CN111378576B - Intestinal tract simulation chip and application thereof - Google Patents
Intestinal tract simulation chip and application thereof Download PDFInfo
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- CN111378576B CN111378576B CN201811624018.3A CN201811624018A CN111378576B CN 111378576 B CN111378576 B CN 111378576B CN 201811624018 A CN201811624018 A CN 201811624018A CN 111378576 B CN111378576 B CN 111378576B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- C—CHEMISTRY; METALLURGY
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- 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
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
Abstract
The invention discloses an intestinal tract simulation chip and application thereof. The intestinal tract simulation chip comprises: the culture layer comprises a culture chamber for culturing cells to be cultured, and a fluff structure is arranged on the inner wall of one side of the culture chamber; an elastic film layer including an elastic film having air permeability; the pneumatic layer comprises a gas chamber and a pneumatic control pipeline communicated with the gas chamber, the elastic membrane is covered on the gas chamber, and the pneumatic layer is at least used for driving the local area of the elastic membrane to generate deformation and/or displacement towards the culture chamber, so that the inner wall of the culture chamber provided with the fluff structure generates corresponding deformation and/or displacement. The intestinal canal simulation chip can simulate intestinal canal villus structure, intestinal peristalsis and intestinal dissolved oxygen environment on the same device.
Description
Technical Field
The invention relates to an intestinal canal simulation chip, in particular to an intestinal canal simulation chip capable of simulating intestinal villus, intestinal peristalsis and an intestinal canal dissolved oxygen environment and application thereof, and belongs to the application field of microfluidic technology in organ chips.
Background
In recent years, research on organ chips has been greatly developed, and has important application prospects in the fields of new drug research and development, stem cell research, tissue organ development, toxicology prediction and the like, and the research is listed as one of the ten emerging technologies in the Darworth forum in 2016. Researchers have implemented the construction of numerous human organs on microfluidic chips, such as chip livers, chip lungs, chip intestines, chip kidneys, chip blood vessels, chip hearts, multi-organ chips, and the like. The netherlands biotechnology company mimeas developed a chip kidney and achieved an application partnership protocol with several pharmaceutical companies for drug screening; in addition, the Qiansheng company also plans to conduct a drug test using a human thrombus simulation chip system of Emulate corporation belonging to the institute of biological engineering of wyss, university of Harvard, and to test the hepatotoxicity of the drug using a liver chip. The study of intestinal microbiota is also a hot spot in recent years of scientific research, and in 2016, three journals Nature, science and Cell issue on the study of the weight of intestinal microbiome, which disclose that the intestinal microbiome plays a vital role in human health and disease from different angles. Scientists at the university of Edinburgh, MRC, research center for inflammation published papers on science, revealed a mechanism by which the immune system prevents bacteria in our intestinal tract from penetrating into the blood to cause sepsis-type systemic inflammation; a breakthrough study called MetaHit, conducted by the european-chinese team, found that a specific intestinal bacterial imbalance could lead to insulin resistance and thus to an increased risk of health problems such as type 2 diabetes, and the results of the related study were published on-line in Nature journal at 2016, 7, 13. In combination with research hotspots in the scientific research world at home and abroad, the development of intestinal chips has become a trend. The intestine is the longest segment of the digestive tract and is also the most important segment of digestive function. Most of the medicines enter the human body through oral administration, and the oral medicines enter the blood circulation through the small intestine, so that research on the absorption of the medicines through intestinal cells becomes an important step of medicine screening. The villus on the wall of the small intestine enables the small intestine to have huge surface area so as to achieve the effect of quickly absorbing nutrient substances, so that the establishment of the villus morphology of the small intestine on the chip has important significance for researching the 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, particularly with respect to the behavior of epithelial cells in response to specific environmental cues. Synthetic in vitro intestinal models can improve the investigation of intestinal function in a well controlled manner, in particular for cell growth and proliferation, drug absorption and host-microorganism interactions.
2012-2015 Donald E.Ingber et al designed a bionic "human intestinal chip" microdevice consisting of two microfluidic channels separated by a porous flexible membrane coated with extracellular matrix (ECM) and arranged by human intestinal epithelium (Caco-2) cells, simulating the complex structure of living intestines and generating low shear stress on the microchannels with low-velocity fluid, reconstructing the intestinal microenvironment by applying cyclic strain (10%, 0.15 Hz) simulating physiological peristaltic movements, under these conditions, columnar epithelium develops rapidly polarized, spontaneously grows into folds, reproducing the structure of intestinal villus and forming a high integrity barrier small molecule, the chip is an integrated chip, the culture layer and the pneumatic layer are integrated and can not be detached, the assembly process is relatively complex, the sample amount is small, the convincing of the biological phenomenon of the reaction is less enough, and although the chip can simulate physiological peristaltic movements, the villus generated under these conditions is not only highly but also has a radius very different from the actual intestines.
In 2014 John c.march et al developed a porous synthetic 3D tissue scaffold with villus features, created 500 μm deep, 200um diameter, high aspect ratio template array holes on polymethyl methacrylate (PMMA) templates using laser engraving, poured onto the PMMA template with PDMS to get villus structure by turnover, poured into PDMS template with agar to get a mold by turnover, finally poured into agar mold with high PLGA/low porogen to finally form the initial villus structure to simulate intestinal environment. The chip simulates intestinal villi structurally, but has relatively complex manufacturing process and relatively high price, and can not simulate the physiological environment of intestinal dynamic change well only by static culture in the chip.
John C.March et al 2017 added a 3D printed bioreactor on a 2014 basis, constructed a small intestine bioreactor using 3D printing and polymer to assemble the small intestine bioreactor, replaced the original PLGA material with PEVA, and added two fluid connectors to the small intestine bioreactor. The chip simulates intestinal fluid mechanics and the structure of villus, but cannot simulate the physiological peristalsis of small intestine, is relatively troublesome to assemble, relatively more complex in manufacturing process, and relatively difficult to operate because culture is needed in the bioreactor.
Disclosure of Invention
The invention mainly aims to provide an intestinal canal simulation chip and application thereof, so as to overcome the defects of the prior art.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides an intestinal canal simulation chip, which comprises
The culture layer comprises a culture chamber for culturing cells to be cultured, and a fluff structure is arranged on the inner wall of one side of the culture chamber;
an elastic film layer including an elastic film having air permeability;
the pneumatic layer comprises a gas chamber and a pneumatic control pipeline communicated with the gas chamber, the elastic membrane is covered on the gas chamber, and the pneumatic layer is at least used for driving the local area of the elastic membrane to generate deformation and/or displacement towards the culture chamber, so that the inner wall of the culture chamber provided with the fluff structure generates corresponding deformation and/or displacement.
In one embodiment, the culture layer further comprises a limiting layer cooperating with the culture chamber, the limiting layer being adapted to fix the amount of deformation/displacement of the inner wall of the culture chamber provided with the fluff structure at a selected height.
Further, the limiting layer comprises a plurality of limiting structures which are arranged opposite to the fluff structures.
The embodiment of the invention also provides an intestinal tract simulation method, which comprises the following steps:
providing the intestinal canal simulation chip;
injecting cells to be cultured into a culture chamber of the intestinal tract simulation chip, and culturing the cells;
and (3) introducing an air source into the air chamber to drive the local area of the elastic membrane to deform and/or displace towards the culture chamber, so that the inner wall of the culture chamber provided with the fluff structure deforms and/or displaces correspondingly, and intestinal peristalsis is simulated.
Compared with the prior art, the invention has the beneficial effects that:
1) The intestinal canal simulation chip provided by the invention can simulate intestinal canal villus structure, intestinal peristalsis and intestinal dissolved oxygen environment on the same device;
2) The villus structure in the intestinal tract simulation chip provided by the invention is not changed due to external force, is more close to human intestinal villus in height and diameter, is also added with simulation of intestinal peristalsis, and is also added with control of oxygen concentration of a culture chamber;
3) The whole intestinal tract simulation chip device can be detached, and the chip is not required to be assembled when cells are cultured in the early stage, so that the operation is easier;
4) The intestinal canal simulation chip can also be used for observing the growth state of cells in the fluffy chip, and is simple and convenient to detach and operate because of the device;
5) The intestinal canal simulation chip device is transparent as a whole, and can monitor the growth state of cells at any time in the culture process, thereby ensuring the progress of experiments.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the following brief description will be given of the drawings used in the embodiments or the description of the prior art, it being obvious that the drawings described below are only examples of the invention herein, and that other drawings can be obtained from these drawings without the inventive effort of a person skilled in the art.
FIG. 1 is a three-dimensional view of an intestinal analog chip in an exemplary embodiment of the invention.
FIGS. 2 a-2 c are three-dimensional views of a culture layer according to an exemplary embodiment of the present invention, respectively.
Fig. 3 is a three-dimensional view of a pneumatic layer in an exemplary embodiment of the present invention.
FIG. 4 is a cross-sectional view of the intestinal analog chip of FIG. 1 along the A-A plane.
Reference numerals illustrate: 110-fixed layer, 1101-screw, 120-culture layer, 1201-limit column, 1202-culture chamber, 1203-fluff structure, 1204-liquid inlet, 1205-liquid outlet, 130-pneumatic layer, 1301-gas chamber, 1302-pneumatic control pipeline, 140-limit layer, 150-elastic membrane layer.
Detailed Description
As described above, in view of the drawbacks of the prior art, the present inventors have long studied and have made a great deal of practical use to propose the technical solution of the present invention. The technical scheme, the implementation process, the principle and the like are further explained as follows.
As one aspect of the invention, an intestinal tract simulation chip comprises
The culture layer comprises a culture chamber for culturing cells to be cultured, and a fluff structure is arranged on the inner wall of one side of the culture chamber;
an elastic film layer including an elastic film having air permeability;
the pneumatic layer comprises a gas chamber and a pneumatic control pipeline communicated with the gas chamber, the elastic membrane is covered on the gas chamber, and the pneumatic layer is at least used for driving the local area of the elastic membrane to generate deformation and/or displacement towards the culture chamber, so that the inner wall of the culture chamber provided with the fluff structure generates corresponding deformation and/or displacement.
In one embodiment, the culture layer further comprises a limiting layer cooperating with the culture chamber, the limiting layer being adapted to fix the amount of deformation/displacement of the inner wall of the culture chamber provided with the fluff structure at a selected height.
Of course, the limiting of the maximum amount of deformation can also be achieved by setting the maximum depth of the culture chamber without providing a limiting layer. Alternatively, a similar function (i.e., the fluff structure and the stopper structure are on the same side) can be achieved by providing a stopper structure.
Further, the limiting layer comprises a plurality of limiting structures which are arranged opposite to the fluff structures.
Further, the limit structure comprises a limit column capable of limiting, so that the deformation of the inner wall of the culture cavity provided with the fluff structure can be fixed on a certain height, and the deformation generated by intestinal peristalsis can be better simulated.
Further, the cross-sectional shape of the culture chamber includes, but is not limited to, a circle, a square, a rectangle, a sector, or an S-shape.
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 limit post is 0.01-19.08 mm, and the diameter is 0.01-49 mm.
In one embodiment, the pile structure has a height of 0.01 to 10mm and a diameter of 0.01 to 3mm. The villus structure in the invention is originally present and is not changed by external force, and is more close to human intestinal villus in height and diameter.
Further, the culture layer is also provided with a liquid inlet and a liquid outlet respectively.
Further, the material of the culture layer and the elastic membrane may be any polymer having air permeability and elasticity, such as PDMS, but not limited thereto.
Further, the mold before the culture layer and the pneumatic layer are overmolded may be any material that can be separated from the 3D printing material, such as agar, but is not limited thereto.
Further, the pneumatic control conduit is in communication with a gas source.
In one embodiment, the gas source introduced into the gas chamber is selected from oxygen-containing gas, nitrogen and carbon dioxide, or can also be introduced into oxygen-containing liquid, oxygen-producing chemical reagent or deoxidizing chemical reagent, etc., so as to better simulate the hypoxia or anaerobic environment of the intestinal tract, and more reasonably study the intestinal flora.
In one embodiment, the intestinal tract simulation chip further comprises a fixing layer, wherein the fixing layer is arranged on the top of the culture layer and/or the bottom of the pneumatic layer.
Further, the material of the fixing layer includes PMMA plate, and the PMMA plate may be replaced by any other material having a certain hardness, such as glass, but not limited thereto.
Further, the upper and lower fixing layers are fixed by screws.
In one embodiment, the whole intestinal canal simulation chip is transparent, and the growth state of cells can be monitored at any time in the culture process, so that the progress of the experiment is ensured.
Further, the intestinal canal simulation chip can also be used for observing the growth state of cells in the fluffy chip, and the operation is simple and convenient because the device is detachable.
Furthermore, each component of the intestinal canal simulation chip is movably connected, the whole device can be detached, the chip is not required to be assembled when the cells are cultured in the early stage, and the operation is easier.
The whole intestinal tract simulation chip device is detachable, cells can be cultured in an incubator by using a culture layer, the pneumatic layers are assembled together after the cells are fully paved, and a certain pressure and frequency are used for inflating the gas chamber, so that an upper elastic die of the intestinal tract simulation chip deforms to drive the upper culture layer to deform, the peristaltic movement is simulated, the maximum deformation of the culture layer is fixed by a limit column of the culture chamber, and the environment with low oxygen content of the intestinal tract is simulated by changing the oxygen content of the gas.
As another aspect of the present invention, it relates to the use of the intestinal tract simulator chip.
For example, as another aspect of the present invention, it relates to an intestinal tract simulation method, which includes:
providing the intestinal canal simulation chip;
injecting cells to be cultured into a culture chamber of the intestinal tract simulation chip, and culturing the cells;
and (3) introducing an air source into the air chamber to drive the local area of the elastic membrane to deform and/or displace towards the culture chamber, so that the inner wall of the culture chamber provided with the fluff structure deforms and/or displaces correspondingly, and intestinal peristalsis is simulated.
The operation principle of the chip is that firstly, a culture medium containing cells is injected into a culture chamber until the cells are fully paved on the bottom surface of the culture chamber and include villus, so that the culture chamber is communicated with the outside, the culture medium in the culture chamber can be continuously replaced, then a pneumatic layer is started, gas is injected into a gas chamber, the upper elastic membrane of the gas chamber can be deformed, the bottom layer of the culture chamber with a villus structure is further driven to be bulged, the intestinal peristalsis simulating action is achieved, the material has air permeability, and the dissolved oxygen condition of the culture chamber can be changed by changing different gases, so that the microenvironment of intestinal tracts is simulated.
The following detailed description of the technical solutions according to the embodiments of the present invention will be given with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-4, an intestinal analog chip according to an exemplary embodiment of the present invention is composed of the following structural layers, the uppermost and lowermost fixing layers 110, and the upper and lower fixing layers 110 are fixed by screws 1101. The second layer is a culture layer 120, wherein each culture layer comprises an upper limit layer 140, specifically, limit columns 1201 can be selected, the height of each limit column is 1.3mm, the diameter is 0.6mm, a lower villus structure 1203, the height of each villus structure is 0.6mm, the diameter is 0.2mm, and a middle culture chamber 1202; the third layer is an elastic film layer 150, i.e. an elastic film, which is disposed on the gas chamber 1301. The fourth layer is the pneumatic layer 130, including a gas chamber 1301, the culture chamber 1202 and the gas chamber 1301 have tubing in communication with the off-chip. The gas chamber 1301 communicates with a pneumatic control line 1302.
The culture layer is also provided with a liquid inlet 1204 and a liquid outlet 1205.
The operation principle and the action relation of the intestinal canal simulation chip are described as follows:
the chip operation principle is that firstly, a culture medium containing cells is injected into a culture chamber 1202 until the cells are fully paved on the bottom surface of the culture chamber 1202 and comprise a villus structure 1203, the culture chamber is communicated with the outside, the culture medium therein can be continuously replaced, then the pneumatic layer 130 is started, gas is injected into the gas chamber 1301, the upper elastic membrane layer 150 of the gas chamber 1301 can be deformed, the bottom layer of the culture chamber with the villus structure is further driven to be bulged, so that the intestinal peristalsis simulating action is achieved, the material itself has air permeability, and the dissolved oxygen condition of the culture chamber can be changed by replacing different gases, so that the microenvironment of an intestinal tract is simulated.
The whole chip device is detachable, cells can be cultured in the incubator by using the culture layer 120, the pneumatic layers are assembled together after the cells are fully paved, and a certain pressure and frequency are used for inflating the gas chamber, so that the upper elastic die is deformed to drive the upper culture layer to deform, the peristaltic motion is simulated, the maximum deformation of the culture layer is fixed through the limit column 1201 of the culture chamber, and the environment with low oxygen content of intestinal tracts is simulated by changing the oxygen content of the gas.
In summary, by the technical scheme of the invention, the intestinal canal simulation chip can simulate intestinal canal villus structure, simulate physiological state of intestinal canal peristalsis, control oxygen concentration in the culture chip and simulate intestinal canal dissolved oxygen environment on the same device.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
It should be understood that the above embodiments are merely for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and implement the same according to the present invention without limiting the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (15)
1. An intestinal tract simulation chip, comprising: a culture layer, an elastic membrane layer and a pneumatic layer which are sequentially stacked along a selected direction;
the culture layer comprises a culture chamber and a limiting layer matched with the culture chamber, wherein the culture chamber is used for culturing cells to be cultured, a fluff structure is arranged on the inner wall of one side of the culture chamber, the limiting layer and the fluff structure are oppositely arranged along the selected direction, the limiting layer comprises a plurality of limiting structures which are oppositely arranged with the fluff structure, and the limiting layer is used for fixing the deformation/displacement of the inner wall of the culture chamber, on which the fluff structure is arranged, at a selected height;
the elastic film layer comprises an elastic film with air permeability;
the pneumatic layer comprises a gas chamber and a pneumatic control pipeline communicated with the gas chamber, the elastic membrane is covered on the gas chamber, and the pneumatic layer is at least used for driving a local area of the elastic membrane to deform and/or displace towards the culture chamber along the selected direction, so that the inner wall of the culture chamber provided with the fluff structure deforms and/or displaces correspondingly.
2. The intestinal tract simulator chip of claim 1, wherein: the limiting structure comprises a limiting column.
3. The intestinal tract simulator chip of claim 1, wherein: the cross-sectional shape of the culture chamber includes a circle, square, rectangle, sector, or S-shape.
4. The intestinal tract simulator chip of claim 3, wherein: the depth of the culture chamber is 0.025-20 mm, and the diameter of the culture chamber is 0.02-50 mm.
5. The intestinal tract simulator chip of claim 3, wherein: the height of the limiting column is 0.01-19.08 mm, and the diameter of the limiting column is 0.01-49 mm.
6. The intestinal tract simulator chip of claim 1, wherein: the height of the fluff structure is 0.01-10 mm, and the diameter of the fluff structure is 0.01-3 mm.
7. The intestinal tract simulator chip of claim 1, wherein: the culture layer is also provided with a liquid inlet and a liquid outlet.
8. The intestinal tract simulator chip of claim 1, wherein: the culture layer and the elastic membrane are made of a polymer with air permeability and elasticity; PDMS is preferred.
9. The intestinal tract simulator chip of claim 1, wherein: the pneumatic control pipeline is communicated with an air source.
10. The intestinal tract simulator chip of claim 9, wherein: the gas source includes an oxygen-containing gas, nitrogen, carbon dioxide, an oxygen-containing liquid, an oxygen-generating chemical, or a deoxidizing chemical.
11. The intestinal tract simulation chip according to claim 1, further comprising a fixation layer arranged on top of the culture layer and/or on bottom of the pneumatic layer.
12. The intestinal tract simulator chip of claim 11, wherein: the fixing layer is made of PMMA plate or glass.
13. The intestinal tract simulation chip according to any of claims 1 to 12, wherein: the intestinal tract simulator chip is transparent.
14. The intestinal tract simulation chip according to any of claims 1 to 12, wherein: each component part of the intestinal canal simulation chip is movably connected.
15. A method of intestinal tract simulation comprising:
providing an intestinal tract simulation chip according to any one of claims 1 to 14;
injecting cells to be cultured into a culture chamber of the intestinal tract simulation chip, and culturing the cells;
and (3) introducing an air source into the air chamber to drive the local area of the elastic membrane to deform and/or displace towards the culture chamber, so that the inner wall of the culture chamber provided with the fluff structure deforms and/or displaces correspondingly, and intestinal peristalsis is simulated.
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