WO2019222870A1

WO2019222870A1 - Bionic intestinal organ-on-a-chip, and preparation method therefor and application thereof

Info

Publication number: WO2019222870A1
Application number: PCT/CN2018/087598
Authority: WO
Inventors: 魏文博; 陈娟娟; 肖亮
Original assignee: 深圳华大生命科学研究院
Priority date: 2018-05-21
Filing date: 2018-05-21
Publication date: 2019-11-28
Also published as: CN111971383B; CN111971383A

Abstract

Disclosed are a bionic intestinal organ-on-a-chip, and a preparation method therefor and application thereof. The bionic intestinal organ-on-a-chip has a fluid passage; a porous membrane is provided in the fluid passage; the porous membrane separates the fluid passage into an upper fluid passage and a lower fluid passage; several projections for simulating the intestinal villus structure and several through holes for simulating the intestinal absorption function are distributed on the porous membrane. The upper fluid passage is used for culturing intestinal cells, and the lower fluid passage is used for collecting metabolites; the projections on the porous membrane are used for simulating the intestinal villus structure, and the through holes are used for simulating the intestinal absorption function. Since a three-dimensional stent structure of the several projections is distributed on the porous membrane in the chip, the difference in intestinal tissue structures generated among different batches of chips can be reduced greatly, and the reproducibility is good. The present bionic intestinal organ-on-a-chip can dynamically culture intestinal cells by using perfusion so as to simulate the dynamic intestinal microenvironment, and is applicable to researches in intestinal diseases, drug screening, food safety, and the like.

Description

Bionic intestinal organ chip, preparation method and application thereof

Technical field

The application relates to the technical field of organ bionics, and in particular, to a bionic intestinal organ chip, and a preparation method and application thereof.

Background technique

Microfluidics, as one of the important frontier science and technology in the 21st century, provides an important platform for simulating human metabolic models in vitro. It is mainly based on micro-nano processing technology, formed by a network of micron-scale channels, and controls the fluid through the entire system, which can realize the routine functions of biology and chemistry laboratories. Because it has micron-sized components that match the cell size, it can perform a variety of cell culture and fluid stimulation in the microchannel of the chip, and build a three-dimensional microenvironment that is close to the physiological environment and has the characteristics of space-time resolution. Important technologies for screening, toxicology, and biomedical research. At this stage, microfluidic technology has been successfully applied to three-dimensional cell co-culture, cell migration, cell sorting, tissue microenvironment, and organoid construction. Among them, Human Organs-on-a-chip is a new cutting-edge interdisciplinary technology developed in recent years. It is a kind of micro-fabrication chip that can simulate human organs using micro-processing technology. The main function of the bionic system. Compared with the traditional two-dimensional static cell culture technology, the cells cultured on the chip have a three-dimensional structure and a spatial distribution structure of a variety of cells. More importantly, the organ chip can provide a dynamic microenvironment for the cells, which is unmatched by traditional methods. . In addition, most of the cells in the chip are based on human-derived cells, which can greatly reduce the interspecific differences produced by animal models. The development of organ chips will help drug development and disease research.

At present, the design and preparation of intestinal chips are mainly focused on Professor Ingber of Harvard University, as shown in Figure 1. The chip is mechanically stretched by caco-2 cells cultured on a porous membrane to differentiate and form a villi structure spontaneously.

Specifically, the existing intestinal chip is sealed by a three-layer PDMS (polydimethylsiloxane) structure, as shown in FIG. 1, wherein the middle layer is a porous membrane 101 for inoculating intestinal cells; The upper and lower layers are fluid channels 02, which are used to simulate the microenvironment of the intestinal fluid; the left and right sides of the channel are vacuum chambers 103, which are used for mechanical movement of stretching the porous membrane.

However, the disadvantage of the intestinal chip of the prior art is that the uniformity of the spontaneous formation of the villi structure cannot be guaranteed, and the chip has a large difference between different batches, which is not convenient for experimental data statistics and result comparison.

Summary of the Invention

The present application provides a bionic intestinal organ chip that can reduce the difference in intestinal tissue structure generated between different batches of chips, and has good reproducibility, and a preparation method and application thereof.

According to a first aspect, an embodiment provides a bionic intestinal organ chip having a fluid channel, and a porous membrane is provided in the fluid channel. The porous membrane divides the fluid channel into an upper fluid channel and a lower fluid channel. There are several protrusions used to simulate the structure of intestinal villi and several through holes used to simulate the intestinal absorption function.

According to a second aspect, an embodiment provides a method for preparing a bionic intestinal organ chip, including the following steps:

Preparing the upper chip and the lower chip;

Preparing a porous membrane template;

Preparation of porous membrane and sealing assembly: The porous membrane is prepared through the porous membrane template, and the porous membrane is sealed between the upper chip and the lower chip to form a bionic intestinal organ chip;

Among them, a plurality of protrusions for simulating the structure of intestinal villi and a plurality of through holes for simulating the intestinal absorption function are distributed on the porous membrane.

According to a third aspect, an embodiment provides a method for preparing a bionic intestinal organ, which is performed by using the above-mentioned bionic intestinal organ chip.

According to the bionic intestinal organ chip and the preparation method and application thereof according to the above embodiments, since there are several protrusions and through holes distributed on the porous membrane, the upper fluid channel is used to culture intestinal cells, and the lower layer is used to collect metabolites. The protrusions are used to simulate the structure of intestinal villi, and the through holes are used to simulate the intestinal absorption function, so that the porous membrane forms a three-dimensional scaffold structure, which can greatly reduce the difference in intestinal tissue structure between different batches of chips, and has good Reproducibility. The bionic intestinal organ chip can use infusion to dynamically culture intestinal cells to simulate the dynamic microenvironment in the intestine, which is suitable for research on intestinal diseases, drug screening, food safety and other research.

BRIEF DESCRIPTION OF THE DRAWINGS

1 is a schematic structural diagram of a bionic intestinal organ chip in the prior art;

2 is a schematic structural diagram of a bionic intestinal organ chip according to the first embodiment;

3 is a flowchart of preparing a bionic intestinal organ chip in Example 2;

4 is a flowchart of preparing an upper-layer chip and a lower-layer chip in Embodiment 2;

5 is a flowchart of preparing a porous membrane template in Example 2;

6 is a schematic structural diagram of a porous membrane template in the second embodiment;

FIG. 7 is a flowchart of preparing a porous membrane and sealing and assembly in the second embodiment; FIG.

8 is a flowchart of preparing a bionic intestinal organ in the third embodiment.

Detailed ways

The present invention will be further described in detail below through specific embodiments in combination with the accompanying drawings.

Embodiment one:

This embodiment provides a bionic intestinal organ chip. The bionic intestinal organ chip has a three-layer structure and simulates the dynamic microenvironment in the intestine. It is used to dynamically cultivate intestinal cells. It is suitable for intestinal diseases, drug screening and food safety. And so on.

As shown in FIG. 2, the bionic intestinal organ chip in this embodiment is provided with a fluid channel. Specifically, the chip includes an upper-layer chip 210 and a lower-layer chip 220. The lower surface of the upper chip 210 has an open upper fluid channel, the upper surface of the lower chip 220 has an open lower fluid channel, a porous film 230 is sealed between the upper chip 210 and the lower chip 220, and the porous film 230 surrounds the upper chip 210. The upper fluid channel 211 is synthesized, and the porous membrane 230 and the lower chip 220 surround a lower fluid channel 221. A plurality of protrusions 231 and through holes 232 are distributed on the porous membrane 230. The protrusions 231 are used to simulate the structure of intestinal villi, the through holes 232 are used to simulate the intestinal absorption function. The through holes 232 connect the upper fluid channel 211 and the lower fluid channel 221. Continuity. The upper chip 210 is punched out at positions corresponding to both ends of the upper fluid channel 211, and the upper chip 210 is punched out at positions corresponding to both ends of the lower fluid channel 221.

In this embodiment, the upper chip 210, the lower chip 220, and the porous film 230 are all made of PDMS.

The protrusions 231 have a conical or circular truncated structure. A plurality of protrusions 231 are arranged in an array on the porous membrane 230. A plurality of through holes 232 are evenly distributed in the area outside the protrusions 231, that is, the protrusions 231 and the through holes 232 are covered together. Porous film 230. The porous membrane 230 forms a three-dimensional three-dimensional scaffold, thereby realizing the three-dimensional spatial structure in the intestine.

The upper fluid passage 211 and the lower fluid passage 221 have a length of 10-15 mm, a width of 1-1.5 mm, and a height of 0.3-0.5 mm. The thickness of the porous film 230 is 30-50 microns. The bottom surface of the protrusion 231 has a diameter of 100-200 microns, a height of 150-200 microns, and a pitch of 150-200 microns. The diameter of the through holes 232 is 10 micrometers, and the pitch between the through holes 232 is 50 micrometers.

For example, the upper fluid passage 211 and the lower fluid passage 221 have a length of 15 mm, a width of 1 mm, and a height of 0.4 mm. The thickness of the porous film 230 is 40 micrometers. The bottom surface of the protrusions 231 has a diameter of 200 μm, a height of 150 μm, and a pitch of 200 μm. In other embodiments, the size of each component can be selected within the above range according to the needs of the simulation.

Since the bionic intestinal organ chip provided in this embodiment has several protrusions 231 and through holes 232 distributed on the porous membrane 230, the upper fluid channel 211 is used for culturing intestinal cells, and the lower layer is used for collecting metabolites. The protrusion 231 is used to simulate the structure of intestinal villi, and the through-hole 232 is used to simulate the absorption function of the intestine, so that the porous membrane 230 forms a three-dimensional scaffold structure, which can greatly reduce the difference in intestinal tissue structure between different batches of chips. With good reproducibility. The bionic intestinal organ chip can use infusion to dynamically culture intestinal cells to simulate the dynamic microenvironment in the intestine, which is suitable for research on intestinal diseases, drug screening, food safety and other research.

Embodiment two:

This embodiment provides a method for preparing a bionic intestinal organ chip. This preparation method mainly uses soft photolithography to prepare the bionic intestinal organ chip in the first embodiment.

As shown in FIG. 3, the method for preparing the bionic intestinal organ chip of this embodiment mainly includes the following steps:

S100: preparing the upper chip and the lower chip;

S200: preparing a porous membrane template;

S300: preparing porous membrane and sealing assembly.

In step S300, a porous membrane is prepared through a porous membrane template, and the porous membrane is sealed between the upper chip and the lower chip to form a bionic intestinal organ chip. Among them, the produced porous membrane has several protrusions for simulating the structure of intestinal villi and several through holes for simulating the intestinal absorption function.

In the preparation method, an organ chip is prepared by separating the upper chip and the lower chip into two monomers, a porous membrane is prepared separately, and the three are sealed together to form a three-layer structure chip with upper and lower flow channels. In this method, steps S100 and S200 are not sequential and can be prepared in sequence or simultaneously.

Specifically, as shown in FIG. 4, step S100 (preparing an upper-layer chip and a lower-layer chip) includes the following steps:

S101: spin-coat a photoresist on a substrate surface of a glass or silicon wafer, and perform pre-baking;

In this embodiment, SU-8 photoresist is preferred. SU-8 photoresist is an epoxy-type, near-ultraviolet negative photoresist. SU-8 photoresist has a low light absorption rate in the near-ultraviolet range. , So that it has better exposure uniformity in the thickness of the photoresist, and can obtain a structure with a nearly vertical edge of the pattern.

The mechanism of SU-8 photoresist photolithography is as follows: the photoinitiator in the photoresist absorbs photons and chemically reacts to produce a strong acid, which acts as an acid catalyst to promote the crosslinking reaction during the pre-baking process. Strong acid is only generated in the photoresist in the exposed area, so the cross-linking reaction only occurs in the exposed area. The cross-linking reaction does not occur in the unexposed area, and the photoresist is insoluble in the developing solution after the cross-linking reaction. The photoresist is soluble in the developing solution without cross-linking reaction, so the developed photoresist forms a pattern opposite to the mask pattern.

In this step, the thickness of the spin-coated SU-8 photoresist is 300-500 microns, which corresponds to the height of the upper fluid channel and the lower fluid channel. The pre-baking temperature is 95 ° C and the time is 2-8 hours.

S102: fixing a mask with a structure pattern of upper and lower fluid channels on a surface of a substrate with a photoresist attached thereto;

The mask has a pattern of an upper fluid channel structure and a lower fluid channel structure. The mask is used to block ultraviolet light, thereby copying the pattern on the mask onto a SU-8 photoresist.

S103: the light source vertically irradiates the glass or silicon wafer with a mask and a photoresist for exposure, and performs post-baking;

The light emitted by the light source passes through the pattern on the mask and irradiates the SU-8 photoresist. The exposed area of the SU-8 photoresist will be crosslinked, and the crosslinked area will not be dissolved in the developing solution. The light sources in this embodiment are all ultraviolet light sources, and are used to emit ultraviolet light for exposure.

In this step, the post-baking temperature is 95 ° C and the time is 10-30 minutes.

S104: After the natural cooling, use ethyl lactate developer to remove the unexposed photoresist, form a template for the upper and lower fluid channel structure, and perform hardening;

In this step, the unexposed SU-8 photoresist is removed by a developing solution. The developed SU-8 photoresist forms a pattern opposite to that of the mask, and then the film is strengthened. The temperature of the film is 180 ° C. For 2 hours.

S105: A template with upper and lower fluid channel structures is used to prepare an upper chip and a lower chip of PDMS material.

In this step, an upper layer chip and a lower layer chip of PDMS material are prepared by using a photoresist template corresponding to the upper layer fluid channel structure and the lower layer fluid channel structure, and entry holes are punched at both ends of the upper layer fluid channel and correspondingly Holes are punched at the entrances and exits of the lower fluid channels, while the lower chips are not punched. Finally, the upper layer chip and the lower layer chip are prepared.

As shown in FIG. 5 and FIG. 6, step S200 (the method for preparing a porous membrane template) includes the following steps:

S201: spin-coat a photoresist on the substrate surface of a glass or silicon wafer, and perform the first pre-baking;

In this step, a SU-8 photoresist 302 having a thickness of 150-200 microns is spin-coated on the glass or silicon wafer 301, and the thickness corresponds to the height of the protrusion on the porous film. The first pre-baking temperature is 95 ° C. The time is 2-4 hours.

S202: fixing a mask having a circular array pattern corresponding to the protrusions on the porous film on the surface of the substrate with the photoresist attached;

The circular array pattern on the mask has a diameter of 100-200 micrometers and a pitch of 150-200 micrometers, corresponding to the protrusions on the porous membrane, and is used to prepare a template on the porous membrane.

S203: Fix the glass or silicon wafer with the mask and photoresist on a rotatable and tiltable platform, and place it under a vertical light source for the first exposure;

In this step, the glass or silicon wafer with photoresist is fixed on the platform, and the platform can adjust the tilt angle and can rotate freely to adjust the exposure angle of the exposure. Before exposure, first tilt the platform by 15-45 °, so that the UV light is tilted by 15-45 ° to expose the SU-8 photoresist 302. During the exposure process, the platform is rotated 360 ° along the normal direction of the table to expose The rear SU-8 photoresist 302 has an exposed area 302a and an unexposed area 302b, and the unexposed area 302b is used to prepare a bump on the porous film.

S204: Remove the mask, spin-coat the photoresist on the exposed photoresist, and perform the second pre-baking;

After the first exposure, the SU-8 photoresist 303 is spin-coated on the SU-8 photoresist 302. The thickness of the SU-8 photoresist 303 is 30-50 microns, which corresponds to the thickness of the porous film. The second pre-baking temperature is 95 ° C and the time is 1-2 hours.

S205: Fix the mask having a circular pattern corresponding to the through hole on the porous film on the surface of the substrate with two layers of photoresist, and the circular pattern is located in the exposed area of the first exposure;

In this step, the diameter of the circular pattern of the mask is 10 micrometers, and the pitch is 50 micrometers. Corresponding to the through holes on the porous film, the circular pattern of the mask is in the first exposure area.

S206: The light source vertically irradiates the glass or silicon wafer with a mask and a photoresist for a second exposure, and performs post-baking;

After the photoresist is exposed twice under ultraviolet light, it is exposed into a plurality of cylindrical 303a, and the cylindrical 303a is used to prepare through holes in the porous film.

S207: After the natural cooling, use a developing solution to remove the unexposed photoresist to form a porous film template.

In this step, the prepared porous membrane template has a structure complementary to that of the porous membrane, and the porous membrane template prepares a porous membrane.

As shown in FIG. 7, step S300 (preparing a porous membrane and sealing assembly) includes the following steps:

S301: Mix PDMS monomer and cross-linking agent to make PDMS pad;

After the PDMS monomer and the cross-linking agent are mixed in a ratio of 15: 1, the air bubbles are removed in a vacuum, poured into a mold, and cured in an oven at 80 ° C. for 2-4 hours to prepare a PDMS pad having a thickness of 5 mm.

S302: performing silanization modification on the surface of the porous membrane template and the PDMS pad;

In this step, trimethylsilane is used to perform silanization modification on the surface of the porous membrane template and the PDMS pad.

S303: spin-coated uncrosslinked PDMS on the porous membrane template after silanization modification;

In this step, the thickness of the uncoated PDMS by spin coating is 30-50 microns.

S304: Place the PDMS pad after silanization modification on a porous membrane template spin-coated with PDMS, and place a static weight on the PDMS pad;

In this step, the weight of the heavy object is 3-6 kg, and the static pressure of the heavy object is 8-12 hours, for example, it is left to stand overnight at room temperature.

S305: heating the porous membrane template, the PDMS pad and the weight together, and curing to form a porous membrane;

In this step, the heating temperature is 80 ° C. and the time is 2-4 hours.

S306: Remove the weight, remove the porous membrane from the porous membrane template, and attach the porous membrane to the PDMS pad;

S307: Align and seal the upper chip and the porous membrane on the PDMS pad;

S308: peel the porous membrane and the upper chip from the PDMS pad together;

S309: Align and seal the lower chip and the porous membrane together to form a bionic intestinal organ chip.

The method for preparing a bionic intestinal organ chip provided in this embodiment mainly uses a soft photolithography technique to prepare a bionic intestinal organ chip. The preparation efficiency is high, and SU-8 photoresist is used for the preparation, which can prepare a chip with high structural accuracy. .

Example three:

This embodiment provides a method for preparing a bionic intestinal organ. The preparation method is performed by using the bionic intestinal organ chip described in Example 1. This method is an application of the bionic intestinal organ chip.

This embodiment constructs a three-dimensional intestinal micro-tissue in a bionic intestinal organ chip and performs dynamic culture, as shown in FIG. 8, which specifically includes the following steps:

S401: sterilize the bionic intestinal organ chip;

Using 70% alcohol and ultraviolet rays to sterilize the bionic intestinal organ chip described in Example 1;

S402: Inject extracellular matrix solution into the fluid channel, modify the porous membrane, and clean the fluid channel after modification;

Inject extracellular matrix (type I collagen, matrigel, matrigel, etc.) solution into the channel from the entrance of the upper chip to modify the PDMS porous membrane. After the modification, wash the channel with serum-free medium or PBS;

S403: Inject the intestinal cell suspension into the upper fluid channel, and let it stand for a certain period of time, so that the intestinal cells adhere to the modified porous membrane;

The intestinal cells (Caco-2) was inoculated in accordance with a concentration of 10 ⁶ / ml into the upper layer of the fluid passage within the chip, still more 2 hours after the porous film is grown with the modified cell adhesion;

S404: The culture medium is continuously injected into the fluid channel at a certain flow rate. After several days of incubation, the intestinal cells are mature and differentiated. The intestinal cells attached to the porous membrane protrusions will mimic the structure of intestinal villi and form three-dimensional intestinal microstructures. .

A syringe pump is used to inject the culture medium into the upper fluid channel and the lower fluid channel of the chip to realize the dynamic culture of perfusion of intestinal cells. After 5-7 days of culture, the intestinal cells (Caco-2) will mature and attach to the three-dimensional protrusions. The structural intestinal cells (Caco-2) will mimic the structure of intestinal villi, forming bionic three-dimensional intestinal microstructures, and can be applied to related research work.

The above uses specific examples to illustrate the present invention, but is only used to help understand the present invention, and is not intended to limit the present invention. For those skilled in the art to which the present invention pertains, according to the idea of the present invention, several simple deductions, deformations, or replacements can also be made.

Claims

A bionic intestinal organ chip has a fluid channel, and a porous membrane is provided in the fluid channel. The porous membrane separates the fluid channel into an upper fluid channel and a lower fluid channel. The porous membrane is characterized in that: There are several protrusions used to simulate the structure of intestinal villi and several through holes used to simulate the intestinal absorption function.
The bionic intestinal organ chip according to claim 1, wherein a plurality of said protrusion arrays are distributed on said porous membrane, and a plurality of said through holes are evenly distributed in a region outside said protrusions .
The bionic intestinal organ chip according to claim 2, wherein the protrusion is a conical or circular truncated structure.
The bionic intestinal organ chip according to claim 1, wherein the upper fluid passage and the lower fluid passage have a length of 10-15 mm, a width of 1-1.5 mm, and a height of 0.3-0.5 mm; The thickness of the porous film is 30-50 microns.
The bionic intestinal organ chip according to claim 4, wherein the diameter of the bottom surface of the protrusion is 100-200 microns, the height is 150-200 microns, and the pitch is 150-200 microns.
The bionic intestinal organ chip according to claim 4, wherein a diameter of the through holes is 10 micrometers, and a distance between the through holes is 50 micrometers.
The bionic intestinal organ chip according to claim 1, comprising an upper chip and a lower chip, wherein a lower surface of the upper chip has an open upper fluid channel, and an upper surface of the lower chip has an open upper fluid. Channel; the porous membrane is sealed between the upper chip and the lower chip, surrounding the upper fluid channel and the lower fluid channel; positions on the upper chip corresponding to both ends of the upper fluid channel and with the lower fluid channel Manholes are punched in the corresponding positions at the two ends.
The bionic intestinal organ chip according to claim 7, wherein the upper chip, the lower chip and the porous membrane having a convex structure are all made of PDMS material.
A method for preparing a bionic intestinal organ chip, which is used to prepare the bionic intestinal organ chip according to any one of claims 1 to 8, which comprises the following steps:

Preparing the upper chip and the lower chip;

Preparing a porous membrane template;

Preparing porous membrane and sealing assembly: preparing a porous membrane through a porous membrane template, and sealing the porous membrane between the upper chip and the lower chip to form a bionic intestinal organ chip;

Wherein, a plurality of protrusions for simulating the structure of intestinal villi and a plurality of through holes for simulating the intestinal absorption function are distributed on the porous membrane.
The method according to claim 9, wherein the preparing the upper chip and the lower chip comprises the following steps:

Spin-coat photoresist on the substrate surface of glass or silicon wafer and perform pre-baking;

Fixing the mask with the structure pattern of the upper and lower fluid channels on the surface of the substrate with the photoresist attached;

The light source vertically irradiates the glass or silicon wafer with a mask and a photoresist for exposure and post-baking;

After being naturally cooled, the developing solution is used to remove the unexposed photoresist to form a template with a structure of upper and lower fluid channels, and a hard film is formed;

An upper layer chip and a lower layer chip made of PDMS material are prepared through a template having an upper and lower fluid channel structure.
The method of claim 10, wherein the thickness of the photoresist is 300-500 microns.
The preparation method according to claim 10, wherein the pre-baking temperature is 95 ° C and the time is 2-8 hours.
The preparation method according to claim 10, wherein the post-baking temperature is 95 ° C and the time is 10-30 minutes.
The method of claim 10, wherein the temperature of the hard film is 180 ° C and the time is 2 hours.
The method according to claim 9, wherein the preparing a porous membrane template comprises the following steps:

Spin-coat photoresist on the substrate surface of glass or silicon wafer, and perform the first pre-baking;

Fixing a mask having a circular array pattern corresponding to the protrusions on the porous film on the surface of the substrate with the photoresist attached;

Fix the glass or silicon wafer with mask and photoresist on a rotatable and tiltable platform, and place it under a vertical light source for the first exposure;

Remove the mask, spin-coat the photoresist on the exposed photoresist, and perform a second pre-bake;

Fixing a mask having a circular pattern corresponding to the through hole on the porous film to the surface of the substrate with two layers of photoresist attached, and the circular pattern is located in an exposed area of the first exposure;

The light source vertically irradiates the glass or silicon wafer with a mask and a photoresist for a second exposure and post-baking;

After being naturally cooled, a developing solution is used to remove the unexposed photoresist to form a porous film template.
The method according to claim 10 or 15, wherein the photoresist is a SU-8 photoresist, and the developing solution is ethyl lactate.
The method according to claim 10 or 15, wherein the light source is an ultraviolet light source.
The method according to claim 15, wherein the first exposure process is: placing a glass or silicon wafer with a mask and a photoresist on an adjustable tilt angle and a freely rotatable On the platform, adjust the tilt angle of the platform, so that the light source is tilted 15-45 ° to irradiate the glass or silicon wafer with the mask and photoresist for the first exposure. During the exposure process, the platform rotates along the normal direction of the table surface. 360 °.
The method according to claim 15, wherein the thickness of the photoresist is 150-200 microns.
The preparation method according to claim 15, wherein the first pre-baking temperature is 95 ° C and the time is 2-4 hours.
The method of claim 15, wherein the circular pattern on the mask has a diameter of 100-200 microns and a pitch of 150-200 microns.
The method of claim 15, wherein the thickness of the photoresist spin-coated again is 30-50 μm.
The preparation method according to claim 15, wherein the temperature of the second pre-baking is 95 ° C, and the time is 1-2 hours.
The method of claim 15, wherein the diameter of the circular pattern on the mask is 10 μm, and the pitch is 50 μm.
The preparation method according to claim 15, wherein the post-baking temperature is 95 ° C and the time is 10-30 minutes.
The method according to claim 9, wherein the preparing a porous membrane and sealing and assembling comprise the following steps:

Mix PDMS monomer with cross-linking agent to make PDMS pad;

Performing silanization modification on the surface of the porous membrane template and the PDMS pad;

Spin-coated uncrosslinked PDMS on the silanized modified porous membrane template;

Place the silanized PDMS pad on a porous membrane template spin-coated with PDMS, and place a heavy object on the PDMS pad to press it statically;

The porous membrane template, the PDMS pad and the weight are heated together and cured to form a porous membrane;

Remove the weight and peel off the porous membrane from the porous membrane template, which is attached to the PDMS pad;

Align and seal the upper fluid channel structure with the porous membrane on the PDMS pad;

Remove the porous membrane and the upper fluid channel structure from the PDMS pad together;

The lower fluid channel structure and the porous membrane are aligned and sealed together to form a bionic intestinal organ chip.
The preparation method according to claim 26, wherein the mixing ratio of the PDMS monomer and the crosslinking agent is 15: 1.
The preparation method according to claim 26, wherein after mixing the PDMS monomer and the cross-linking agent, remove bubbles in a vacuum, pour it into a mold, and place it in an 80 ° C oven for 2-4 hours for curing to make a thickness 5mm PDMS pad.
The method of claim 26, wherein the thickness of the spin-coated uncrosslinked PDMS is 30-50 microns.
The method according to claim 26, wherein the weight of the weight is 3-6 kg, and the static pressure time of the weight is 8-12 hours.
The preparation method according to claim 26, wherein the heating temperature is 80 ° C and the time is 2-4 hours.
The method according to claim 26, wherein the sealing method uses plasma treatment and bonding.
A method for preparing a bionic intestinal organ, which is performed by using the bionic intestinal organ chip according to any one of claims 1 to 8.
The method according to claim 33, comprising the steps of:

Sterilizing the bionic intestinal organ chip according to any one of claims 1 to 8;

Inject extracellular matrix solution into the fluid channel, modify the porous membrane, and clean the fluid channel after modification;

Inject the intestinal cell suspension into the upper fluid channel and let it stand for a certain period of time to make the intestinal cells adhere to the modified porous membrane;

The medium is then continuously injected into the upper and lower fluid channels at a certain flow rate. After several days of incubation, the intestinal cells are mature and differentiated. The intestinal cells attached to the porous membrane protrusions will mimic the structure of intestinal villi, forming a three-dimensional intestinal microstructure organization.
The method according to claim 34, wherein the bionic intestinal organ chip is sterilized by using 70% alcohol and ultraviolet light.
The method according to claim 34, wherein the extracellular matrix solution is type I collagen or matrigel solution.
The method according to claim 34, wherein the fluid channel is washed with serum-free medium or PBS after modification.
The method according to claim 34, wherein the intestinal cell concentration is 10 6 cells / ml.
The method according to claim 34, wherein the intestinal cell suspension is allowed to stand for more than two hours after being injected into the fluid channel.
The method according to claim 34, wherein the culture medium is continuously injected into the upper and lower fluid channels at a certain flow rate, and the intestinal cells are cultured for 5-7 days.