CN219363671U - High-flux gas exposure bionic lung micro-fluidic chip device for drug screening - Google Patents

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 inlet and a liquid outlet which are gathered together. The microfluidic chip device can be used for performing in-vitro high-flux drug screening by injecting culture mediums containing different types of drugs into different channels to interfere chip gas exposure; 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

Technical Field

The utility model discloses a high-flux gas-exposed bionic lung micro-fluidic chip device capable of carrying out drug screening, 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 utility model aims at overcoming the defects of the traditional bionic lung chip, and provides a high-flux gas exposure bionic lung micro-fluidic chip device capable of carrying out drug screening, which has the following design mechanism: 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 utility model 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 microfluidic chip device provided by the utility model can be used for screening medicines of different medicine components when different gas concentration gradients are exposed. 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 utility model 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 lower chip channels is 4, the upper chip gas channels are used for culturing human bronchial epithelial cells in advance, 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 Deferoxamine mesylate (DFO) and a blank culture medium without medicinal components are respectively introduced into the lower chip channels in experiments, 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.

In the chip device, 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 utility model, 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.

Compared with the prior art, the utility model 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 a single-layer lung source epithelial cell 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

FIG. 1 is a schematic diagram of the components of the apparatus of the present utility model;

FIG. 2 is a top view and partial cross-sectional view of the device of the present utility model;

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 fabrication of the present chip device.

Detailed Description

The utility model 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 utility model is further described in detail below in connection with chip design, fabrication process, etc:

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. Bending of the porous membrane 2 should be avoided in the experimental procedure, wherein the gas flow rate should be reduced during nitrogen purging to avoid folds left when too strong a gas flow causes bending of the porous membrane 2.

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 the composite chipAnd bubbles between the contact surfaces of the porous films 2, and putting the composite chip into a 70-90 ℃ oven to heat for about 1-3 hours to complete bonding between the composite chip and the porous films 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.

Claims (4)

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.