WO2019222870A1 - 一种仿生肠道器官芯片及其制备方法和应用 - Google Patents

一种仿生肠道器官芯片及其制备方法和应用 Download PDF

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WO2019222870A1
WO2019222870A1 PCT/CN2018/087598 CN2018087598W WO2019222870A1 WO 2019222870 A1 WO2019222870 A1 WO 2019222870A1 CN 2018087598 W CN2018087598 W CN 2018087598W WO 2019222870 A1 WO2019222870 A1 WO 2019222870A1
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chip
porous membrane
intestinal
photoresist
fluid channel
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PCT/CN2018/087598
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English (en)
French (fr)
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魏文博
陈娟娟
肖亮
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深圳华大生命科学研究院
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Priority to PCT/CN2018/087598 priority Critical patent/WO2019222870A1/zh
Priority to CN201880092368.1A priority patent/CN111971383B/zh
Publication of WO2019222870A1 publication Critical patent/WO2019222870A1/zh

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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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/16—Apparatus for enzymology or microbiology containing, or adapted to contain, solid media
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • protrusions used to simulate the structure of intestinal villi and several through holes used to simulate the intestinal absorption function.
  • an embodiment provides a method for preparing a bionic intestinal organ chip, including the following steps:
  • 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;
  • 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.
  • an embodiment provides a method for preparing a bionic intestinal organ, which is performed by using the above-mentioned bionic intestinal organ chip.
  • the bionic intestinal organ chip 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.
  • FIG. 1 is a schematic structural diagram of a bionic intestinal organ chip in the prior art
  • FIG. 2 is a schematic structural diagram of a bionic intestinal organ chip according to the first embodiment
  • Example 3 is a flowchart of preparing a bionic intestinal organ chip in Example 2.
  • Example 5 is a flowchart of preparing a porous membrane template in Example 2.
  • FIG. 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.
  • 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.
  • the bionic intestinal organ chip in this embodiment is provided with a fluid channel.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m, a height of 150 ⁇ m, and a pitch of 200 ⁇ m. In other embodiments, the size of each component can be selected within the above range according to the needs of the simulation.
  • 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.
  • 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.
  • the method for preparing the bionic intestinal organ chip of this embodiment mainly includes the following steps:
  • 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.
  • the produced porous membrane has several protrusions for simulating the structure of intestinal villi and several through holes for simulating the intestinal absorption function.
  • 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.
  • steps S100 and S200 are not sequential and can be prepared in sequence or simultaneously.
  • 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;
  • 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.
  • 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.
  • 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.
  • the post-baking temperature is 95 ° C and the time is 10-30 minutes.
  • 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.
  • 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.
  • 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
  • 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.
  • 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;
  • 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.
  • first tilt the platform by 15-45 ° so that the UV light is tilted by 15-45 ° to expose the SU-8 photoresist 302.
  • 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;
  • 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;
  • the diameter of the circular pattern of the mask is 10 micrometers, and the pitch is 50 micrometers.
  • the circular pattern of the mask is in the first exposure area.
  • the light source vertically irradiates the glass or silicon wafer with a mask and a photoresist for a second exposure, and performs post-baking;
  • the photoresist 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.
  • the prepared porous membrane template has a structure complementary to that of the porous membrane, and the porous membrane template prepares a porous membrane.
  • step S300 (preparing a porous membrane and sealing assembly) includes the following steps:
  • 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.
  • trimethylsilane is used to perform silanization modification on the surface of the porous membrane template and the PDMS pad.
  • the thickness of the uncoated PDMS by spin coating is 30-50 microns.
  • 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.
  • the heating temperature is 80 ° C. and the time is 2-4 hours.
  • the method for preparing a bionic intestinal organ chip 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. .
  • 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
  • S402 Inject extracellular matrix solution into the fluid channel, modify the porous membrane, and clean the fluid channel after modification;
  • 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.
  • 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.

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Abstract

本申请公开了一种仿生肠道器官芯片及其制备方法和应用，仿生肠道器官芯片具有流体通道，在流体通道内设有多孔膜，多孔膜将流体通道分隔成上层流体通道和下层流体通道，多孔膜上分布有若干个用于模拟肠绒毛结构的凸起和若干个用于模拟肠道吸收功能的通孔。上层流体通道用于培养肠细胞，下层用于收集代谢产物，多孔膜上的凸起用于模拟肠绒毛结构，通孔用于模拟肠道的吸收功能。由于该芯片内的多孔膜上分布有若干个凸起的三维支架结构，可极大减小不同批次芯片之间产生的肠道组织结构的差异，具有良好的重现性。本仿生肠道器官芯片可利用灌流对肠细胞进行动态培养，以模拟肠道内动态微环境，适用于肠道疾病、药物筛选、食品安全等研究。

Description

一种仿生肠道器官芯片及其制备方法和应用 技术领域
本申请涉及器官仿生技术领域，具体涉及一种仿生肠道器官芯片及其制备方法和应用。
背景技术
微流控芯片技术(Microfluidics)作为21世纪重要前沿科学技术之一，为体外模拟人体代谢模型提供了一种重要平台。它主要以微纳加工技术为基础，由微米级通道形成网络，以可控流体贯穿整个系统，可实现生物学与化学实验室的常规功能。因其具有与细胞大小相匹配的微米尺寸构件，可在芯片微通道内进行多种细胞培养与流体刺激，构建与生理环境接近并具有时空分辨特点的三维微环境，已成为组织器官构建、药物筛选、毒理学以及生物医学研究的重要技术。现阶段微流控技术已成功应用于三维细胞共培养、细胞迁移、细胞分选、组织微环境与类器官构建等。其中，人体器官芯片(Organs-on-a-chip)是近几年发展起来的一种新兴前沿交叉学科技术，是一种利用微加工技术，在微流控芯片上制造出能够模拟人类器官的主要功能的仿生系统。与传统二维静态细胞培养技术相比，芯片内培养的细胞具有三维结构以及多种细胞的空间分布结构，更重要的是器官芯片可以为细胞提供动态的微环境，这是传统手段无法比拟的。此外，芯片内的细胞大部分都是基于人源的细胞，可以极大的降低动物模型所产生的种间差异。器官芯片的发展将有助于药物研发、疾病研究等。
目前，肠道芯片的设计与制备主要集中在哈佛大学的Ingber教授，如图1所示。该芯片通过多培养在多孔膜上的caco-2细胞进行机械拉伸，使其分化并自发形成绒毛结构。
具体的，现有的肠道芯片由三层PDMS(聚二甲基硅氧烷)结构封接而成，如图1所示，其中，中间层为多孔多孔膜101，用于接种肠细胞；上下两层为流体通道02，用于模拟肠道流体微环境；通道左右两侧为真空腔103，用于对多孔膜进行拉伸的机械运动。
但现有技术的肠道芯片的缺点在于细胞自发形成绒毛结构的均一性 不能得到保证，使芯片在不同批次间产生较大的差异，不便于实验数据统计和结果对比。
发明内容
本申请提供一种可减少不同批次芯片之间产生的肠道组织结构的差异，具有良好的重现性的仿生肠道器官芯片及其制备方法和应用。
根据第一方面，一种实施例中提供一种仿生肠道器官芯片，具有流体通道，在流体通道内设有多孔膜，多孔膜将流体通道分隔成上层流体通道和下层流体通道，多孔膜上分布有若干个用于模拟肠绒毛结构的凸起和若干个用于模拟肠道吸收功能的通孔。
根据第二方面，一种实施例中提供一种仿生肠道器官芯片的制备方法，包括如下步骤：
制备上层芯片和下层芯片；
制备多孔膜模板；
制备多孔膜及封接组装：通过多孔膜模板制备多孔膜，再将多孔膜封接在上层芯片和下层芯片之间，形成仿生肠道器官芯片；
其中，多孔膜上分布有若干个用于模拟肠绒毛结构的凸起和若干个用于模拟肠道吸收功能的通孔。
根据第三方面，一种实施例中提供了一种制备仿生肠道器官的方法，利用上述的仿生肠道器官芯片进行。
依据上述实施例的仿生肠道器官芯片及其制备方法和应用，由于多孔膜上分布有若干个凸起和通孔，上层流体通道用于培养肠细胞，下层用于收集代谢产物，多孔膜上的凸起用于模拟肠绒毛结构，通孔用于模拟肠道的吸收功能，使得多孔膜形成三维支架结构，可极大减小不同批次芯片之间产生的肠道组织结构的差异，具有良好的重现性。本仿生肠道器官芯片可利用灌流对肠细胞进行动态培养，以模拟肠道内动态微环境，适用于肠道疾病、药物筛选、食品安全等研究。
附图说明
图1为现有技术中仿生肠道器官芯片的结构示意图；
图2为实施例一中仿生肠道器官芯片的结构示意图；
图3为实施例二中制备仿生肠道器官芯片的流程图；
图4为实施例二中制备上层芯片和下层芯片的流程图；
图5为实施例二中制备多孔膜模板的流程图；
图6为实施例二中多孔膜模板制备过程中的结构示意图；
图7为实施例二中制备多孔膜及封接组装的流程图；
图8为实施例三中制备仿生肠道器官的流程图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
实施例一：
本实施例提供了一种仿生肠道器官芯片，本仿生肠道器官芯片为三层结构，模拟肠道内动态微环境，用于动态培养肠道细胞，适用于肠道疾病、药物筛选、食品安全等研究。
如图2所示，本实施例的仿生肠道器官芯片内设有流体通道。具体的，芯片包括上层芯片210和下层芯片220。上层芯片210的下表面具有开放的上层流体通道，下层芯片220的上表面具有开放的下层流体通道，多孔膜230封接在上层芯片210和下层芯片220之间，多孔膜230与上层芯片210围合成上层流体通道211，多孔膜230与下层芯片220围合成下层流体通道221。多孔膜230上分布有若干个凸起231和通孔232，凸起231用于模拟肠绒毛结构，通孔232用于模拟肠道吸收功能，通孔232将上层流体通道211和下层流体通道221导通。上层芯片210上与上层流体通道211两端对应的位置打出入孔，上层芯片210上与下层流体通道221两端对应的位置打出出入孔。
本实施例中，上层芯片210、下层芯片220和多孔膜230均为PDMS材质。
凸起231为圆锥或圆台结构，若干个凸起231阵列分布在多孔膜230上，若干个通孔232均匀分布在凸起231之外的区域内，即凸起231和通孔232一起布满多孔膜230。多孔膜230形成一个立体的三维支架，从而实现模拟肠道内三维空间结构。
上层流体通道211和下层流体通道221的长度为10-15毫米，宽度为1-1.5毫米，高度为0.3-0.5毫米。多孔膜230的厚度为30-50微米。凸起231的底面直径为100-200微米，高度为150-200微米，间距为150-200微米。通孔232的直径为10微米，通孔232之间的间距为50 微米。
例如，上层流体通道211和下层流体通道221的长度为15毫米，宽度为1毫米，高度为0.4毫米。多孔膜230的厚度为40微米。凸起231的底面直径为200微米，高度为150微米，间距为200微米。在其他实施例中，各组成部分的尺寸可根据模拟需要在上述范围内选取。
本实施例提供的仿生肠道器官芯片，由于多孔膜230上分布有若干个凸起231和通孔232，上层流体通道211用于培养肠细胞，下层用于收集代谢产物，多孔膜230上的凸起231用于模拟肠绒毛结构，通孔232用于模拟肠道的吸收功能，使得多孔膜230形成三维支架结构，可极大减小不同批次芯片之间产生的肠道组织结构的差异，具有良好的重现性。本仿生肠道器官芯片可利用灌流对肠细胞进行动态培养，以模拟肠道内动态微环境，适用于肠道疾病、药物筛选、食品安全等研究。
实施例二：
本实施例提供了一种仿生肠道器官芯片的制备方法，本制备方法主要采用软光刻技术制备上述实施例一中的仿生肠道器官芯片。
如图3所示，本实施例的仿生肠道器官芯片的制备方法主要包括如下步骤：
S100：制备上层芯片和下层芯片；
S200：制备多孔膜模板；
S300：制备多孔膜及封接组装。
步骤S300中，通过多孔膜模板制备多孔膜，并将所述多孔膜封接在所述上层芯片和下层芯片之间，形成仿生肠道器官芯片。其中，生产的多孔膜上分布有若干个用于模拟肠绒毛结构的凸起和若干个用于模拟肠道吸收功能的通孔。
本制备方法将器官芯片分开成上层芯片和下层芯片两个单体制备，再单独制备多孔膜，最后将三者封接在一起，形成具有上下层流通通道的三层结构芯片。本方法中，步骤S100和S200无先后顺序，可先后制备，也可同时制备。
具体的，如图4所示，步骤S100(制备上层芯片和下层芯片)包括如下步骤：
S101：在玻璃或硅片的基底表面旋涂光刻胶，并进行前烘；
本实施例中优选SU-8光刻胶，SU-8光刻胶是一种环氧型的、近紫 外光负光刻胶，SU-8光刻胶在近紫外光范围内光吸收率低，使得在光刻胶厚度上都具有较好的曝光均匀性，能够得到图形边缘近乎垂直的结构。
SU-8光刻胶光刻的机理如下：光刻胶中的光引发剂吸收光子发生了化学反应，生产一种强酸，其作用是前烘过程中作为酸催化剂促进交联反应。只有在曝光区域的光刻胶中才会产生强酸，故只有在曝光区域内才发生交联反应，未曝光的区域不发生交联反应，而光刻胶发生交联反应后不溶于显影液，而光刻胶未发生交联反应的溶于显影液，因此显影后的光刻胶形成与掩膜图案相反的图形。
本步骤中，旋涂SU-8光刻胶的厚度为300-500微米，与上层流体通道和下层流体通道的高度对应。前烘的温度为95℃，时间为2-8小时。
S102：将具有上下层流体通道结构图案的掩膜固定于附有光刻胶的基底表面；
掩膜具有上层流体通道结构和下层流体通道结构的图案，掩膜用于隔档紫外光，从而将掩膜上图案复制到SU-8光刻胶上。
S103：光源垂直照射附有掩膜和光刻胶的玻璃或硅片进行曝光，并进行后烘；
光源发射的光穿过掩膜上的图案照射到SU-8光刻胶上，SU-8光刻胶被曝光的区域将发生交联，交联后的区域不溶于显影液。本实施例中的光源均为紫外光光源，用于发射紫外光进行曝光。
本步骤中，后烘的温度为95℃，时间为10-30分钟。
S104：自然冷却后，采用乳酸乙酯显影液去除未曝光的光刻胶，形成上下层流体通道结构的模板，并进行坚膜；
本步骤中，通过显影液去除未曝光的SU-8光刻胶，显影后的SU-8光刻胶形成与掩膜相反的图形结构，再坚膜加固，坚膜的温度为180℃，时间为2小时。
S105：具有上下层流体通道结构的模板制备PDMS材质的上层芯片和下层芯片。
本步骤中，通过具有与上层流体通道结构和下层流体通道结构相对应的光刻胶模板制备出PDMS材质的上层芯片和下层芯片，并且在上层芯片流体通道的两端打出入孔，并在对应下层流体通道的出入口处打孔，而下层芯片不打孔。最终完成制备成上层芯片和下层芯片。
如图5和图6所示，步骤S200(多孔膜模板的制备方法)包括如下 步骤：
S201：在玻璃或硅片的基底表面旋涂光刻胶，并进行第一次前烘；
本步骤中，在玻璃或硅片301上旋涂厚度为150-200微米的SU-8光刻胶302，此厚度与多孔膜上的凸起高度对应，第一前烘的温度为95℃，时间为2-4小时。
S202：将具有与多孔膜上凸起对应的圆形阵列图案的掩膜固定于附有光刻胶的基底表面；
掩膜上的圆形阵列图案的直径为100-200微米，间距为150-200微米，与多孔膜上凸起对应，用于制备多孔膜上凸起的模板。
S203：将附有掩膜和光刻胶的玻璃或硅片固定于可旋转及可倾斜的平台上，并置于垂直光源下进行第一次曝光；
本步骤中，将附有光刻胶的玻璃或硅片固定在平台上，平台可调节倾斜角度及可自由旋转，用于调节曝光的光照角度。在曝光前，先将平台的倾斜15-45°，使得紫外光倾斜15-45°对SU-8光刻胶302进行曝光，在曝光过程中，平台沿着台面的法向旋转360°，曝光后SU-8光刻胶302具有曝光区域302a和未曝光区域302b，未曝光区域302b用于制备多孔膜上的凸起。
S204：去除掩膜，在经过曝光的光刻胶上再旋涂光刻胶，并进行第二次前烘；
第一曝光后，在SU-8光刻胶302上旋涂SU-8光刻胶303，SU-8光刻胶303的厚度为30-50微米，与多孔膜的厚度对应。第二次前烘的温度为95℃，时间为1-2小时。
S205：将具有与多孔膜上通孔对应的圆形图案的掩膜固定于附有两层光刻胶的基底表面，并且圆形图案位于第一次曝光的曝光区域内；
本步骤中，掩膜的圆形图案的直径为10微米，间距为50微米，与多孔膜上的通孔对应，掩膜的圆形图案在第一次曝光的区域内。
S206：光源垂直照射附有掩膜和光刻胶的玻璃或硅片进行第二次曝光，并进行后烘；
紫外光对光刻胶进行二次曝光后，曝光成若干个圆柱形303a，圆柱形303a用于制备多孔膜上的通孔。
S207：自然冷却后，采用显影液去除未曝光的光刻胶，形成多孔膜模板。
本步骤中，制备的多孔膜模板具有与多孔膜相反互补的结构，多孔膜模板制备多孔膜。
如图7所示，步骤S300(制备多孔膜及封接组装)包括如下步骤：
S301：将PDMS单体与交联剂混合制成PDMS垫板；
将PDMS单体与交联剂按照15:1的比例混合后，真空除气泡，倒在模具中，置于80℃烘箱内2-4小时固化，制成厚度为5毫米的PDMS垫板。
S302：在多孔膜模板和PDMS垫板的表面进行硅烷化修饰；
本步骤中，采用三甲基硅烷在多孔膜模板和PDMS垫板的表面进行硅烷化修饰。
S303：在硅烷化修饰后的多孔膜模板上旋涂未交联的PDMS；
本步骤中，旋涂未交联的PDMS的厚度为30-50微米。
S304：将硅烷化修饰后的PDMS垫板置于旋涂有PDMS的多孔膜模板上，并在PDMS垫板上放置重物静压；
本步骤中，重物的重量为3-6kg，重物静压的时间为8-12小时，例如置于室温环境下静止过夜。
S305：将多孔膜模板、PDMS垫板和重物一起加热，固化制成多孔膜；
本步骤中，加热的温度为80℃，时间为2-4小时。
S306：移去重物，从多孔膜模板上揭下多孔膜，多孔膜附着在PDMS垫板上；
S307：将上层芯片与PDMS垫板上的多孔膜对准封接在一起；
S308：将多孔膜和上层芯片一起从PDMS垫板剥离；
S309：将下层芯片与多孔膜对准封接在一起，形成仿生肠道器官芯片。
本实施例提供的仿生肠道器官芯片的制备方法，主要采用软光刻技术制备仿生肠道器官芯片，制备效率高，并选用SU-8光刻胶进行制备，能够制备出结构精度高的芯片。
实施例三：
本实施例提供了一种制备仿生肠道器官的方法，本制备方法通过实施例一所述的仿生肠道器官芯片进行，本方法为对仿生肠道器官芯片的应用。
本实施例在仿生肠道器官芯片内构建三维肠道微组织并进行动态培养，如图8所示，具体包括如下步骤：
S401：对仿生肠道器官芯片进行灭菌处理；
分别利用70％的酒精和紫外线射线对实施例一所述的仿生肠道器官芯片进行灭菌处理；
S402：将细胞外基质溶液注入到流体通道内，对多孔膜进行修饰，修饰后清洗流体通道；
将细胞外基质(I型胶原、基质胶matrigel等)溶液从上层芯片的入口注入到通道内对PDMS多孔膜进行修饰，修饰后用无血清的培养基或PBS清洗通道；。
S403：将肠细胞悬液注入到上层流体通道内，静止一定时间，以使肠细胞黏附在修饰后的多孔膜上；
将肠细胞(Caco-2)按照10 6个/ml的浓度接种到芯片内的上层流体通道内，静止2小时以上，使细胞黏附与修饰后的多孔膜上生长；
S404：再将培养基按照一定的流速连续注入到流体通道内，培育若干个天后，肠细胞分化成熟，附着在多孔膜凸起上的肠细胞将具有模拟肠绒毛结构，形成三维肠道微组织。
利用注射泵将培养基注入到芯片的上层流体通道与下层流体通道内，实现对肠细胞进行灌流的动态培养，培养5-7天后肠细胞(Caco-2)将分化成熟，附着在三维凸起结构上的肠细胞将(Caco-2)具有模拟肠绒毛结构，形成仿生三维肠道微组织，并可以应用于相关的研究工作。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (40)

  1. 一种仿生肠道器官芯片，具有流体通道，在所述流体通道内设有多孔膜，所述多孔膜将所述流体通道分隔成上层流体通道和下层流体通道，其特征在于，所述多孔膜上分布有若干个用于模拟肠绒毛结构的凸起和若干个用于模拟肠道吸收功能的通孔。
  2. 如权利要求1所述的仿生肠道器官芯片，其特征在于，若干个所述凸起阵列分布在所述多孔膜上，若干个所述通孔均匀分布在所述凸起之外的区域内。
  3. 如权利要求2所述的仿生肠道器官芯片，其特征在于，所述凸起为圆锥或圆台结构。
  4. 如权利要求1所述的仿生肠道器官芯片，其特征在于，所述上层流体通道和下层流体通道的长度为10-15毫米，宽度为1-1.5毫米，高度为0.3-0.5毫米；所述多孔膜的厚度为30-50微米。
  5. 如权利要求4所述的仿生肠道器官芯片，其特征在于，所述凸起的底面直径为100-200微米，高度为150-200微米，间距为150-200微米。
  6. 如权利要求4所述的仿生肠道器官芯片，其特征在于，所述通孔的直径为10微米，所述通孔之间的间距为50微米。
  7. 如权利要求1所述的仿生肠道器官芯片，其特征在于，包括上层芯片和下层芯片，所述上层芯片的下表面具有开放的上层流体通道，所述下层芯片的上表面具有开放的上层流体通道；所述多孔膜封接在所述上层芯片和下层芯片之间，围合成所述上层流体通道和下层流体通道；所述上层芯片上与上层流体通道两端对应的位置以及与下层流体通道两端对应的位置分别打有出入孔。
  8. 如权利要求7所述的仿生肠道器官芯片，其特征在于，所述上层芯片、下层芯片以及具有凸起结构的多孔膜均为PDMS材质。
  9. 一种仿生肠道器官芯片的制备方法，用于制备如权利要求1至8中任一项所述的仿生肠道器官芯片，其特征在于，包括如下步骤：
    制备上层芯片和下层芯片；
    制备多孔膜模板；
    制备多孔膜及封接组装：通过多孔膜模板制备多孔膜，并将所述多孔膜封接在所述上层芯片和下层芯片之间，形成仿生肠道器官芯片；
    其中，所述多孔膜上分布有若干个用于模拟肠绒毛结构的凸起和若干个用于模拟肠道吸收功能的通孔。
  10. 如权利要求9所述的制备方法，其特征在于，所述制备上层芯片和下层芯片包括如下步骤：
    在玻璃或硅片的基底表面旋涂光刻胶，并进行前烘；
    将具有上下层流体通道结构图案的掩膜固定于附有光刻胶的基底表面；
    光源垂直照射附有掩膜和光刻胶的玻璃或硅片进行曝光，并进行后烘；
    自然冷却后，采用显影液去除未曝光的光刻胶，形成具有上下层流体通道结构的模板，并进行坚膜；
    通过具有上下层流体通道结构的模板制备PDMS材质的上层芯片和下层芯片。
  11. 如权利要求10所述的制备方法，其特征在于，所述光刻胶的厚度为300-500微米。
  12. 如权利要求10所述的制备方法，其特征在于，所述前烘的温度为95℃，时间为2-8小时。
  13. 如权利要求10所述的制备方法，其特征在于，所述后烘的温度为95℃，时间为10-30分钟。
  14. 如权利要求10所述的制备方法，其特征在于，所述坚膜的温度为180℃，时间为2小时。
  15. 如权利要求9所述的制备方法，其特征在于，所述制备多孔膜模板包括如下步骤：
    在玻璃或硅片的基底表面旋涂光刻胶，并进行第一次前烘；
    将具有与多孔膜上凸起对应的圆形阵列图案的掩膜固定于附有光刻胶的基底表面；
    将附有掩膜和光刻胶的玻璃或硅片固定于可旋转及可倾斜调节的平台上，并置于垂直光源下进行第一次曝光；
    去除掩膜，在经过曝光的光刻胶上再旋涂光刻胶，并进行第二次前烘；
    将具有与多孔膜上通孔对应的圆形图案的掩膜固定于附有两层光刻胶的基底表面，并且所述圆形图案位于第一次曝光的曝光区域内；
    光源垂直照射附有掩膜和光刻胶的玻璃或硅片进行第二次曝光，并进行后烘；
    自然冷却后，采用显影液去除未曝光的光刻胶，形成多孔膜模板。
  16. 如权利要求10或15所述的制备方法，其特征在于，所述光刻胶为SU-8光刻胶，所述显影液为乳酸乙酯。
  17. 如权利要求10或15所述的制备方法，其特征在于，所述光源为紫外光光源。
  18. 如权利要求15所述的制备方法，其特征在于，所述第一次曝光的过程具体为：将附有掩膜和光刻胶的玻璃或硅片置于可调节倾斜角度及可自由旋转的平台上，调节平台的倾斜角度，使光源以倾斜15-45°照射附有掩膜和光刻胶的玻璃或硅片进行第一次曝光，在曝光过程中，平台沿着台面的法向旋转360°。
  19. 如权利要求15所述的制备方法，其特征在于，所述光刻胶的厚度为150-200微米。
  20. 如权利要求15所述的制备方法，其特征在于，所述第一次前烘的温度为95℃，时间为2-4小时。
  21. 如权利要求15所述的制备方法，其特征在于，所述掩膜上的圆形图案的直径为100-200微米，间距为150-200微米。
  22. 如权利要求15所述的制备方法，其特征在于，所述再次旋涂的光刻胶的厚度为30-50微米。
  23. 如权利要求15所述的制备方法，其特征在于，所述第二次前烘的温度为95℃，时间为1-2小时。
  24. 如权利要求15所述的制备方法，其特征在于，所述掩膜上的圆形图案的直径为10微米，间距为50微米。
  25. 如权利要求15所述的制备方法，其特征在于，所述后烘的温度为95℃，时间为10-30分钟。
  26. 如权利要求9所述的制备方法，其特征在于，所述制备多孔膜及封接组装包括如下步骤：
    将PDMS单体与交联剂混合制成PDMS垫板；
    在所述多孔膜模板和PDMS垫板的表面进行硅烷化修饰；
    在硅烷化修饰后的多孔膜模板上旋涂未交联的PDMS；
    将硅烷化修饰后的PDMS垫板置于旋涂有PDMS的多孔膜模板上，并 在PDMS垫板上放置重物静压；
    将多孔膜模板、PDMS垫板和重物一起加热，固化制成多孔膜；
    移去重物，从多孔膜模板上揭下多孔膜，所述多孔膜附着在PDMS垫板上；
    将所述上层流体通道结构与PDMS垫板上的多孔膜对准封接在一起；
    将多孔膜和上层流体通道结构一起从PDMS垫板剥离；
    将所述下层流体通道结构与多孔膜对准封接在一起，形成仿生肠道器官芯片。
  27. 如权利要求26所述的制备方法，其特征在于，所述PDMS单体与交联剂的混合比为15:1。
  28. 如权利要求26所述的制备方法，其特征在于，所述PDMS单体与交联剂混合后，真空除气泡，倒在模具中，置于80℃烘箱内2-4小时固化，制成厚度为5毫米的PDMS垫板。
  29. 如权利要求26所述的制备方法，其特征在于，所述旋涂未交联的PDMS的厚度为30-50微米。
  30. 如权利要求26所述的制备方法，其特征在于，所述重物的重量为3-6kg，重物静压的时间为8-12小时。
  31. 如权利要求26所述的制备方法，其特征在于，所述加热的温度为80℃，时间为2-4小时。
  32. 如权利要求26所述的制备方法，其特征在于，所述封接方法采用等离子处理，并进行键合。
  33. 一种制备仿生肠道器官的方法，其特征在于，利用权利要求1至8中任一项所述的仿生肠道器官芯片进行。
  34. 如权利要求33所述的方法，其特征在于，包括如下步骤：
    对如权利要求1至8中任一项所述的仿生肠道器官芯片进行灭菌处理；
    将细胞外基质溶液注入到流体通道内，对多孔膜进行修饰，修饰后清洗流体通道；
    将肠细胞悬液注入到上层流体通道内，静止一定时间，以使肠细胞黏附在修饰后的多孔膜上；
    再将培养基以一定的流速连续注入到上层和下层流体通道内，培育若干个天后，肠细胞分化成熟，附着在多孔膜凸起上的肠细胞将具有模 拟肠绒毛结构，形成三维肠道微组织。
  35. 如权利要求34所述的制备方法，其特征在于，采用浓度为70％的酒精和紫外线对仿生肠道器官芯片进行灭菌处理。
  36. 如权利要求34所述的方法，其特征在于，所述细胞外基质溶液为I型胶原或基质胶matrigel溶液。
  37. 如权利要求34所述的方法，其特征在于，修饰后采用无血清的培养基或PBS清洗流体通道。
  38. 如权利要求34所述的方法，其特征在于，所述肠细胞浓度为10 6个/ml。
  39. 如权利要求34所述的方法，其特征在于，所述肠细胞悬液注入到流体通道内后，静止两小时以上。
  40. 如权利要求34所述的方法，其特征在于，将培养基以一定的流速连续注入到上层和下层流体通道内，培育肠细胞5-7天。
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