CN111971383B - Bionic intestinal organ chip and preparation method and application thereof - Google Patents
Bionic intestinal organ chip and preparation method and application thereof Download PDFInfo
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- CN111971383B CN111971383B CN201880092368.1A CN201880092368A CN111971383B CN 111971383 B CN111971383 B CN 111971383B CN 201880092368 A CN201880092368 A CN 201880092368A CN 111971383 B CN111971383 B CN 111971383B
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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
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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
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Abstract
The application discloses bionic intestinal organ chip and a preparation method and application thereof, the bionic intestinal organ chip is provided with a fluid channel, a porous membrane is arranged in the fluid channel, the fluid channel is divided into an upper fluid channel and a lower fluid channel by the porous membrane, and a plurality of bulges used for simulating intestinal villus structures and a plurality of through holes used for simulating intestinal absorption functions are distributed on the porous membrane. The upper fluid channel is used for culturing intestinal cells, the lower fluid channel is used for collecting metabolites, the protrusions on the porous membrane are used for simulating an intestinal villus structure, and the through holes are used for simulating the absorption function of the intestinal tract. Because the porous membrane in the chip is distributed with a plurality of raised three-dimensional support structures, the difference of intestinal tract tissue structures generated among different batches of chips can be greatly reduced, and the chip has good reproducibility. The bionic intestinal organ chip can dynamically culture intestinal cells by perfusion so as to simulate a dynamic microenvironment in the intestinal tract, and is suitable for researches on intestinal diseases, drug screening, food safety and the like.
Description
Technical Field
The application relates to the technical field of organ bionics, in particular to a bionic intestinal organ chip and a preparation method and application thereof.
Background
The microfluidic chip technology (Microfluidics) is one of important advanced science technologies in the 21 st century, and provides an important platform for simulating a human body metabolism model in vitro. The system is mainly based on a micro-nano processing technology, a network is formed by micron-sized channels, and a controllable fluid penetrates through the whole system, so that the conventional functions of biology and chemical laboratories can be realized. Because the micro-scale micro-component has the micron-scale component matched with the size of the cells, various cell culture and fluid stimulation can be carried out in the micro-channel of the chip, and a three-dimensional microenvironment which is close to the physiological environment and has the characteristic of space-time resolution is constructed, so that the micro-scale micro-component becomes an important technology for tissue organ construction, drug screening, toxicology and biomedical research. At present, the microfluidic technology has been successfully applied to three-dimensional cell co-culture, cell migration, cell sorting, tissue microenvironment, organoid construction and the like. Among them, the human organ chip (organ-on-a-chip) is an emerging leading-edge interdisciplinary technology developed in recent years, and is a bionic system capable of simulating main functions of human Organs and manufactured on a micro-fluidic chip by using a micro-processing technology. Compared with the traditional two-dimensional static cell culture technology, the cells cultured in the chip have a three-dimensional structure and a space distribution structure of various cells, and more importantly, the organ chip can provide a dynamic microenvironment for the cells, which is incomparable with the traditional means. In addition, most of cells in the chip are based on human cells, so that the interspecies difference generated by an animal model can be greatly reduced. The development of organ chips will contribute to drug development, disease research, and the like.
Currently, the design and fabrication of enterochips is mainly focused on professor Ingber at harvard university, as shown in fig. 1. The chip is mechanically stretched by multiple caco-2 cells cultured on a porous membrane to differentiate and spontaneously form a villus structure.
Specifically, the existing intestinal chip is formed by sealing three layers of PDMS (polydimethylsiloxane) structures, as shown in fig. 1, wherein the middle layer is a porous membrane 101 for inoculating intestinal cells; the upper layer and the lower layer are fluid channels 02 for simulating intestinal fluid microenvironment; the left and right sides of the channel are vacuum chambers 103 for mechanical movement of stretching the porous membrane.
However, the intestinal chip in the prior art has the disadvantages that the homogeneity of the hair structure formed by the cells spontaneously can not be guaranteed, so that the chip generates larger difference among different batches, and the statistics of experimental data and the comparison of results are inconvenient.
Disclosure of Invention
The application provides a bionic intestinal organ chip which can reduce the difference of intestinal tissue structures generated among 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 tract organ chip, which has a fluid channel, a porous membrane is arranged in the fluid channel, the porous membrane divides the fluid channel into an upper fluid channel and a lower fluid channel, and a plurality of protrusions for simulating an intestinal villus structure and a plurality of through holes for simulating an intestinal absorption function are distributed on the porous membrane.
According to a second aspect, an embodiment provides a method for preparing a bionic intestinal organ chip, comprising the following steps:
preparing an upper chip and a lower chip;
preparing a porous membrane template;
preparing a porous membrane and sealing and assembling: preparing a porous membrane through a porous membrane template, and sealing the porous membrane between an upper chip and a lower chip to form a bionic intestinal organ chip;
wherein, a plurality of bulges used for simulating an intestinal villus structure and a plurality of through holes used for simulating an 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 bionic intestinal organ chip.
According to the bionic intestinal organ chip and the preparation method and the application thereof of the embodiment, the plurality of protrusions and the through holes are distributed on the porous membrane, the upper fluid channel is used for culturing intestinal cells, the lower fluid channel is used for collecting metabolites, the protrusions on the porous membrane are used for simulating an intestinal villus structure, and the through holes are used for simulating the absorption function of intestinal tracts, so that the porous membrane forms a three-dimensional support structure, the difference of intestinal tract tissue structures generated among different batches of chips can be greatly reduced, and the bionic intestinal organ chip has good reproducibility. The bionic intestinal organ chip can dynamically culture intestinal cells by perfusion so as to simulate a dynamic microenvironment in the intestinal tract, and is suitable for researches on intestinal diseases, drug screening, food safety and the like.
Drawings
FIG. 1 is a schematic structural diagram of a bionic intestinal tract organ chip in the prior art;
FIG. 2 is a schematic structural diagram of a bionic intestinal tract chip according to a first embodiment;
FIG. 3 is a flow chart of the preparation of a bionic intestinal tract organ chip according to the second embodiment;
FIG. 4 is a flowchart of the preparation of the upper chip and the lower chip in example two;
FIG. 5 is a flow chart of the preparation of a porous membrane template in example two;
FIG. 6 is a schematic structural view of a porous membrane template in the process of preparation according to example two;
FIG. 7 is a flow chart of the preparation of a porous membrane and sealing assembly in example two;
fig. 8 is a flow chart of the preparation of the biomimetic intestinal organ in the third embodiment.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
The first embodiment is as follows:
the embodiment provides a bionic intestinal organ chip, and the bionic intestinal organ chip is of a three-layer structure, simulates a dynamic microenvironment in an intestinal tract, is used for dynamically culturing intestinal cells, and is suitable for researches such as intestinal diseases, drug screening and food safety.
As shown in fig. 2, a fluid channel is disposed in the bionic intestinal tract chip of the embodiment. Specifically, the chip includes an upper chip 210 and a lower 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, the porous membrane 230 is sealed between the upper chip 210 and the lower chip 220, the porous membrane 230 and the upper chip 210 enclose the upper fluid channel 211, and the porous membrane 230 and the lower chip 220 enclose the lower fluid channel 221. The porous membrane 230 is distributed with a plurality of protrusions 231 and through holes 232, the protrusions 231 are used for simulating intestinal villus structures, the through holes 232 are used for simulating intestinal absorption functions, and the through holes 232 conduct the upper fluid channel 211 and the lower fluid channel 221. The upper chip 210 has access holes opened at positions corresponding to both ends of the upper fluid channel 211, and the upper chip 210 has access holes opened 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 made of PDMS.
The protrusions 231 are in a cone or truncated cone structure, a plurality of protrusions 231 are distributed on the porous membrane 230 in an array, and a plurality of through holes 232 are uniformly distributed in the area outside the protrusions 231, i.e. the protrusions 231 and the through holes 232 are distributed on the porous membrane 230 together. The porous membrane 230 forms a three-dimensional scaffold to simulate the three-dimensional spatial structure in the intestine.
The upper fluid channel 211 and the lower fluid channel 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 porous membrane 230 has a thickness of 30-50 microns. The bottom surface diameter of the protrusion 231 is 100-. 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 channel 211 and the lower fluid channel 221 have a length of 15 mm, a width of 1 mm, and a height of 0.4 mm. The porous membrane 230 has a thickness of 40 microns. The protrusions 231 had a bottom surface with a diameter of 200 microns, a height of 150 microns and a pitch of 200 microns. In other embodiments, the dimensions of the various components may be selected within the ranges described above according to simulation needs.
According to the bionic intestinal organ chip provided by the embodiment, the plurality of protrusions 231 and the through holes 232 are distributed on the porous membrane 230, the upper fluid channel 211 is used for culturing intestinal cells, the lower fluid channel is used for collecting metabolites, the protrusions 231 on the porous membrane 230 are used for simulating an intestinal villus structure, and the through holes 232 are used for simulating an absorption function of an intestinal tract, so that the porous membrane 230 forms a three-dimensional support structure, the difference of intestinal tract tissue structures generated among different batches of chips can be greatly reduced, and the bionic intestinal organ chip has good reproducibility. The bionic intestinal organ chip can dynamically culture intestinal cells by perfusion so as to simulate a dynamic microenvironment in the intestinal tract, and is suitable for researches on intestinal diseases, drug screening, food safety and the like.
Example two:
the embodiment provides a preparation method of a bionic intestinal organ chip, and the preparation method mainly adopts a soft lithography technology to prepare the bionic intestinal organ chip in the first embodiment.
As shown in fig. 3, the method for preparing the bionic intestinal tract organ chip of the embodiment mainly comprises the following steps:
s100: preparing an upper chip and a lower chip;
s200: preparing a porous membrane template;
s300: preparing a porous membrane and sealing and assembling.
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 the bionic intestinal organ chip. Wherein, a plurality of bulges used for simulating the intestinal villus structure and a plurality of through holes used for simulating the intestinal absorption function are distributed on the produced porous membrane.
The preparation method comprises the steps of separating an organ chip into an upper chip and a lower chip, preparing the upper chip and the lower chip, preparing the porous membrane separately, and finally sealing the upper chip, the lower chip and the porous membrane together to form the three-layer chip with the upper and lower flow channels. In the method, the steps S100 and S200 are not in sequence, and can be prepared sequentially or simultaneously.
Specifically, as shown in fig. 4, the step S100 (preparing the upper chip and the lower chip) includes the following steps:
s101: spin-coating a photoresist on the surface of a substrate of a glass or silicon wafer, and pre-baking;
in the embodiment, the SU-8 photoresist is preferably selected, the SU-8 photoresist is an epoxy type negative photoresist for near ultraviolet light, and the SU-8 photoresist has low light absorption rate in the near ultraviolet light range, so that the photoresist has good exposure uniformity in the thickness, and a structure with nearly vertical pattern edges can be obtained.
The mechanism of SU-8 photoresist lithography is as follows: the photoinitiator in the photoresist absorbs photons to perform a chemical reaction to produce a strong acid which acts as an acid catalyst to promote the crosslinking reaction during the pre-baking process. The strong acid is generated only in the photoresist in the exposure region, so that the crosslinking reaction is generated only in the exposure region, the unexposed region does not generate the crosslinking reaction, the photoresist is insoluble in a developing solution after the crosslinking reaction, and the photoresist is soluble in the developing solution without the crosslinking reaction, so that 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-. The temperature of the pre-baking is 95 ℃ and the time is 2-8 hours.
S102: fixing a mask with upper and lower fluid channel structure patterns on the surface of the substrate attached with the photoresist;
the mask has a pattern of upper and lower fluid channel structures, and is used to block ultraviolet light, thereby replicating the pattern on the mask onto the SU-8 photoresist.
S103: vertically irradiating glass or silicon wafer attached with a mask and photoresist by a light source for exposure, and post-baking;
the light emitted by the light source irradiates the SU-8 photoresist through the pattern on the mask, the exposed area of the SU-8 photoresist is crosslinked, and the crosslinked area is insoluble in a developing solution. The light sources in this embodiment are all ultraviolet light sources, and are used for emitting ultraviolet light for exposure.
In the step, the post-baking temperature is 95 ℃ and the time is 10-30 minutes.
S104: after natural cooling, removing unexposed photoresist by adopting ethyl lactate developing solution to form a template with an upper and lower fluid channel structure, and hardening;
in the step, the unexposed SU-8 photoresist is removed by the developing solution, the developed SU-8 photoresist forms a pattern structure opposite to the mask, and then the film is hardened at the temperature of 180 ℃ for 2 hours.
S105: and preparing an upper chip and a lower chip made of PDMS materials by using the template with the upper and lower fluid channel structures.
In the step, an upper chip and a lower chip made of PDMS are prepared through a photoresist template corresponding to an upper fluid channel structure and a lower fluid channel structure, holes are punched at two ends of the upper chip fluid channel, holes are punched at the positions corresponding to the inlet and the outlet of the lower fluid channel, and the lower chip is not punched. And finally, preparing an upper chip and a lower chip.
As shown in fig. 5 and 6, the step S200 (a method of preparing a porous membrane template) includes the steps of:
s201: spin-coating a photoresist on the surface of a substrate of a glass or silicon wafer, and performing primary prebaking;
in this step, SU-8 photoresist 302 with a thickness of 150-.
S202: fixing a mask having a circular array pattern corresponding to the protrusions on the porous film on the surface of the substrate attached with the photoresist;
the diameter of the circular array pattern on the mask is 100-200 microns, the distance is 150-200 microns, the circular array pattern corresponds to the bulges on the porous membrane, and the circular array pattern is used for preparing the template of the bulges on the porous membrane.
S203: fixing the glass or silicon chip attached with the mask and the photoresist on a rotatable and inclinable platform, and placing the platform under a vertical light source for first exposure;
in the step, the glass or silicon wafer attached with the photoresist is fixed on a platform, and the platform can adjust the inclination angle and can freely rotate and is used for adjusting the illumination angle of exposure. Before exposure, the platform is inclined by 15-45 degrees, so that ultraviolet light is inclined by 15-45 degrees to expose the SU-8 photoresist 302, in the exposure process, the platform rotates 360 degrees along the normal direction of the table, the exposed SU-8 photoresist 302 is provided with an exposed area 302a and an unexposed area 302b, and the unexposed area 302b is used for preparing the protrusion on the porous membrane.
S204: removing the mask, spin-coating photoresist on the exposed photoresist, and performing secondary prebaking;
after the first exposure, SU-8 photoresist 303 is spin coated on the SU-8 photoresist 302, and the thickness of the SU-8 photoresist 303 is 30-50 μm, which corresponds to the thickness of the porous film. The temperature of the second pre-drying is 95 ℃ and the time is 1-2 hours.
S205: fixing a mask with a circular pattern corresponding to the through hole on the porous membrane on the surface of the substrate attached with the two layers of photoresist, wherein the circular pattern is positioned in an exposure area of the first exposure;
in this step, the circular pattern of the mask, which has a diameter of 10 micrometers and a pitch of 50 micrometers, corresponds to the through holes on the porous film, and is in the first exposure region.
S206: vertically irradiating the glass or silicon wafer attached with the mask and the photoresist by a light source for secondary exposure, and post-baking;
after the photoresist is exposed for the second time by ultraviolet light, a plurality of cylindrical shapes 303a are formed by exposure, and the cylindrical shapes 303a are used for preparing through holes on the porous membrane.
S207: and after natural cooling, removing the unexposed photoresist by adopting a developing solution to form the porous membrane template.
In this step, the prepared porous membrane template has a structure complementary to the porous membrane, and the porous membrane template prepares the porous membrane.
As shown in fig. 7, step S300 (preparing a porous membrane and sealing assembly) includes the following steps:
s301: mixing a PDMS monomer and a cross-linking agent to prepare a PDMS cushion plate;
mixing PDMS monomer and cross-linking agent according to the ratio of 15:1, removing air bubbles in vacuum, pouring into a mould, and curing in an oven at 80 deg.C for 2-4 hr to obtain a PDMS pad with a thickness of 5 mm.
S302: performing silanization modification on the surfaces of the porous membrane template and the PDMS base plate;
in the step, trimethylsilane is used for silanization modification on the surfaces of the porous membrane template and the PDMS base plate.
S303: spin-coating uncrosslinked PDMS on the silanized and modified porous membrane template;
in this step, the thickness of the spin-coated uncrosslinked PDMS was 30-50 μm.
S304: placing the PDMS base plate which is subjected to silanization modification on a porous membrane template which is spin-coated with PDMS, and placing a weight on the PDMS base plate for static pressure;
in this step, the weight is 3-6kg, and the weight is statically pressed for 8-12 hours, for example, left to stand overnight at room temperature.
S305: heating the porous membrane template, the PDMS base plate and the weight together, and curing to prepare a porous membrane;
in the step, the heating temperature is 80 ℃ and the heating time is 2-4 hours.
S306: removing the weight, and uncovering the porous membrane from the porous membrane template, wherein the porous membrane is attached to the PDMS base plate;
s307: aligning and sealing the upper chip and the porous membrane on the PDMS backing plate together;
s308: peeling the porous membrane and the upper chip together from the PDMS backing plate;
s309: and aligning and sealing the lower chip and the porous membrane together to form the bionic intestinal organ chip.
The preparation method of the bionic intestinal tract organ chip provided by the embodiment mainly adopts the soft lithography technology to prepare the bionic intestinal tract organ chip, the preparation efficiency is high, and the chip with high structure precision can be prepared by selecting SU-8 photoresist for preparation.
Example three:
the embodiment provides a method for preparing a bionic intestinal organ, which is carried out by the bionic intestinal organ chip described in the embodiment I, and the method is an application of the bionic intestinal organ chip.
In this embodiment, a three-dimensional intestinal micro-tissue is constructed in a bionic intestinal organ chip and dynamically cultured, as shown in fig. 8, which specifically includes the following steps:
s401: sterilizing the bionic intestinal organ chip;
sterilizing the bionic intestinal organ chip of the first embodiment by using 70% alcohol and ultraviolet rays respectively;
s402: injecting an extracellular matrix solution into the fluid channel, modifying the porous membrane, and cleaning the fluid channel after modification;
injecting an extracellular matrix (type I collagen, matrix gelatin and the like) solution into the channel from the inlet of the upper chip to modify the PDMS porous membrane, and then washing the channel by using a serum-free culture medium or PBS; .
S403: injecting the intestinal cell suspension into the upper fluid channel, and standing for a certain time to make the intestinal cells adhere to the modified porous membrane;
intestinal cells (Caco-2) were treated as 106Inoculating the cells/ml into an upper fluid channel in the chip, standing for more than 2 hours to ensure that the cells are adhered to and grow on the modified porous membrane;
s404: and then continuously injecting the culture medium into the fluid channel according to a certain flow rate, after culturing for a plurality of days, differentiating and maturing the intestinal cells, and enabling the intestinal cells attached to the porous membrane protrusions to have a simulated intestinal villus structure to form a three-dimensional intestinal tract micro-tissue.
The culture medium is injected into the upper fluid channel and the lower fluid channel of the chip by using an injection pump to realize dynamic culture of perfusion on intestinal cells, the intestinal cells (Caco-2) are differentiated and matured after 5-7 days of culture, the intestinal cells (Caco-2) attached to the three-dimensional bulge structure have a simulated intestinal villus structure to form a bionic three-dimensional intestinal micro-tissue, and the bionic three-dimensional intestinal micro-tissue can be applied to related research work.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (39)
1. A bionic intestinal organ chip is provided with a fluid channel, a porous membrane is arranged in the fluid channel, and the porous membrane divides the fluid channel into an upper fluid channel and a lower fluid channel.
2. The biomimetic intestinal organ chip of claim 1, wherein the protrusions are in a cone or truncated cone structure.
3. The biomimetic intestinal organ chip according to claim 1, wherein the upper fluid channel and the lower fluid channel have a length of 10-15 mm, a width of 1-1.5 mm, and a height of 0.3-0.5 mm; the porous membrane has a thickness of 30-50 microns.
4. The bionic intestinal tract organ chip as claimed in claim 3, wherein the bottom surface diameter of the protrusion is 100-.
5. The biomimetic intestinal organ chip according to claim 3, wherein the through holes have a diameter of 10 microns and a pitch between the through holes is 50 microns.
6. The biomimetic 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 to form the upper fluid channel and the lower fluid channel, and the positions of the upper chip corresponding to the two ends of the upper fluid channel and the positions of the upper chip corresponding to the two ends of the lower fluid channel are respectively provided with an access hole.
7. The biomimetic intestinal organ chip of claim 6, wherein the upper chip, the lower chip and the porous membrane with the protrusion structure are all made of PDMS.
8. A method for preparing a bionic intestinal organ chip according to any one of claims 1 to 7, comprising the steps of:
preparing an upper chip and a lower chip;
preparing a porous membrane template;
preparing a porous membrane and sealing and assembling: 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 bulges used for simulating an intestinal villus structure and a plurality of through holes used for simulating an intestinal absorption function are distributed on the porous membrane.
9. The method of claim 8, wherein the preparing the upper chip and the lower chip comprises the steps of:
spin-coating a photoresist on the surface of a substrate of a glass or silicon wafer, and pre-baking;
fixing a mask with upper and lower fluid channel structure patterns on the surface of the substrate attached with the photoresist;
vertically irradiating glass or silicon wafer attached with a mask and photoresist by a light source for exposure, and post-baking;
after natural cooling, removing the unexposed photoresist by adopting a developing solution to form a template with an upper and lower fluid channel structure, and hardening;
and preparing an upper chip and a lower chip made of PDMS materials through a template with an upper fluid channel structure and a lower fluid channel structure.
10. The method as claimed in claim 9, wherein the photoresist has a thickness of 300-500 μm.
11. The method of claim 9, wherein the pre-baking is at a temperature of 95 ℃ for a period of 2 to 8 hours.
12. The method of claim 9, wherein the post-baking is at a temperature of 95 ℃ for a time of 10 to 30 minutes.
13. The method of claim 9, wherein the hardening temperature is 180 ℃ and the hardening time is 2 hours.
14. The method of claim 8, wherein the preparing a porous membrane template comprises the steps of:
spin-coating a photoresist on the surface of a substrate of a glass or silicon wafer, and performing primary prebaking;
fixing a mask having a circular array pattern corresponding to the protrusions on the porous film on the surface of the substrate attached with the photoresist;
fixing the glass or silicon chip with the mask and the photoresist on a platform which can rotate and be obliquely adjusted, and placing the platform under a vertical light source for first exposure;
removing the mask, spin-coating photoresist on the exposed photoresist, and performing secondary prebaking;
fixing a mask with a circular pattern corresponding to the through hole on the porous membrane on the surface of the substrate attached with the two layers of photoresist, wherein the circular pattern is positioned in an exposure area of the first exposure;
vertically irradiating the glass or silicon wafer attached with the mask and the photoresist by a light source for secondary exposure, and post-baking;
and after natural cooling, removing the unexposed photoresist by adopting a developing solution to form the porous membrane template.
15. The production method according to claim 9 or 14, wherein the resist is SU-8 resist, and the developing solution is ethyl lactate.
16. The method of claim 9 or 14, wherein the light source is an ultraviolet light source.
17. The method according to claim 14, wherein the first exposure comprises: the glass or silicon chip attached with the mask and the photoresist is placed on a platform which can adjust the inclination angle and can rotate freely, the inclination angle of the platform is adjusted, so that a light source irradiates the glass or silicon chip attached with the mask and the photoresist at an inclination angle of 15-45 degrees for first exposure, and in the exposure process, the platform rotates 360 degrees along the normal direction of the table board.
18. The method as claimed in claim 14, wherein the photoresist has a thickness of 150-200 μm.
19. The method of claim 14, wherein the first prebaking temperature is 95 ℃ for 2-4 hours.
20. The method as claimed in claim 14, wherein the diameter of the circular pattern on the mask is 100-200 μm, and the pitch is 150-200 μm.
21. The method of claim 14, wherein the spin-on photoresist has a thickness of 30 to 50 microns.
22. The method of claim 14, wherein the second pre-bake is at a temperature of 95 ℃ for a period of 1 to 2 hours.
23. The method of claim 14, wherein the circular pattern on the mask has a diameter of 10 microns and a pitch of 50 microns.
24. The method of claim 14, wherein the post-baking is at a temperature of 95 ℃ for a time of 10 to 30 minutes.
25. The method of claim 8, wherein the steps of preparing the porous membrane and sealing the assembly comprise:
mixing a PDMS monomer and a cross-linking agent to prepare a PDMS cushion plate;
performing silanization modification on the surfaces of the porous membrane template and the PDMS base plate;
spin-coating uncrosslinked PDMS on the silanized and modified porous membrane template;
placing the PDMS base plate which is subjected to silanization modification on a porous membrane template which is spin-coated with PDMS, and placing a weight on the PDMS base plate for static pressure;
heating the porous membrane template, the PDMS base plate and the weight together, and curing to prepare a porous membrane;
removing the weight, and uncovering the porous membrane from the porous membrane template, wherein the porous membrane is attached to the PDMS backing plate;
aligning and sealing the upper fluid channel structure with the porous membrane on the PDMS backing plate;
peeling the porous membrane and the upper fluid channel structure together from the PDMS backing plate;
and aligning and sealing the lower-layer fluid channel structure and the porous membrane together to form the bionic intestinal organ chip.
26. The method of claim 25, wherein the PDMS monomer and the cross-linking agent are mixed at a ratio of 15: 1.
27. The method of claim 26, wherein the PDMS monomer is mixed with the cross-linking agent, vacuumed to remove air bubbles, poured into a mold, and cured in an oven at 80 ℃ for 2-4 hours to form a PDMS shim plate with a thickness of 5 mm.
28. The method of claim 25, wherein the spin-on uncrosslinked PDMS has a thickness of 30-50 microns.
29. The method of claim 25, wherein the weight is 3 to 6kg and the weight is statically pressed for 8 to 12 hours.
30. The method of claim 25, wherein the heating is at a temperature of 80 ℃ for a period of 2 to 4 hours.
31. The method of claim 25, wherein the sealing is performed by plasma treatment and bonding.
32. A method for preparing a biomimetic intestinal organ, characterized in that the method is performed by using the biomimetic intestinal organ chip according to any one of claims 1 to 7.
33. The method of claim 32, comprising the steps of:
sterilizing the biomimetic intestinal organ chip according to any one of claims 1 to 7;
injecting an extracellular matrix solution into the fluid channel, modifying the porous membrane, and cleaning the fluid channel after modification;
injecting the intestinal cell suspension into the upper fluid channel, and standing for a certain time to make the intestinal cells adhere to the modified porous membrane;
and then continuously injecting the culture medium into the upper layer fluid channel and the lower layer fluid channel at a certain flow rate, culturing for a plurality of days, wherein the intestinal cells are differentiated and mature, and the intestinal cells attached to the porous membrane protrusions have a simulated intestinal villus structure to form a three-dimensional intestinal micro-tissue.
34. The method of claim 33, wherein the bionic intestinal organ chip is sterilized by using alcohol with a concentration of 70% and ultraviolet rays.
35. The method of claim 33, wherein the extracellular matrix solution is collagen type I or matrigel solution.
36. The method of claim 33, wherein the fluid channel is washed with serum-free medium or PBS after the modifying.
37. The method of claim 33, wherein the intestinal cell concentration is 106One per ml.
38. The method of claim 33, wherein the suspension of intestinal cells is allowed to rest for more than two hours after injection into the fluid passageway.
39. The method of claim 33, wherein the intestinal cells are incubated for 5-7 days with continuous injection of culture medium into the upper and lower fluid passageways at a flow rate.
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| CN113088452A (en) * | 2021-04-25 | 2021-07-09 | 复旦大学附属中山医院 | Aorta organoid chip, preparation method, chip system and application |
| CN113814010B (en) * | 2021-08-30 | 2022-10-11 | 复旦大学 | Multi-cell and multi-tissue co-culture bionic micro-fluidic chip and preparation method thereof |
| CN113926498B (en) * | 2021-11-04 | 2022-11-29 | 田甜 | Preparation method of laminar flow low-shear force micro-fluidic chip capable of promoting brain-like organ maturation |
| CN114107175B (en) * | 2021-11-22 | 2024-01-02 | 北京大学 | Organoid culture method for inducing growth of intestinal organoids by mechanical stretching |
| CN114196537A (en) * | 2021-11-30 | 2022-03-18 | 北京大学 | Uniaxial cell stretching chip with topological structure and preparation method thereof |
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