CN111378576A - Intestinal tract simulation chip and application thereof - Google Patents

Abstract

The invention discloses an intestinal simulation chip and its application. The intestinal simulation chip includes: a culture layer, including a culture chamber, for culturing cells to be cultured, and a villi structure is arranged on the inner wall of one side of the culture chamber; an elastic film layer, including an elastic film with air permeability a pneumatic layer, comprising a gas chamber and a pneumatic control pipe communicating with the gas chamber, the elastic membrane is covered on the gas chamber, and the pneumatic layer is at least used to drive a local area of the elastic membrane Deformation and/or displacement tending to the culturing chamber is generated, thereby causing corresponding deformation and/or displacement of the inner wall of the culturing chamber provided with the villi structure. The intestinal simulation chip of the present invention can realize the simulation of the intestinal villi structure, the simulation of the intestinal peristalsis, and the simulation of the intestinal dissolved oxygen environment on the same device.

Description

A kind of intestinal simulation chip and its application

technical field

The invention relates to an intestinal simulation chip, in particular to an intestinal simulation chip capable of simulating intestinal villi, intestinal peristalsis and intestinal dissolved oxygen environment and its application, belonging to the application field of microfluidic technology in organ chips.

Background technique

In recent years, the research on organ chips has made great progress, and it has important application prospects in the fields of new drug research and development, stem cell research, tissue and organ development, and toxicology prediction. one. Researchers have realized the construction of many human organs on microfluidic chips, such as liver-on-chip, lung-on-chip, intestine-on-chip, kidney-on-chip, blood vessel-on-chip, heart-on-chip, and multi-organ-on-chip. Mimetas, a Dutch biotech company, has developed a kidney-on-a-chip and has an application partnership agreement with several pharmaceutical companies to use it for drug screening; in addition, Johnson & Johnson plans to use a human thrombus from Emulate, a subsidiary of Harvard's wyss Institute for Bioengineering. The simulation chip system is used for drug testing, and the liver chip is used to test the liver toxicity of drugs. The study of gut microbiota has also become a hotspot of scientific research in recent years. In 2016, three major journals, Nature, Science and Cell, published blockbuster research articles on the gut microbiome, which revealed the gut microbiome from different perspectives. It plays a vital role in human health and disease. Scientists from the University of Edinburgh's MRC Inflammation Research Centre published a paper in science revealing the mechanism by which the immune system prevents bacteria in our gut from infiltrating the bloodstream, causing systemic inflammation such as sepsis; a European-Chinese team called MetaHit A breakthrough study by researchers found that a specific imbalance of gut bacteria can lead to insulin resistance, which can lead to an increased risk of health problems such as type 2 diabetes, the findings were published online July 13, 2016 in the journal Nature. Combined with the current research hotspots in the scientific research community at home and abroad, the development of intestinal chips has become a trend. The intestine is the longest section of the digestive tract and the most important section for digestive function. Most drugs enter the human body orally, and oral drugs must enter the blood circulation through the small intestine. Therefore, studying the absorption of drugs through intestinal cells has become an important step in drug screening. The villi on the small intestine wall make the small intestine have a huge surface area, so as to achieve the effect of rapid absorption of nutrients, so the establishment of the morphology of the small intestine villi on the chip is of great significance for the study of intestinal function.

In vivo animal models can be used to study many intestinal diseases and phenomena, including inflammatory genomics, irritable bowel syndrome, short bowel syndrome, gastroenteritis, etc. However, many intestinal processes are difficult to control using in vivo intestinal models, especially on the behavior of epithelial cells in response to specific environmental cues. Synthetic in vitro gut models can improve the study of gut function, especially cell growth and proliferation, drug absorption, and host-microbe interactions, in a well-controlled manner.

In 2012-2015, Donald E. Ingber et.al designed a biomimetic "human gut-on-a-chip" microdevice consisting of two microfluidic channels separated by a porous flexible membrane coated with an extracellular matrix (ECM). open, and lined by human intestinal epithelial (Caco-2) cells, mimicking the complex structure of the living intestine, and flowing fluid at low velocity to generate low shear stress on the microchannels by applying cyclic strain (10%, 0.15 Hz) to reconstruct the intestinal microenvironment, under these conditions, the columnar epithelium develops rapidly polarized, spontaneously grows into folds, reproduces the structure of intestinal villi, and forms a high-integrity barrier small molecule, the chip is an all-in-one chip , the culture layer and the pneumatic layer are integrated and cannot be separated, the assembly process is relatively complicated, the sample volume is small, and the biological phenomenon of the reaction is less convincing, and although the chip can simulate physiological peristalsis, it is produced under such conditions. The villi are very different not only in height but also in radius from actual intestinal villi.

In 2014, John C. March et.al developed porous synthetic 3D tissue scaffolds with villi features, using laser engraving to create 500 μm deep, 200 μm diameter, high aspect ratio template array holes on polymethyl methacrylate (PMMA) templates. , pour PDMS onto the PMMA template to obtain the villi structure by turning the mold, then pour the agar into the PDMS template to obtain the mold by turning the mold, and finally pour the high PLGA/low porogen into the agar mold to finally form the initial villi structure with to simulate the intestinal environment. Although the chip simulates intestinal villi in structure, the manufacturing process is relatively complicated, the price is relatively high, and it is only statically cultured in the chip, which cannot well simulate the physiological environment of intestinal dynamic changes.

In 2017, John C.March et.al added a 3D printed bioreactor on the basis of 2014, using 3D printing and polymers to build a small intestinal bioreactor to assemble the small intestinal bioreactor, the original PLGA. The material was changed to PEVA, and two fluid connectors were added to the small intestinal bioreactor. Although the chip simulates the gut hydrodynamics and the structure of its villi, it cannot simulate the physiological peristalsis of the small intestine, and the assembly is relatively troublesome, the manufacturing process is relatively more complicated, and the cultivation must be carried out in this bioreactor, which is relatively difficult to operate.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to provide an intestinal simulation chip and its application to overcome the deficiencies of the prior art.

In order to realize the foregoing invention purpose, the technical scheme adopted in the present invention includes:

An embodiment of the present invention provides an intestinal simulation chip, which includes

a culture layer, comprising a culture chamber for culturing the cells to be cultured, and a villi structure is provided on the inner wall of one side of the culture chamber;

an elastic film layer, including a breathable elastic film;

a pneumatic layer, comprising a gas chamber and a pneumatic control pipe communicating with the gas chamber, the elastic membrane is covered on the gas chamber, and the pneumatic layer is at least used to drive a local area of the elastic membrane to generate The deformation and/or displacement of the culture chamber leads to corresponding deformation and/or displacement of the inner wall of the culture chamber provided with the villi structure.

In one embodiment, the culture layer further includes a limit layer matched with the culture chamber, and the limit layer is used to fix the deformation/displacement of the inner wall of the culture chamber provided with the villi structure. at the selected height.

Further, the limiting layer includes a plurality of limiting structures disposed opposite to the fluff structure.

The embodiment of the present invention also provides an intestinal simulation method, which includes:

Provide the aforementioned intestinal simulation chip;

injecting the cells to be cultured into the culture chamber of the intestinal simulation chip to carry out cell culture;

Passing a gas source into the gas chamber to drive the local area of the elastic membrane to produce deformation and/or displacement towards the culture chamber, thereby causing corresponding deformation of the inner wall of the culture chamber provided with the villi structure and/or displacement, thereby simulating intestinal peristalsis.

Compared with the prior art, the beneficial effects of the present invention include:

1) The intestinal simulation chip provided by the present invention can realize the simulation of the intestinal villi structure, the simulation of intestinal peristalsis, and the simulation of the intestinal dissolved oxygen environment on the same device;

2) The villi structure in the intestinal simulation chip provided by the present invention does not change due to external force, and is closer to human intestinal villi in height and diameter, and also adds the simulation of intestinal peristalsis, and also adds to the culture. Control of chamber oxygen concentration;

3) The whole device of the intestinal simulation chip of the present invention can be disassembled, and the chip does not need to be assembled when culturing cells in the early stage, and the operation is easier;

4) The intestinal simulation chip of the present invention can also be used to observe the growth state of cells in the villous chip, and the operation is simple and convenient because the device can be disassembled;

5) The intestinal simulation chip device of the present invention is transparent as a whole, and the growth state of the cells can be monitored at any time during the culturing process to ensure the progress of the experiment.

Description of drawings

In order to illustrate the technical solutions of the present invention more clearly, the following will briefly introduce the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

FIG. 1 is a three-dimensional view of an intestinal simulation chip in an exemplary embodiment of the present invention.

2a-2c are three-dimensional views of a culture layer in an exemplary embodiment of the present invention, respectively.

Figure 3 is a three-dimensional view of the aerodynamic layer in an exemplary embodiment of the present invention.

FIG. 4 is a cross-sectional view of the intestinal simulation chip in FIG. 1 along the A-A plane.

Description of reference numerals: 110-fixed layer, 1101-screw, 120-culture layer, 1201-limiting column, 1202-culture chamber, 1203-villus structure, 1204-liquid inlet, 1205-liquid outlet, 130- Pneumatic layer, 1301-gas chamber, 1302-pneumatic control pipeline, 140-limiting layer, 150-elastic membrane layer.

Detailed ways

As mentioned above, in view of the defects of the prior art, the inventor of the present application has been able to propose the technical solution of the present invention after long-term research and extensive practice. The technical solution, its implementation process and principle will be further explained as follows.

As an aspect of the technical solution of the present invention, it relates to an intestinal simulation chip, which includes

a culture layer, comprising a culture chamber for culturing the cells to be cultured, and a villi structure is provided on the inner wall of one side of the culture chamber;

an elastic film layer, including a breathable elastic film;

a pneumatic layer, comprising a gas chamber and a pneumatic control pipe communicating with the gas chamber, the elastic membrane is covered on the gas chamber, and the pneumatic layer is at least used to drive a local area of the elastic membrane to generate The deformation and/or displacement of the culture chamber leads to corresponding deformation and/or displacement of the inner wall of the culture chamber provided with the villi structure.

In one embodiment, the culture layer further includes a limit layer matched with the culture chamber, and the limit layer is used to fix the deformation/displacement of the inner wall of the culture chamber provided with the villi structure. at the selected height.

Of course, without setting the limiting layer, the maximum deformation amount can also be limited by setting the maximum depth of the culture chamber. Alternatively, similar functions can also be achieved by setting the limiting structure (ie, the fluff structure and the limiting structure are on the same side).

Further, the limiting layer includes a plurality of limiting structures disposed opposite to the fluff structure.

Further, the limiting structure includes a limiting column that can limit the position, so that the deformation amount of the inner wall of the culture chamber provided with the villi structure can be fixed at a certain height, which better simulates the deformation amount generated by intestinal peristalsis.

Further, the cross-sectional shape of the culture chamber includes a circle, a square, a rectangle, a fan shape or an S shape, but is not limited thereto.

Further, the depth of the culture chamber is 0.025-20 mm, and the diameter is 0.02-50 mm.

Further, the height of the limiting column is 0.01-19.08 mm, and the diameter is 0.01-49 mm.

In one embodiment, the height of the fluff structure is 0.01-10 mm, and the diameter is 0.01-3 mm. The villi structure in the present invention is not changed by external force, and is closer to human intestinal villi in height and diameter.

Further, a liquid inlet and a liquid outlet are respectively opened on the culture layer.

Further, the materials of the culture layer and the elastic film can be any polymer with air permeability and elasticity, such as PDMS, etc., but not limited thereto.

Further, the mold before the culture layer and the pneumatic layer are overturned can be any material that can be separated from the 3D printing material, such as agar, but not limited thereto.

Further, the pneumatic control pipeline is communicated with the air source.

In one embodiment, the gas source introduced into the gas chamber is selected from the group consisting of oxygen-containing gas, nitrogen, carbon dioxide, or can also be introduced into oxygen-containing liquid, oxygen-generating chemical reagent or deoxygenating agent. Chemical reagents, etc., can better simulate the hypoxic or anaerobic environment of the intestinal tract, which is more reasonable for the study of intestinal flora.

In one embodiment, the intestinal simulation chip further includes a fixed layer, and the fixed layer is disposed on the top of the culture layer and/or the bottom of the pneumatic layer.

Further, the material of the fixed layer includes a PMMA board, and the PMMA board can also be replaced with any other material having a certain hardness, such as glass, etc., but not limited to this.

Further, the upper and lower fixed layers are fixed by screws.

In one embodiment, the whole intestinal simulation chip is transparent, and the growth state of the cells can be monitored at any time during the culture process to ensure the progress of the experiment.

Further, the intestinal simulation chip can also be used to observe the growth state of cells in the villiated chip, and the operation is simple and convenient because the device is detachable.

Further, each component of the intestinal simulation chip is movably connected, and the whole device can be disassembled, and the chip does not need to be assembled when culturing cells in the early stage, and the operation is easier.

The whole device of the intestinal simulation chip of the present invention can be disassembled, and the cells can be cultured in the incubator with the culture layer first, and after the cells are covered, the pneumatic layer is assembled together, and the gas chamber is inflated with a certain pressure and frequency. , to deform the upper elastic mold and then drive the upper culture layer to deform to simulate peristalsis, and fix the maximum deformation amount of the culture layer through the limit column of the culture chamber, and simulate intestinal hypoxia by changing the oxygen content of the gas environment of.

As another aspect of the technical solution of the present invention, it relates to the application of the aforementioned intestinal simulation chip.

For example, as another aspect of the technical solution of the present invention, it relates to an intestinal simulation method, which includes:

Provide the aforementioned intestinal simulation chip;

injecting the cells to be cultured into the culture chamber of the intestinal simulation chip to carry out cell culture;

Passing a gas source into the gas chamber to drive the local area of the elastic membrane to produce deformation and/or displacement towards the culture chamber, thereby causing corresponding deformation of the inner wall of the culture chamber provided with the villi structure and/or displacement, thereby simulating intestinal peristalsis.

The operating principle of the chip of the present invention is to inject the culture medium containing cells into the culture chamber first, and wait until the cells are covered with the bottom surface of the culture chamber including the villi, so that the culture chamber is communicated with the outside world, so that the culture medium can be continuously replaced, Then activate the pneumatic layer and inject gas into the gas chamber, so that the upper elastic membrane of the gas chamber can be deformed, and then the bottom layer with the villi structure of the culture chamber is also inflated, so as to simulate the behavior of intestinal peristalsis. , and the material itself is breathable, and changing the different gases can change the dissolved oxygen in the culture chamber to simulate the microenvironment of the intestinal tract.

The technical solutions in the embodiments of the present invention will be described in detail below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

Referring to FIGS. 1-4 , an intestinal simulation chip according to an exemplary embodiment of the present invention is composed of the following structural layers. The uppermost and the lowermost are fixed layers 110 , and the upper and lower fixed layers 110 are fixed by screws 1101 . The second layer is the culture layer 120. Each culture layer includes the upper limit layer 140. Specifically, limit columns 1201 can be selected. The height of each limit column is 1.3 mm and the diameter is 0.6 mm. , the height of each villi structure is 0.6mm, the diameter is 0.2mm, and the culture chamber 1202 in the middle; the third layer is the elastic film layer 150 , namely the elastic film, which is covered on the gas chamber 1301 . The fourth layer is the pneumatic layer 130, which includes a gas chamber 1301. The culture chamber 1202 and the gas chamber 1301 are connected with the outside of the chip by pipes. The gas chamber 1301 communicates with the pneumatic control pipe 1302 .

The culture layer is also provided with a liquid inlet 1204 and a liquid outlet 1205 .

The operation principle and action relationship of the intestinal simulation chip of the present invention are described as follows:

The operating principle of the chip is to first inject the culture medium containing cells into the culture chamber 1202, and wait until the cells are covered with the bottom surface of the culture chamber 1202 including the villi structure 1203, so that the culture chamber is communicated with the outside world, so that the culture medium can be continuously Then, the pneumatic layer 130 is activated again, and the gas is injected into the gas chamber 1301, so that the upper elastic film layer 150 of the gas chamber 1301 can be deformed, thereby driving the bottom layer of the culture chamber with the villi structure to be bulged, Therefore, the behavior of simulating intestinal peristalsis can be achieved, and the material itself is breathable. Changing different gases can change the dissolved oxygen in the culture chamber to simulate the microenvironment of the intestinal tract.

The entire chip device can be disassembled. The cells can be cultured in the incubator with the culture layer 120 first. After the cells are filled, the pneumatic layers are assembled together, and the gas chamber is inflated with a certain pressure and frequency to make the upper layer elastic. The mold deforms and then drives the upper culture layer to deform to simulate peristalsis, and the maximum deformation amount of the culture layer is fixed by the limit column 1201 of the culture chamber, and the oxygen content of the gas is changed to simulate the intestinal hypoxic environment.

To sum up, with the above technical solutions of the present invention, the intestinal simulation chip of the present invention can simulate the structure of intestinal villi on the same device, and can also simulate the physiological state of intestinal peristalsis, and can control the culture chip The oxygen concentration in the intestinal tract can be simulated to simulate the intestinal dissolved oxygen environment.

It should be noted that, herein, the terms "comprising", "comprising" or any other variation thereof are intended to encompass non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements, It also includes other elements not expressly listed or inherent to such a process, method, article or apparatus. Without further limitation, an element qualified by the phrase "comprising a..." does not preclude the presence of additional identical elements in a process, method, article or apparatus that includes the element.

It should be understood that the above-mentioned embodiments are only intended to illustrate the technical concept and characteristics of the present invention, and the purpose thereof is to enable those who are familiar with the art to understand the content of the present invention and implement it accordingly, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included within the protection scope of the present invention.

Claims (15)

1. an intestinal simulation chip is characterized in that comprising: a culture layer, comprising a culture chamber for culturing the cells to be cultured, and a villi structure is provided on the inner wall of one side of the culture chamber; an elastic film layer, including a breathable elastic film; a pneumatic layer, comprising a gas chamber and a pneumatic control pipe communicating with the gas chamber, the elastic membrane is covered on the gas chamber, and the pneumatic layer is at least used to drive a local area of the elastic membrane to generate The deformation and/or displacement of the culture chamber leads to corresponding deformation and/or displacement of the inner wall of the culture chamber provided with the villi structure. 2 . The intestinal simulation chip according to claim 1 , wherein the culture layer further comprises a limit layer matched with the culture chamber, and the limit layer is used for setting the culture chamber. 3 . The deformation/displacement amount of the inner wall of the fluff structure is fixed at a selected height; preferably, the limiting layer includes a plurality of limiting structures arranged opposite to the fluff structure; preferably, the limiting structure includes a limiting structure column. 3 . The intestinal simulation chip according to claim 1 , wherein the cross-sectional shape of the culture chamber comprises a circle, a square, a rectangle, a fan shape or an S shape. 4 . 4 . The intestinal simulation chip according to claim 3 , wherein the culture chamber has a depth of 0.025-20 mm and a diameter of 0.02-50 mm. 5 . 5 . The intestinal simulation chip according to claim 2 , wherein the limit post has a height of 0.01-19.08 mm and a diameter of 0.01-49 mm. 6 . 6 . The intestinal simulation chip according to claim 1 , wherein the height of the villi structure is 0.01-10 mm, and the diameter is 0.01-3 mm. 7 . 7 . The intestinal simulation chip according to claim 1 , wherein the culture layer is further provided with a liquid inlet and a liquid outlet. 8 . 8 . The intestinal simulation chip according to claim 1 , wherein the materials of the culture layer and the elastic film comprise polymers with air permeability and elasticity; preferably PDMS. 9 . 9 . The intestinal simulation chip according to claim 1 , wherein the pneumatic control pipeline is communicated with an air source. 10 . 10 . The intestinal simulation chip according to claim 9 , wherein the gas source comprises oxygen-containing gas, nitrogen gas, carbon dioxide, oxygen-containing liquid, oxygen-generating chemical reagents or deoxidizing chemical reagents. 11 . 11 . The intestinal simulation chip according to claim 1 , further comprising a fixed layer, and the fixed layer is arranged on the top of the culture layer and/or the bottom of the pneumatic layer. 12 . 12 . The intestinal simulation chip according to claim 11 , wherein the material of the fixed layer comprises PMMA plate or glass. 13 . 13. The intestinal simulation chip according to any one of claims 1-12, wherein the intestinal simulation chip is transparent. 14. The intestinal simulation chip according to any one of claims 1-12, wherein each component of the intestinal simulation chip is an active connection. 15. An intestinal simulation method, characterized in that it comprises: providing the intestinal simulation chip according to any one of claims 1-14; injecting the cells to be cultured into the culture chamber of the intestinal simulation chip to carry out cell culture; Passing a gas source into the gas chamber to drive the local area of the elastic membrane to produce deformation and/or displacement toward the culture chamber, thereby causing corresponding deformation of the inner wall of the culture chamber provided with the villi structure and/or displacement, thereby simulating intestinal peristalsis.

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