WO2024066251A1 - 一种集成器官芯片及其制备方法 - Google Patents

一种集成器官芯片及其制备方法 Download PDF

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WO2024066251A1
WO2024066251A1 PCT/CN2023/083938 CN2023083938W WO2024066251A1 WO 2024066251 A1 WO2024066251 A1 WO 2024066251A1 CN 2023083938 W CN2023083938 W CN 2023083938W WO 2024066251 A1 WO2024066251 A1 WO 2024066251A1
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layer
electrode
hole
bare
microporous
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PCT/CN2023/083938
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English (en)
French (fr)
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朱楚洪
柯明
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中国人民解放军陆军军医大学
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Publication of WO2024066251A1 publication Critical patent/WO2024066251A1/zh

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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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • 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
    • C12M23/22—Transparent or translucent parts
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

Definitions

  • the present invention relates to biomedicine and microfluidic technology, and in particular to an integrated organ chip and a preparation method thereof.
  • cells may need external electrical or mechanical stimulation to regulate the cells to make their functions mature.
  • Integrated organ chips with cells as the main culture medium that can be stimulated and regulated by multi-factors based on microfluidic technology in vitro can achieve this function.
  • the integrated organ chip system assembled by microfluidic devices that can achieve a single function can meet the needs of dynamic and static culture of the culture, and perform electrical and mechanical stimulation during the culture process.
  • integrated microfluidic chips have been widely used in the culture and regulation of cells, cell clusters and organoids.
  • the existing patent application with publication number CN113667603A discloses a liver organoid culture chip and its preparation method and application, wherein the liver organoid culture chip includes: a cell culture plate; a biomaterial with a microporous array; a culture method including digesting human embryonic stem cells or human induced pluripotent stem cells into single cells and inoculating them in a culture medium for culture to obtain foregut embryonic cells; then digesting the foregut embryonic cells into single cells and inoculating them in the microporous array in the liver organoid culture chip for culture to obtain liver organoids.
  • liver organoid culture from different sources or different tissue types, it is not combined with regulatory technology to promote organoid maturation, and an external perfusion device is required. In practical applications, promoting the functional maturation of organoids cultured in vitro is the key to further culture and development of organoids.
  • the present invention provides an integrated organ chip and a method for preparing the same.
  • the main technical solutions adopted by the present invention include:
  • an embodiment of the present invention provides an integrated organ chip, wherein the integrated organ chip is used to apply electrical stimulation and mechanical stimulation as required during the process of culturing cells, cell clusters or organoids; the integrated organ chip comprises:
  • An electrode layer for providing electrical stimulation a microporous layer located above the electrode layer, a flow chamber layer for providing mechanical stimulation and interacting with an external device; a peripheral circuit;
  • the electrode layer comprises: a substrate, and bare electrodes located on the substrate corresponding to each micropore in the micropore layer; the bare electrodes are electrically connected to the peripheral circuit by means of conductive components embedded on the surface of the substrate; and the non-bare electrode area on the surface of the substrate is an insulating area;
  • the microporous layer comprises: a structure having a plurality of mutually independent single-hole chambers attached to the electrode layer, wherein the single hole in each single-hole chamber is transparent and the bottom thereof corresponds to one or more bare electrodes of the electrode layer, so that the bare electrodes after being energized can exert adjustable electrical stimulation on the culture in the single-hole chamber;
  • the flow chamber layer is a structure having multiple cavity structures pasted on top of the microporous layer for carrying the culture medium belonging to the culture.
  • the culture medium flows in the respective cavity structures with the assistance of auxiliary equipment to obtain controllable fluid shear stress.
  • the cavity of each cavity structure is connected to multiple single-pore chambers of the microporous layer and provides culture medium for the culture in the single-pore chamber.
  • each bare electrode is a sheet-shaped circular bare electrode; each bare electrode on the substrate is independently connected to a peripheral circuit; and the diameter of the circular bare electrode is smaller than the aperture of each single hole in the microporous layer;
  • the substrate includes: a glass base, a conductive component layer laminated on the glass base, and a non-bare electrode.
  • the conductive component of the conductive component layer is a metal laminated wire, one end of the wire is connected to a bare electrode, and the other end is provided with a metal pad with a mark for electrical connection with a peripheral circuit.
  • the glass base is a 4-inch round high-transmittance glass with a thickness of 500+-20um;
  • the diameter of the circular bare electrode is 70+-10um
  • the material of the insulating layer is polyimide, with a thickness less than or equal to 2um;
  • microporous layer and the flow chamber layer are made of the same material.
  • the microporous layer is a PDMS porous membrane layer formed by punching holes after curing the polydimethylsiloxane polymer PDMS;
  • the diameter of each single hole is 1mm to 6mm;
  • the multiple single-pore chambers of the microporous layer are arranged in multiple rows, each row has N separate single-pore chambers, N is greater than or equal to 2, and the bottom of each single-pore chamber corresponds to one or more bare electrodes.
  • the cavity structure of the flow cavity layer is a single cavity structure having a rectangular parallelepiped shape; each single cavity structure having a rectangular parallelepiped shape corresponds to a row of single-hole chambers; and a culture medium inlet is provided at the end of the single cavity structure having a rectangular parallelepiped shape, and a culture medium outlet is provided at the end;
  • the flow cavity layer is a negative mold formed by pouring and solidifying a PDMS prepolymer on a tungsten steel bar positive mold to form a single cavity structure with multiple rectangular shapes; each single cavity structure in the shape of a rectangular block corresponds to a row of single-hole chambers; and the end of the single cavity structure in the shape of a rectangular block is provided with a culture medium inlet, and the end is provided with a culture medium outlet.
  • the single cavity structure in the shape of a rectangular parallelepiped has a length of 15 mm, a width of 6 mm, and a depth of 1 mm;
  • the aperture of the medium inlet/medium outlet is 1mm-5mm;
  • Auxiliary equipment includes: peripheral perfusion equipment or rocker shaker for achieving culture medium flow.
  • an embodiment of the present invention provides a method for preparing an integrated organ chip according to any one of the first aspects, comprising:
  • the manufacturing of the microporous layer in S01 includes:
  • the liquid polydimethylsiloxane prepolymer is cured in an oven at 60 degrees Celsius for 4 hours or in an oven at 85 degrees Celsius for 2 hours to form a PDMS polymer, and then a microfluidic puncher is used to punch holes in the PDMS polymer according to a preset structure to form a single-hole chamber with a matrix-like single-hole structure;
  • At least one circular bare electrode of the electrode layer is exposed on the bottom surface of the single-hole chamber of each single-hole structure;
  • the manufacturing of the flow chamber layer in S01 includes:
  • the PDMS prepolymer with bubbles removed is covered on the tungsten steel strip positive mold and cured in an oven at 60 degrees Celsius for 4 hours or at 85 degrees Celsius for 2 hours to form a single cavity structure in the shape of multiple cuboids.
  • a hole is punched at both ends of each rectangular single cavity structure to form a culture medium inlet and a culture medium outlet.
  • the S02 includes:
  • the PDMS prepolymer with bubbles removed is coated on the upper surface of the microporous layer, the flow cavity layer is covered on the surface of the microporous layer, and the layer is placed in an oven at 60 degrees Celsius and hot-pressed and cured overnight for packaging, so that the microporous layer and the flow cavity layer are bonded together to form a reversible packaging structure.
  • the manufacturing of the electrode layer in S01 includes:
  • a metal conductive layer with a thickness of 150 nm is formed on the surface of the first glass layer by magnetron sputtering a metal layer;
  • the intermediate structure is cut into a single piece including 16 bare electrodes and pads according to a predetermined size to obtain an electrode layer.
  • the integrated organ chip of the present invention can realize electrical and mechanical stimulation of the culture, and realize the cultivation of appropriate culture according to experimental requirements.
  • each single hole in the microporous layer of the present invention can be used to dynamically suspend and statically culture organoids or cell clusters separately, and each single hole has one or more electrode points, which can accurately stimulate the culture with current or voltage.
  • the electrode layer uses high-transmittance glass, which is harder than stainless steel or plastic and has good light transmittance, is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture.
  • the flow chamber layer in a specific application may not be connected to an external perfusion device, and a stable flow can be formed in the microchamber through a seesaw shaker, and a fluid shear force around the surface of the suspended culture can be formed in a single cavity.
  • FIG1A is a schematic top view of an integrated organ chip carrying peripheral circuits provided by an embodiment of the present invention.
  • FIG1B is a schematic top view of an integrated organ chip without peripheral circuits provided by an embodiment of the present invention.
  • FIG2 is a schematic top view of the second microporous layer in FIG1 ;
  • FIG3 is a schematic top view of the third flow chamber layer in FIG1 ;
  • FIG4 is a schematic top view of the first electrode layer in FIG1 ;
  • FIG5 is a schematic cross-sectional view of an integrated organ chip electrically stimulated spherical culture of the present invention
  • FIG6 is a schematic cross-sectional view of dynamic suspension culture in an integrated organ chip of the present invention.
  • the present invention provides an integrated organ chip and a preparation method thereof, which can realize dynamic and static culture and regulation of organoids in vitro.
  • the present embodiment provides an integrated organ chip, which is used to apply electrical stimulation and mechanical stimulation as required during the process of culturing cells, cell clusters or organoids.
  • the integrated organ chip includes: an electrode layer for providing electrical stimulation, a microporous layer located above the electrode layer for culture, a flow chamber layer for providing mechanical stimulation and interacting with the outside; a peripheral circuit;
  • the electrode layer includes: a substrate, and bare electrodes located in each micropore in the corresponding micropore layer on the substrate; the bare electrodes are electrically connected to the peripheral circuit by means of conductive components embedded on the surface of the substrate; the non-bare electrode area on the surface of the substrate is an insulating area; the peripheral circuit of this embodiment can be a printed circuit board, as shown in Figure 1A.
  • the microporous layer comprises: a structure having a plurality of mutually independent single-hole chambers attached to the electrode layer, each single hole being transparent and having one or more bare electrodes corresponding to the bottom of the electrode layer, so that the bare electrodes after being energized can exert adjustable electrical stimulation on the culture in the single-hole chamber;
  • the flow chamber layer is a plurality of cavity structures (such as a plurality of single cavity structures in a rectangular shape) pasted on top of the microporous layer for carrying the culture medium belonging to the culture.
  • the culture medium flows in the respective cavity structures (i.e., flows above each microporous layer) with the assistance of auxiliary equipment to obtain controllable fluid shear stress.
  • Each cavity structure connects to the plurality of single-pore chambers of the microporous layer and provides culture medium for the culture in the single-pore chamber.
  • the cavity structure of the flow cavity layer can be set to a single cavity structure with a rectangular shape, so that each single cavity structure with a rectangular shape corresponds to a row of single-hole chambers; and the end of the single cavity structure with a rectangular shape is provided with a culture medium inlet, and the end is provided with a culture medium outlet.
  • the flow cavity layer in this embodiment is not limited to a rectangular shape. In this embodiment, a rectangular shape is used as an example for illustration, and in practical applications, it can be selected according to actual needs.
  • each single-hole chamber in the microporous layer of the present invention can independently perform dynamic suspension and static culture on the organoid or cell cluster, and each single-hole chamber has one or more electrode points, i.e., bare electrodes, which can accurately perform current or voltage stimulation on the culture.
  • each single-hole chamber in the microporous layer corresponds to a bare electrode; in other embodiments, a single-hole chamber may correspond to three bare electrodes.
  • the following figures and embodiments are all described as a single-hole chamber corresponding to one bare electrode.
  • 111 is the first layer of glass base
  • 112 is a metal pad
  • 113 is a metal extension wire
  • 114 is a metal circular electrode, i.e., a circular bare electrode/electrode point
  • 211 is the second layer, i.e., a microporous layer
  • 311 is the third layer, i.e., a flow chamber layer
  • 312 is a single cavity structure in the shape of a rectangular parallelepiped
  • 313 is the inlet and outlet of the culture medium;
  • each bare electrode is a sheet-shaped circular bare electrode; each bare electrode on the substrate is independently connected to the peripheral circuit; and the diameter of the circular bare electrode is smaller than the aperture of each single hole in the microporous layer;
  • the circle in FIG4 represents a circular metal electrode point or a circular bare electrode or an electrode of the electrode layer, the line of the circle is a metal extension line, and the rectangle at the other end of the metal extension line represents a metal pad.
  • the substrate of this embodiment may include: a glass base, a conductive component layer stacked on the glass base, and an insulating layer in the non-bare electrode area; the conductive component of the conductive component layer is a wire (such as a metal extension wire/metal laminated wire), one end of the wire is connected to the bare electrode, and the other end is provided with a metal pad with an identification for electrical connection with the peripheral circuit.
  • a wire such as a metal extension wire/metal laminated wire
  • a glass base can be selected in the electrode layer shown in Figures 1 and 4.
  • the glass base can be a 4-inch or 6-inch round high-transmittance glass with a thickness of 500+-50um; the round glass can be cut according to the structure of the electrode layer to form multiple electrode layers, and the size of each cut electrode layer is about 4cm*5cm.
  • the diameter of the circular bare electrode is 70um; the material of the insulating layer can be polyimide, with a thickness of less than or equal to 2um; usually, 16 circular metal points can be set on the substrate (in practice, the design size can be adjusted to set more than 16, such as 16 to 400), and its wires such as metal extension wires are extended to the edge of the glass as needed, and a rectangular metal pad is set on the edge of the glass to connect with each metal extension wire.
  • the peripheral circuit can be a PCB board, which is electrically connected to the metal pad through the wiring pins on the PCB board, such as welding.
  • the insulating layer can be coated on the surface of the glass base to achieve non-
  • the insulation of the bare electrode area the insulating layer is made of polyimide, and the thickness is less than or equal to 2 um.
  • polyimide is not limited and can be selected according to actual needs.
  • FIG. 2 shows a schematic top view of a microporous layer.
  • the microporous layer of this embodiment can be a PDMS porous membrane layer formed by punching holes in a PDMS polymer formed by curing a polydimethylsiloxane prepolymer (PDMS); the PDMS polymer has good biocompatibility and high light transmittance, and is used for cell growth and observation; the porous structure (i.e., multiple single-hole chambers) is arranged in a row and covered on the electrode layer, and each row has 4 single-hole chambers.
  • PDMS polydimethylsiloxane prepolymer
  • a circular metal point i.e., a bare electrode
  • the aperture of each single hole is 1 mm to 6 mm, such as an aperture of 3 mm.
  • the bare electrode can directly contact the culture in the single hole to achieve current or voltage stimulation of the culture.
  • the porous structure of the microporous layer shown in FIG2 can be arranged in multiple rows, each row having N (N is greater than or equal to 2) separate single-hole chambers, and the bottom of each single-hole chamber corresponds to a bare electrode.
  • N is greater than or equal to 2
  • the arrangement of the porous structure and the number of pores may not be limited and may be selected according to actual needs.
  • FIG3 shows a schematic top view of a flow cavity layer.
  • the flow cavity layer of this embodiment can be formed by covering a metal positive mold (such as a tungsten steel bar positive mold) with liquid polydimethylsiloxane (PDMS) prepolymer and solidifying it to form multiple single cavity structures with a rectangular shape.
  • a metal positive mold such as a tungsten steel bar positive mold
  • PDMS liquid polydimethylsiloxane
  • a hole is punched at both ends of each single cavity structure as an inlet and outlet, that is, a culture medium inlet is provided at the end of each single cavity structure in a rectangular shape, and a culture medium outlet is provided at the end; the aperture of the culture medium inlet/culture medium outlet is 5 mm.
  • each rectangular single cavity structure in FIG. 3 corresponds to a row of single-hole chambers.
  • the cavity structure of the flow chamber layer may not be limited to a rectangular shape, but may be other shapes, which can be selected and arranged according to actual needs, such as being able to correspond to more than one single-hole chamber of the microporous layer and achieve cultivation. Provision of culture medium and flow shear force.
  • the third flow chamber layer is attached to the surface of the second porous layer, and the three-layer structure is superimposed and packaged to form an integrated organ chip.
  • the materials of the microporous layer and the flow chamber layer are consistent. In other embodiments, the materials of these two layers may not be restricted and can be selected according to actual needs.
  • the integrated organ chip when used for dynamic suspension and static culture of organoids or cell clusters, the organoids or cell clusters are placed in the single-hole chamber of the second microporous layer, the first electrode layer can apply electrical stimulation with adjustable voltage or current to the culture, and the flow of culture medium in the third flow chamber layer can apply controllable fluid shear stress to the culture.
  • the auxiliary equipment of this embodiment may include: a peripheral perfusion device or a seesaw shaker for realizing the flow of culture medium.
  • a seesaw shaker is used, thereby eliminating the need for connecting a restrictive structure such as a pipeline, and better realizing the cultivation of the culture. That is, the entire cultivation process can be carried out without connecting a peripheral perfusion device, and the chip can be placed on a seesaw shaker to realize flow cultivation.
  • the above chip can perform precise electrical stimulation (including current and voltage) and stable fluid shear force stimulation on organoids or cell clusters.
  • the first layer uses high-transmittance glass, which is harder than stainless steel or plastic and has good light transmittance, which is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture.
  • organoids or cell clusters can be dynamically suspended and statically cultured separately.
  • Each single-hole chamber has one or more electrode points, which can accurately stimulate the culture with current or voltage.
  • the third layer does not need to be connected to the peripheral perfusion equipment, and a stable flow can be formed in the microchamber through a seesaw shaker, and a fluid shear force can be formed around the surface of the suspended culture in the single-hole chamber.
  • This embodiment provides a method for preparing an integrated organ chip, which is mainly an illustration of the preparation process of the integrated organ chip shown in the first embodiment.
  • making the microporous layer may include:
  • a universal microfluidic chip puncher was used to punch holes in the PDMS polymer according to a preset structure to form multiple single-hole chambers; the bottom surface of each single-hole chamber exposed one or more circular bare electrodes of the electrode layer.
  • fabricating the flow chamber layer may include:
  • Liquid polydimethylsiloxane prepolymer is poured and cured on the tungsten steel bar positive mold to form a negative mold with a single cavity structure of multiple rectangular parallelepiped shapes.
  • the PDMS prepolymer with bubbles removed is covered on the tungsten steel bar positive mold, poured and cured at 60 degrees Celsius for 4 hours to form a single cavity structure with multiple rectangular parallelepiped structures, and a hole is punched at both ends of each cavity structure to form a culture medium inlet and a culture medium outlet.
  • This step may specifically include: using PDMS prepolymer to coat the upper surface of the microporous layer, covering the flow chamber layer on the surface of the microporous layer, placing in a 60 degree Celsius oven, and hot pressing and curing overnight to perform encapsulation, so that the microporous layer and the flow chamber layer are bonded together.
  • the formed encapsulation structure is a reversible structure. It should be noted that this reversible encapsulation structure can remove the flow chamber layer after the experiment is completed, which is convenient for taking out the experimental culture, and the microporous layer and the flow chamber layer can be reused after cleaning.
  • the bonding process of the microporous layer and the flow chamber layer on the electrode layer is the same as the bonding process of the microporous layer and the flow chamber layer in S02. That is, PDMS prepolymer is applied to the upper surface of the electrode layer, the microporous layer is covered on the surface of the electrode layer, and the electrode layer is placed in a 60 In a Celsius oven, the packaging is performed by hot pressing and curing overnight, so that the bonded microporous layer and the flow chamber layer are superimposed on the electrode layer. At this time, the packaging structure formed there is also a reversible structure.
  • the first, second and third layers of the integrated organ chip's structures are stacked and pasted together in sequence to achieve reversible packaging.
  • M01 Choose 4-inch/6-inch glass with a thickness of 500 ⁇ m and high light transmittance.
  • M02 Clean the glass surface with acetone, pure water, anhydrous ethanol and pure water in sequence, with the cleaning time being no less than 5 minutes.
  • a metal conductive layer (Au) with a thickness of 150nm is formed on the surface of the first layer of glass by magnetron sputtering.
  • M04 Spin-coat the insulating material polyimide on the surface of the first metal layer (except for the circular metal dots and metal pads) with a thickness not exceeding 2 ⁇ m, and form an insulating layer after baking.
  • M05 Cut the first layer of glass-metal conductive layer structure into electrode layer sheets containing 16 electrode points and pads according to the designed outer dimensions.
  • the size of the electrode layer sheets is about 4cm*5cm.
  • microporous layer is prepared, which specifically includes the following steps:
  • M06 Prepare PDMS prepolymer solution, pour it into a glass dish and remove bubbles in a vacuum box with a vacuum degree of -15 kPa and the extraction time is not less than 30 minutes.
  • M07 PDMS prepolymer was cured at 85°C for 2 hours or 60°C for 4 hours.
  • M08 Use a knife to cut off the long strips of PDMS, corresponding to the arrangement of the circular metal dots, and use a puncher to punch holes to form multiple microporous structures, that is, multiple independent single-hole chambers.
  • M09 Paste the second layer of PDMS with microporous structure on the surface of the first layer, exposing the circular electrode points and metal pads.
  • the flow chamber layer is prepared, which specifically includes the following steps:
  • M10 Cover the PDMS prepolymer with bubbles removed on the smooth tungsten steel alloy positive mold, and cure the PDMS prepolymer at 85°C for 2 hours or 60°C for 4 hours.
  • M11 Use a knife to cut off the PDMS female mold with a rectangular cavity structure covering the male mold.
  • M12 Punch a hole at each end of a rectangular cavity structure (i.e., a single cavity structure in a rectangular shape), and then stick one cavity side of the structure onto the second microporous layer.
  • a rectangular cavity structure i.e., a single cavity structure in a rectangular shape
  • M13 By welding a single strand of ultra-fine copper wire, the metal pads on the organ chip are connected one by one to the wiring pins on the printed circuit board. Finally, the welding points are tested with the help of a multimeter to obtain an integrated organ chip.
  • the integrated organ chip prepared in the above manner mainly includes a first electrode layer, a second microporous layer, and a third flow chamber layer.
  • the first electrode layer can apply electrical stimulation with adjustable voltage or current to the culture
  • the flow of culture medium in the third flow chamber layer can apply controllable fluid shear stress to the culture; thereby, precise electrical stimulation (including current and voltage) and stable fluid shear force stimulation of the organoids or cell clusters are achieved.
  • the entire culture process can be carried out without connecting to peripheral perfusion equipment, and the chip can be placed on a seesaw shaker to achieve flow culture.
  • the circular metal electrode points of the first layer can provide precise current or voltage stimulation to the organoid or cell cluster through external electrical stimulation signals, ensuring that the culture in each single hole cavity obtains a consistent stimulation signal, which can reduce the errors caused by experimental conditions between the same experimental batches.
  • the device When the culture in a single cavity is subjected to dynamic suspension culture, the device is placed on a seesaw shaker, and different rocking frequencies and amplitudes are set.
  • the liquid flow in the third-layer microcavity can form a stable fluid shear force around the culture surface in the single microwell.
  • Static electrical stimulation experiments and dynamic suspension culture can be carried out simultaneously or step by step according to experimental needs.
  • the entire experimental device does not need to be connected to an external perfusion device, which is convenient for Place the sample in an incubator for long periods of time to conduct flow experiments and avoid contamination from external equipment.
  • the circular metal electrode points can contact the bottom surface of the circular culture with a diameter of 200 ⁇ m-2mm, and the electrode points electrically stimulate the culture.
  • the microporous structure is designed as a single-hole cavity structure that is not interconnected.
  • the flow of the culture fluid can cause the culture to be in a suspended state, forming a stable wall shear force on the surface of the culture.
  • the magnitude of the wall shear force can be adjusted.
  • This embodiment is illustrated by taking 16 circular metal electrode points in the electrode layer as an example, and this embodiment is not limited to it.
  • the structure of each chamber in the microporous layer and the flow cavity layer is also illustrated by example. With reference to the technology in this embodiment, it can be expanded to more. The size and shape of the entire device can be changed according to the experimental requirements, and the peripheral circuit can be expanded according to the number of metal pads.
  • any reference numerals placed between brackets shall not be construed as limiting the claims.
  • the word “comprising” does not exclude the presence of components or steps not listed in the claims.
  • the word “a” or “an” preceding a component does not exclude the presence of a plurality of such components.
  • the invention may be implemented by means of hardware comprising several different components and by means of a suitably programmed computer. In the claims enumerating several means, several of these means may be embodied by the same hardware.
  • the use of the words first, second, third, etc. is for convenience of expression only and does not indicate any order. These words may be understood as part of the component name.

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Abstract

本申请提供一种集成器官芯片及其制备方法,涉及生物医学和微流技术领域。该集成器官芯片用于在培养细胞、细胞团或类器官的过程中按照需求施加电学刺激和力学刺激;该集成器官芯片包括:用于提供电学刺激的电极层、位于电极层上方的用于培养的微孔层、用于提供力学刺激且与外接交互的流动腔层;外围电路;电极层包括:基底,位于基底上的对应微孔层中各微孔的裸电极;微孔层包括:粘贴于电极层上的具有多个相互独立的单孔腔室的结构,每一单孔为通透的且底部对应电极层的一裸电极或多个电极组成的多电极;流动腔层为粘贴在微孔层上方的用于承载培养物所属培养基的具有多个单一空腔结构。该方法可以将多种调控技术结合来满足类器官或细胞团培养和促进其功能成熟。

Description

一种集成器官芯片及其制备方法 技术领域
本发明涉及生物医学和微流技术，尤其涉及一种集成器官芯片及其制备方法。
背景技术
体外培养类器官或细胞团时，因细胞可能需要外界的电学或者力学刺激，调控细胞的使其功能趋于成熟。体外基于微流控技术的多因素刺激调控以细胞为主要培养物的集成器官芯片能够实现这一功能。通过能够实现单一功能的微流控器件组装而成的集成器官芯片系统能够满足对培养物进行动态和静态培养，并在培养过程中进行电学刺激和力学刺激目前，集成化的微流控芯片已广泛应用于细胞、细胞团和类器官的培养和调控。
现有公开号CN113667603A的专利申请公开了一种肝脏类器官培养芯片及其制备方法与应用，所述肝脏类器官培养芯片包括：细胞培养板；具有微孔阵列的生物材料；培养方法包括将人胚胎干细胞或人诱导多功能干细胞消化成单个细胞接种于培养基中培养，获得前肠胚细胞；再将前肠胚细胞消化成单个细胞接种于所述肝脏类器官培养芯片中的所述微孔阵列中培养，获得肝脏类器官。虽然上述方法可用于不同来源或者是不同组织类型的均一且高通量的肝脏类器官培养，但未结合促进类器官成熟的调控技术，且需要外接灌流装置。在实际应用中，促进体外培养的类器官的功能成熟是类器官进一步培养和发育的关键。
为此，如何将多种调控技术结合来满足类器官培养和促进其功能成熟的集成器官芯片成为当前亟需解决的技术问题。
发明内容
(一)要解决的技术问题
鉴于现有技术的上述缺点、不足，本发明提供一种集成器官芯片及其制备方法。
(二)技术方案
为了达到上述目的，本发明采用的主要技术方案包括：
第一方面，本发明实施例提供一种集成器官芯片，所述集成器官芯片用于在培养细胞、细胞团或类器官的过程中按照需求施加电学刺激和力学刺激；集成器官芯片包括：
用于提供电学刺激的电极层、位于电极层上方的微孔层、用于提供力学刺激且与外接交互的流动腔层；外围电路；
所述电极层包括：基底，位于基底上的对应微孔层中各微孔的裸电极；所述裸电极借助于基底表面嵌套的导电组件与外围电路电连接；所述基底表面的非裸电极区域为绝缘区域；
所述微孔层包括：粘贴于电极层上的具有多个相互独立的单孔腔室的结构，每一单孔腔室中的单孔为通透且底部对应电极层的一个以上的裸电极，以使通电后的裸电极对单孔腔室内的培养物施加可调的电学刺激；
所述流动腔层为粘贴在所述微孔层上方的用于承载培养物所属培养基的具有多个空腔结构，所述培养基在辅助设备的辅助下在各自的空腔结构内流动，以获得可控的流体剪切应力，所述每一个空腔结构的空腔连通微孔层的多个单孔腔室且为单孔腔室内的培养物提供培养基。
可选地，每一个裸电极为片状的圆形裸电极；基底上的各个裸电极均相互独立的连接外围电路；且圆形裸电极的直径小于微孔层中每一单孔的孔径；
基底包括：玻璃底座、层积在玻璃底座上的导电组件层、非裸电极 区域的绝缘层；所述导电组件层的导电组件为金属层积导线，导线一端连接裸电极，另一端设置具有标识的金属焊盘用于与外围电路电连接。
可选地，所述玻璃底座为4英寸圆形高透光玻璃，厚度为500+-20um；
圆形裸电极的直径为70+-10um；
绝缘层的材质为聚酰亚胺，厚度小于等于2um；
和/或，所述微孔层和所述流动腔层的材质一致。
可选地，所述微孔层为采用聚二甲基硅氧烷聚合物PDMS固化后打孔形成的PDMS多孔膜层；
每一单孔的孔径为1mm至6mm；
所述微孔层的多个单孔腔室排成多行，每一行具有N个单独的单孔腔室，N大于等于2，每一单孔腔室的底部对应一个或多个裸电极。
可选地，所述流动腔层的空腔结构为具有长方体形状的单一空腔结构；每一个长方体形状的单一空腔结构对应单孔腔室的一行；且长方体形状的单一空腔结构的端头设有培养基进口，端尾设有培养基出口；
或者，
所述流动腔层为PDMS预聚物在钨钢条阳模上倒膜固化形成具有多个长方体形状的单一空腔结构的阴模；每一个长方体形状的单一空腔结构对应单孔腔室的一行；且长方体形状的单一空腔结构的端头设有培养基进口，端尾设有培养基出口。
可选地，长方体形状的单一空腔结构的长15mm，宽6mm，深1mm；
培养基进口/培养基出口的孔径为1mm-5mm；
或者，
辅助设备包括：用于实现培养基流动的外围灌流设备或翘板摇床。
第二方面，本发明实施例提供一种针对第一方面任一所述的集成器官芯片的制备方法，其包括：
S01、依次制作所述电极层、所述微孔层和所述流动腔层；
S02、将所述微孔层和所述流动腔层粘结在一起，且使流动腔层的培养基对应到微孔层的各单孔腔室；
S03、将粘结后的所述微孔层和所述流动腔层叠拼在所述电极层上，且将所述电极层与外围电路电连接，得到集成器官芯片。
可选地，S01中的制作所述微孔层包括：
将液态聚二甲基硅氧烷预聚物在60摄氏度烘箱中固化4小时或者85摄氏度烘箱中固化2小时，形成PDMS聚合物，然后用微流控打孔器在PDMS聚合物上按照预设结构进行打孔形成矩阵式的多个单孔结构的单孔腔室；
每个单孔结构的单孔腔室底面暴露电极层的一个以上圆形裸电极；
S01中的制作所述流动腔层包括：
去除气泡的PDMS预聚物覆盖在钨钢条阳模上，在60摄氏度烘箱中固化4小时或者85摄氏度固化2个小时，形成多个长方体形状的单一空腔结构，
在每一个长方体形状的单一空腔结构两端各打一个孔，形成培养基进口和培养基出口。
可选地，所述S02包括：
使用去除气泡的PDMS预聚物涂于微孔层上表面，将所述流动腔层覆盖于微孔层表面，放入60摄氏度烘箱中，通过热压的方式热压固化过夜进行封装，使得所述微孔层和所述流动腔层粘结在一起且形成可逆的封装结构。
可选地，S01中的制作所述电极层包括：
选择厚度为500μm高透光4英寸玻璃片，依次用丙酮、纯水、无水乙醇、纯水对玻璃表面进行清洗，清洗时间不少于5分钟；
在第一层玻璃表面通过磁控溅射金属层，形成厚度150nm的金属导电层；
通过旋涂法在第一层金属层表面的非圆形金属点和金属焊盘位置外，旋涂小于等于2μm的绝缘材料聚酰亚胺，烘烤后形成绝缘层，得到中间结构；
对所述中间结构按照预定尺寸切割成包含16个裸电极和焊盘的单片，得到电极层。
(三)有益效果
本发明的集成器官芯片可以实现对培养物的电学刺激和力学刺激，实现根据实验需求培养合适的培养物。另外，本发明的微孔层可中每个单一孔腔内都可单独对类器官或细胞团进行动态悬浮和静态培养，且每个单一孔腔内都有一个或多个电极点，可精准对培养物进行电流或电压刺激。
进一步地，在电极层采用高透光玻璃，比不锈钢或者塑料硬度大，透光性好，便于显微镜下观测和高温灭菌，更适合细胞培养。
另外，具体应用中的流动腔层可不接外围灌注设备，通过翘板摇床即可在微腔内形成稳定的流动，并且在单一孔腔内形成围绕悬浮培养物表面的流体剪切力。
附图说明
图1A为本发明一实施例提供的集成器官芯片的携带外围电路的俯视示意图；
图1B为本发明一实施例提供的集成器官芯片的无外围电路的俯视示意图；
图2为图1中的第二层微孔层的俯视示意图；
图3为图1中的第三层流动腔层的俯视示意图；
图4为图1中的第一层电极层的俯视示意图；
图5为本发明的集成器官芯片电刺激球形培养物剖面示意图；
图6为本发明的集成器官芯片内动态悬浮培养剖面示意图。
具体实施方式
为了更好的解释本发明，以便于理解，下面结合附图，通过具体实施方式，对本发明作详细描述。
为了更好的理解上述技术方案，下面将参照附图更详细地描述本发明的示例性实施例。虽然附图中显示了本发明的示例性实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更清楚、透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
目前，也有文献报道单一调控技术和培养相结合的类器官芯片，如：调控诱导的心肌细胞成熟和促进血管类器官管腔样结构成熟等类器官芯片。但多种调控技术结合来满足类器官培养和促进其功能成熟且无需外接灌注设备的集成器官芯片尚未见报道。为解决以上不足，本发明提供集成器官芯片及其制备方法，可在体外实现类器官的动静态培养和调控。
实施例一
本实施例提供一种集成器官芯片，本实施例的集成器官芯片用于在培养细胞、细胞团或类器官的过程中按照需求施加电学刺激和力学刺激。
其中，集成器官芯片包括：用于提供电学刺激的电极层、位于电极层上方用于培养的微孔层、用于提供力学刺激且与外接交互的流动腔层；外围电路；
所述电极层包括：基底，位于基底上的对应微孔层中各微孔的裸电极；所述裸电极借助于基底表面嵌套的导电组件与外围电路电连接；所述基底表面的非裸电极区域为绝缘区域；本实施例的外围电路可为印刷电路板，如图1A所示。
所述微孔层包括：粘贴于电极层上具有多个相互独立的单孔腔室的结构，每一单孔为通透的且底部对应电极层的一或多个裸电极，以使通电后的裸电极对单孔腔室内的培养物施加可调的电学刺激；
所述流动腔层为粘贴在所述微孔层上方的用于承载培养物所属培养基的具有多个空腔结构(如多个长方体形状的单一空腔结构)，所述培养基在辅助设备的辅助下在各自的空腔结构内流动(即在各微孔层上方流动)，以获得可控的流体剪切应力，所述每一个空腔结构连通微孔层的多个单孔腔室且为单孔腔室内的培养物提供培养基。
在实际应用中，可将上述流动腔层的空腔结构设置为具有长方体形状的单一空腔结构，以便每一个长方体形状的单一空腔结构对应单孔腔室的一行；且长方体形状的单一空腔结构的端头设有培养基进口，端尾设有培养基出口。当然，本实施例中流动腔层不限定长方体形状，本实施例中以长方体形状进行举例说明，在实际应用中根据实际需要选择。
本实施例的集成器官芯片可以实现对培养物的电学刺激和力学刺激，实现根据实验需求培养合适的培养物。另外，本发明的微孔层中每个单孔腔室内都可单独对类器官或细胞团进行动态悬浮和静态培养，且每个单孔腔室内都有一个或多个电极点即裸电极，可精准对培养物进行电流或电压刺激。
通常，微孔层中每一个单孔腔室对应一个裸电极；在其他实施例中，还可以是一个单孔腔室对应三个裸电极，下面附图和实施例中均以单孔腔室对应一个裸电极进行说明。
以下结合图1A至图4所示，对一种具体的集成器官芯片进行举例说明。在图1B中，111为第一层玻璃底座，112为金属焊盘，113为金属延长线，114为金属圆形电极即圆形裸电极/电极点，211为第二层即微孔层，311为第三层即流动腔层，312为长方体形状的单一空腔结构，313为培养基的进出口；
针对电极层，如图4所示，每一个裸电极为片状的圆形裸电极；基底上的各个裸电极均相互独立的连接外围电路；且圆形裸电极的直径小于微孔层中每一单孔的孔径；图4中圆圈代表圆形金属电极点或圆形裸电极或电极层的电极，圆圈的线条为金属延长线，金属延长线的另一端的长方形代表金属焊盘。
可理解的是，本实施例的基底可包括：玻璃底座、层积在玻璃底座上的导电组件层、非裸电极区域的绝缘层；所述导电组件层的导电组件为导线(如金属延长线/金属层积导线)，导线一端连接裸电极，另一端设置具有标识的金属焊盘用于与外围电路电连接。
由于，透明玻璃便于观测培养物生长状态，故在图1和图4中所示的电极层中可选择玻璃底座，本实施例中，玻璃底座可为4英寸或6英寸圆形高透光玻璃，厚度为500+-50um；圆形玻璃上可按照电极层的结构进行切割，形成多个电极层，切割的每一电极层尺寸在4cm*5cm左右。
圆形裸电极的直径为70um；绝缘层的材质可为聚酰亚胺，厚度小于等于2um；通常，基底上可设置16个圆形金属点(实际中可调整设计尺寸设置超过16个，如16至400个均可)，其导线如金属延长线根据需要延长至玻璃边缘，玻璃边缘上设置有长方形金属焊盘，与每一根金属延长线衔接。外围电路可为PCB板，其通过PCB板上的排线引脚与金属焊盘电连接如焊接。
在实际应用中，绝缘层可采用镀膜方式覆盖玻璃底座表面实现对非 裸电极区域的绝缘，绝缘层的材质为聚酰亚胺，厚度小于等于2um。本实施例中不限定聚酰亚胺，根据实际需要选择。
如图2所示，图2示出了一种微孔层的俯视示意图，本实施例的微孔层可为采用聚二甲基硅氧烷预聚物(PDMS)固化后形成PDMS聚合物打孔形成的PDMS多孔膜层；PDMS聚合物具有很好的生物相容性，高透光度，用于细胞的生长和观测；多孔结构(即多个单孔腔室)排列成一行覆盖在电极层上，每行具有4个单孔腔室，每个孔腔底面暴露一个圆形金属点即裸电极，每一单孔的孔径为1mm至6mm，如孔径3mm。本实施例中裸电极可直接接触单孔中的培养物，实现对培养物的电流或电压刺激。
图2所示的微孔层的多孔结构可排成多行，每一行具有N(N大于等于2)个单独的单孔腔室，每一单孔腔室的底部对应一个裸电极。在其他实施例中，可不限定多孔结构的排列方式和多孔数量，根据实际需要选择。
如图3所示，图3示出了一种流动腔层的俯视示意图，本实施例的流动腔层可采用液态聚二甲基硅氧烷(PDMS)预聚物覆盖在金属阳模(如钨钢条阳模)上固化后形成多个具有长方体形状的单一空腔结构，每一个单一空腔结构长15mm，宽6mm，深1mm。在每一个单一空腔结构两端各打一个孔作为进出口，即每一个长方体形状的单一空腔结构的端头设有培养基进口，端尾设有培养基出口；培养基进口/培养基出口的孔径为5mm。
因为图2中所示的4个单一孔腔组成一行，故，在图3中每一个长方体形状的单一空腔结构对应单孔腔室的一行。当然，在其他实施例中，流动腔层的空腔结构可不限定为长方体形状，还可为其他形状，根据实际需要选择和设置，如能够对应微孔层的一个以上的单孔腔室且实现培 养基和流动剪切力的提供。
本实施例中第三层流动腔层粘贴在第二层多孔层表面，三层结构叠加封装形成集成器官芯片。为更好的实现集成器官芯片的制备或可逆封装，本实施例中使微孔层和所述流动腔层的材质一致，在其他实施例中，可不对这两层的材质限制，根据实际需要选择。
本实施例中使用集成器官芯片动态悬浮和静态培养类器官或细胞团时，把类器官或细胞团放在第二层微孔层的单孔腔室内，第一层电极层可对培养物施加电压或电流可调的电刺激，第三层流动腔层内培养基的流动可对培养物施加可控的流体剪切应力。
需要说明的是，本实施例的辅助设备可包括：用于实现培养基流动的外围灌流设备或翘板摇床。优选使用翘板摇床，由此无需连接管路之类的限制行结构，较好的实现培养物的培养。即整个培养过程可以不连接外围灌流设备，将该芯片放置在翘板摇床上即可实现流动培养。
上述芯片可对类器官或细胞团进行精确的电刺激(包括电流和电压)和稳定的流体剪切力刺激。第一层采用高透光玻璃，比不锈钢或者塑料硬度大，透光性好，便于显微镜下观测和高温灭菌，更适合细胞培养。第二层中每个单孔腔室内都可单独对类器官或细胞团进行动态悬浮和静态培养。每个单孔腔室内都有一个或多个电极点，可精准对培养物进行电流或电压刺激。第三层可不接外围灌注设备，通过翘板摇床即可在微腔内形成稳定的流动，并且在单孔腔室内形成围绕悬浮培养物表面的流体剪切力。
实施例二
本实施例提供一种集成器官芯片的制备方法，其主要是对实施例一所示的集成器官芯片的制备过程的说明。
本实施例的集成器官芯片的制备方法，包括：
S01、依次制作所述电极层、所述微孔层和所述流动腔层。
举例来说，制作所述微孔层可包括：
将液态聚二甲基硅氧烷(PDMS)预聚物在60℃烘箱中固化4小时后，用微流控芯片通用打孔器对PDMS聚合物上按照预设结构进行打孔后形成多个单孔腔室；每个单孔腔室底面暴露电极层的一个或多个圆形裸电极。
另外，制作所述流动腔层可包括：
将液态聚二甲基硅氧烷预聚物在钨钢条阳模上倒膜固化后形成具有多个长方体形状的单一空腔结构的阴模。具体地，去除气泡的PDMS预聚物覆盖在钨钢条阳模上，在60摄氏度倒膜固化4个小时，形成多个长方体结构的单一空腔结构，在每一个空腔结构两端各打一个孔，形成培养基进口和培养基出口。
S02、将所述微孔层和所述流动腔层粘结在一起，且使流动腔层的培养基对应到微孔层的各单孔腔室。
该步骤可具体包括：使用PDMS预聚物涂于微孔层上表面，将流动腔层覆盖于微孔层表面，放入60摄氏度烘箱中，通过热压的方式热压固化过夜进行封装，使得所述微孔层和所述流动腔层粘结在一起。此时，形成的封装结构为可逆结构，需要说明的是，此可逆封装的结构可在实验完成后拆除流动腔层，便于实验培养物的取出，且可将对微孔层和流动腔层进行清洁后重复使用。
S03、将粘结后的所述微孔层和所述流动腔层叠拼在所述电极层上，且将所述电极层与外围电路电连接，得到集成器官芯片。
需要说明是，粘结后的所述微孔层和所述流动腔层叠拼在所述电极层上的拼叠方式与S02中微孔层和流动腔层的粘结过程相同。即，使用PDMS预聚物涂于电极层上表面，将微孔层覆盖于电极层表面，放入60 摄氏度烘箱中，通过热压的方式热压固化过夜进行封装，使得粘结后的所述微孔层和所述流动腔层叠拼在所述电极层上。此时，该处形成的封装结构也为可逆结构。
也就是说，集成器官芯片的第一层、第二层和第三层的结构依次叠拼并粘贴在一起，实现可逆封装。
为更好的理解，下面提供一种上述图1所示的集成器官芯片的制备方法，具体步骤如下：
M01：选择厚度为500μm高透光4英寸/6英寸玻璃片。
M02：依次用丙酮、纯水、无水乙醇、纯水对玻璃表面进行清洗，清洗时间不少于5分钟。
M03：在第一层玻璃表面通过磁控溅射金属层(Au)，厚度150nm，形成金属导电层。
M04：通过旋涂法在第一层金属层表面(圆形金属点和金属焊盘位置除外)，旋涂绝缘材料聚酰亚胺，厚度不超过2μm，烘烤后形成绝缘层。
M05：将第一层玻璃金属导电层结构体按照设计外尺寸切割成包含16个电极点和焊盘的电极层片，电极层片的尺寸范围在4cm*5cm左右。
接着，制备微孔层，具体包括如下步骤：
M06：配制PDMS预聚物溶液，倒入玻璃皿中在真空箱中去气泡，真空度为-15kPa，抽取时间不少于30分钟。
M07：PDMS预聚物在85℃固化2小时或60摄氏度固化4小时。
M08：用刀切割下长条形的PDMS，对应圆形金属点排列位置，用打孔器打孔，形成多个微孔结构即多个相互独立的单孔腔室。
M09：将第二层具有微孔结构的PDMS粘贴在第一层表面，露出圆形电极点和金属焊盘。
然后，制备流动腔层，具体包括如下步骤：
M10：将去除气泡的PDMS预聚物覆盖在光滑的钨钢合金阳模上，PDMS预聚物在85℃固化2小时或60摄氏度固化4小时。
M11：用刀切割下覆盖在阳模上具有长方体空腔结构的PDMS阴模。
M12：在长方体空腔结构(即长方体形状的单一空腔结构)两端各打一个孔，然后将其空腔一面粘贴在第二层微孔层上。
M13：通过焊接单股超细铜线将器官芯片上的金属焊盘与印制电路板上的排线引脚一一对应连接，最后借助于万用表测试焊接点即可得到集成器官芯片。
上述方式制备的集成器官芯片主要包括第一层电极层、第二层微孔层、第三层流动腔层。使用该芯片进行动态悬浮和静态培养类器官或者细胞团时，把类器官或者细胞团放在第二层微孔层的单孔腔室内，第一层电极层可对培养物施加电压或电流可调的电刺激，第三层流动腔层内培养基的流动可对培养物施加可控的流体剪切应力；由此，实现对类器官或细胞团进行精确的电刺激(包括电流和电压)和稳定的流体剪切力刺激。并且整个培养过程可以不连接外围灌流设备，将该芯片放置在翘板摇床上即可实现流动培养。
在实际使用中，一个类器官或细胞团接种在第二层的单一孔腔内静态培养时，通过外界电刺激信号，第一层圆形金属电极点可对类器官或细胞团进行精准的电流或者电压刺激，保证每个单一孔腔内的培养物获得一致的刺激信号，可降低同一实验批次间的实验条件导致的误差。
当对单一孔腔内的培养物进行动态悬浮培养时，将设备放置在翘板摇床上，设置不同的摇摆频率和幅度，第三层微腔内的液体流动可在单一微孔内围绕培养物表面形成稳定的流体剪切力。
静态电刺激实验和动态悬浮培养可根据实验需要同时进行或者分步进行。单独进行单台悬浮培养时整个实验装置可不外接灌注设备，便于 放入孵箱内长时间进行流动实验，避免外接设备引入污染。
如图5和图6所示，其中圆形金属电极点可与200μm-2mm直径的圆形培养物底面接触，电极点对培养物进行电刺激。其中微孔结构设计为不互通的单孔腔结构，当流动培养时，培养液的流动可是培养物处于悬浮状态，在培养物的表面形成稳定的壁面剪切力，通过调整流动的速度，可调整壁面剪切力的大小。本实施例各以电极层中的16个圆形金属电极点进行举例说明，本实施例不对其限定，同时微孔层和流动腔层中各个腔室的结构也为举例说明，参照本实施实例中的技术进行扩展至更多，整个设备的尺寸可根据实验需求更改尺寸和形状，外围电路可依据金属焊盘的数量进行扩展。
应当注意的是，在权利要求中，不应将位于括号之间的任何附图标记理解成对权利要求的限制。词语“包含”不排除存在未列在权利要求中的部件或步骤。位于部件之前的词语“一”或“一个”不排除存在多个这样的部件。本发明可以借助于包括有若干不同部件的硬件以及借助于适当编程的计算机来实现。在列举了若干装置的权利要求中，这些装置中的若干个可以是通过同一个硬件来具体体现。词语第一、第二、第三等的使用，仅是为了表述方便，而不表示任何顺序。可将这些词语理解为部件名称的一部分。
此外，需要说明的是，在本说明书的描述中，术语“一个实施例”、“一些实施例”、“实施例”、“示例”、“具体示例”或“一些示例”等的描述，是指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书 中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管已描述了本发明的优选实施例，但本领域的技术人员在得知了基本创造性概念后，则可对这些实施例作出另外的变更和修改。所以，权利要求应该解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种修改和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也应该包含这些修改和变型在内。

Claims (10)

  1. 一种集成器官芯片，其特征在于，所述集成器官芯片用于在培养细胞、细胞团或类器官的过程中按照需求施加电学刺激和力学刺激；集成器官芯片包括：
    用于提供电学刺激的电极层、位于电极层上方的微孔层、用于提供力学刺激且与外接交互的流动腔层；外围电路；
    所述电极层包括：基底，位于基底上的对应微孔层中各微孔的裸电极；所述裸电极借助于基底表面嵌套的导电组件与外围电路电连接；所述基底表面的非裸电极区域为绝缘区域；
    所述微孔层包括：粘贴于电极层上的具有多个相互独立的单孔腔室的结构，每一单孔腔室中的单孔为通透且底部对应电极层的一个以上的裸电极，以使通电后的裸电极对单孔腔室内的培养物施加可调的电学刺激；
    所述流动腔层为粘贴在所述微孔层上方的用于承载培养物所属培养基的具有多个空腔结构，所述培养基在辅助设备的辅助下在各自的空腔结构内流动，以获得可控的流体剪切应力，每一个所述空腔结构的空腔连通微孔层的多个单孔腔室且为单孔腔室内的培养物提供培养基。
  2. 根据权利要求1所述的集成器官芯片，其特征在于，
    每一个裸电极为片状的圆形裸电极；基底上的各个裸电极均相互独立的连接外围电路；且圆形裸电极的直径小于微孔层中每一单孔的孔径；
    基底包括：玻璃底座、层积在玻璃底座上的导电组件层、非裸电极区域的绝缘层；所述导电组件层的导电组件为金属层积导线，导线一端连接裸电极，另一端设置具有标识的金属焊盘用于与外围电路电连接。
  3. 根据权利要求2所述的集成器官芯片，其特征在于，
    所述玻璃底座为4英寸圆形高透光玻璃，厚度为500+-20um；
    圆形裸电极的直径为70+-10um；
    绝缘层的材质为聚酰亚胺，厚度小于等于2um；
    和/或，所述微孔层和所述流动腔层的材质一致。
  4. 根据权利要求1所述的集成器官芯片，其特征在于，
    所述微孔层为采用聚二甲基硅氧烷聚合物PDMS固化后打孔形成的PDMS多孔膜层；
    每一单孔的孔径为1mm至6mm；
    所述微孔层的多个单孔腔室排成多行，每一行具有N个单独的单孔腔室，N大于等于2，每一单孔腔室的底部对应一个或多个裸电极。
  5. 根据权利要求1或4所述的集成器官芯片，其特征在于，
    所述流动腔层的空腔结构为具有长方体形状的单一空腔结构；每一个长方体形状的单一空腔结构对应单孔腔室的一行；且长方体形状的单一空腔结构的端头设有培养基进口，端尾设有培养基出口；
    或者，
    所述流动腔层为PDMS预聚物在钨钢条阳模上倒膜固化形成具有多个长方体形状的单一空腔结构的阴模；每一个长方体形状的单一空腔结构对应单孔腔室的一行；且长方体形状的单一空腔结构的端头设有培养基进口，端尾设有培养基出口。
  6. 根据权利要求5所述的集成器官芯片，其特征在于，
    长方体形状的单一空腔结构的长15mm，宽6mm，深1mm；
    培养基进口/培养基出口的孔径为1mm-5mm；
    或者，
    辅助设备包括：用于实现培养基流动的外围灌流设备或翘板摇床。
  7. 一种针对权利要求1至6任一所述的集成器官芯片的制备方法，其特征在于，包括：
    S01、依次制作所述电极层、所述微孔层和所述流动腔层；
    S02、将所述微孔层和所述流动腔层粘结在一起，且使流动腔层的培养基对应到微孔层的各单孔腔室；
    S03、将粘结后的所述微孔层和所述流动腔层叠拼在所述电极层上，且将所述电极层与外围电路电连接，得到集成器官芯片。
  8. 根据权利要求7所述的制备方法，其特征在于，S01中的制作所述微孔层包括：
    将液态聚二甲基硅氧烷预聚物在60摄氏度烘箱中固化4小时或者85摄氏度烘箱中固化2小时，形成PDMS聚合物，然后用微流控打孔器在PDMS聚合物上按照预设结构进行打孔形成矩阵式的多个单孔结构的单孔腔室；
    每个单孔结构的单孔腔室底面暴露电极层的一个以上圆形裸电极；
    S01中的制作所述流动腔层包括：
    去除气泡的PDMS预聚物覆盖在钨钢条阳模上，在60摄氏度烘箱中固化4小时或者85摄氏度固化2个小时，形成多个长方体形状的单一空腔结构；
    在每一个长方体形状的单一空腔结构两端各打一个孔，形成培养基进口和培养基出口。
  9. 根据权利要求7所述的制备方法，其特征在于，所述S02包括：
    使用去除气泡的PDMS预聚物涂于微孔层上表面，将所述流动腔层覆盖于微孔层表面，放入60摄氏度烘箱中，通过热压的方式热压固化过夜进行封装，使得所述微孔层和所述流动腔层粘结在一起且形成可逆的封装结构。
  10. 根据权利要求7至9任一所述的制备方法，其特征在于，S01中的制作所述电极层包括：
    选择厚度为500μm高透光4英寸玻璃片，依次用丙酮、纯水、无水 乙醇、纯水对玻璃表面进行清洗，清洗时间不少于5分钟；
    在第一层玻璃表面通过磁控溅射金属层，形成厚度150nm的金属导电层；
    通过旋涂法在第一层金属层表面的非圆形金属点和金属焊盘位置外，旋涂小于等于2μm的绝缘材料聚酰亚胺，烘烤后形成绝缘层，得到中间结构；
    对所述中间结构按照预定尺寸切割成包含16个裸电极和焊盘的单片，得到电极层。
PCT/CN2023/083938 2022-09-26 2023-03-26 一种集成器官芯片及其制备方法 WO2024066251A1 (zh)

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