WO2022067959A1 - 3d多器官共培养芯片 - Google Patents

3d多器官共培养芯片 Download PDF

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Publication number
WO2022067959A1
WO2022067959A1 PCT/CN2020/126309 CN2020126309W WO2022067959A1 WO 2022067959 A1 WO2022067959 A1 WO 2022067959A1 CN 2020126309 W CN2020126309 W CN 2020126309W WO 2022067959 A1 WO2022067959 A1 WO 2022067959A1
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WIPO (PCT)
Prior art keywords
culture
hole
liquid storage
organ
fluid
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2020/126309
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English (en)
French (fr)
Inventor
肖荣荣
周宇
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Beijing Daxiang Biotech Co Ltd
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Beijing Daxiang Biotech Co Ltd
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Priority to EP20938520.2A priority Critical patent/EP3998332A4/en
Priority to US17/537,525 priority patent/US12480078B2/en
Publication of WO2022067959A1 publication Critical patent/WO2022067959A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes

Definitions

  • the present application relates to the technical field of biological tissue engineering, for example, to a 3D multi-organ co-culture chip.
  • 3D cell models constructed in vitro can better represent models of the spatial and chemical complexity of living tissue. Compared with traditional 2D models, 3D cell models have great advantages in studying molecular mechanisms of tissue function, collecting signaling pathways, and drug responses in certain diseases. However, traditional 3D models also have some limitations. For example, organoids are variable in size and shape, and it is difficult to fix cells in a consistent position for further analysis. In addition, many 3D model systems lack microscale structures and tissue-tissue interfaces for multi-organ co-culture.
  • vascular endothelial cell layer and surrounding connective tissue and parenchymal cells are critical for all organs. Also, cells are often not exposed to normal mechanical signals such as fluid shear, tension, and compression, which can affect organ development and function. The lack of fluid flow affects the interactions between cultured tissue cells, circulating blood cells, and immune cells.
  • Organ-on-a-chip technology is an emerging technology that can overcome these limitations.
  • Organ-on-a-chip includes continuously perfused cell culture channels that mimic tissue and organ-level physiology that can reproduce multicellular architecture, tissue-tissue interfaces, physicochemical microenvironments, and vascular perfusion of the body. These devices can reproduce tissue and organ perfusion. functional level.
  • high-resolution, real-time imaging of live cells, and monitoring of biochemical, genetic, and metabolic activities can be achieved. This technique has great potential to facilitate the study of tissue development, organ physiology and disease pathology.
  • organ-on-a-chip technology plays an important role in lead compound research, drug toxicity testing, and biomarker identification.
  • Organ-on-a-chip technology enables the construction of low-cost in vitro models that recapitulate tissue and organ-level functions.
  • the existing co-culture microfluidic organ chip has a narrow application range, poor universality, complicated model building operations, and complicated fluid control methods.
  • the embodiments of the present disclosure provide a 3D multi-organ co-culture chip to solve the problems of the existing co-culture microfluidic organ chip with narrow application range, poor universality, complicated model building operations, and complicated fluid control methods.
  • the 3D multi-organ co-culture chip includes: a chip body on which one or more groups of culture modules are arranged; wherein, each group of culture modules includes:
  • a liquid storage hole one end is open and located on the upper surface of the chip body
  • the first culture micropore is located below the liquid storage hole and communicated with the liquid storage hole;
  • the second culture micropore is located below the first culture micropore and communicated with the first culture micropore;
  • the plurality of second fluid operation holes are open at one end and located on the upper surface of the chip body, and the other ends are respectively communicated with the second culture micropores through channels.
  • the 3D multi-organ co-culture chip provided by the embodiments of the present disclosure can achieve the following technical effects:
  • the 3D multi-organ co-culture chip provided by the embodiments of the present disclosure has a simple structure, the second fluid manipulation hole is communicated with the second culture micropore through a channel (for example, a through channel), and simple operations are performed through the liquid storage hole and the fluid manipulation hole, It is convenient for cell inoculation, liquid change, sampling and other operations. It is simple to operate and does not require professional and technical personnel, which expands the application scope of the culture chip and improves the universality.
  • the first and second culture microwells provide flexibility for 2D and 3D organ culture alone or co-culture.
  • Gravity-based fluid drive is a simple and accurate fluid control method, which can realize real-time dynamic update of the culture environment in the second culture microwell, and can realize long-term in vitro dynamic co-culture of multiple organs.
  • Multiple culture modules are arranged according to the multi-well plate spacing for high-throughput, compatible with high-throughput, automated equipment.
  • Physical forces including physiologically relevant levels of fluid shear, circulatory pressure, and mechanical compressive forces can also be combined to achieve organ-specific responses such as aggregation of circulating immune cells, responses to drugs, toxins, and other environmental perturbations.
  • long-term in vitro dynamic co-culture of multiple organs can be achieved.
  • FIG. 1 is a schematic exploded view of the structure of a culture module of a 3D multi-organ co-culture chip provided in an embodiment of the present disclosure
  • FIG. 2 is a schematic structural exploded view of a culture module of another 3D multi-organ co-culture chip provided in an embodiment of the present disclosure
  • FIG. 3 is a schematic top-view structural diagram of a culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure
  • FIG. 4 is a schematic exploded view of the structure of a culture module of another 3D multi-organ co-culture chip provided in an embodiment of the present disclosure
  • Fig. 5 is the top-view structure schematic diagram of the culture module shown in Fig. 4;
  • FIG. 6 is a schematic partial structure diagram of a culture module of another 3D multi-organ co-culture chip provided in an embodiment of the present disclosure
  • FIG. 7 is a schematic partial structural diagram of a culture module of another 3D multi-organ co-culture chip provided in an embodiment of the present disclosure
  • FIG. 8 is a schematic exploded view of the structure of a culture module of another 3D multi-organ co-culture chip provided in an embodiment of the present disclosure
  • Fig. 9 is the top-view structure schematic diagram of the culture module shown in Fig. 8.
  • FIG. 10 is a schematic exploded view of the structure of a culture module of another 3D multi-organ co-culture chip provided by an embodiment of the present disclosure
  • Figure 11 is a schematic top view of the culture module shown in Figure 10;
  • FIG. 12 is a schematic exploded view of the structure of a culture module of another 3D multi-organ co-culture chip provided in an embodiment of the present disclosure
  • FIG. 13 is a schematic top view of the structure of the culture module shown in FIG. 12 .
  • 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.
  • 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.
  • 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.
  • an embodiment of the present disclosure provides a 3D multi-organ co-culture chip, including a chip body on which one or more culture modules are disposed.
  • each culture module includes a liquid storage hole 21 , a first culture micro hole 22 , a second culture micro hole 23 and a plurality of second fluid operation holes 24 .
  • One end of the liquid storage hole 21 is open and is located on the upper surface of the chip body.
  • the first culture micropore 22 is used for culturing the cells of the first organ; it is located below the liquid storage hole 21 and communicated with the liquid storage hole 21 .
  • the second culture microwell 23 is used for culturing the cells of the second organ; it is located below the first culture microwell 22 and communicates with the first culture microwell 22 .
  • One end of the plurality of second fluid manipulation holes 24 is open and located on the upper surface of the chip body, and the other ends are respectively communicated with the second culture micropores 23 through channels.
  • the 3D multi-organ co-culture chip provided by the embodiments of the present disclosure has a simple structure, the second fluid manipulation hole is communicated with the second culture micropore through a channel (for example, a through channel), and simple operations are performed through the liquid storage hole and the fluid manipulation hole, It is convenient for cell inoculation, liquid change, sampling and other operations. It is simple to operate and does not require professional and technical personnel, which expands the application scope of the culture chip and improves the universality.
  • the first and second culture microwells provide flexibility for 2D and 3D organ culture alone or co-culture.
  • Gravity-based fluid drive is a simple and accurate fluid control method, which can realize real-time dynamic update of the culture environment in the second culture microwell, and can realize long-term in vitro dynamic co-culture of multiple organs.
  • Multiple culture modules are arranged according to the multi-well plate spacing for high-throughput, compatible with high-throughput, automated equipment.
  • Physical forces including physiologically relevant levels of fluid shear, circulatory pressure, and mechanical compressive forces can also be combined to achieve organ-specific responses such as aggregation of circulating immune cells, responses to drugs, toxins, and other environmental perturbations.
  • long-term in vitro dynamic co-culture of multiple organs can be achieved.
  • the 3D multi-organ co-culture chip of the embodiment of the present disclosure can be used to construct a multi-organ model in vitro.
  • the cells of the second organ cultured in the second culture microwell 23 may be one or more types of cells related to the same organ, for example, 2 types, 3 types or even more types.
  • the co-culture chip enables in situ 2D or 3D co-culture of multiple organ types. Models for the study of multiple organ interactions, such as blood vessel-tumor models, blood-brain barrier-tumor models, etc., can be constructed to study angiogenesis, drug penetration, cell polarization, cell migration, and drug activity evaluation.
  • the liquid storage hole 21 holds a culture medium or a drug diluent, etc., to provide the first organ cells with a required culture medium or a drug to be tested.
  • the first organ cells can be cultured in the first culture microwell 22 in 3D.
  • the cells of the second organ are cultured in the second culture microwell 23 , that is, the liquid storage well 21 , the first culture microwell 22 and the second culture microwell 23 are coaxially communicated with each other.
  • the second fluid manipulation hole 24 and the channel realize the microfluidic control of the culture fluid in the second culture micropore 23, and simultaneously perform fluid manipulation through the liquid storage hole 21 and the second fluid manipulation hole 24, which is a simple and accurate fluid. way to control.
  • the shapes of the liquid storage hole 21 , the first culture microwell 22 , the second culture microwell 23 and the second fluid operation hole 24 are not limited, and may be a circular hole, an oval, a square, a rectangle, a fan, or Polygons (such as hexagons, octagons, etc.) and other geometric shapes, under the condition that the design requirements are met, the shape of the liquid storage hole 21 and the second fluid operation hole 24 can hold as much culture medium or drug dilution as possible
  • the shapes of the first culture micropores 22 and the second culture micropores 23 are designed to facilitate growth.
  • the shape of the liquid storage hole 21 and the second fluid operation hole 24 includes a circle or an ellipse.
  • the shape of the first culture micropores 22 is circular.
  • the shape of the second culture microwell 23 is square, rectangle or hexagon.
  • the planting and culturing of the first organ cells in the first culture microwell 22 adopts the method of open through-holes, which facilitates subsequent on-machine detection and cell recovery for analysis such as RNA and protein extraction.
  • the number and arrangement of the culture modules provided on the chip body are not limited, and can be determined according to needs.
  • the second culture micropores 23 may be through holes or grooves.
  • the second culture micropores 23 are through-holes. Then, the liquid storage hole 21 , the first culture micro-hole 22 and the second culture micro-hole 23 form a through hole, and the chip body can be used with the bottom plate when in use.
  • the second culture micropores 23 are grooves. Then the liquid storage hole 21 , the first culture micro-hole 22 and the second culture micro-hole 23 constitute non-through holes, and need not be used with a bottom plate.
  • the number of multiple culture modules set on the chip body is 12, 24, 36, or 96, etc.
  • the arrangement of the multiple culture modules is the same as that of existing commercialized sample adding equipment and detectors. (eg, microplate reader, high-content imaging system, etc.) compatible matching. Enables high-throughput organ culture and detection.
  • the cross section of the liquid storage hole 21 is larger than the cross section of the first culture microwell 22 ; the cross section of the second culture microwell 23 is greater than or equal to the cross section of the first culture microwell 22 .
  • the cross-section of the liquid storage hole 21 is larger than the cross-section of the first culture micro-hole 22 , so that the liquid exchange process does not affect the cells in the first culture micro-hole 22 .
  • the cross section of the second culture microwell 23 is larger than that of the first culture microwell 22 , so that the cells growing at the edge of the second culture microwell 23 do not or less contact the cells in the first culture microwell 22 .
  • the relative size of the cross-section of the liquid storage hole 21 and the cross-section of the second culture micro-hole 23 is not limited, and can be set according to actual needs.
  • the cross-section of the liquid storage hole 21 is larger than the cross-section of the first culture micro-hole 22
  • the cross-section of the liquid-storage hole 21 is made larger than the cross-section of the second culture micro-hole 23 . It is convenient for cultivation operation and observation.
  • the specific size parameters of the liquid storage hole 21 , the first culture micro-hole 22 and the second culture micro-hole 23 can be determined according to actual needs, which are not limited herein.
  • the liquid storage hole 21 adopts a column hole design, which reduces the risk of cross-contamination between the holes and reduces the edge effect caused by evaporation.
  • the first culture micropore 22 is communicated with the second culture micropore 23, and the communication here is understood in a broad sense, and not only includes the direct communication between the first culture micropore 22 and the second culture micropore 23 (as shown in FIG. 1 ). shown), also including the communication between the two through a diaphragm.
  • a thin film layer 14 is arranged between the first culture microwell 22 and the second culture microwell 23, but the thin film layer 14 is distributed with Micro-nano-sized holes, through these micro-nano-sized holes, the culture fluid in the first culture micro-hole 22 and the second culture micro-hole 23 can exchange substances, which is also considered to be the first culture micro-hole 22 and the second culture micro-hole 23.
  • the holes 23 communicate with each other.
  • different organ models can be constructed and related studies can be carried out.
  • the communication mode between the first culture microwell 22 and the second culture microwell 23 is direct connection, that is, no diaphragm is provided.
  • the 3D multi-organ co-culture chip composed of this kind of culture module is defined as a membraneless chip.
  • the two organs are physically separated by a 3D matrix, and the cells and secreted factors of the first organ and the second organ cultured in 3D can interact through the matrix.
  • the organ model constructed by the membraneless chip can be used to study the interaction of two organ-derived cells, to study the induced migration of cells, to study the growth and sprouting of blood vessels during the co-culture of blood vessels with various tissues and organs.
  • the communication mode between the first culture microwell 22 and the second culture microwell 23 is a diaphragm connection.
  • the 3D multi-organ co-culture chip composed of this kind of culture module is defined as a membrane chip.
  • the cells of the first organ in the first culturing microwell 22 and the cells of the second organ in the second culturing microwell 23 communicate with each other through a diaphragm, which can not only achieve physical isolation, but also achieve biological and chemical isolation. factor co-communication.
  • the organ model constructed by this membrane chip can be used to simulate the interaction of the barrier model with other organs, such as the blood-brain barrier, and the membrane layer provides mechanical support for the adhesion of human brain microvascular endothelial cells.
  • Channels may enable fluid flow to provide the shear forces necessary for barrier formation.
  • tumor-based drugs and related research for example, anti-glioma compounds for the blood-brain barrier.
  • the second fluid operation hole 24 is a stepped hole, and the diameter of the stepped hole becomes smaller from the upper surface of the chip body to the inside. Settling of the seeded cells at the bottom of the second fluid manipulation well 24 is reduced.
  • the stepped holes include first-order stepped holes or second-order stepped holes. It can be determined according to actual needs. For details, please refer to the relevant content about the stepped holes in the specific structural form of a 3D multi-organ co-culture chip below.
  • the center distance between the liquid storage hole 21 and the second fluid operation hole 24 is 1 ⁇ 10 mm.
  • the center-to-center distance refers to the distance between the center of the liquid storage hole 21 and the center of the second fluid operation hole 24 .
  • the center distance between the liquid storage hole 21 and the second fluid operation hole 24 is 3 ⁇ 8 mm.
  • a plurality of second fluid operation holes 24 are arranged in pairs; each pair of second fluid operation holes 24 is communicated through a through channel 25 ; the through channel 25 includes a first side through channel 251 and a second through channel 25 . Side through-channels 252 , and the first side through-channels 251 and the second side through-channels 252 are disposed on opposite sides of the second culture microwell 23 .
  • the second fluid operation hole 24 communicating with the first side through channel 251 is defined as the first side second fluid operation hole 2401
  • the second fluid operation hole 24 communicating with the second side through channel 252 is defined as The second fluid manipulation hole 2402 on the second side.
  • the second fluid manipulation hole 2401 on the first side and the through channel 251 on the first side can be used as a sample injection channel
  • the second fluid manipulation hole 2402 on the second side and the through channel 252 on the second side can be used as a sample output channel; It can also be exchanged, not limited.
  • the culture medium in the second fluid manipulation hole 2401 on the first side or the second fluid manipulation hole 2402 on the second side can be manipulated, so that the first side through channel 251 (or the second side through channel 251) 252) into the second culture micropore 23, and the culture solution in the second culture micropore 23 flows into the second side through channel 252 (or the first side through channel 251) to realize the second culture micropore The communication and flow of the culture medium in 23.
  • the first culture module includes a liquid storage well 21, a first culture microwell 22, a second culture microwell 23, and two second fluid operation wells 24 ( A pair of second fluid operation holes 24), and a through channel 25;
  • the liquid storage hole 21, the first culture micro-hole 22 and the second culture micro-hole 23 are connected in sequence from top to bottom and are arranged coaxially;
  • two second fluid The operation holes 24 are respectively located on both sides of the liquid storage hole 21;
  • the second fluid operation holes 2401 on the first side communicate with the second culture micropores 23 through the first side through-channels 251, and the second fluid operation holes 2402 on the second side pass through the second culturing micropores 23.
  • the side through-channels 252 communicate with the second culture micropores 23 .
  • the culture module further includes a plurality of side fluid operation holes 29 and a bypass channel 26, one end of the side fluid operation hole 29 is open and is located on the upper surface of the chip body; the bypass channel 26 The two ends are respectively communicated with a side fluid manipulation hole 29 , and the middle portion is communicated with the second culture micropore 23 .
  • other types of cells belonging to the same organ as the cells in the second culture microwell 23 can be added to the bypass channel 26 , and co-culture is realized at the connection between the bypass channel 26 and the second culture microwell 23 .
  • the side fluid operation holes 29 at both ends of a bypass channel 26 can be filled with cell culture fluid, and microfluidic culture can be realized.
  • connection between the bypass channel 26 and the second culture micropore 23 is set as one or more microchannels.
  • the microchannel may be one or more microchannels formed by disposing a barrier (one or more) on the communication port of the communication place.
  • the size of the microchannel is not limited, and is based on the fact that the cells in the bypass channel 26 cannot pass into the second culture micropore 23, but can achieve material exchange.
  • the cells in the bypass channel 26 are physically separated from the cells in the second culture micro-well 23 , and at the same time, the exchange of secreted factors between the cells in the bypass channel 26 and the second culture micro-well 23 is achieved.
  • a plurality of fences are provided at the connection between the bypass channel 26 and the second culture microwell 23 to form a plurality of microchannels.
  • the configuration of the plurality of fences and the microchannels is not limited.
  • one or more vertical fences 261 are arranged at set intervals on the side wall of the middle part of the bypass channel 26 and the communication port of the second culture micro-hole 23 .
  • the communication port is divided into a plurality of vertical and parallel vertical microchannels 262 .
  • Independent growth of cells or 3D matrix in the second culture microwell 23 and bypass channel 26 can be controlled by controlling the vertical microchannel height and width while maintaining factor exchange.
  • one or more transverse fences 263 are arranged laterally on one side wall of the middle of the bypass channel 26 and the communication port of the second culture microwell 23 .
  • the transverse fence is arranged in the vertical middle of the communication port, that is, a transverse microchannel 264 is formed above and below the one transverse fence, respectively.
  • the connection between the bypass channel 26 and the second culture micropore 23 is set as a microchannel.
  • the structure of a microchannel is not limited.
  • a lateral fence 262 is arranged laterally on the sidewall of the middle of the bypass channel 26 and the communication port of the second culture microwell 23, and the lower part of the communication port (such as As shown in FIG. 7 ) or upper shielding, a lateral microchannel 264 is formed above or below the one lateral fence 262 (as shown in FIG. 7 ).
  • the bypass channel 26 is provided on both sides or on one side of the other two opposite sides of the second culture microwell 23 that are not communicated with the through channel 25 . Then, the number of the second fluid operating holes 24 corresponding to the number of the bypass passages 26 is increased correspondingly, and the two ports of the bypass passages 26 are respectively communicated with each other.
  • the second type of culture module which is based on the first type of culture module, adds two bypass passages 26, which are respectively arranged in the passages that are not communicated with the through passages 25.
  • the other two opposite sides of the second culture microwell 23 ; and the communication ports at the connection between the bypass channel 26 and the second culture microwell 23 are separated by a plurality of vertical fences 261 to form a plurality of vertical microchannels 262 .
  • two pairs of side fluid operating holes 29 are added, which correspond to the four ports of the two bypass passages 26 respectively.
  • a deformation of the second type of culture module which is based on the first type of culture module, only a bypass channel 26 is added, which is arranged in the second culture micropore 23 that is not communicated with the through channel 25.
  • One side of the other two opposite sides; and the communication port at the connection between the bypass channel 26 and the second culture microwell 23 is separated and arranged into a plurality of vertical microchannels 262 by a plurality of vertical fences 261 .
  • a pair of side fluid operating holes 29 are added, which correspond to two ports of a bypass passage 26 . Referring to Figures 4 and 5, only one bypass channel 26 can be added on one of the sides.
  • the communication port at the connection between the bypass channel 26 and the second culture micropore 23 is set as one microchannel.
  • the one microchannel refer to the above-mentioned related content, which will not be repeated here.
  • the extension direction of the bypass channel 26 is the same as the extension direction of the through channel 25 .
  • the flow of the culture liquid in the second culture micro-hole 23 is regular, and the culture effect is good. For example, using gravity driven bidirectional flow or unidirectional driven flow.
  • the second culture microwell is square and has a first side 231 , a second side 232 , a third side 233 and a third side 233 .
  • the first side 231 and the second side 232 are opposite sides
  • the third side 233 and the fourth side 234 are opposite sides
  • the through channel 25 is arranged on the side of the first side 231
  • the first side through channel 251, the second side through channel 252 of the through channel 25 is set on the second side 232 side
  • a communication port is set on the third side 233 and/or the fourth side 234 for connecting with the bypass channel
  • the middle of 26 is connected.
  • the culture module further includes a plurality of first fluid manipulation holes 27 ; one end of the plurality of first fluid manipulation holes 27 is open and located on the upper surface of the chip body, The other ends are communicated with the liquid storage holes 21 through the first communication channels 28 respectively.
  • the communication and flow of the fluid in the liquid storage hole 21 and the first fluid operation hole 27 are realized through the first communication channel 28, and the culture liquid in the first fluid operation hole 27 can be operated to realize the culture liquid in the liquid storage hole 21. Even if the culture environment in the first culture micropore 22 surrounded by the culture medium in the liquid storage hole 21 is in a flowing state, a dynamic culture environment is formed.
  • the arrangement position and number of the plurality of first fluid manipulation holes 27 are not limited, as long as the cultured fluid in the liquid storage holes 21 can be realized by manipulating the culture fluid therein.
  • the plurality of first fluid operation holes 27 are divided into two groups, which are respectively disposed on opposite sides of the liquid storage hole 21 . That is, the plurality of first fluid manipulation holes 27 are divided into a first side first fluid manipulation hole 271 and a second side first fluid manipulation hole 272 .
  • the first side and the second side here may be the same as the first side and the second side of the aforementioned second fluid manipulation hole 24 portion.
  • a plurality of first fluid operation holes 27 are arranged in pairs, and each pair of first fluid operation holes 27 is arranged on opposite sides of the liquid storage hole 21 respectively.
  • the culture module includes two first fluid operation holes 27 , which are respectively disposed on opposite sides of the liquid storage hole 21 .
  • the first fluid operating holes 27 are disposed on opposite sides of the liquid storage hole 21 through the diameter, and are parallel to the through channel 25 .
  • the two first fluid operation holes 27 and the center line of the liquid storage hole 21 form a predetermined angle, that is, the first fluid operation holes 27 are arranged on opposite sides of the non-diameter.
  • the set angle is not limited. That is, the arrangement position of the first fluid operation hole 27 in the embodiment of the present disclosure is not limited.
  • a plurality of first fluid operation holes 27 are respectively added to pass through the first communication channels 28 respectively. It is communicated with the liquid storage hole 21 to form the corresponding fourth type of culture module, fifth type of culture module and sixth type of culture module.
  • the specific structure is not limited.
  • a specific structural form of a 3D multi-organ co-culture chip is given below, but is not limited to this specific structural form.
  • the chip body includes:
  • the first liquid storage layer 11 is provided with a liquid storage hole 21 and a second operation hole I241 of the second fluid operation hole 24;
  • the first culture layer 12 is provided with the first culture micropore 22 and the second operation hole II 242 of the second fluid operation hole 24; the first culture layer 12 is stacked under the first liquid storage layer 11, and the first The culture micropore 22 communicates with the liquid storage hole 21; the second operation hole II 242 communicates with the second operation hole I 241;
  • the second culture layer 13 is provided with second culture micropores 23 and channels (eg, through-channels 25); the second culture layer 13 is stacked under the first culture layer 12, and the second culture micropores 23 and The first culture micropore 22 communicates with each other, and the channel communicates with the second culture micropore 23 and the second operation hole II 242 .
  • the chip body includes a three-layer chip structure, and the three-layer chip structure can be stacked and connected in sequence.
  • the various layers of chips can be assembled together by bonding processes such as double-sided tape, ultrasonic, thermal bonding, plasma, and hot pressing.
  • the first liquid storage layer 11 and the first culture layer 12 can be combined to form a one-layer structure for processing, which can be determined according to the actual situation.
  • each layer of chip structure in the chip body is polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), PS, COC, COP one or more.
  • PMMA polymethyl methacrylate
  • PDMS polydimethylsiloxane
  • PC polycarbonate
  • PS COC
  • COP one or more.
  • the fabrication of each layer structure may adopt soft lithography, molding method, laser etching, machining, LIGA or one-time injection molding to obtain the chip structure of each layer.
  • the second fluid operation hole 24 is a stepped hole; and the diameter of the second operation hole I241 is larger than that of the second operation hole II242.
  • the second fluid operating hole 24 in this embodiment is a first-step stepped hole.
  • the channel (through channel 25 ) on the second culture layer 13 communicates with the second operation hole II 242 on the first culture layer 12 .
  • the connection method is not limited.
  • one end of the channel is extended into the second operation hole II 242 to communicate the two.
  • the second culture layer 13 is further provided with a hole groove, and a port of the channel is arranged on the side wall of the hole groove, that is, the hole groove can be regarded as the second operation hole III 243 of the second fluid operation hole 24 .
  • the diameter of the second operation hole I241 is larger than the diameter of the second operation hole II242, and the diameter of the second operation hole II242 is larger than the diameter of the second operation hole III243. Reduces cell retention in wells when seeding cells.
  • the second fluid operating hole 24 in this embodiment is a second-step stepped hole.
  • the diameter of the second operation hole I 241 is equal to the diameter of the second operation hole II 242 , and the diameter of the second operation hole II 242 is larger than the diameter of the second operation hole III 243 .
  • the second operation hole I 241 and the second operation hole II 242 have the same size, which reduces the generation of air bubbles, and the second operation hole III 243 has the smallest pore size, which reduces the deposition of cells in the holes when cells are seeded.
  • the second fluid operating hole 24 in this embodiment is a first-step stepped hole.
  • the first liquid storage layer 11 is further provided with side operation holes I291 of the side fluid operation holes 29
  • the first culture layer 12 is further provided with side operation holes I 291 .
  • the side operation hole II 292 of the side fluid operation hole 29, the side operation hole II 292 is communicated with the side operation hole I 291;
  • the second culture layer 13 is also provided with a bypass channel 26, and the bypass channel 26 communicates with the second culture micropore 23 With side operation hole II 292.
  • the bypass channel 26 on the second culture layer 13 communicates with the side operation hole II 292 on the first culture layer 12 .
  • the connection method is not limited.
  • one end of the bypass passage 26 is extended into the side operation hole II 292 to communicate the two.
  • the second culture layer 13 is also provided with a hole groove, and a port of the bypass channel 26 is arranged on the side wall of the hole groove, that is, the hole groove can be regarded as the side operation hole III 293 of the side fluid operation hole 29. .
  • the structural form of the side fluid operation hole 29 is the same as that of the aforementioned second fluid operation hole 24 .
  • the side fluid operating holes 29 are stepped holes; the configuration form of the stepped holes may refer to the second fluid operating holes 24 .
  • the culture module when the culture module includes the first fluid operation hole 27 , the first fluid operation hole 27 and the first communication channel 28 are further provided on the first liquid storage layer 11 .
  • the first fluid manipulation hole 27 is a straight hole.
  • the first communication channel 28 is disposed through the side wall between the liquid storage hole 21 and the first fluid operation hole 27 .
  • the chip body further includes a thin film layer 14 on which micro-nano-sized pores are distributed; That is, on the basis of the aforementioned three-layer chip body, the film layer 14 is added to form a four-layer chip body.
  • the film layer 14 may be a transparent polyester (polyethylene terephthalate, PET) film with apertures.
  • PET polyethylene terephthalate
  • the thin film layer 14 can be assembled with the first liquid storage layer 11 and the first culture layer 12 first, and then assembled with the second culture layer 13 as a whole.
  • the disposition of the thin film layer 14 needs to allow for the communication between the second fluid operation hole 24 and the microchannel (through channel 25 and bypass channel 26 ).
  • the 3D multi-organ co-culture chip is divided into a filmless chip and a filmed chip according to whether the film layer 14 is provided between the first culture microwell 22 and the second culture microwell 23 (same as the aforementioned non-membrane chip). and membrane chips).
  • the liquid storage holes 21 and the second operation holes I241 provided on the first liquid storage layer 11 are column holes.
  • the side operation holes I provided on the first liquid storage layer are column holes.
  • the culture module includes the first fluid manipulation hole the first fluid manipulation hole is a column hole.
  • the first liquid storage layer 11 is provided with 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.
  • An embodiment of the present disclosure is an application of the aforementioned 3D multi-organ co-culture chip for constructing a multi-organ model in vitro.
  • a specific in vitro construction method of a multi-organ model is given below, which is for reference only.
  • the 3D multi-organ co-culture chip of the embodiment of the present disclosure is used to construct a multi-organ model in vitro, it is not limited to the following construction method. .
  • the method for constructing a 3D multi-organ model of a blood vessel and a tumor using a co-culture chip includes the following steps:
  • the chip was sterilized by ultraviolet light. After the sterilization, 20 ⁇ L of low-concentration rat tail type I collagen was added to the second fluid operation hole 24 , incubated at 37° C., then discarded, and washed three times with PBS. Add 2uL of fibrin (2.5mg/mL)-collagen (0.2mg/mL) gel along the first culture microwell 22 to the second culture microwell 23 for culture, to ensure that the gel can only fill the second culture microwell 23 , 37°C solidified for 0.5h.
  • Digest and centrifuge HUVECs resuspend them with complete medium to a certain concentration (1 ⁇ 106 cells/mL) of single-cell suspension, and pass through the port of the first side through channel 251 (including through channel 25 and bypass channel 26) (which can be defined as injection port), add 20 ⁇ L of endothelial cell suspension. And tilt the chip to make the endothelial cells fully adhere to the surface of the solidified fibrin and collagen gel.

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Abstract

本申请公开一种3D多器官共培养芯片，该3D多器官共培养芯片包括：芯片本体，其上设置有一组或多组培养模块；其中，每组培养模块，包括：储液孔，一端为敞口且位于芯片本体的上表面；第一培养微孔，位于储液孔下方且与其连通；第二培养微孔，位于第一培养微孔下方且与其连通；多个第二流体操作孔，一端呈敞口且位于芯片本体的上表面，另一端分别通过通道与第二培养微孔连通。该3D多器官共培养芯片结构简单，通过通道将第二流体操作孔与第二培养微孔连通，通过对流体操作孔内的培养液进行操作，可实现第二培养微孔内培养环境的改变，方便操作。

Description

3D多器官共培养芯片
本申请基于申请号为202011064607.8、申请日为2020年09月30日的中国专利申请提出，并要求该中国专利申请的优先权，该中国专利申请的全部内容在此引入本申请作为参考。
技术领域
本申请涉及生物组织工程技术领域，例如涉及一种3D多器官共培养芯片。
背景技术
常规的2D细胞培养已经发展了将近一个世纪，且在生物医药领域贡献了巨大的价值，但是，2D培养方式，不能实现多种细胞类型的组织特异性、以及分化功能或者精确预测体内的组织功能和药物活性。体外构建的3D细胞模型可以更好地代表活体组织的空间和化学复杂性模型。与传统2D模型相比，3D细胞模型在研究组织功能的分子机制、收集信号通路、以及某些疾病药物响应中具有很大优势。然而传统3D模型也有一些限制性，比如，类器官是大小和形状多变的，很难将细胞固定在一致的位置上进行下一步的分析。除此之外，很多3D模型系统，缺乏微小尺度结构以及多器官共培养的组织-组织界面。比如，血管内皮细胞层以及周围结缔组织和实质细胞，这个对所有器官来说都是至关重要的。而且，细胞通常无法暴露在正常的机械信号如流体剪切力、张力、压缩力中，这些都会影响器官的发育和功能。缺乏流体流动，会影响培养的组织细胞、循环血细胞以及免疫细胞之间的相互作用。
微流控器官芯片技术是一种新兴的技术，则可以克服这些限制。器官芯片包括持续灌流的模拟组织和器官水平的生理结构的细胞培养通道，可以重现多细胞的结构、组织-组织界面、物理化学微环境以及身体的血管灌注，这些装置可以再现组织和器官的功能水平。而且，可以实现活细胞高分辨率、实时成像，以及监测生物化学、基因、代谢活性。这个技术有很大的潜力，促进组织发育、器官生理和疾病病理学的研究。在药物发现和开发过程中，器官芯片技术在先导化合物研究、药物毒性测试以及生物标志物鉴定过程中有重要作用。器官芯片技术可以构建低成本的体外模型，重现组织和器官水平的功能。
在实现本公开实施例的过程中，发现相关技术中至少存在如下问题：现有的共培养微流控器官芯片应用范围狭窄，普适性差，模型构建操作复杂，流体控制方式复杂。
发明内容
为了对披露的实施例的一些方面有基本的理解，下面给出了简单的概括。所述概括不是泛泛评述，也不是要确定关键/重要组成元素或描绘这些实施例的保护范围，而是作为后面的详细说明的序言。
本公开实施例提供一种3D多器官共培养芯片，以解决现有的共培养微流控器官芯片应用范围狭窄，普适性差，模型构建操作复杂，流体控制方式复杂的问题。
在一些实施例中，所述3D多器官共培养芯片，包括：芯片本体，其上设置有一组或多组培养模块；其中，每组培养模块，包括：
储液孔，一端为敞口且位于芯片本体的上表面；
第一培养微孔，位于储液孔下方且与储液孔连通；
第二培养微孔，位于第一培养微孔下方且与第一培养微孔连通；
多个第二流体操作孔，一端呈敞口且位于芯片本体的上表面，另一端分别通过通道与第二培养微孔连通。
本公开实施例提供的3D多器官共培养芯片，可以实现以下技术效果：
本公开实施例提供的3D多器官共培养芯片，结构简单，通过通道(例如，贯通通道)将第二流体操作孔与第二培养微孔连通，通过储液孔和流体操作孔进行简单操作，方便细胞接种、换液、取样等操作，操作简单，不需要专业技术人员，扩大了培养芯片的应用范围，普适性提高。第一和第二培养微孔可灵活实现2D和3D器官单独或共培养。基于重力的流体驱动是是一种简单、精准的流体控制方式，可实现第二培养微孔内培养环境的实时动态更新，而且，可实现多器官的长时间体外动态共培养。多个培养模块按照多孔板间距高通量布局，与高通量、自动化设备兼容。还可以结合物理作用力包括生理水平相关的流体剪切力、循环压力和机械压缩力，可以实现器官特异性的响应如聚集循环免疫细胞、响应药物、毒素以及其他环境干扰。而且，可实现多器官的长时间体外动态共培养。
以上的总体描述和下文中的描述仅是示例性和解释性的，不用于限制本申请。
附图说明
一个或多个实施例通过与之对应的附图进行示例性说明，这些示例性说明和附图并不构成对实施例的限定，附图中具有相同参考数字标号的元件示为类似的元件，附图不构成比例限制，并且其中：
图1是本公开实施例提供的一种3D多器官共培养芯片的培养模块的结构爆炸示意图；
图2是本公开实施例提供的另一种3D多器官共培养芯片的培养模块的结构爆炸示意图；
图3是本公开实施例提供的一种3D多器官共培养芯片的培养模块的俯视结构示意图；
图4是本公开实施例提供的另一种3D多器官共培养芯片的培养模块的结构爆炸示意图；
图5是图4所示的培养模块的俯视结构示意图；
图6是本公开实施例提供的另一种3D多器官共培养芯片的培养模块的局部结构示意图；
图7是本公开实施例提供的另一种3D多器官共培养芯片的培养模块的局部结构示意 图；
图8是本公开实施例提供的另一种3D多器官共培养芯片的培养模块的结构爆炸示意图；
图9是图8所示的培养模块的俯视结构示意图；
图10是本公开实施例提供的另一种3D多器官共培养芯片的培养模块的结构爆炸示意图；
图11是图10所示的培养模块的俯视结构示意图；
图12是本公开实施例提供的另一种3D多器官共培养芯片的培养模块的结构爆炸示意图；
图13是图12所示的培养模块的俯视结构示意图。
附图标记：
11、第一储液层；12、第一培养层；13、第二培养层；14、薄膜层；21、储液孔；22、第一培养微孔；23、第二培养微孔；231、第一侧边；232、第二侧边；233、第三侧边；234、第四侧边；24、第二流体操作孔；2401、第一侧第二流体操作孔；2402、第二侧第二流体操作孔；241、第二操作孔Ⅰ；242、第二操作孔Ⅱ；243、第二操作孔Ⅲ；25、贯通通道；251、第一侧贯通通道；252、第二侧贯通通道；26、旁通通道；261、竖向栅栏；262、竖向微通道；263、横向栅栏；264、横向微通道；27、第一流体操作孔；271、第一侧第一流体操作孔；272、第二侧第一流体操作孔；28、第一连通通道；29、边侧流体操作孔；291、边侧操作孔Ⅰ；292、边侧操作孔Ⅱ；293、边侧操作孔Ⅲ。
具体实施方式
为了能够更加详尽地了解本公开实施例的特点与技术内容，下面结合附图对本公开实施例的实现进行详细阐述，所附附图仅供参考说明之用，并非用来限定本公开实施例。在以下的技术描述中，为方便解释起见，通过多个细节以提供对所披露实施例的充分理解。然而，在没有这些细节的情况下，一个或多个实施例仍然可以实施。在其它情况下，为简化附图，熟知的结构和装置可以简化展示。
本公开实施例的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本公开实施例的实施例。此外，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含。
本公开实施例中，术语“上”、“下”、“内”、“中”、“外”、“前”、“后”等指示的方位或位置关系为基于附图所示的方位或位置关系。这些术语主要是为了更好地描述本公开实施例及其实施例，并非用于限定所指示的装置、元件或组成部分必须具有特定方位，或以特定方位进行构造和操作。并且，上述部分术语除了可以用于表示方位或位置关系以外，还可能用于表示其他含义，例如术语“上”在某些情况下也可能用于表示某 种依附关系或连接关系。对于本领域普通技术人员而言，可以根据具体情况理解这些术语在本公开实施例中的具体含义。
另外，术语“设置”、“连接”、“固定”应做广义理解。例如，“连接”可以是固定连接，可拆卸连接，或整体式构造；可以是机械连接，或电连接；可以是直接相连，或者是通过中间媒介间接相连，又或者是两个装置、元件或组成部分之间内部的连通。对于本领域普通技术人员而言，可以根据具体情况理解上述术语在本公开实施例中的具体含义。
除非另有说明，术语“多个”表示两个或两个以上。
需要说明的是，在不冲突的情况下，本公开实施例中的实施例及实施例中的特征可以相互组合。
结合图1-11所示，本公开实施例提供一种3D多器官共培养芯片，包括芯片本体，其上设置有一个或多个培养模块。其中，每个培养模块包括储液孔21，第一培养微孔22，第二培养微孔23和多个第二流体操作孔24。储液孔21的一端为敞口且位于芯片本体的上表面。第一培养微孔22用于培养第一器官细胞；位于储液孔21下方且与储液孔21连通。第二培养微孔23用于培养第二器官细胞；位于第一培养微孔22下方且与第一培养微孔22连通。多个第二流体操作孔24的一端呈敞口且位于芯片本体的上表面，另一端分别通过通道与第二培养微孔23连通。
本公开实施例提供的3D多器官共培养芯片，结构简单，通过通道(例如，贯通通道)将第二流体操作孔与第二培养微孔连通，通过储液孔和流体操作孔进行简单操作，方便细胞接种、换液、取样等操作，操作简单，不需要专业技术人员，扩大了培养芯片的应用范围，普适性提高。第一和第二培养微孔可灵活实现2D和3D器官单独或共培养。基于重力的流体驱动是是一种简单、精准的流体控制方式，可实现第二培养微孔内培养环境的实时动态更新，而且，可实现多器官的长时间体外动态共培养。多个培养模块按照多孔板间距高通量布局，与高通量、自动化设备兼容。还可以结合物理作用力包括生理水平相关的流体剪切力、循环压力和机械压缩力，可以实现器官特异性的响应如聚集循环免疫细胞、响应药物、毒素以及其他环境干扰。而且，可实现多器官的长时间体外动态共培养。
本公开实施例的3D多器官共培养芯片可用于体外构建多器官模型。其中，第二培养微孔23中培养的第二器官细胞可以是同一器官相关的一种或多种细胞，例如，2种、3种甚至更多种。该共培养芯片可实现多种器官类型的原位2D或3D共培养。可构建用于研究多种器官相互作用的模型，如血管-肿瘤模型、血脑屏障-肿瘤模型等，用于研究血管发生、药物渗透、细胞极化、细胞迁移和药物活性评价等。
本公开实施例中，培养模块中，储液孔21中盛放的是培养基或者药物稀释液等，为第一器官细胞提供所需的培养液或者需要测试的药物等。第一培养微孔22内可以3D培养第一器官细胞。第二培养微孔23内培养第二器官细胞，即储液孔21、第一培养微孔22和第二培养微孔23同轴连通设置。而第二流体操作孔24和通道实现第二培养微孔23内 的培养液的微流控制，同时通过储液孔21和第二流体操作孔24进行流体操作，是一种简单、精准的流体控制方式。
本公开实施例中，储液孔21、第一培养微孔22、第二培养微孔23和第二流体操作孔24的形状不限定，可以是圆孔、椭圆形、正方形、长方形、扇形或多边形(例如六边形、八边形等)等几何形状，在满足设计要求的情况下，储液孔21和第二流体操作孔24的形状以尽量盛放更多的培养基或者药物稀释液为设计依据，第一培养微孔22和第二培养微孔23的形状以便于生长为设计依据。
可选地，储液孔21和第二流体操作孔24的形状包括圆形或椭圆形。
可选地，第一培养微孔22的形状为圆形。
可选地，第二培养微孔23的形状为正方形、长方形或六边形。
另外，第一培养微孔22内第一器官细胞的种植和培养采用开放通孔的方式，方便后续上机检测以及细胞回收进行RNA、蛋白提取等分析。芯片本体上设置的培养模块的数量和排布形式不限定，依据需要确定即可。
本公开实施例中，第二培养微孔23可以是通孔，也可以是凹槽。
可选地，第二培养微孔23为通孔。则储液孔21、第一培养微孔22和第二培养微孔23构成通孔，该芯片本体在使用时，配合底板使用即可。
可选地，第二培养微孔23为凹槽。则储液孔21、第一培养微孔22和第二培养微孔23构成非通孔，无需配合底板使用。
可选地，芯片本体上设置的多个培养模块的数量为12个、24个、36个或96个等，多个培养模块中的排布方式与现有商品化的加样设备、检测器(如，酶标仪、高内涵成像系统等)兼容匹配。实现高通量器官培养和检测。
在一些实施例中，储液孔21的横截面大于第一培养微孔22的横截面；第二培养微孔23的横截面大于或等于第一培养微孔22的横截面。本实施例中，储液孔21的横截面大于第一培养微孔22的横截面，使换液过程不影响第一培养微孔22内的细胞。第二培养微孔23的横截面大于第一培养微孔22的横截面，使第二培养微孔23边缘生长的细胞不与或少与第一培养微孔22中的细胞接触。
本公开实施例中，储液孔21的横截面与第二培养微孔23的横截面的相对大小不限定，依据实际需要设定即可。
可选地，当储液孔21的横截面大于第一培养微孔22的横截面时，使储液孔21的横截面大于所述第二培养微孔23的横截面。便于培养操作和观察。
本公开实施例中，培养模块中，储液孔21、第一培养微孔22和第二培养微孔23的具体尺寸参数可依据实际需要确定，在此不作限定。
本公开实施例中，储液孔21采用柱孔设计，降低孔间交叉污染的风险，减少蒸发导致的边缘效应。
本公开实施例中，第一培养微孔22与第二培养微孔23连通，此处的连通作广义理解， 不但包括第一培养微孔22与第二培养微孔23直接连通(如图1所示)，也包括两者之间通过隔膜连通，例如，如图2所示，第一培养微孔22与第二培养微孔23之间设置薄膜层14，但该薄膜层14上分布有微纳尺寸的孔，通过这些微纳尺寸的孔，第一培养微孔22与第二培养微孔23内的培养液可以进行物质交换，也认为是第一培养微孔22与第二培养微孔23连通。依据第一培养微孔22与第二培养微孔23之间是直接连通，还是隔膜连通，可以构建不同的器官模型，进行相关的研究。
可选地，第一培养微孔22与第二培养微孔23之间的连通方式为直接连接，即没有设置隔膜。由该种培养模块构成的3D多器官共培养芯片定义为无膜芯片。针对该无膜芯片，在进行器官细胞种植时，通过3D基质将两种器官进行物理隔离，3D培养的第一器官和第二器官细胞、分泌因子可以通过基质产生互作。由无膜芯片构建的器官模型可用于研究两种器官来源细胞的相互作用，研究细胞的诱导迁移、研究血管与各种组织器官的共培养过程中，血管的生长、出芽等。
可选地，第一培养微孔22与第二培养微孔23之间的连通方式为隔膜连接。由该种培养模块构成的3D多器官共培养芯片定义为有膜芯片。针对该种有膜芯片，第一培养微孔22内的第一器官细胞与第二培养微孔23内的第二器官细胞之间通过隔膜相通，既可以实现物理隔离，又可以实现生物和化学因子的共通讯。由该种有膜芯片构建的器官模型可用于模拟屏障类模型与其他器官的相互作用，如血脑屏障，隔膜层为人脑微血管内皮细胞粘附提供机械支撑。通道(例如，贯通通道)可以实现流体流动为屏障形成提供必需的剪切力。用于基于血脑屏障的神经类药物、肿瘤类药物筛选及相关研究，例如，用于血脑屏障的抗脑胶质瘤化合物。
在一些实施例中，结合图3所示，第二流体操作孔24呈阶梯孔，且由芯片本体的上表面至内部，阶梯孔的孔径变小。减少接种的细胞在第二流体操作孔24的底部的沉降。可选地，阶梯孔包括一阶阶梯孔或者二阶阶梯孔。依据实际需要确定即可。具体可参见下述的一种3D多器官共培养芯片的具体结构形式中关于阶梯孔的相关内容。
在一些实施例中，储液孔21与第二流体操作孔24的中心间距是1～10mm。与跨膜电阻仪电极兼容，可实时测量TEER值。其中，中心间距是指的储液孔21的中心与第二流体操作孔24的中心之间的距离。
可选地，储液孔21与第二流体操作孔24的中心间距是3～8mm。
在一些实施例中，培养模块中，多个第二流体操作孔24成对设置；每对第二流体操作孔24通过一个贯通通道25连通；贯通通道25包括第一侧贯通通道251和第二侧贯通通道252，且第一侧贯通通道251与第二侧贯通通道252相对设置于第二培养微孔23的相对两侧。本公开实施例中，与第一侧贯通通道251连通的第二流体操作孔24定义为第一侧第二流体操作孔2401，与第二侧贯通通道252连通的第二流体操作孔24定义为第二侧第二流体操作孔2402。在3D器官培养过程中，第一侧第二流体操作孔2401和第一侧贯通通道251可以作为进样通道，第二侧第二流体操作孔2402和第二侧贯通通道252作 为出样通道；也可以交换，不限定。在芯片本体的上表面，即可对第一侧第二流体操作孔2401或第二侧第二流体操作孔2402中的培养液进行操作，使第一侧贯通通道251(或者第二侧贯通通道252)内的培养液流入第二培养微孔23中，第二培养微孔23中的培养液再流入第二侧贯通通道252(或者第一侧贯通通道251)内，实现第二培养微孔23内的培养液的连通和流动。
在一些实施例中，如图1和图2所示，第一种培养模块，包括储液孔21、第一培养微孔22，第二培养微孔23，两个第二流体操作孔24(一对第二流体操作孔24)，以及一个贯通通道25；储液孔21、第一培养微孔22和第二培养微孔23由上至下依次连通且同轴设置；两个第二流体操作孔24分别位于储液孔21的两侧；第一侧第二流体操作孔2401通过第一侧贯通通道251与第二培养微孔23连通，第二侧第二流体操作孔2402通过第二侧贯通通道252与第二培养微孔23连通。
当然，还可以在前述的第一种培养模块的基础上，增加贯通通道25的数量，并相应地增加第二流体操作孔24对数，即一个贯通通道25对应一对第二流体操作孔24，来获得不同变形的培养模块，在此不再赘述。
在一些实施例中，培养模块中，还包括，多个边侧流体操作孔29和旁通通道26，边侧流体操作孔29的一端呈敞口且位于芯片本体的上表面；旁通通道26的两端分别与一个边侧流体操作孔29连通，中部与第二培养微孔23连通。本公开实施例中，旁通通道26内可添加与第二培养微孔23内的细胞属于同一器官的其他类型细胞，在旁通通道26与第二培养微孔23的连通处实现共培养。而一个旁通通道26的两端的边侧流体操作孔29内可以填充细胞培养液，且实现微流培养。
可选地，旁通通道26与第二培养微孔23的连通处设置为一个或多个微通道。此处，微通道可以通过在连通处的连通口上设置栅栏(一个或多个)而形成的一个或多个微通道。微通道的尺寸不限定，以能够使得旁通通道26内的细胞不能通过进入第二培养微孔23内，但能够实现物质交换为依据。实现旁通通道26内的细胞与第二培养微孔23内的细胞进行物理分离，同时实现旁通通道26与第二培养微孔23的细胞分泌因子交换。
结合图4和图5所示，旁通通道26与第二培养微孔23的连通处设置有多个栅栏，形成多个微通道。多个栅栏以及微通道的构造方式不限定。
可选地，如图4和图5所示，在旁通通道26的中部的一侧侧壁与第二培养微孔23的连通口上以设定间隔设置一个或多个竖向栅栏261，将连通口分隔为多个竖向且平行的竖向微通道262。可以通过控制竖向微通道高度和宽度控制第二培养微孔23和旁通通道26内的细胞或3D基质独立生长又保持因子交换。
可选地，在旁通通道26的中部的一侧侧壁与第二培养微孔23的连通口上沿横向设置一个或多个横向栅栏263。当沿横向设置一个横向栅栏263时，该一个横向栅栏设置于连通口的竖向上的中间，即在该一个横向栅栏的上方和下方分别形成一个横向微通道264。当沿横向设置多个横向栅栏时，以设定间隔设置即可。
结合图7所示，旁通通道26与第二培养微孔23的连通处设置为一个微通道。一个微通道的构造方式不限定，可选地，在旁通通道26的中部的一侧侧壁与第二培养微孔23的连通口上沿横向设置一个横向栅栏262，将连通口的下部(如图7所示)或上部遮挡，从而在该一个横向栅栏262的上方(如图7所示)或下方形成一个横向微通道264。
在一些实施例中，旁通通道26设置在未与贯通通道25连通的第二培养微孔23的另外两个相对侧的两侧或单侧。则，相应地增加与旁通通道26的数量相同的对数的第二流体操作孔24，分别对应与旁通通道26的两端口连通。
可选地，如图4和图5所示，第二种培养模块，其是在第一种培养模块的基础上，增加了两个旁通通道26，分别设置在未与贯通通道25连通的第二培养微孔23的另外两个相对侧；且旁通通道26与第二培养微孔23的连通处的连通口利用多个竖向栅栏261分隔设置为多个竖向微通道262。并相应地增加了两对边侧流体操作孔29，分别对应与两个旁通通道26的四个端口连通。
可选地，第二种培养模块的一种变形，其是在第一种培养模块的基础上，只增加了一个旁通通道26，设置在未与贯通通道25连通的第二培养微孔23的另外两个相对侧中的单侧；且旁通通道26与第二培养微孔23的连通处的连通口利用多个竖向栅栏261分隔设置为多个竖向微通道262。并相应地增加了一对边侧流体操作孔29，对应与一个旁通通道26的两个端口连通。参考图4和图5，仅在其中一个侧边上增加一个旁通通道26即可。
在一些实施例中，如图7所示，第三种培养模块，其是第二种培养模块的基础上，将旁通通道26与第二培养微孔23的连通处的连通口设置为一个微通道。该一个微通道的构建参见前述相关内容，在此不再赘述。
在一些实施例中，参见图4至图7所示，旁通通道26的延伸方向与贯通通道25的延伸方向一致。使第二培养微孔23中的培养液的流动呈一定规律，培养效果好。例如，利用重力驱动的双向流动或单向驱动流动。
可选地，如图7所示，所述第二培养微孔呈方形，具有第一侧边231、第二侧边232、第三侧边233和第三侧边233。其中，第一侧边231和第二侧边232为相对的两侧边，第三侧边233和第四侧边234为相对的两侧边；在第一侧边231侧设置贯通通道25的第一侧贯通通道251，第二侧边232侧设置贯通通道25的第二侧贯通通道252，在第三侧边233和/或第四侧边234上开设连通口，用于与旁通通道26的中部连通。
在一些实施例中，结合图8至图13所示，培养模块，还包括多个第一流体操作孔27；多个第一流体操作孔27的一端呈敞口且位于芯片本体的上表面，另一端分别通过第一连通通道28与储液孔21连通。通过第一连通通道28实现储液孔21与第一流体操作孔27内的流体的连通和流动，则对第一流体操作孔27内培养液进行操作，可实现储液孔21内的培养液的流动，即使处于储液孔21内培养液包围的第一培养微孔22内的培养环境处于流动状态，形成动态培养环境。
本公开实施例中，多个第一流体操作孔27的设置位置和数量不限定，只要通过操作 其内的培养液可实现储液孔21内的培养的流体即可。可选地，多个第一流体操作孔27分为两组，分别设置于储液孔21的相对两侧。即多个第一流体操作孔27分为第一侧第一流体操作孔271和第二侧第一流体操作孔272。其中，此处的第一侧和第二侧可以与前述第二流体操作孔24部分的第一侧和第二侧相同。
可选地，培养模块中，多个第一流体操作孔27成对设置，每对第一流体操作孔27分别设置于储液孔21的相对两侧。
可选地，培养模块中，包括两个第一流体操作孔27，分别设置于储液孔21的相对两侧。如图10至图13所示，第一流体操作孔27设置于储液孔21的过直径的相对两侧，且与贯通通道25平行。或者，如图8和图9所示，两个第一流体操作孔27与储液孔21中心连线呈设定夹角，即第一流体操作孔27设置于非过直径的相对两侧。其中，设定夹角不限定。即，本公开实施例中的第一流体操作孔27的设置位置不限定。
本公开实施例中，在前述的第一种培养模块、第二种培养模块和第三种培养模块的基础上，分别增加多个第一流体操作孔27，使之分别通过第一连通通道28与储液孔21连通，形成对应的第四种培养模块、第五种培养模块和第六种培养模块。
本公开实施例的3D多器官共培养芯片中，只要具有前述的一个或多个培养模块即可，具体的构成形式不限定。下面给出一种3D多器官共培养芯片的具体结构形式，但不限于该种具体结构形式。
在一些实施例中，芯片本体，包括：
第一储液层11，其上设置有储液孔21、第二流体操作孔24的第二操作孔Ⅰ241；
第一培养层12，其上设置有第一培养微孔22、第二流体操作孔24的第二操作孔Ⅱ242；第一培养层12叠置于第一储液层11下方，且使得第一培养微孔22与储液孔21连通；第二操作孔Ⅱ242与第二操作孔Ⅰ241连通；
第二培养层13，其上设置有第二培养微孔23和通道(例如，贯通通道25)；第二培养层13叠置于第一培养层12下方，且使得第二培养微孔23与第一培养微孔22连通，通道连通第二培养微孔23与第二操作孔Ⅱ242。
本公开实施例中，芯片本体包括三层芯片结构，将三层芯片结构按顺序叠置连接即可。可以利用双面胶、超声、热键合、plasma、热压等封接工艺将各层芯片粘结组装到一起。其中，第一储液层11和第一培养层12可合并设置为一层结构进行加工，依据实际情况确定即可。
本公开实施例中，芯片本体中的各层芯片结构的材质为聚甲基丙烯酸甲酯(PMMA)、聚二甲基硅氧烷(PDMS)、聚碳酸酯(PC)、PS、COC、COP等一种或几种。各层结构的制作可以采用软光刻、塑模法、激光刻蚀、机加工、LIGA或者一次性注塑等方式获得各层芯片结构。
可选地，第二流体操作孔24呈阶梯孔；且第二操作孔Ⅰ241的孔径大于第二操作孔Ⅱ242的孔径。本实施例的第二流体操作孔24为一阶阶梯孔。
本公开实施例中，第二培养层13上的通道(贯通通道25)与第一培养层12上的第二操作孔Ⅱ242连通。连通方式不限定。可选地，使通道的一端延伸至第二操作孔Ⅱ242内，使两者连通。
可选地，第二培养层13上还设置有孔槽，通道的一端口设置于该孔槽的侧壁，即孔槽可视为第二流体操作孔24的第二操作孔Ⅲ243。
可选地，第二操作孔Ⅰ241的孔径大于第二操作孔Ⅱ242的孔径，第二操作孔Ⅱ242的孔径大于第二操作孔Ⅲ243的孔径。减少接种细胞时细胞在孔内的驻留。本实施例的第二流体操作孔24为二阶阶梯孔。
可选地，第二操作孔Ⅰ241的孔径等于第二操作孔Ⅱ242的孔径，第二操作孔Ⅱ242的孔径大于第二操作孔Ⅲ243的孔径。第二操作孔Ⅰ241和第二操作孔Ⅱ242大小相同，减少气泡的产生，第二操作孔Ⅲ243的孔径最小，减少接种细胞时细胞在孔内的沉积。本实施例的第二流体操作孔24为一阶阶梯孔。
在一些实施例中，培养模块包括边侧流体操作孔29时，第一储液层11上还设置有边侧流体操作孔29的边侧操作孔Ⅰ291，第一培养层12上还设置有边侧流体操作孔29的边侧操作孔Ⅱ292，边侧操作孔Ⅱ292与边侧操作孔Ⅰ291连通；第二培养层13上还设置有旁通通道26，旁通通道26连通第二培养微孔23与边侧操作孔Ⅱ292。
本公开实施例中，第二培养层13上的旁通通道26与第一培养层12上的边侧操作孔Ⅱ292连通。连通方式不限定。可选地，使旁通通道26的一端延伸至边侧操作孔Ⅱ292内，使两者连通。
可选地，第二培养层13上还设置有孔槽，旁通通道26的一端口设置于该孔槽的侧壁，即孔槽可视为边侧流体操作孔29的边侧操作孔Ⅲ293。
本公开实施例中，边侧流体操作孔29的结构形式同前述的第二流体操作孔24。可选地，边侧流体操作孔29呈阶梯孔；阶梯孔的构成形式参考第二流体操作孔24即可。
在一些实施例中，培养模块包括第一流体操作孔27时，第一储液层11上还设置有第一流体操作孔27和第一连通通道28。
可选地，第一流体操作孔27为直孔。
可选地，第一连通通道28贯通设置于储液孔21与第一流体操作孔27之间的侧壁上。
在一些实施例中，芯片本体，还包括薄膜层14，薄膜层14上分布微纳尺寸的孔；薄膜层14设置于第一培养微孔22与第二培养微孔23之间。即在前述的三层芯片本体的基础上，增加薄膜层14构成四层芯片本体。本公开实施例中，薄膜层14可以采用透明、带有孔径的聚酯(polyethylene terephthalate，PET)薄膜。在组装本公开实施例的四层芯片本体时，可先将薄膜层14与与第一储液层11和第一培养层12组装后，作为整体再与第二培养层13进行组装。本公开实施例中，薄膜层14的设置需让开第二流体操作孔24与微通道(贯通通道25和旁通通道26)的连通处。
本公开实施例中，依据第一培养微孔22与第二培养微孔23之间是否设置薄膜层14 将3D多器官共培养芯片区分为无膜芯片和有膜芯片(同前述的无膜芯片和有膜芯片)。
在一些实施例中，第一储液层11上设置的储液孔21和第二操作孔Ⅰ241呈柱孔。当培养模块包括边侧流体操作孔时，第一储液层上设置的边侧操作孔Ⅰ呈柱孔。当培养模块包括第一流体操作孔时，第一流体操作孔呈柱孔。本实施例中，第一储液层11呈上具有多个凸出其表面的柱孔，避免不同孔内的流体相互影响，避免污染。
本公开实施例一种前述的3D多器官共培养芯片用于体外构建多器官模型的应用。下面给出一种具体的多器官模型的体外构建方法，仅作参考，在本公开实施例的3D多器官共培养芯片用于体外构建多器官模型的应用时，不限于采用下述的构建方法。
本公开实施例的共培养芯片构建3D血管与肿瘤多器官模型的构建方法，包括以下步骤：
将芯片紫外灭菌，灭菌结束后于第二流体操作孔24中加入20μL的低浓度鼠尾I型胶原，37℃孵育，然后弃去，并用PBS清洗三遍。将2uL纤维蛋白(2.5mg/mL)-胶原(0.2mg/mL)凝胶沿第一培养微孔22加入到第二培养微孔23中培养，保证凝胶仅能充满第二培养微孔23，37℃凝固0.5h。
消化离心HUVECs，用完全培养基重悬成一定浓度(1×106cells/mL)的单细胞悬液，在第一侧贯通通道251(包括贯通通道25和旁通通道26)的端口(可定义为进样口)处，加入20μL内皮细胞悬液。并倾斜芯片使内皮细胞充分贴壁于凝固的纤维蛋白和胶原凝胶表面上。
在HUVECs接种后的第二天消化离心U251细胞，用含有10％血清的培养基重悬成200w/ml的单细胞悬液，在1.5ml EP管中，按照比例加入特定体积的5mg/mL胶原或基质胶等其它基质材料，保证该浓度下3D材料形成良好的三维结构。再按比例加入细胞悬液，移液枪吹打混和均匀，快速高通量转移接种于第一培养微孔22中，根据微孔尺寸每孔6～12μL；细胞种植结束后，将整块芯片转移至37℃培养箱培养。
以上描述和附图充分地示出了本公开的实施例，以使本领域的技术人员能够实践它们。其他实施例可以包括结构的以及其他的改变。实施例仅代表可能的变化。除非明确要求，否则单独的部件和功能是可选的，并且操作的顺序可以变化。一些实施例的部分和特征可以被包括在或替换其他实施例的部分和特征。本公开的实施例并不局限于上面已经描述并在附图中示出的结构，并且可以在不脱离其范围进行各种修改和改变。本公开的范围仅由所附的权利要求来限制。

Claims (10)

  1. 一种3D多器官共培养芯片，其特征在于，包括：芯片本体，其上设置有一组或多组培养模块；其中，每组所述培养模块，包括：
    储液孔，一端为敞口且位于所述芯片本体的上表面；
    第一培养微孔，位于所述储液孔下方且与所述储液孔连通；
    第二培养微孔，位于所述第一培养微孔下方且与所述第一培养微孔连通；
    多个第二流体操作孔，一端呈敞口且位于所述芯片本体的上表面，另一端通过通道与所述第二培养微孔连通。
  2. 根据权利要求1所述的3D多器官共培养芯片，其特征在于，
    所述多个第二流体操作孔成对设置；每对所述第二流体操作孔通过一个贯通通道连通；所述贯通通道包括第一侧贯通通道和第二侧贯通通道，且所述第一侧贯通通道与所述第二侧贯通通道相对设置于所述第二培养微孔的相对两侧。
  3. 根据权利要求2所述的3D多器官共培养芯片，其特征在于，所述培养模块，还包括：
    多个边侧流体操作孔，一端呈敞口且位于所述芯片本体的上表面；
    旁通通道，两端分别与一个所述边侧流体操作孔连通，中部与所述第二培养微孔连通。
  4. 根据权利要求3所述的3D多器官共培养芯片，其特征在于，
    所述旁通通道与所述第二培养微孔的连通处设置有一个或多个微通道。
  5. 根据权利要求1中任一项所述的3D多器官共培养芯片，其特征在于，所述培养模块，还包括：
    多个第一流体操作孔，一端呈敞口且位于所述芯片本体的上表面，另一端分别通过第一连通通道与所述储液孔连通。
  6. 根据权利要求1至5中任一项所述的3D多器官共培养芯片，其特征在于，
    所述储液孔的横截面大于所述第一培养微孔的横截面；所述第二培养微孔的横截面大于或等于所述第一培养微孔的横截面；所述储液孔的横截面大于所述第二培养微孔的横截面。
  7. 根据权利要求1至5中任一项所述的3D多器官共培养芯片，其特征在于，所述储液孔与所述第二流体操作孔的中心间距是1～10mm。
  8. 根据权利要求1至5中任一项所述的3D多器官共培养芯片，其特征在于，
    所述芯片本体，包括：
    第一储液层，其上设置有所述储液孔和所述第二流体操作孔的第二操作孔Ⅰ；
    第一培养层，其上设置有所述第一培养微孔和所述第二流体操作孔的第二操作孔Ⅱ；所述第一培养层叠置于所述第一储液层下方，且使得所述第一培养微孔与所述储液孔连通；所述第二操作孔Ⅱ与所述第二操作孔Ⅰ连通；
    第二培养层，其上设置有所述第二培养微孔和所述通道；所述第二培养层叠置于所述 第一培养层下方，且使得所述第二培养微孔与所述第一培养微孔连通，所述通道连通所述第二培养微孔与所述第二操作孔Ⅱ；
    其中，当所述培养模块包括边侧流体操作孔和旁通通道时，所述第一储液层上还设置有所述边侧流体操作孔的边侧操作孔Ⅰ，所述第一培养层上还设置有所述边侧流体操作孔的边侧操作孔Ⅱ，所述边侧操作孔Ⅱ与所述边侧操作孔Ⅰ连通；所述第二培养层上还设置有所述旁通通道，所述旁通通道连通所述第二培养微孔与所述边侧操作孔Ⅱ；
    当所述培养模块包括第一流体操作孔和第一连通通道时，所述第一储液层上还设置有所述第一流体操作孔和所述第一连通通道。
  9. 根据权利要求8所述的3D多器官共培养芯片，其特征在于，所述芯片本体，还包括：
    薄膜层，其上分布微纳尺寸的孔；设置于所述第一培养微孔与所述第二培养微孔之间。
  10. 根据权利要求8所述的3D多器官共培养芯片，其特征在于，所述第一储液层上设置的所述储液孔和所述第二操作孔Ⅰ呈柱孔；
    当所述培养模块包括边侧流体操作孔时，所述第一储液层上设置的所述边侧操作孔Ⅰ呈柱孔；
    当所述培养模块包括所述第一流体操作孔时，所述第一流体操作孔呈柱孔。
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111826284A (zh) * 2019-04-19 2020-10-27 北京大橡科技有限公司 高通量培养板、高通量多器官共培养芯片及其应用
CN115301302A (zh) * 2022-07-29 2022-11-08 江苏艾玮得生物科技有限公司 微流控芯片
CN115466676A (zh) * 2022-09-02 2022-12-13 丹望医疗科技(上海)有限公司 用于体外器官微球制备的温控装置
CN116004388A (zh) * 2022-12-27 2023-04-25 山东师范大学 一种微流控芯片及体外三维类器官模型构建方法
CN116478817A (zh) * 2023-05-11 2023-07-25 江苏艾玮得生物科技有限公司 一种自重力高通量膜式芯片
WO2024113488A1 (zh) * 2022-11-30 2024-06-06 苏州大学 一种用于药效、毒性和药代检测的迷你哺乳动物模型及其应用
WO2024140283A1 (zh) * 2022-12-27 2024-07-04 江苏艾玮得生物科技有限公司 一种器官芯片

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022116406A1 (zh) * 2020-12-02 2022-06-09 北京大橡科技有限公司 敞开式共培养器官芯片及其应用
CN114574361B (zh) * 2020-12-02 2026-03-24 北京大橡科技有限公司 敞开式共培养器官芯片及其应用
CN112680348B (zh) * 2020-12-31 2022-08-16 北京大橡科技有限公司 基于器官芯片的器官模型构建方法及器官模型
CN113388516B (zh) * 2021-06-25 2022-03-11 上海睿钰生物科技有限公司 培养装置
CN114276930B (zh) * 2021-12-16 2024-06-21 北京大橡科技有限公司 气液培养式器官芯片及其应用
USD1028280S1 (en) 2021-12-27 2024-05-21 Molecular Devices (Austria) GmbH Organoid microplate
US12544754B2 (en) 2022-01-18 2026-02-10 Molecular Devices (Austria) GmbH 3D printing of organoid passaging plate
EP4493670A1 (en) * 2022-03-18 2025-01-22 Molecular Devices (Austria) GmbH Cell culture application methods of using a separation well microplate
CN114456936A (zh) * 2022-04-12 2022-05-10 北京大橡科技有限公司 芯片、类器官模型及其构建方法和构建装置以及应用
CN118530833B (zh) * 2024-04-02 2024-11-29 武汉大学 一种同步共分化的多组织或多类器官模型及其构建方法
CN119351217A (zh) * 2024-12-27 2025-01-24 北京大橡科技有限公司 神经-血脑屏障共培养模型及其构建方法和应用
CN121022586A (zh) * 2025-08-21 2025-11-28 株洲市中心医院 一种仿生运动器官芯片系统及使用方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103502426A (zh) * 2011-02-28 2014-01-08 哈佛大学校长及研究员协会 细胞培养系统
CN210103904U (zh) * 2019-03-29 2020-02-21 北京大橡科技有限公司 一种3d多层高通量器官芯片及基于其构建的生物屏障模型
CN210528951U (zh) * 2019-04-19 2020-05-15 北京大橡科技有限公司 高通量悬置培养板和高通量多器官共培养芯片
CN212476781U (zh) * 2020-09-30 2021-02-05 北京大橡科技有限公司 3d多器官共培养芯片

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11118150B2 (en) * 2011-10-20 2021-09-14 Research Foundation Of The City University Of New York Layered microfluidic array
CN103981085B (zh) * 2014-05-27 2016-01-06 东南大学 一种自设浓度梯度药物筛选器官芯片及其制备方法
US10481150B2 (en) * 2014-10-27 2019-11-19 The Governing Council Of The University Of Toronto Microfluidic device for cell-based assays
CN105420103A (zh) * 2015-12-07 2016-03-23 内江师范学院 一种简易的微流控芯片及细胞分析方法
WO2017175236A1 (en) * 2016-04-06 2017-10-12 Dandekar Jain Prajakta Microfluidic platform for developing in-vitro co-cultures of mammalian tissues.
SG10201606627QA (en) * 2016-08-10 2018-03-28 Agency Science Tech & Res Microfluidic chip
US10982181B2 (en) * 2016-09-07 2021-04-20 Triad National Security, Llc Devices for cell culture and methods of making and using the same
CN111269830B (zh) * 2018-12-05 2022-07-15 中国科学院大连化学物理研究所 一种基于微流控技术的多器官芯片及其应用
CN110106081B (zh) * 2019-05-13 2022-10-14 大连医科大学附属第一医院 用于构建脑功能单元模型的微流控芯片及构建方法
CN110923138A (zh) * 2019-11-22 2020-03-27 华东理工大学 一种可实现氧气浓度梯度的细胞共培养微流控芯片及其应用
CN111548939B (zh) * 2020-06-18 2024-10-25 苏州济研生物医药科技有限公司 一种类器官灌流培养芯片及其使用方法
CN112646713A (zh) * 2020-12-25 2021-04-13 中国科学院广州生物医药与健康研究院 一种整合性肿瘤细胞行为实验用芯片

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103502426A (zh) * 2011-02-28 2014-01-08 哈佛大学校长及研究员协会 细胞培养系统
CN210103904U (zh) * 2019-03-29 2020-02-21 北京大橡科技有限公司 一种3d多层高通量器官芯片及基于其构建的生物屏障模型
CN210528951U (zh) * 2019-04-19 2020-05-15 北京大橡科技有限公司 高通量悬置培养板和高通量多器官共培养芯片
CN212476781U (zh) * 2020-09-30 2021-02-05 北京大橡科技有限公司 3d多器官共培养芯片

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111826284A (zh) * 2019-04-19 2020-10-27 北京大橡科技有限公司 高通量培养板、高通量多器官共培养芯片及其应用
CN111826284B (zh) * 2019-04-19 2025-08-29 北京大橡科技有限公司 高通量培养板、高通量多器官共培养芯片及其应用
CN115301302A (zh) * 2022-07-29 2022-11-08 江苏艾玮得生物科技有限公司 微流控芯片
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WO2024113488A1 (zh) * 2022-11-30 2024-06-06 苏州大学 一种用于药效、毒性和药代检测的迷你哺乳动物模型及其应用
CN116004388A (zh) * 2022-12-27 2023-04-25 山东师范大学 一种微流控芯片及体外三维类器官模型构建方法
WO2024140283A1 (zh) * 2022-12-27 2024-07-04 江苏艾玮得生物科技有限公司 一种器官芯片
CN116478817A (zh) * 2023-05-11 2023-07-25 江苏艾玮得生物科技有限公司 一种自重力高通量膜式芯片

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