CN116640666A - High-flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof - Google Patents

High-flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof Download PDF

Info

Publication number
CN116640666A
CN116640666A CN202310361831.0A CN202310361831A CN116640666A CN 116640666 A CN116640666 A CN 116640666A CN 202310361831 A CN202310361831 A CN 202310361831A CN 116640666 A CN116640666 A CN 116640666A
Authority
CN
China
Prior art keywords
chip
gas
channel
channels
different
Prior art date
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.)
Pending
Application number
CN202310361831.0A
Other languages
Chinese (zh)
Inventor
谢复炜
李泽之
李翔
薛靖贤
冯博洋
华辰凤
尚平平
赵俊伟
刘克建
聂聪
刘惠民
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhengzhou Tobacco Research Institute of CNTC
Original Assignee
Zhengzhou Tobacco Research Institute of CNTC
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Zhengzhou Tobacco Research Institute of CNTC filed Critical Zhengzhou Tobacco Research Institute of CNTC
Priority to CN202310361831.0A priority Critical patent/CN116640666A/en
Publication of CN116640666A publication Critical patent/CN116640666A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12M25/02—Membranes; Filters
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Sustainable Development (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The utility model provides a high flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof, the device includes upper chip, porous film, lower floor's chip and bottom base plate, is equipped with four gas channel and four liquid channel that are parallel distribution in upper and lower floor's chip respectively, characterized by: the gas channels in the upper chip and the lower chip are vertically and correspondingly arranged with the liquid channels, and the four liquid channels in the lower chip are respectively provided with an independent liquid channel inlet and a liquid channel outlet which are gathered together. According to the invention, the culture mediums containing different kinds of medicines are injected into different channels to interfere the gas exposure of the chip, so that the in-vitro high-flux medicine screening is performed; and a high throughput test scenario can be formed. The chip can be used for screening the drug effect of different drug components when the different drug components are exposed in different gas concentration gradients.

Description

High-flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof
Technical Field
The invention discloses a high-flux gas-exposed bionic lung micro-fluidic chip device capable of carrying out drug screening and a preparation method thereof, and belongs to the technical field of biomedical engineering.
Background
The human body is a living body taking cells as units, researches the cells, knows the characteristics of the composition, the structure, the morphology, the functions and the like of the cells, and has important significance for human to know life activities. The research on human diseases in the fields of biology, medicine, pharmacy and the like often does not leave out the cell culture technology, and the construction of a disease model on a cell level can help researchers to know the occurrence and development of the diseases deeply from the pathophysiology angle. To date, conventional two-dimensional cell culture techniques have been extended for centuries and are still the most widely used cell culture methods today, but with many studies involving conventional cell culture techniques entering the bottleneck period, researchers have increasingly recognized that cells cultured in two dimensions are quite different from the three-dimensional dynamic in vivo physiological environment.
As the demand for dynamic three-dimensional multicellular co-culture has been raised for in vitro cell research, microfluidic chip technology has received increasing attention. The concept of an 'organ chip' becomes a hot spot field in recent years due to the micro environment of three-dimensional dynamic culture of in-vitro cells created by a microfluidic chip technology and the micro-fluid control capability of a microfluidic system with enough accuracy at a micro scale. Among them, many innovations of "lung chip" provide important support for the study of pulmonary diseases and the exploration of pulmonary organ biomimetics.
A "lung chip" is a device that highly reduces the local microenvironment of a lung organ from physiological or physical environmental factors by introducing different types of cells (e.g., lung epithelial cells, vascular endothelial cells, lung fibroblasts, etc.) into the chip or by applying different influencing factors (e.g., chemical concentration gradients, fluid shear stress, circulatory mechanical forces, gas-liquid interfaces, etc.), which are not possible with conventional two-dimensional static cell culture techniques. However, the existing bionic lung chip still has limitations, such as that part of the bionic lung chip does not establish a gas-liquid interface, which limits the reduction capability of the bionic lung chip on the physiological level of lung organs; as another example, the bionic lung chip test flux is generally low, which will bring heavy experimental burden to researchers and prolong the experimental period.
The Chinese patent also discloses some technologies related to micro-fluidic chips, and the structures, the compositions, the characteristics and the functions of the technologies are different. The applicant has previously filed a microchip device (ZL 202220927432.7) capable of realizing two-dimensional exposure of aerosol and liquid, which comprises an upper chip, a middle porous film, a lower chip and a bottom substrate, wherein the arrangement mode of four liquid channels of the lower chip and the limitation of the arranged channels cannot be simultaneously used for testing different cells or different pharmaceutical compositions, the research and test purposes of different cells or different pharmaceutical compositions cannot be met, and the improvement of structure and arrangement is needed.
Disclosure of Invention
The invention aims at overcoming the defects of the existing bionic lung chip and provides a high-flux gas exposure bionic lung micro-fluidic chip device capable of carrying out drug screening, and a preparation method and application thereof. The design mechanism of the invention is as follows: the formation of the gas-liquid interface and the stimulation of the fluid shear stress in the chip summarize the main physiological functions of lung organs, the arrangement of a plurality of liquid channels allows a single experiment to test a plurality of medicine components or different concentrations of the same medicine component, the arrangement of the gas concentration gradient can increase the test flux, reduce the overall workload of related researches on biological effects and chemical concentration dependency, greatly improve the efficiency of scientific research experiments and lighten the experimental burden of researchers.
The aim of the invention is realized by the following technical scheme:
a high flux gas exposes bionical lung micro-fluidic chip device for drug screening, includes upper chip, porous film, lower floor's chip and bottom base plate that from top to bottom laminating in proper order together, is equipped with four gas channel and two gas channel inlets and a gas channel export that are parallel distribution in the upper chip, is equipped with four liquid channel that are parallel distribution in the lower floor's chip, wherein: the gas channels in the upper chip and the lower chip are vertically and correspondingly arranged with the liquid channels, and the four liquid channels in the lower chip are respectively provided with an independent liquid channel inlet and a liquid channel outlet which are gathered together.
The depth of the gas channel in the upper chip is 800-1500 μm, preferably 800 μm. The depth of the liquid channel in the lower chip is 50-150 μm (preferably 100 μm), and a plurality of elliptical cell culture chambers are arranged in each channel.
The four liquid channels in the lower chip can be replaced by six liquid channels, so that tests of more drug types or more concentration gradients can be performed.
The device of the invention can be used for drug screening of different drug components when exposed to different gas concentration gradients. The application mode is as follows: the upper chip can be subjected to cell culture in advance and then subjected to gas exposure, and the lower chip can be introduced with culture mediums containing different drug components or different concentrations of the same drug component for researching the intervention effect of different drugs or different concentrations of the same drug on the gas exposure and screening the drug components effective for the intervention.
The invention is more specifically described as follows:
the high-flux gas exposure bionic lung micro-fluidic chip device for screening the medicine components comprises an upper chip capable of carrying out cell culture and simultaneously carrying out gas exposure, a lower chip capable of being introduced with culture mediums containing different medicine components or the same medicine components, a porous film for separating the upper chip and the lower chip and simultaneously providing attachment points for cells, and a bottom substrate for providing fixation and support for the chips; the upper chip can be used for culturing cells in advance, sucking out the culture medium in the upper chip channel when the cells are attached and grow with the porous film, and introducing gas components into the upper chip for exposure; different channels of the lower chip can realize perfusion of culture mediums with different drug components or different concentrations of the same drug component according to different research requirements; the upper chip and the lower chip are separated by a porous film, cells can be inoculated on the upper side of the porous film, and the exposure of a gas end and the intervention of a liquid end are received when the chips form a gas-liquid interface.
The upper chip is provided with a gas concentration gradient generation unit, and the unit comprises a plurality of gas channels, two inlets and an outlet which are arranged in parallel; the gas channels are horizontally parallel arrays, can be used for culturing lung epithelial cells in a cell culturing stage, and are used for forming a gas-liquid interface, forming a gas concentration gradient and exposing the gas to the cultured cells in a gas exposing stage; two different gases are introduced into the two inlets, the two gases are diluted with each other, so that concentration gradients of the two gases can be formed, and exposure experiments of gas components with different concentrations can be realized on the same chip. For example, when the two inlets of the gas concentration gradient generating unit are respectively filled with cigarette smoke and air in an experiment, if the number of the gas channels is 4, 4 concentration gradients from air (namely 0% of cigarette smoke) to cigarette smoke (namely 100% of cigarette smoke) can be formed. The two gases are sucked into the gas channel of the chip, and the two gases can be instantaneously and uniformly mixed and spontaneously form a gas concentration gradient due to the large diffusion coefficient of the gases, and the gas concentration is determined by the distance between the gas channel and the gas inlet.
Similarly, the lower chip comprises an array of channels for fluid communication, an oval cell culture chamber for cell culture, and a plurality of channel inlets and an outlet; the number of liquid channel arrays and oval cell culture chambers can be pre-designed according to requirements; the oval cell culture chamber is designed to reduce the flow rate of fluid from the channel into the cell culture chamber, creating a cell culture microenvironment with low shear stress for the fluid. During experiments, culture mediums containing different medicinal components are respectively introduced into the liquid channels to intervene on cells in a gas exposure environment, and medicinal components effective for intervention are screened according to experimental purposes; meanwhile, under the gas exposure environment, the liquid channel can be also filled with culture mediums containing the same medicinal components with different concentrations, so as to screen the medicinal effects of the medicinal concentrations. For example, when the number of the lower chip channels is 4, the upper chip gas channels are used for culturing human bronchial epithelial cells in advance, and when experiments are carried out, different types of inhibitors, namely 10 mu M necrosis inhibitor Necrostatin-1, 10 mu M apoptosis inhibitor Z-VAD-FMK, 10 mu M iron death inhibitor Deferoxaminemesylate (DFO) and a blank culture medium without medicine components are respectively introduced into the lower chip channels, and medicines which are most effective in inhibiting cell death caused by the exposure of cigarette smoke with different concentrations are screened; for example, when the number of lower chip channels is 6, the upper chip gas channels are used for culturing human bronchial epithelial cells in advance, and when experiments, a medium containing 20, 10, 5, 2.5 and 1.25 mu M DFO and a blank medium containing no drug component are prepared by using a double dilution method, and 6 channels of the lower chip are respectively introduced, so that the concentration of DFO which is most effective for inhibiting cell death caused by the exposure of cigarette smoke with different concentrations is screened. The above two examples screen drugs for specific drug effects by introducing different kinds or concentrations of drug components, respectively.
The bionic structure can be used for researching the intervention effect of different concentrations of non-drugs or the same drugs on gas exposure, and is beneficial to in-vitro high-flux drug screening on diseases caused by harmful gas exposure.
The bottom substrate is used for fixing and supporting the whole chip, and the material and the thickness of the substrate can be selected, purchased and processed according to experimental requirements.
In the invention, the porous film plays a role in spacing and communicating the upper chip and the lower chip, and the film separates the upper area and the lower area when the cells are inoculated, so that the cells cultured on the upper side of the film only appear in a fixed area and cannot migrate into a liquid channel of the lower chip through the film; when the gas is exposed, the liquid in the lower chip cannot overflow to the upper chip through the micropores of the film, but cells cultured on the upper side of the film can receive the nourishment of the lower culture medium and the intervention of the medicine through the micropores, and simultaneously receive the exposure of the gas components in the gas channel of the upper chip; after gas exposure, cells cultured on the upper side of the membrane can undergo real-time intercellular substance exchange and signaling through the membrane pores, which helps to create a complex physiological microenvironment.
In the invention, the channel directions in the upper chip and the lower chip are mutually perpendicular. In the upper chip, the gas concentration conditions formed by each channel are different, and the medicine components introduced by each channel in the lower chip are different, namely, the chip test flux = the number of the upper chip array channels x the number of the lower chip array channels. Taking 4 x 4 array as an example, the upper chip contains 4 gas channels to form 4 concentration gradients of two gases, 4 liquid channels are respectively introduced with culture mediums containing different medicinal components, the upper chip channel and the lower chip channel are vertical, and 16 test environments with different scenes can be respectively generated. Likewise, the number of corresponding arrays of gas and/or liquid channels may be varied as desired, such as in a 6 x 6 array, an 8 x 8 array, a 10 x 10 array, etc.
The upper chip, the lower chip, the porous film and the bottom substrate can be compounded by different materials. Wherein, the upper and lower chips are made of Polydimethylsiloxane (PDMS); the porous film can be made of various materials such as Polycarbonate (PC), polyethylene terephthalate (PET), PDMS and the like; the substrate may be glass or organic polymer material such as polymethyl methacrylate (PMMA), PC, PET, etc.
In the present invention, cells of the lung epithelial type such as alveolar epithelial cells and lung bronchial epithelial cells can be cultured in the upper chip channel array. After forming a gas-liquid interface, introducing gas components required by research, such as formaldehyde, acetaldehyde, acrolein, cigarette smoke, automobile exhaust or kitchen oil smoke and the like, into a gas exposure experiment to realize concentration gradient gas exposure of the cultured cells; culture mediums containing different kinds of medicinal components or different concentrations of the same medicinal components can be introduced into the lower chip channel array. Under the condition of simulating in-vivo cell growth environment (gas-liquid interface, fluid shear stress and the like), the chip is subjected to concentration gradient gas exposure so as to observe the conditions of cell survival, metabolism, injury, oxidative stress, inflammatory reaction, cell interaction and the like or study the effect, mechanism or target point of action of different medicine components on a lung pathological model and the like.
Compared with the prior art, the invention has the following advantages:
1. the bionic lung micro-fluidic chip comprises an upper chip structure and a lower chip structure; the upper chip can be filled with gas components for gas concentration gradient exposure experiments; different medicine components can be introduced into the lower chip channel array to intervene on the cultured cells, and the effect of the different medicine components on the lung disease model caused by the exposure of the gas components is researched. The high-flux test is achieved through superposition of test fluxes of the upper chip and the lower chip, and the method is extremely suitable for research of the types such as efficient drug high-flux screening of lung diseases caused by chemical concentration gradient and biological effect dependency and gas component exposure.
2. The cells cultured in the bionic lung microfluidic chip are inoculated on the upper side and the lower side of the porous film, and single-layer lung source epithelial cells can be cultured on the upper side of the porous film; on the basis, the cells on the upper side of the porous film can be in direct contact with the gas components in the gas channel and the liquid components in the liquid channel, and can perform substance exchange and signal transmission between the cells to generate a series of biological effect crosstalk, so that more biological information can be obtained.
3. The establishment of the gas-liquid interface summarizes main physiological functions of lung organs, the liquid culture medium dynamically cultures the chips to introduce fluid shear stress so as to further improve the bionic capacity of the chips on a physiological level, but the fluid shear stress is too large and can damage cells, the cell culture chamber of the lower chip is designed into an elliptical shape, so that the flow rate of the liquid culture medium is reduced when entering the cell culture chamber from a channel, the most direct purpose of reducing the flow rate of the liquid culture medium is to create a lower fluid shear stress condition for the cultured cells, and the interference of the fluid shear stress on experiments is eliminated.
Drawings
Figure 1 is a schematic diagram of the components of the device of the present invention.
FIG. 2 is a top view and partial cross-sectional view of the device of the present invention;
in fig. 1-2: 1. 1-1 parts of upper chip, 1-2 parts of gas channel, 1-3 parts of gas channel inlet and 1-3 parts of gas channel outlet; 2. a porous film; 3. a lower chip, 3-1, a liquid channel, 3-2, a liquid channel inlet, 3-3 and a liquid channel outlet; 4. a base substrate.
FIG. 3 is a flow chart of the preparation of the device of the present invention.
FIG. 4 is a flow chart of co-cultured cell loading of the device of the present invention.
FIG. 5 is a graph depicting the gas concentration gradients in the apparatus of the present invention.
FIG. 6 is a representation of the independence of the fluid channels of the device of the present invention.
FIG. 7 is a graph representing the gas exposure test of the device of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings (examples):
as shown in fig. 1-2: a high flux gas exposes bionical lung micro-fluidic chip device for drug screening, including from top to bottom laminating upper chip 1, porous film 2, lower floor's chip 3 and bottom base plate 4 together in proper order, be equipped with in upper chip 1 and be parallel distribution's four gas channel 1-1 and two gas channel inlets 1-2 and a gas channel export 1-3, be equipped with in the 3 piece of lower floor's chip and be parallel distribution's four liquid channel 3-1, wherein: the gas channels 1-1 and the liquid channels 3-1 in the upper chip and the lower chip are vertically and correspondingly arranged, and the four liquid channels in the lower chip are respectively provided with an independent liquid channel inlet 3-2 and a liquid channel outlet 3-3 which are gathered together.
The invention is further described in detail below in connection with the design, fabrication process, cell loading and culturing, functional verification, etc. of the chip:
1. design of bionic lung micro-fluidic chip
1. Upper chip: the upper chip 1 is mainly composed of a concentration gradient generating unit which is composed of four parallel gas channels 1-1, two gas inlets 1-2 and one gas outlet 1-3. Two different gases are respectively connected into the two gas inlets 1-2, and meanwhile, a certain negative pressure is applied to the gas outlets 1-3, so that the two gases can be freely diffused in the gas channels of the chip under the driving of the negative pressure, and the different gases can be fully and uniformly mixed in a short time usually due to the large gas diffusion coefficient, so that concentration gradients of the two gases can be formed in the four gas channels. The channel depth of the upper chip 1 in this example is set to 800-1500 μm. In order to facilitate the injection of the culture medium into the channels of the lower chip 3, the upper chip should leave access openings to the channels of the lower chip in pre-designed positions.
2. Porous film: the material, thickness and pore diameter of the porous film 2 are selected according to the experimental requirements, and a PC film with the pore diameter of 5 μm and the thickness of 25 μm is selected in the example. In order to facilitate the injection of the culture medium into the liquid channel 3-1 of the lower chip 3, the porous membrane 2 should be left with the inlet and outlet of the liquid channel 3-1 of the lower chip 3 at a position designed in advance.
3. The lower chip: the lower chip 3 comprises four liquid channels 3-1, which correspond to the four independent liquid channel inlets 3-2 and are converged at the same liquid channel outlet 3-3. The separate inlets allow different types of cells or different types of drugs to be injected into different channels, respectively, sharing one outlet can further save space and help reduce the overall size of the chip. The depth of the underlying chip channels in this example was set to 50-150 μm.
4. A substrate: the material and thickness of the base plate need to be selected according to experimental requirements, and PMMA plates with the thickness of 2mm are selected.
2. Manufacturing process of bionic lung micro-fluidic chip
1. Upper chip: the commercial PDMS prepolymer and the curing agent sold in the matching way are fully mixed according to a certain proportion (10:1 in this example), and are subjected to vacuum degassing, then poured into an upper chip manufacturing die, and are placed into a 50-80 ℃ oven for heating and curing for about 1-3 hours, after PDMS is completely cured, the PDMS chip is carefully removed from the die, the redundant part of the chip is cut off by a surgical knife, and holes are punched at a designated position, so that the upper chip 1 is obtained, and the upper chip 1 is shown in fig. 3A.
2. Porous film: and opening a laser cutting machine, cutting the PC porous film with the thickness of 25 mu m by using the laser cutting machine according to the drawing design in software in advance, and obtaining the porous film 2 required by preparing the chip, wherein the drawing design is shown in fig. 3B.
3. The lower chip: the commercial PDMS prepolymer and the curing agent sold in the matching way are fully mixed according to a certain proportion (10:1 in this example), and are subjected to vacuum degassing, then poured into a die for manufacturing the lower chip 3, and are placed into a 50-80 ℃ oven for heating and curing for about 1-3 hours, after PDMS is fully cured, the PDMS chip is carefully removed from the die, and the redundant part of the chip is cut off by a surgical knife, so that the lower chip 3 is obtained, and the lower chip 3 is shown in figure 3C.
4. A substrate: and cutting the PMMA plate with the thickness of 2mm by using a laser cutting machine according to the drawing design in software in advance, so as to obtain the bottom substrate 4, see fig. 3D.
5. The prepared base substrate 4 and porous film 2 are modified by a plasma surface treatment instrument (gas: O) 2 Power: 100W, pressure: 600mTorr, time: 60-120 s), then immediately immersing in an ATPES aqueous solution with the temperature of 80 ℃ being 5%, taking out the bottom substrate 3 and the porous film 3 after 20min, repeatedly flushing with distilled water, and drying the surface moisture by using compressed nitrogen for later use. The porous film 2 should be avoided in the experimental procedureWherein the nitrogen sweep should reduce the gas flow rate to avoid folds in the porous membrane 2 caused by too strong a gas flow.
6. The bottom surface of the lower chip 3 faces upwards, and the modified surface of the lower substrate 4 is put into a plasma surface treatment instrument for modification (gas: O) 2 Power: 100W, pressure: 600mTorr, time: 30-60 s), the lower chip 3 and the lower substrate 4 are taken out, aligned and contacted under an alignment mirror, air bubbles between the lower chip 3 and the lower substrate 4 are driven by using a roller, and the composite chip is put into a 70 ℃ oven to be heated for about 2 hours to finish bonding between the lower chip 3 and the lower substrate 4, as shown in fig. 3E.
7. Taking out the composite chip from the oven, cooling at room temperature, placing the composite chip cooled to room temperature and the treated porous film 2 with bonding surface facing upwards into an ion surface treatment instrument for modification (gas: O) 2 Power: 100W, pressure: 600mTorr, time: 30-60 s), carefully taking out the composite chip and the porous film 2 after plasma treatment, aligning and contacting under an alignment mirror, repeatedly pressing with a roller to drive bubbles between contact surfaces of the composite chip and the porous film 2, and placing the composite chip into a baking oven at 70-90 ℃ to heat for about 1-3h to finish bonding between the composite chip and the porous film 2, as shown in fig. 3F.
8. Taking out the composite chip from the oven, cooling at room temperature, placing the composite chip cooled to room temperature and the bonding surface of the upper chip 1 upwards into an ion surface treatment instrument for modification (gas: O) 2 Power: 100W, pressure: 600mTorr, time: 30-60 s), carefully taking out the composite chip and the upper chip 1, aligning and contacting under an alignment mirror, repeatedly pressing with a roller to drive bubbles between contact surfaces of the composite chip and the upper chip 1, putting the composite chip into a 70 ℃ oven, and heating for about 1-3h to complete bonding between the composite chip and the upper chip 1, thereby obtaining the designed chip and completing a preparation process, as shown in fig. 3G.
3. Cell loading and culturing of bionic lung micro-fluidic chip
Taking the example of co-culture of two kinds of vascular endothelial cells HUVEC and bronchial epithelial cells BEAS-2B.
1. The prepared chip was irradiated with ultraviolet light for about 2 hours, 100-200mg/mL of type I rat tail collagen solution was introduced into the upper and lower channels of the chip, and the chip was placed in a cell incubator overnight.
2. The channels of the microfluidic chip treated with type i rat tail collagen solution were repeatedly rinsed with PBS.
3. Treatment of vascular endothelial cells HUVEC to give 2X 10 7 cell suspension of cells/mL.
4. Using a syringe pump, the culture medium containing HUVEC cells was passed into the four channels of the lower chip at a flow rate of 25. Mu.L/min, and the chip was placed in the cell incubator upside down for 1-3 hours.
5. After HUVEC cells adhere to the wall, bronchial epithelial cells BEAS-2B cells were treated to give 5X 10 6 cell suspension of cells/mL.
6. The microfluidic chip was removed, and the culture medium containing BEAS-2B cells was introduced into the upper chip channel at a flow rate of 50-80. Mu.L/min using a syringe pump, and the chip was placed in a cell incubator for about 24 hours.
7. Taking out the microfluidic chip, carefully sucking out the culture medium in the upper chip channel, and introducing fresh culture medium into the lower chip channel at a flow rate of 1-2 mu L/min by a syringe pump for 24h.
8. The flow diagram of the on-chip co-cultured cell loading is shown in FIG. 4.
4. Bionic lung microfluidic chip function verification
1. And (3) gas concentration gradient verification: here we introduce two gases, CO 2 And synthetic air, in combination with a color change of bromothymol blue pH indicator, to characterize the gas concentration gradient. The specific principle is that the bromothymol blue pH indicator is blue under alkaline condition, yellow under acidic condition and CO 2 When dissolved in water, carbonic acid is generated, and the amount of carbonic acid and CO 2 The concentration is proportional, so that bromothymol blue pH indicator is in CO 2 The gas tends to be acidic gradually and the color changes from blue to yellow. Before the experiment started, CO was collected using a gas capture bag 2 Gas and synthetic air. Introducing bromothymol blue pH indicator into the lower layer channel of the chip at a flow rate of 1-2 μL/min by using a syringe pump, and packagingWith CO 2 The gas trapping bags of the gas and the synthetic air are respectively connected into two inlets of the chip gas concentration gradient generator, the negative pressure pump is regulated to have the flow rate of 1-2mL/min and is connected to an outlet of the chip gas concentration gradient generator, and the color change of the bromothymol blue pH indicator is recorded by using the mobile phone Mate 30 Pro. The concentration gradient characterization result is shown in figure 5.
2. And (3) liquid channel independence verification: four kinds of ink with different colors and obvious difference are prepared before the experiment starts, the four kinds of ink with different colors are respectively introduced into different channels of the lower chip by using a syringe pump at the flow rate of 1-2 mu L/min, and the colors of the channels of the lower chip are recorded by using the mobile phone Mate 30 Pro. The independent verification result of the liquid channel is shown in figure 6.
3. And (3) verifying the gas exposure function in the chip: the upper chip is used for culturing bronchial epithelial cells BEAS-2B, and the lower chip is used for introducing different medicinal components, and cigarette smoke and synthetic air are used for gas exposure. The necrosis inhibitor Necrostatin-1, the apoptosis inhibitor Z-VAD-FMK, the iron death inhibitor Ferrostatin-1 or a blank culture medium is respectively introduced into 4 liquid channels of the lower chip two hours before the experiment starts, and the flow rate is 1-2 mu L/min. And respectively collecting cigarette smoke and synthetic air. When the exposure experiment starts, the cigarette smoke and the synthetic air are respectively connected into 2 gas inlets of the gas concentration gradient generating unit of the upper chip, and a negative pressure pump with the flow rate of 1-2mL/min is connected into an outlet of the gas concentration gradient generating unit, and the exposure experiment lasts for 10-20min. After the exposure experiment was completed, the chip was continuously cultured for 24 hours. After the end of the continuous culture, the cell viability in the chip was measured using the Live/read assay kit and recorded by photographing with a fluorescence inverted microscope, where we defined the gas channels named Line1, line 2, line 3 and Line 4, and the smoke concentration was gradually decreased from Line1 to Line 4. Live/read experiments are shown in FIG. 7.

Claims (6)

1. A high flux gas exposes bionical lung micro-fluidic chip device for drug screening, includes upper chip, porous film, lower floor's chip and bottom base plate that from top to bottom laminating together in proper order is equipped with four gas channel and two gas channel inlets and a gas channel export that are parallel distribution in the upper chip, is equipped with four liquid channel that are parallel distribution in the lower floor's chip, its characterized in that: the gas channels in the upper chip and the lower chip are vertically and correspondingly arranged with the liquid channels, and the four liquid channels in the lower chip are respectively provided with an independent liquid channel inlet and a liquid channel outlet which are gathered together.
2. The high-throughput gas-exposed biomimetic pulmonary microfluidic chip device for drug screening of claim 1, wherein: the depth of the gas channel in the upper chip is 800-1500 μm.
3. The high-throughput gas-exposed biomimetic pulmonary microfluidic chip device for drug screening of claim 1, wherein: the depth of the liquid channel in the lower chip is 50-150 mu m, and a plurality of elliptical cell culture chambers are arranged in each channel.
4. The high-throughput gas-exposed biomimetic pulmonary microfluidic chip device for drug screening of claim 1, wherein: the four liquid channels in the lower chip may be replaced with six.
5. Use of the high-throughput gas-exposed biomimetic pulmonary microfluidic chip device according to claim 1, wherein: the device can be used for screening medicines of different medicine components when different gas concentration gradients are exposed.
6. The use of the high-throughput gas-exposed biomimetic pulmonary microfluidic chip device according to claim 5, wherein: the application mode is as follows: the upper chip can be subjected to cell culture in advance and then subjected to gas exposure, and the lower chip can be introduced with culture mediums containing different drug components or different concentrations of the same drug component for researching the intervention effect of different drugs or different concentrations of the same drug on the gas exposure and screening the drug components effective for the intervention.
CN202310361831.0A 2023-04-07 2023-04-07 High-flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof Pending CN116640666A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310361831.0A CN116640666A (en) 2023-04-07 2023-04-07 High-flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310361831.0A CN116640666A (en) 2023-04-07 2023-04-07 High-flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof

Publications (1)

Publication Number Publication Date
CN116640666A true CN116640666A (en) 2023-08-25

Family

ID=87623607

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310361831.0A Pending CN116640666A (en) 2023-04-07 2023-04-07 High-flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof

Country Status (1)

Country Link
CN (1) CN116640666A (en)

Similar Documents

Publication Publication Date Title
US7785883B2 (en) Automatable artificial immune system (AIS)
EP2335370B1 (en) Organ-on-a-chip-device
CN111218404A (en) Bionic multi-organ chip and preparation method and application thereof
US20070141552A1 (en) Automatable artificial immune system (AIS)
Wei et al. Organs-on-chips and its applications
KR20190136132A (en) Engineered liver tissues, arrays thereof, and methods of making the same
CN112680348B (en) Organ model construction method based on organ chip and organ model
Ahmed Organ-on-a-chip microengineering for bio-mimicking disease models and revolutionizing drug discovery
CN212316139U (en) Bionic multi-organ chip
CN114317272B (en) Culture device for multicellular co-culture and cell culture method
Marx Trends in cell culture technology
Farshidfar et al. The feasible application of microfluidic tissue/organ-on-a-chip as an impersonator of oral tissues and organs: a direction for future research
CN116478819B (en) Microfluidic system for constructing three-dimensional organ microenvironment model, and preparation method and application thereof
CN116640666A (en) High-flux gas exposure bionic lung micro-fluidic chip device for drug screening and application thereof
CN219363671U (en) High-flux gas exposure bionic lung micro-fluidic chip device for drug screening
Victorious Current applications of organ-on-a-chip: a step closer to personalized medicine
CN219409759U (en) Co-culture micro-fluidic chip gas exposure device capable of forming gas-liquid interface
CN116496897A (en) Co-culture micro-fluidic chip gas exposure device capable of forming gas-liquid interface and application thereof
CN219409758U (en) Three-dimensional culture chip
CN113814010B (en) Multi-cell and multi-tissue co-culture bionic micro-fluidic chip and preparation method thereof
CN117965301A (en) Multi-organ chip, preparation method and use method
CN114703139B (en) Construction method and application of in-vitro lung cancer model
CN116445282B (en) Microfluidic system and application thereof in constructing bionic organ microenvironment
WO2018143831A1 (en) Method of manufacturing a multilayer microfluidic device for dynamic tissue culture
CN109456934B (en) Preparation method of three-dimensional glomerulus model

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination