CN219409759U - Co-culture micro-fluidic chip gas exposure device capable of forming gas-liquid interface - Google Patents

Abstract

The device comprises an upper chip, a porous film, a lower chip and a bottom substrate, and is characterized in that: the gas channels and the liquid channels of the upper chip and the lower chip are correspondingly arranged in an up-down parallel manner, two gas inlets and outlets are respectively arranged at two ends of the four channels of the upper chip, and independent liquid inlets and outlets are correspondingly arranged at two ends of the four channels of the lower chip. The upper chip gas channel of the device can be used for cell culture in the cell culture stage, and one or two gases are introduced for single gas or mixed gas exposure during gas exposure; the four mutually independent liquid channels of the lower chip can be respectively inoculated with the same cells, different cells or non-inoculated cells, and can also perform the intervention of the same or different medicinal components; the porous film is positioned between the upper chip and the lower chip, which not only prevents liquid from overflowing to the upper chip, but also is convenient for separating and culturing upper cells and lower cells to keep good intercellular signal transmission.

Description

Co-culture micro-fluidic chip gas exposure device capable of forming gas-liquid interface

Technical Field

The utility model provides a gas exposure device for a co-culture microfluidic chip, in particular relates to a microfluidic device capable of forming a gas-liquid interface, and belongs to the technical field of biology and analytical chemistry.

Background

Research in various fields such as tissue engineering, bionic organs, three-dimensional cell culture and the like relates to the construction of in vitro gas-liquid interfaces which exist in various organs and tissues of a human body, such as respiratory organs, digestive organs, skin and the like. From the in vitro bionic point of view, the gas-liquid interface summarizes the main functions of many tissues and organs, such as oral cavity, esophagus, trachea, lung, intestinal tract, skin, etc., so the establishment of the in vitro gas-liquid interface is always the key and difficult point of the above tissue or organ bionic. Based on the establishment of the gas-liquid interface, the bionic factors such as different types of cells and different physical stimulus factors (such as mechanical circulating force, fluid shear stress and the like) are introduced for summation, so that the external bionic organ has great reducing capability on the internal physiological microenvironment.

The dependence of biological effect and chemical concentration gradient is one of the most basic and common operations in scientific experiments of biology, medicine, pharmacy and the like, the establishment of liquid concentration gradient is already a very mature method and is widely applied, and the research of various gas components, especially gas environmental pollutants such as kitchen oil smoke, factory exhaust gas, automobile exhaust gas and cigarette smoke, is usually carried out by collecting the gas components and the gas components in a glass fiber filter disc, dissolving the gas components in the liquid to prepare a solution with a certain concentration, and further diluting the solution to prepare the solution with the concentration gradient. Such studies are extremely common in biology, but the gas components lose a considerable part of the gas phase components during the processes of capturing, dissolving, configuring and diluting, which undoubtedly leads to the bias and even the error of experimental data, so that the most ideal study on biological effects and gas concentration gradients is to directly use gas pollutants for exposing in a gas form, and meanwhile, the premise of exposing the cultured cells in vitro is to establish a gas-liquid interface.

The microfluidic chip technology has the advantages of high analysis efficiency, accurate quantification, high reproducibility, small reagent consumption and the like, and in recent years, as the microfluidic chip technology receives more and more attention, a concentration gradient generating device based on the microfluidic technology has been widely applied to the fields of drug activity screening, cell and microorganism culture, tissue organ bionics, trace substance detection, gas pollutant risk assessment and the like. Compared with the traditional manual concentration preparation, the concentration gradient generation device based on the microfluidic technology has great advantages. The microfluidic concentration gradient generating device can realize accurate control of fluids (gas and liquid) according to the application requirements, and the principle is that fluids with different concentrations or different components meet, mix and separate in a channel network inside the concentration gradient generating device, and finally form a concentration gradient through a diffusion effect, so that an equal ratio, an equal difference, an index ratio or any expected concentration gradient can be formed, and the generated temporal and spatial instantaneous gradients are accurate, controllable, easy to quantify and good in reproducibility, thereby helping researchers to better develop scientific research experiments.

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 experimental tests between different cells cannot be simultaneously performed and the influence of each concentration of aerosol exposure on cell secretion cannot be analyzed due to the arrangement mode of four liquid channels of the lower chip and the limitation caused by the mutual perpendicularity of the upper chip channel and the lower chip channel, so that the more complicated and deep research test contents cannot be satisfied, and the improvement of the structure and the arrangement is needed.

In biomimetic chips, a strategy commonly adopted for gas exposure is to expose a single concentration of gas in a single experiment, and such a chip device with lower test flux necessarily increases the overall experimental burden. Meanwhile, most bionic chips aiming at tissues or organs with gas-liquid interfaces are usually only used for performing the bionic operation on the liquid level, and the reduction degree of the bionic operation on the physiological and pathological environments is reduced due to the lack of the bionic operation on the physical environment. In addition, most biomimetic chips use a single cell type to biomimetic a tissue or organ, and thus the experimental conclusion is often far from in vivo experiments. Therefore, development of a gas exposure device capable of forming a gas concentration gradient at a gas-liquid interface for laboratory use is urgently needed, a gas concentration gradient generation unit is integrated with a chip device, so that the dependence between the gas concentration gradient and biological effects can be obtained in one experiment, and meanwhile, the introduction of multiple types of cells is beneficial to increasing the environmental complexity of a bionic chip and improving the bionic capacity to in-vivo physiological microenvironment.

Disclosure of Invention

The utility model aims at providing a co-culture microfluidic chip gas exposure device capable of forming a gas-liquid interface based on the prior art, which has the following design mechanism: the gas-liquid interface is established, and meanwhile, the gas concentration gradient is formed, so that the test flux of a single experiment can be increased; the gas and liquid layers of the chip are separated by a porous membrane, which allows co-cultivation of different cells to be established on both sides of the porous membrane; the liquid channels are independent of each other, which allows different types of cells to be introduced into the different channels; the liquid channel is parallel to the gas channel, so that culture medium effluent of the liquid channel corresponding to each smoke concentration is allowed to be collected for further detection and analysis, the practicability of the device is definitely greatly increased, the application range is expanded, and the efficiency of scientific research experiments is greatly improved.

The aim of the utility model is realized by the following technical scheme:

the utility model provides a can form gaseous exposure device of co-culture micro-fluidic chip of gas-liquid interface, includes upper chip, porous film, lower floor's chip and bottom base plate that from top to bottom coincide together in proper order, is equipped with four gas channel 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 and the liquid channels in the upper chip and the lower chip are correspondingly arranged in an up-down parallel mode, two ends of the four channels in the upper chip are respectively provided with two mutually communicated gas inlets and two mutually communicated gas outlets, and two ends of the four liquid channels in the lower chip are respectively provided with an independent liquid inlet and an independent liquid outlet.

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 the channel.

The distance between the gas inlet and the gas outlet of the gas channel is smaller than the distance between the liquid inlet and the liquid outlet of the liquid channel, so that the process assembly is facilitated.

Four liquid inlets and four liquid outlets in the lower chip are distributed in an arc shape, so that the conduit access and the development of subsequent experiments are facilitated.

The co-culture microfluidic chip gas exposure device capable of forming a gas-liquid interface can be applied to the following situations:

(1) The four liquid channels are mutually independent and can be used for respectively introducing cells of the same type or different types to construct the same or different physiological environments.

(2) The four mutually independent liquid channels are respectively provided with an independent liquid inlet and an independent liquid outlet, and can be used for respectively collecting and detecting bioactive components including inflammatory factors, lactate dehydrogenase and superoxide dismutase from effluent liquid of each liquid channel.

(3) The human lung epithelial cells are cultured in the gas channel of the upper chip, the cells (namely blank) are not cultured in the four liquid channels of the lower chip, human vascular endothelial cells, human macrophages and human fibroblasts are cultured, and cigarette smoke is introduced into the gas channel, so that the synergistic or antagonistic effect of different cell types in cigarette smoke exposure during co-culture can be compared.

(4) Human lung epithelial cells are cultured in the gas channel of the upper chip, human vascular endothelial cells are cultured in the four liquid channels of the lower chip, and the concentration gradient of cigarette smoke is formed in the gas channel, so that biological effects on different cigarette smoke concentrations when the human lung epithelial cells and the human vascular endothelial cells are co-cultured are compared.

The technical scheme of the utility model is further described as follows:

the utility model relates to a microfluidic chip gas exposure device capable of performing cell co-culture at a gas-liquid interface, which comprises an upper chip capable of performing cell culture and gas exposure, a lower chip capable of performing cell culture and liquid perfusion, a porous film for providing cell adhesion and separating the upper chip from the lower chip, and a bottom substrate for providing fixed support for the chips; the upper chip and the lower chip are separated by a porous film, and cells can be inoculated on the upper side and the lower side of the porous film; and culturing cells in the upper chip and the lower chip in advance, sucking out the culture medium in the upper chip channel when the cells are fully adhered to the porous film, and introducing gas into the upper chip for exposure experiments.

The upper chip comprises 4 gas channels, 2 inlets and 2 outlets which are arranged in parallel, and the depth of the channels is 800-1500 mu m; the gas channels are parallel in horizontal direction and can be used for cell culture in a cell culture stage and gas exposure in an exposure experiment; for example, a culture medium containing cells is introduced into two inlets of a chip, when the cells are fully adhered to a porous film, the culture medium in a channel of an upper chip is sucked out from an outlet, and gases (such as formaldehyde gas and air) are introduced into the two inlets of the upper chip, and if the gases are two gases with different components, the two gases are diluted with each other to form concentration gradients of the two gas components, and if the same gas is introduced, the exposure of the same gas can be carried out in 4 channels, so that multiple groups of results can be obtained through one experiment.

Similarly, the lower chip comprises 4 liquid channels, 4 inlets and 4 outlets, and the depth of the channels is 50-150 mu m; a plurality of elliptical cell culture chambers can be arranged in the channel so as to reduce the flow rate of the fluid when the fluid enters the cell culture chambers from the channel, and a cell culture environment with low fluid shear stress is created; the 4 liquid channels are mutually independent and can be respectively introduced into cells of the same type or different types to construct the same or different physiological environments; the 4 mutually independent liquid channels are respectively provided with 1 inlet and 1 outlet, so that the effluent liquid of each liquid channel is convenient to respectively collect and detect bioactive components such as inflammatory factors, lactate dehydrogenase, superoxide dismutase and the like. For example, culturing human lung epithelial cells in an upper chip channel, respectively culturing no cells, human vascular endothelial cells, human macrophages and human fibroblasts in 4 channels of a lower chip, and introducing cigarette smoke into a gas channel, thereby comparing the synergistic or antagonistic effect of different cell types in cigarette smoke exposure during co-culture; for another example, human lung epithelial cells are cultured in the upper chip channel, human vascular endothelial cells are cultured in the lower chip 4 channels, and a concentration gradient of cigarette smoke is formed in the gas channel, so that biological effects on different cigarette smoke concentrations when the human lung epithelial cells and the human vascular endothelial cells are co-cultured are compared.

The upper and lower layers of chips are separated by the porous film, the film material, thickness and aperture can be selected according to the requirement, different cells can be cultured on the upper and lower sides of the film respectively, the cells on the upper and lower sides of the film can react with the gas component exposure substances of the upper layer of chips, the three-dimensional culture can realize the instant conduction of signals between the upper and lower layers of cells, and the three-dimensional culture can be used for researching the problems of the synergistic effect of different cells on the stimulation of different physical, chemical, physiological and other factors.

The bottom substrate is used for fixing and supporting the whole chip, and the materials, the sizes and the thicknesses can be selected and processed according to the requirements.

The porous film plays a role of spacing and communicating the upper chip and the lower chip, is a key for effectively establishing a gas-liquid interface, the film cultures two cell separation areas, liquid in the lower chip cannot flow into the upper chip through the micropores, but cells cultured on two sides of the film can receive nourishment of a lower culture medium through the micropores and simultaneously respond to exposure of upper gas components, and bioactive components secreted by the cells on two sides of the film can generate complex crosstalk among the cells.

The upper chip, the lower chip, the porous film and the bottom substrate can be compounded by selecting different materials according to application requirements. Wherein, the upper and lower chips can be 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 can be made of glass, polymethyl methacrylate, polycarbonate, polyethylene terephthalate, polystyrene, polypropylene and other materials.

In the using process, the same kind of cells can be cultured in the upper chip, and different gas condition stimulation can be implemented on co-cultured or independent cultured cells by introducing gas components such as formaldehyde, acetaldehyde, acrolein, cigarette smoke, automobile tail gas and the like; the same or different cell types can be introduced into the lower chip to co-culture with the upper cell. Under the condition of simulating the in-vivo cell growth environment, the cells are subjected to gas exposure, so that the conditions of cell survival, metabolism, damage, cell interaction and the like are observed, and meanwhile, the screening of the drug effect of the drug can be realized, so that the effect of the drug on a disease model can be evaluated, and the method has extremely important significance for drug research and development.

The 4 liquid channels of the lower chip are mutually independent, so that culture medium effluent of the liquid channel seeds can be collected and detected respectively. In-situ fluorescence detection of cells in a bionic chip is always a main method for chip detection, detection of chip effluent is increased, further deep analysis and understanding of gas can be further carried out on co-cultured cells in the chip, and expansion of the detection method also provides stronger applicability for more types of research.

Compared with the prior art, the utility model has the advantages that:

the microfluidic chip in the device comprises an upper chip structure and a lower chip structure, wherein a gas-liquid interface is established on a porous film between the upper chip and the lower chip; on the gas-liquid interface, the upper chip can culture the same kind of cells, and in addition, the upper chip can be exposed by introducing a concentration gas or a concentration gradient gas; the lower chip is used for culturing the same or different cells, and simultaneously can be used for filling culture medium to nourish the whole chip.

The cells cultured in the microfluidic chip are positioned at the upper side and the lower side of the porous film, and different cells can be respectively cultured at the upper side and the lower side of the porous film or cultured at one side of the porous film for different research contents and application scenes; on the basis, the upper cells of the porous film can be in direct contact with the gas stimulating components, and the lower cells can conduct instant signal transmission with the upper cells, so that the related biological information of the gas stimulus acting on the co-cultured cells in a complex culture environment is obtained.

The liquid channels of the lower chip are mutually independent and respectively provided with independent outlets, so that culture medium effluent liquid of each channel can be respectively collected and analyzed, and the analysis of the effluent liquid further enhances the detection capability of biological effects of cells in the chip after the cells are exposed by gas components.

The lower chip cell culture chamber is designed into an elliptical shape, so that the flow rate of the liquid culture medium is reduced when the liquid culture medium enters the cell culture chamber from the channel, and 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, so that the interference of the fluid shear stress on the test is eliminated.

Drawings

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

in the figure: 1. 1-1 parts of upper chip, 1-2 parts of gas channel, 1-3 parts of gas inlet and 1-3 parts of gas 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. 2 is a geometry of the present device.

Detailed Description

The utility model is further described below with reference to the accompanying drawings:

as shown in fig. 1: the utility model discloses a co-culture micro-fluidic chip gas exposure device capable of forming a gas-liquid interface, which comprises an upper chip 1, a porous film 2, a lower chip 3 and a bottom substrate 4 which are sequentially overlapped from top to bottom, wherein four gas channels 1-1 which are distributed in parallel are arranged in the upper chip 1, and four liquid channels 3-1 which are distributed in parallel are arranged in the lower chip 3, wherein: the gas channels 1-1 and the liquid channels 3-1 in the upper chip and the lower chip are correspondingly arranged in an up-down parallel mode, two ends of the four channels 1-1 in the upper chip are respectively provided with two mutually communicated gas inlets 1-2 and two mutually communicated gas outlets 1-3, and two ends of the four liquid channels 3-1 in the lower chip are respectively provided with an independent liquid inlet 3-2 and an independent liquid outlet 3-3.

The upper chip and the lower chip are made of PDMS through inverse molding, the PC porous film between the upper chip and the lower chip is made of laser cutting machine, the bottom substrate is ultra-thin high-permeability glass, all parts of the chip are aligned and bonded under the alignment mirror after being modified by oxygen plasma, the structure is stable, and liquid leakage is not easy.

Claims (5)

1. The utility model provides a can form gaseous exposure device of co-culture micro-fluidic chip at gas-liquid interface, includes upper chip, porous film, lower floor's chip and the bottom base plate that from top to bottom laminating together in proper order, is equipped with four gas passage that are parallel distribution in the upper chip, is equipped with four liquid passage that are parallel distribution in the lower floor's chip, its characterized in that: the gas channels and the liquid channels in the upper chip and the lower chip are correspondingly arranged in an up-down parallel mode, two ends of the four channels in the upper chip are respectively provided with two mutually communicated gas inlets and two mutually communicated gas outlets, and two ends of the four liquid channels in the lower chip are respectively provided with an independent liquid inlet and an independent liquid outlet.

2. The gas exposure device for a co-culture microfluidic chip capable of forming a gas-liquid interface according to claim 1, wherein the gas exposure device is characterized in that: the depth of the gas channel in the upper chip is 800-1500 μm.

3. The gas exposure device for a co-culture microfluidic chip capable of forming a gas-liquid interface according to claim 1, wherein the gas exposure device is characterized in that: the depth of the liquid channel in the lower chip is 50-150 μm, and a plurality of cell culture chambers with elliptical areas are arranged in the channel.

4. The gas exposure device for a co-culture microfluidic chip capable of forming a gas-liquid interface according to claim 1, wherein the gas exposure device is characterized in that: the distance between the gas inlet and the gas outlet of the gas channel is smaller than the distance between the liquid inlet and the liquid outlet of the liquid channel, so that the process assembly is facilitated.

5. The gas exposure device for a co-culture microfluidic chip capable of forming a gas-liquid interface according to claim 1, wherein the gas exposure device is characterized in that: four liquid inlets and four liquid outlets in the lower chip are distributed in an arc shape, so that the conduit access and the development of subsequent experiments are facilitated.