open-type co-culture organ chip according to the embodiment of the present disclosure;

FIG. 5 is a schematic cross-sectional view taken along line B-B of FIG. 6;

FIG. 6 is a schematic top view of a culture unit of another open-type co-culture organ chip according to the embodiment of the present disclosure;

FIG. 7 is a schematic cross-sectional view taken along line C-C of FIG. 6;

FIG. 8 is a schematic top view of a culture unit of another open-type co-culture organ chip according to the embodiment of the present disclosure;

FIG. 9 is a schematic top view of a culture unit of another open-type co-culture organ chip according to the embodiment of the present disclosure;

FIG. 10 is a schematic top view showing a structure of a culture unit of another open-type co-culture organ chip according to the embodiment of the present disclosure;

FIG. 11 is a schematic top view showing a structure of a culture unit of another open-type co-culture organ chip according to the embodiment of the present disclosure;

FIG. 12 is a schematic cross-sectional view taken along line D-D of FIG. 11;

FIG. 13 is a schematic cross-sectional view of a culture unit of another open type co-cultured organ chip according to the embodiment of the present disclosure;

FIG. 14 is a schematic view showing an exploded structure of a culture unit of another open type co-culture organ chip according to the embodiment of the present disclosure;

FIG. 15 is a schematic cross-sectional view of a culture unit of another open type co-cultured organ chip according to the embodiment of the present disclosure;

FIG. 16 is a schematic view showing an exploded structure of a culture unit of another open type co-culture organ chip according to the embodiment of the present disclosure;

FIG. 17 is a schematic cross-sectional view of a culture unit of another open type co-cultured organ chip according to the embodiment of the present disclosure;

FIG. 18 is a schematic structural view of an open type co-cultured organ chip according to an embodiment of the present disclosure;

FIG. 19 is a graph representing the fluorescence staining results of tumor cells in a tumor-liver co-model constructed according to the embodiments of the present disclosure;

FIG. 20 is a graph representing the fluorescence staining results of human primary liver cells in tumor-liver comorbid type constructed according to the embodiments of the present disclosure;

FIG. 21 is a graph of the results of drug sensitivity detection of tumor cells in a tumor-liver co-model constructed according to the embodiments of the present disclosure;

fig. 22 is a graph of hepatotoxicity test results of human primary liver cells in tumor-liver comorbid type constructed in the examples of the present disclosure.

Reference numerals:

10. a central liquid storage hole; 11. a step surface; 12. a large pore diameter section; 13. a small bore section; 20. a culture well; 30. a co-culture channel; 301. an upper liquid storage section; 302. a lower layer culture section; 31. co-culture reservoir channels; 32. co-culturing the planting channel; 40. a lateral liquid storage hole; 41. a first side liquid storage hole; 42. a second side liquid storage hole; 50. a communication channel; 51. a first communicating passage; 52. a second communicating passage; 100. an organ chip body; 101. fencing; 102. a liquid containing groove; 110. a first liquid storage layer; 120. a second liquid storage layer; 130. a culture layer; 131. a planting area; 140. a base plate.

Detailed Description

So that the manner in which the features and elements of the disclosed embodiments can be understood in detail, a more particular description of the disclosed embodiments, briefly summarized above, may be had by reference to the embodiments, some of which are illustrated in the appended drawings. In the following description of the technology, for purposes of explanation, numerous details are set forth in order 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 in simplified form in order to simplify the drawing.

The terms "first," "second," and the like in the description and in the claims, and the above-described drawings of embodiments of the present disclosure, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances such that embodiments of the present disclosure described herein may be made. Furthermore, the terms "comprising" and "having," as well as any variations thereof, are intended to cover non-exclusive inclusions.

In the embodiments of the present disclosure, the terms "upper", "lower", "inner", "middle", "outer", "front", "rear", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings. These terms are used primarily to better describe the disclosed embodiments and their examples and are not intended to limit the indicated devices, elements or components to a particular orientation or to be constructed and operated in a particular orientation. Moreover, some of the above terms may be used to indicate other meanings besides the orientation or positional relationship, for example, the term "on" may also be used to indicate some kind of attachment or connection relationship in some cases. The specific meanings of these terms in the embodiments of the present disclosure can be understood by those of ordinary skill in the art as appropriate.

In addition, the terms "disposed," "connected," and "secured" are to be construed broadly. For example, "connected" may be a fixed connection, a detachable connection, or a unitary construction; can be a mechanical connection, or an electrical connection; may be directly connected, or indirectly connected through intervening media, or may be in internal communication between two devices, elements or components. Specific meanings of the above terms in the embodiments of the present disclosure can be understood by those of ordinary skill in the art according to specific situations.

The term "plurality" means two or more unless otherwise specified.

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

Referring to fig. 1 to 18, an open-type co-culture organ chip according to an embodiment of the present disclosure includes one or more culture units, each culture unit including a central liquid storage hole 10 in the form of a stepped blind hole; one or more culture holes 20 are arranged on the bottom wall of the central liquid storage hole 10, and one or more co-culture channels 30 are axially and extendedly arranged on the stepped surface 11 of the central liquid storage hole 10.

The co-culture organ chip of the embodiment of the disclosure is open, the openings of the culture holes 20 and the co-culture channels 30 face to the same side, and cell planting can be completed on the same side (e.g., upper side) of the organ chip, thereby greatly reducing the operation difficulty. By carrying out standardized design on the arrangement layout of the central liquid storage holes 10 on the co-culture organ chip, the open type operation channel has strong compatibility with the commercial operation and detection equipment, is convenient for subsequent on-machine detection and cell recovery for RNA, protein extraction and other analysis, is simpler and more suitable for industrial popularization. The method is simple to operate, does not need professional technicians, enlarges the application range of the organ chip and improves the universality.

In the embodiment of the disclosure, the cells/organs planted in different culture holes 20 in the central liquid storage hole 10 can be independently cultured, and different cells/organs planted in the culture holes 20 and the co-culture channel 30 can be co-cultured in a non-contact manner, so that the open type co-culture organ chip can realize the non-contact co-culture of cells or tissue micro-organs, can be used for in-vitro construction and long-time culture of multi-cell and multi-organ models, and further can be used for in-vitro construction of multi-cell co-culture models or multi-organ co-culture models.

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 aperture section 12 and a small aperture section 13 that are communicated with each other, and the large aperture section 12 is located on the upper layer. Then, when the large pore diameter section 12 of the central reservoir 10 is filled with culture medium or drug diluent, it can provide the required nutrient solution or drug to be tested for the cells/organs within the culture well 20 and co-culture channel 30; when only the small-aperture section 12 of the central reservoir 10 is filled with the culture medium or the drug diluent, it only provides the required nutrient solution or the drug to be tested for the cells/organs in the culture well 20, i.e. the small-aperture section 12 of the central reservoir 10 is the exclusive reservoir of the culture well 20. When the liquid level of the culture medium or the drug diluent filled in the co-culture channel 30 does not exceed the stepped surface 11 of the central liquid storage hole 10, the upper layer of the co-culture channel 30 is the exclusive liquid storage hole of the culture hole section of the lower layer.

The plurality of culture wells 20 at the bottom of the central reservoir 10 can be seeded with the same or different types of cells, and if the same cells are seeded, selective seeding can be performed according to the amount of the sample, for example, seeding only one of the regions (one culture well 20) can be used. The co-culture channel 30 is used for culturing the cells of the second organ, the upper layer of the co-culture channel is used as a dedicated stock solution channel, and the bottom part/bottom wall of the co-culture channel is used for planting and culturing the cells of the second organ. The co-culture channel 30 may also be seeded with cells of the same or different types, depending on the actual requirements.

In the disclosed embodiment, the culture well 20 and the co-culture channel 30 are through holes or blind holes. In the case where the culture well 20 and the co-culture channel 30 are through holes, the co-culture organ chip further comprises a bottom plate 140 disposed at the bottom of the culture well and the co-culture channel. In use, rests on the base plate 140.

In the embodiment of the present disclosure, the shapes of the central liquid storage hole 10 and the culture holes 20 are not limited, and may be geometric shapes such as a circular hole, an ellipse, a square, a rectangle, a sector, or a polygon (e.g., a hexagon, an octagon, etc.), and under the condition that the design requirements are met, the shape of the central liquid storage hole 10 is designed to contain as much culture medium or drug diluent as possible, and the shape of the culture holes 20 is designed to facilitate growth.

Optionally, the diameter of the large-aperture section 12 of the central liquid storage hole 10 is in the range of 15-20 mm. Optionally, the large-aperture section 12 of the central liquid storage hole 10 is a circular hole with a diameter range of 15-18 mm. Optionally, the large pore diameter section 12 of the central reservoir bore 10 is 16.5mm in diameter.

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 central reservoir hole 10 is 17.4mm deep.

Alternatively, as shown in FIG. 2, the central reservoir well 10 is a cylindrical well. Has pillars protruding from the surface of the organ chip body 100, and holes are formed on the pillars.

Alternatively, as shown in FIG. 3, the central reservoir 10 is a recessed well. Opened in the organ chip body 100.

Optionally, as shown in fig. 2 and 5, the radial width d of the stepped surface 11 of the central liquid storage hole 10 is 0.5-5 mm. Namely, the radius of the small-aperture section 13 of the central liquid storage hole 10 is 0.5-5 mm smaller than that of the large-aperture section 12.

The culture wells 20 are disposed at the bottom of the central liquid storage well 10, and the number and the arrangement manner thereof are not limited, and may be determined according to the area and the shape of the bottom of the central liquid storage well 10. For example, the number of the culture wells 20 is 1, 2, 3, 4, 5 or more, and the specific number may be determined according to actual needs. The plurality of culture wells 20 may be arranged in an array, for example, a circular array or a rectangular array.

Alternatively, the volume of the culture well 20 is 1 to 50. mu.L. The dimension of the culture well 20 on the side is not limited as long as the volume requirement is satisfied. Of course, other values are possible, and cells may be cultured.

For co-culture channel 30, within which culture of a second organ is performed, it comprises, in the axial direction, communicating upper reservoir channel 301 and lower culture channel 302. And the open end of the co-culture channel 30 is positioned on the stepped surface 11 of the central liquid storage hole 10, so that the open end of the co-culture channel 30 is higher than the culture hole 20 for non-contact co-culture. The structure is not limited as long as the culture well 20 at the bottom of the central liquid storage well 10 is kept in non-contact with the liquid storage well.

In some embodiments, as shown in conjunction with FIGS. 1 and 7, co-cultivation channel 30 comprises a closed/non-closed annular slot along the circumference of stepped surface 11 of central reservoir well 10. I.e., co-cultivation channel 30 is a connected integral structure. In this embodiment, when the co-cultivation channel 30 comprises a closed circular slot, it is a blind hole. The structural integrity is ensured.

Alternatively, as shown in FIGS. 1 and 4, a non-closed annular slot is axially extended from the stepped surface 11 of the central liquid storage hole 10 to serve as a co-culture channel 30.

Alternatively, as shown in FIG. 6 and FIG. 7, a closed ring-shaped slot is axially extended from the stepped surface 11 of the central liquid storage hole 10 to serve as the co-culture channel 30. In this embodiment, the annular slot may be a circular slot, which is a blind hole. The integrity of the organ chip body is ensured.

In some embodiments, as shown in conjunction with fig. 8-10, co-culture channel 30 comprises a plurality of channels. Namely a plurality of pore channels which are arranged on the stepped wall surface of the central liquid storage hole 10 along the axial direction. In this embodiment, the plurality of channels are circumferentially distributed along the stepped surface 11 of the central liquid storage hole 10. The cross section of the pore canal is not limited, and can be in a geometrical shape such as a circle, an ellipse, a square, a rectangle, a fan ring or a polygon (such as a hexagon, an octagon and the like), and the shape of the pore canal is designed according to the condition that the design requirement is met, so that more culture medium or medicament diluent can be contained as much as possible.

Alternatively, as shown in fig. 8, a plurality of channels with sector-ring-shaped cross section are axially extended from the stepped surface 11 of the central liquid storage hole 10 to serve as the co-culture channels 30.

Alternatively, as shown in fig. 9, a plurality of channels with a circular cross section are axially extended from the stepped surface 11 of the central liquid storage hole 10 to serve as the co-culture channels 30.

Alternatively, as shown in fig. 10, a plurality of pore channels with oval cross-section are axially extended from the stepped surface 11 of the central liquid storage hole 10 to serve as the co-culture channels 30.

In some embodiments, the volume of the co-culture channel 30 is 20-500. mu.L. Ensuring a certain inoculation amount. Here, the volume of the co-culture channel 30 means the entire volume of one closed/non-closed annular slot or the total volume of a plurality of tunnels. Of course, the volume of the co-culture channel 30 is not limited to this range, and if the structure allows, the volume of the co-culture channel 30 can be enlarged to meet different inoculum size requirements.

In some embodiments, as shown in conjunction with FIGS. 4-7, co-culture channel 30 comprises an upper reservoir section 301 and a lower culture section 302 in communication; the size of the lower culture well section 302 is smaller than or equal to the size of the upper reservoir well section 301. When the ratio is less than the above range, the loss of the inoculated cells can be avoided, and the inoculation rate can be improved.

As shown in FIGS. 4 and 5, the co-culture channel 30 is a non-closed annular groove hole extending in the axial direction on the stepped surface 11; the radial width of the lower culture section 302 is less than the radial width of the upper reservoir 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 FIGS. 11 and 12, the co-culture organ chip further comprises side wells 40 and a communication channel 50, the side wells 40 being disposed in pairs around the wells; the communicating channel 50 communicates the central liquid storage hole 10 and the pair of side liquid storage holes 40 around the central liquid storage hole, and/or communicates the co-culture channel 30 and the pair of side liquid storage holes 40 around the central liquid storage hole 10. The co-culture chip of the present embodiment is defined as a dynamic co-culture organ chip, which can realize dynamic culture or dynamic co-culture of the culture wells 20 and/or the co-culture channels 30, and is compatible with various fluid manipulation methods. Real-time dynamic updates of the culture environment within culture well 20 and/or co-culture channel 30 can be achieved.

The dynamic co-culture organ chip of the present embodiment has three communication modes, the first is that the first communication channel 51 communicates only with the central liquid storage hole 10 and the paired side liquid storage holes 40 around it; the second is that the second communicating channel 52 only communicates the coculture channel 30 and the paired side liquid storage holes 40 around the central liquid storage hole 10; the third is that the communicating channel 50 communicates with the central liquid storage hole 10 and the pair of side liquid storage holes 40 around the central liquid storage hole, and communicates with the coculture channel 30 and the pair of side liquid storage holes 40 around the central liquid storage hole 10. In the third communication mode, the communication passage 50 may be the first communication passage 51 and the second communication passage 52 which are independently provided, or may be one communication passage 50 in which the first communication passage 51 and the second communication passage 52 are integrated (as shown in fig. 12).

In this embodiment, a plurality of pairs of side liquid storing holes 40 may be formed around one central liquid storing hole 10, and the side liquid storing holes are not limited to the pair of side liquid storing holes 40 shown in fig. 11. A communication passage (e.g., a first communication passage 51) is provided between the central reservoir hole 10 and each of the side reservoir holes 40 to communicate with each other. Furthermore, the number of the communication channels between the central liquid storage hole 10 and each of the side liquid storage holes 40 is not limited to one as shown in FIG. 11, and a plurality of communication channels may be provided to improve the dynamic culture effect.

Optionally, the side reservoirs 40 are provided in pairs on opposite sides of the reservoir. Improve the balance of fluid flow and improve the dynamic culture effect.

In this embodiment, the communicating passages 50 are also provided in pairs based on the paired arrangement of the side liquid storage holes 40. Wherein, when the communicating channel 50 communicates the co-culture channel 30 with the paired side liquid storage holes 40 around the central liquid storage hole 10, the paired communicating channels 50 are arranged in corresponding number according to the structure of the co-culture channel 30, thereby ensuring that the micro-flow control can be realized in the co-culture channel 30. For example, referring to fig. 8 to 9, when the co-culture channel 30 includes a plurality of wells, each well is provided with one or more pairs of side wells, and each side well is connected to the corresponding well.

Alternatively, as shown in fig. 12, the side reservoir holes 40 are post holes. Like the central liquid storage hole 10 in the column hole.

Alternatively, the side reservoir hole 40 is a concave hole. Reference is made to the aforementioned central reservoir 10 in the form of a recess.

In this embodiment, the dimensions of the communicating channel 50 are designed to allow microfluidic control of the medium in the central reservoir 10 and/or co-culture. The 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 mm to 100mm². Within the area range of the cross section, the microfluidic dynamic culture can be better realized.

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

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

Alternatively, the communication channel 50 is square in cross-section, with a width in the range of 2mm and a height in the range of 2 mm.

In this embodiment, the shape of the side liquid storage hole 40 is not limited, and may be a circular hole, an oval, a square, a rectangle, a sector, or a polygon (e.g., a hexagon, an octagon, etc.), and the like, and the shape of the side liquid storage hole 40 is designed based on the fact that more culture media or drug diluents are contained as much as possible when the design requirements are satisfied.

Alternatively, the side reservoir wells 40 are of the well size of a standard 48-well plate or a standard 96-well plate. The method is convenient for subsequent on-machine detection and cell recovery to perform analysis such as RNA and protein extraction, is simpler and more suitable for industrial popularization.

In the open type co-culture organ chip according to the embodiment of the present disclosure, the culture unit or units may be provided, and the specific molding method is not limited.

In some embodiments, the open coculture organ chip is integrally injection molded. For example, Polystyrene (PS) and Polymethyl Methacrylate (PMMA) are integrally injection molded, and these materials have low cost, are easy to injection mold, have no toxicity to cells, and have no specific adsorption.

In some embodiments, the open coculture organ chips are obtained by layered processing and assembling a construct. The assembly is carried out after the layered processing, the whole is broken into parts, and the molding process is simplified.

The following provides a specific structure of the open type co-cultured organ chip, but is not limited to this specific structure.

In some embodiments, as shown in connection with fig. 13 to 17, the open type co-culture organ chip includes a first reservoir layer 110, a second reservoir layer 120 and a culture layer 130, which are sequentially stacked. One or more first liquid storage holes are formed in the first liquid storage layer 110; the second liquid storage layer 120 is provided with one or more second liquid storage holes and one or more co-culture liquid storage channels 31 arranged around each second liquid storage hole; the culture layer 130 is provided with one or more planting areas 131 and one or more co-culture planting channels 32 surrounding each planting area 131, and one or more culture holes 20 are provided in each planting area 131. The first liquid storage hole, the second liquid storage hole and the planting area 131 are coaxially arranged to form a central liquid storage hole 10; the co-culture liquid storage channel 31 and the co-culture planting channel 32 are communicated in a one-to-one correspondence mode to form a co-culture channel 30.

In the embodiment of the disclosure, the open type co-culture organ chip comprises a three-layer chip structure, and the three-layer chip structure is stacked and connected in sequence. The chips of each layer can be bonded and assembled together by using sealing processes such as double-sided adhesive tape, ultrasonic, thermal bonding, plasma, hot pressing and the like.

In the embodiment of the disclosure, the material of each layer of chip structure is PMMA, PS, or the like. The chip structure of each layer can be manufactured by soft lithography, a molding method, laser etching, machining, LIGA (laser induced cracking) or one-time injection molding.

In this embodiment, the organ chip body 100 is processed in layers, and then divided and processed by combining 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 that of the second liquid storage hole, and after the first liquid storage hole and the second liquid storage hole are stacked, a stepped hole is formed. In this embodiment, the first liquid storage hole is a large-aperture section 12 of the central liquid storage hole 10, and the second liquid storage hole is a small-aperture section 13 of the central liquid storage hole 10. A plurality of through holes are processed on the first liquid storage layer 110 and the second liquid storage layer 120 respectively, and the forming/processing is simple.

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

In some embodiments, as shown in fig. 15 and 16, for the dynamic co-culture organ chip, the first stock solution layer 110 is further provided with a plurality of first edge-side stock solution holes 41 and first communication channels 51, the plurality of first edge-side stock solution holes 41 being provided in pairs around (e.g., on both sides of) the first stock solution holes; the first communication passage 51 communicates the first reservoir and the pair of first side reservoir 41 on both sides thereof, respectively; and/or, the second liquid storage layer 120 is further provided with a plurality of second side liquid storage holes 42 and a plurality of second communication channels 52, and the plurality of second side liquid storage holes 42 are arranged in pairs around (e.g., on both sides of) the second liquid storage holes; the second communicating passage 52 communicates the second reservoir holes and the pair of second side reservoir holes 42 on both sides thereof, respectively.

The present embodiment corresponds to the aforementioned three communication manners, wherein for the third communication manner, the first communication passage 51 and the second communication passage 52 are independently disposed or can be buckled to form one communication passage 50. The independent arrangement means that the first communicating channel 51 and the second communicating channel 52 are still in an independent state and do not generate mixed flow even after the first liquid storage layer 110 and the second liquid storage layer 120 are buckled.

For the embodiment in which culture wells 20 and/or co-culture channels 30 are through-holes, as shown in FIG. 17, the open type co-culture organ chip further comprises a bottom plate 140, and a culture layer 130 is stacked on the bottom plate 140. Convenient for inoculation and culture. The substrate 140 may be a glass or PS substrate 140.

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 reservoir hole 40 is provided, the first side reservoir hole 41 is also a pillar hole (as shown in fig. 16). In this embodiment, the first liquid storage layer 110 has a plurality of pillar holes protruding from the surface thereof, so as to prevent the fluid in different holes from affecting each other and avoid contamination.

In some embodiments, as shown in fig. 18, the central reservoir well 10 (first reservoir well) is a column well; or, when the central liquid storage hole 10 and the side liquid storage holes 40 are both pillar holes, the surrounding barrier 101 is disposed on the peripheral edge of the surface of the organ chip body 100, that is, the surrounding barrier 101 is disposed on the peripheral edge of the surface of the organ chip body 100. A liquid storage tank is formed between the enclosure 101 and the column hole, so that the evaporation of the culture medium in the culture process is reduced, and the culture effect is improved.

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) form a through-hole central liquid storage hole, the first liquid storage layer 110 and the second liquid storage layer 120 are sequentially stacked on the culture layer 130, the through-hole central liquid storage hole and the surface of the culture layer 130 enclose the central liquid storage hole 10 forming a stepped blind hole, at this time, the area enclosed by the through-hole central liquid storage hole on the surface of the culture layer 130 is the culture layer planting area 131, the size of the culture layer planting area 131 may be the same as the size of the second liquid storage hole, or may be smaller than the size of the second liquid storage hole, without limitation, and it is sufficient to set the culture hole 31 in the culture layer planting area 131.

The embodiment of the disclosure provides an application of a co-culture organ chip for constructing a multi-cell co-culture organ model.

In this example, in the multi-cell co-culture organ model, the multi-cells may be one or more cells derived from the same organ, or may be a plurality of cells derived from different organs. The constructed organ model is thus a single organ model or a multiple organ model depending on the kind of cells.

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

s11, respectively inoculating the first cells into the culture wells 20 of the co-culture organ chip, and inoculating the second cells into the co-culture channels 30 of the co-culture organ chip;

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

Wherein in steps S11 and S12, the first cell comprises one or more cells of the first organ and the second cell comprises one or more cells of the second organ; the first organ and the second organ may be the same or different. And when the first organ and the second organ are the same, constructing to obtain a single organ model. When the first organ and the second organ are different, a multi-organ model is constructed.

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

Optionally, when different, that is, the first culture medium is added into the small-aperture section 13 of the central liquid storage hole 10, and the second culture medium is added into the co-culture channel 30, the liquid level of the first culture medium and the liquid level of the second culture medium are controlled not to exceed the step surface 11 of the central liquid storage hole 10. Ensuring the independent culture of the first cell and the second cell.

Alternatively, when the same, only the central reservoir 10 needs to be filled with the first culture medium, the co-culture channel 30 is also filled with the first culture medium. Co-culturing the first cell and the second cell is achieved.

Alternatively, when the co-cultured organ chip employs the aforementioned dynamic co-cultured organ chip, the culture employs dynamic culture. The dynamic culture parameters are not limited.

s21, inoculating the first cell into the culture hole 20 of the co-culture organ chip, then adding the first culture medium into the central liquid storage hole 10, culturing and constructing the first organ model.

S22, after the first module is constructed for a first set time (e.g., 72 hours), removing the first culture medium, then inoculating the second cells into the co-culture channel 30, then 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-culture channel 30, and culturing to construct the first organ-second organ model.

Wherein in steps S21 and S22, the first cell comprises one or more cells of the first organ and the second cell comprises one or more cells of the second organ; the first organ and the second organ may be the same or different. And when the first organ and the second organ are the same, constructing to obtain a single organ model. When the first organ and the second organ are different, a multi-organ model is constructed.

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

In step S22, the first set time is not limited and may be determined depending on factors such as the type and the inoculation amount of the first cell. Alternatively, the first set time is 72 h.

In some embodiments, the use further comprises the use of the constructed multi-cell co-cultured organ model for interaction studies of a plurality of cells; alternatively, the application of the method is to perform a co-evaluation study of organ injury and drug efficacy or an in vitro efficacy evaluation study of an organ metabolizing drug.

Specifically, after the organ model is constructed in step S12 or the first organ-second organ model is constructed in step S22, the method further includes a step of performing detection.

Optionally, the method further comprises: and S30, continuously culturing the organ model/the first-second organ module for a second set time, adding a mixed culture medium solution containing the drug to be researched into the central liquid storage hole 10, and then culturing/dynamically culturing to obtain the organ model to be detected. And (5) carrying out various detections on the organ model to be detected.

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

In an embodiment of the disclosure, the first cell comprises a tumor cell; such as HCT-116, NCI-H460, or MDA-MB-231, etc. The second cell comprises one or a combination of any of human primary liver cells, liver cell lines LO2, HapRG and the like.

Optionally, the constructed multi-cell co-culture organ model is subjected to research of contact/non-contact immune response of various cells; alternatively, studies were conducted for non-contact co-culture of cells with feeder cells.

The co-culture organ chip of the embodiment of the present disclosure will be specifically described below by taking as an example the application of constructing a tumor-liver common mode type to study organ injury and drug efficacy or study in vitro drug efficacy of organ metabolism drugs.

Example 1

The dynamic co-culture organ chip of the culture unit shown in FIG. 16 is adopted to construct a tumor-liver co-model, and comprises the following steps:

s41, sterilizing the open dynamic co-cultured organ chip, adding mixed cell suspension containing tumor cells and matrix material into the culture hole 20, culturing at 37 deg.C, and gelatinizing; obtaining a gelled tumor organ chip; then, a first culture medium (e.g., DMEM + serum or 1640 basic medium + serum) is added to the central well 10 of the gelled organ microchip, and then culture/dynamic culture is performed at 37 ℃ to construct a tumor model. The mixed cell suspension containing the tumor cells and the matrix material is a single cell suspension containing the matrix material and the model cells, and the pH value is 6.5-7.5. The tumor cell refers to a cell line which responds to an anticancer drug, such as HCT 116. The adding amount of the first culture medium can fill the central liquid storage hole 10, and the side liquid storage holes 40 are also filled with the first culture medium; alternatively, the first culture medium may fill a portion of the central reservoir 10 to meet the culture requirements.

S42, forming single cell suspension from the human primary liver cells, centrifuging and resuspending, and preparing the cell suspension with a specific density according to requirements. Adding collagen (pH is adjusted by NaOH and hepes buffer solution) or other matrix materials such as matrigel and the like in a specific volume into a 1.5ml EP tube according to a proportion to obtain a mixed cell suspension, and ensuring that the 3D material forms a good three-dimensional structure under the concentration (the collagen is 1-2mg/ml, and the matrigel is 30% -80%). After the construction culture is carried out for 72h in the step S41, the first culture medium is removed, then the mixed cell suspension is added into the lower layer culture section 302 of the co-culture channel 30, the mixed cell suspension is blown and beaten by a pipette and is uniformly mixed, the mixed cell suspension is quickly transferred and inoculated into the lower layer culture section 302 of the co-culture channel 30 by pipetting, and after the cell planting is finished, the mixed cell suspension is placed at 37 ℃ for 10 minutes to ensure that the matrix material can be well gelatinized. The small-pore section 13 of the central reservoir 10 (the second reservoir of the second reservoir layer 120) is added with the tumor cell line culture medium, and the co-culture channel 30 is added with the primary liver culture medium. Then continuously dynamically culturing for 24h at the temperature of 37 ℃; constructing and obtaining a tumor-liver co-model.

S43, performing drug stimulation on the tumor-liver common mode: after the liver-tumor common model is constructed and co-culture is continued for 24h, a prepared culture medium containing a specific type and concentration of the drug to be screened (taking the 10 mu M antitumor drug sunitinib as an example) is filled in the central liquid storage hole 10. The dynamic culture was continued at 37 ℃ for 120 h.

S431, detection of a drug sensitive result: the 3D cultured cells will be evaluated for ATP by removing the drug-loaded media after drug stimulation for 120h at step S43 and adding Cell titer glo to the small pore section 13 (or second reservoir) of the central reservoir 10. The method is applicable to, but not limited to, the above characterization method, and other characterization reagents or methods are also compatible, such as cell titer blue, high content imaging technology, to perform imaging characterization on the number of living and dead cells.

S432, hepatotoxicity detection: the culture medium with the drug is removed after drug stimulation for 120h in step S43, an evaluation reagent is added into the co-culture channel 30, and the part can use a commercial kit to characterize the liver function response after drug treatment by albumin, alpha-GST or metabolic enzyme, wherein the characterization mode can be Elasa kit or mRNA extraction to quantify the expression amount of the main metabolic enzyme. In addition, the activity of the liver cells after the action of the medicine can be characterized by using the existing ATP and metabolic capability detection means, and the mitochondrial function of the cells can also be characterized by using a mitochondrial membrane potential kit or a mitochondrial reactive oxygen radical kit. Thus, the hepatotoxic effect outcome of a drug can be characterized using a single parameter as well as multiple parameters.

In this example 1, F-actin and nuclear staining characterization was performed on the tumor cells and the human primary liver cells in the tumor-liver co-model constructed in step S42, respectively, to obtain a fluorescence staining result characterization map of the tumor cells as shown in fig. 19 and a fluorescence staining result characterization map of the human primary liver cells as shown in fig. 20, which shows that the tumor cells and the human primary liver cells are highly expressed and highly biomimetic in tight junction protein.

In example 1, as shown in the drug sensitivity test result graph of step S431 shown in fig. 21, it can be seen that the tumor inhibition rate is only 30% after the drug (sunitinib) is added, indicating that the drug has almost no anti-tumor effect at this concentration.

In this example 1, as shown in the graph of the hepatotoxicity test result in step S432 shown in fig. 22, it can be seen that the inhibition rate after adding the drug (sunitinib) is close to 100%, indicating that the drug has a strong liver injury effect at this concentration.

The above description and drawings sufficiently illustrate embodiments of the disclosure to enable those skilled in the art to practice them. Other embodiments may include structural and other changes. The examples merely typify possible variations. Individual components and functions are optional unless explicitly required, and the sequence of operations may vary. Portions and features of some embodiments may be included in or substituted for those of others. The embodiments of the present disclosure are not limited to the structures that have been described above and shown in the 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

1. An open type co-cultured organ chip, comprising: one or more culture units, each of said culture units comprising:

the central liquid storage hole is a stepped blind hole; be provided with one or more culture holes on the diapire in center liquid storage hole, be provided with the coculture passageway along axial extension on the ladder face in center liquid storage hole.

2. The co-culture organ chip according to claim 1, wherein the co-culture channel comprises a closed/non-closed annular slot along a circumference of the stepped surface of the reservoir well;

alternatively, the co-culture channel comprises a plurality of pores.

3. The co-culture organ chip of claim 2, wherein the volume of the co-culture channel is 20 to 500. mu.L.

4. The co-culture organ chip of claim 1, wherein the volume of the culture well is 1 to 50. mu.L.

5. The co-cultured organ chip according to claim 1, further comprising:

the side liquid storage holes are arranged around the central liquid storage hole in pairs;

and the communicating channel is communicated with the central liquid storage hole and the peripheral liquid storage holes thereof, and/or is communicated with the coculture channel and the peripheral liquid storage holes of the central liquid storage hole where the coculture channel is located.

6. The co-culture organ chip of claim 5, wherein the communication channel has a cross-sectional area ranging from 0.01 to 100mm²。

7. The co-cultured organ chip according to claim 1,

the culture hole and/or the co-culture channel are/is a through hole or a blind hole; when the culture well and/or the co-culture channel is a through-hole, the co-culture organ chip further comprises:

a bottom plate disposed at the bottom of the culture well and the co-culture channel.

8. The co-cultured organ chip according to claim 1,

the co-culture channel comprises an upper liquid storage section and a lower culture section which are communicated; the size of the lower culture hole section is smaller than or equal to that of the upper liquid storage hole section.

9. The co-culture organ chip according to any one of claims 1 to 8, comprising a first stock solution layer, a second stock solution layer and a culture layer which are stacked in order;

the first liquid storage layer is provided with one or more first liquid storage holes;

a second liquid storage layer provided with one or more second liquid storage holes and one or more co-culture liquid storage channels arranged around each of the second liquid storage holes;

the cultivation layer is provided with one or more planting areas and one or more co-cultivation planting channels surrounding each planting area, and one or more cultivation holes are formed in each planting area;

the first liquid storage hole, the second liquid storage hole and the planting area are coaxially arranged to form the central liquid storage hole; the co-culture liquid storage channels and the co-culture planting channels are communicated in a one-to-one correspondence mode to form the co-culture channels.

10. The co-cultured organ chip according to claim 9,

the first liquid storage layer is also provided with a plurality of first side liquid storage holes and a first communication channel, and the first side liquid storage holes are arranged on two sides of the first liquid storage holes in pairs; the first communication channel is communicated with the first liquid storage hole and the first side liquid storage holes on two sides of the first liquid storage hole; and/or the presence of a gas in the gas,

the second liquid storage layer is also provided with a plurality of second side liquid storage holes and second communication channels, and the plurality of second side liquid storage holes are arranged on two sides of the second liquid storage holes in pairs; the second communicating channel is communicated with the second liquid storage hole and the second side liquid storage holes on two sides of the second liquid storage hole.