CN115806876A - Microorganism-intestine-brain axis multi-organ chip and preparation method thereof - Google Patents

Abstract

The invention relates to a microorganism-intestine-brain axis multi-organ chip and a preparation method thereof, belonging to the technical field of multi-organ chip preparation. The invention discloses a microorganism-intestine-brain axis multi-organ chip, which can be used for carrying out experiments by human cells or organoids, has good correlation between the experimental results and human, and does not have the difference between species of animal experiments and human; meanwhile, the microorganism-intestine-brain axis multi-organ chip adopts a micro-fluidic chip technology, and continuously perfuses fluid in a micro-channel and a cavity to culture cells and tissues, so that the experimental process can be obviously accelerated, and the experimental time can be shortened; in addition, the microorganism-intestine-brain axis multi-organ chip of the invention adopts the multi-organ chip processed by PDMS to replace animal experiments by combining the micro-fluidic technology and the fluid mechanics, which can greatly save the experiment cost, and has no dispute of experiment ethics, so that the invention can be expanded to large-scale standardized processing production, thereby facilitating high-throughput and large-scale experimental research.

Description

A microbe-gut-brain axis multi-organ chip and its preparation method

technical field

The invention belongs to the technical field of multi-organ chip preparation, and relates to a microorganism-gut-brain axis multi-organ chip and a preparation method thereof.

Background technique

There is a complex interaction between gut microbes and the brain. Changes in the number and types of gut microbiota will affect key processes in brain development, such as the production and maturation of neurons, thereby changing brain immune function and blood-brain barrier permeability. Sex, brain structure and neural circuits; at the same time, changes in the central nervous system (CNS) can also induce changes in the gut microbiome. The above physiological changes involve complex interactions across organ systems, and this in vivo pathway through which gut microbes generate bidirectional communication through the gut and the brain is known as the "microbe-gut-brain axis." The current research on the "microbe-gut-brain axis" is mainly carried out through animal models and clinical experiments.

Animal model experiments are currently mainly carried out on model animals, mainly including Caenorhabditis elegans, Drosophila, zebrafish and mice. Model animal models have a high degree of individual standardization, repeatable experiments, and provide a certain degree of scalability in experimental results, but these models also have great limitations, as shown in: First, the composition of gut microbiota between different species exists The gut microbes of simple model animals are mostly aerobic, while obligate anaerobic bacteria dominate in the human gut, and the gut microbes of model animal models cannot summarize human beings in terms of quantity, type and complexity. The characteristics of intestinal microorganisms; secondly, model animal experiments cannot replicate the genetic diversity of humans, and the cell composition, structure and electrophysiological characteristics of the central nervous system of model animals are difficult to fully summarize the complexity of the human brain. Therefore, the results of model animal experiments cannot be directly translated into experimental protocols for humans, and the expensive experimental costs and experimental ethics are also unavoidable problems. Therefore, under the guidance of the "3Rs" (Replacement, Refinement, Reduction) principle, it is also a future trend to reduce, replace and optimize animal experiments.

In addition, in the clinical research on the microbiome-gut-brain axis, the sample size is too small, and there are differences in the age, region, gender, gastrointestinal symptoms, diet and supplements of the subjects. The gut microbiome is easily disturbed by various external factors, and the research time span is generally long. In addition, in clinical research, the symptom representation of the patient's brain can only be carried out through extremely limited means, such as cognitive assessment, medical imaging (in vivo), etc. Cognitive assessment is easily affected by various subjective and objective factors, and only Imaging examinations for living individuals cannot directly perform various research and analysis on the brain that needs to be achieved through anatomy.

Whether using animal models or conducting clinical research, there are great limitations. Therefore, there is an urgent need to establish an in vitro model of the gut-brain axis that is highly relevant to human physiopathological characteristics in order to study and analyze the mechanism of gut microbes' influence on the brain.

Contents of the invention

In view of this, one of the objectives of the present invention is to provide a microbe-gut-brain axis multi-organ chip; the second purpose of the present invention is to provide a method for preparing a microbe-gut-brain axis multi-organ chip.

To achieve the above object, the present invention provides the following technical solutions:

1. A microbe-gut-brain axis multi-organ chip, the chip comprises an upper chip and a lower chip from top to bottom;

The upper chip includes microorganism-intestinal symbiosis module I, blood-brain barrier (BBB) module II, brain organoid module III, liquid mixing module IV, and the lower chip includes microorganism-intestinal symbiosis module I, blood-brain barrier (BBB) module ) Module II, Brain Organoid Module III;

Among them, the brain organoid module III is surrounded by the microorganism-intestinal symbiosis module I, the liquid mixing module IV and the blood-brain barrier (BBB) module II in turn.

Preferably, the microorganism-intestinal symbiosis module I of the upper chip includes an upper microchannel a and two linear upper vacuum chambers b respectively located on both sides of the upper microchannel a, and the two ends of the upper microchannel a are connected with upper microchannels respectively. The entrance and exit of the channel ② and ⑤,

The two upper vacuum chambers b are respectively connected with connection ports ③ and ④;

Described upper layer microchannel a comprises linear upper layer microchannel 1a-1, linear upper layer microchannel 2a-2 and linear upper layer microchannel 3a-3 connected successively by one end thereof, wherein linear upper layer microchannel 2a-2 and linear upper layer microchannel 3a-3 The outer end angle formed between the linear upper layer microchannel 1a-1 and the linear upper layer microchannel 3a-3 is 135°, the length of the linear upper layer microchannel 2a-2 is 2cm, the width is 1mm, The thickness is 0.5mm, and the length of the linear upper layer microchannel 1a-1 and the linear upper layer microchannel 3a-3 is 5mm, the width is 1mm, and the thickness is 0.5mm;

The dimensions of the upper vacuum chamber b are 1.5cm in length, 1mm in width and 0.5mm in thickness.

Preferably, the blood-brain barrier (BBB) module II of the upper chip includes a linear upper microchannel c, and one end of the upper microchannel c is connected to an upper microchannel inlet ⑥;

The linear upper microchannel c has a medium length of 2cm, a width of 1mm and a thickness of 0.5mm.

Preferably, the liquid mixing module IV of the upper chip includes a zigzag microchannel d, one end of the zigzag microchannel d is connected to the other end of the upper microchannel c, and the zigzag microchannel d is connected to the upper layer The junction of the microchannel c is connected to the inlet of the brain organoid culture medium ⑧, and the other end of the zigzag microchannel d is connected to the outlet of the brain organoid culture medium ⑨;

The zigzag microchannel d has a width of 1 mm, a thickness of 0.5 mm, and a length of the zigzag part of 1.5 cm.

直线形出口通道g连接有上层腔室的出口

Preferably, the brain organoid module III of the upper layer chip contains a circular upper chamber e, a linear inlet channel f and an upper chamber connected to the circular upper chamber e with an upper chamber consistent with its horizontal linear direction The linear outlet channel g, the linear inlet channel f is connected with the inlet of the upper chamber

The linear outlet channel g is connected to the outlet of the upper chamber

The circular upper chamber e has a diameter of 1.5 cm and a thickness of 5 mm;

The length of the linear inlet channel f is 1.5 cm, the width is 1 mm, and the thickness is 2 mm; the length of the linear outlet channel g is 1.3 cm, the width is 1 mm, and the thickness is 2 mm.

Preferably, the lower chip includes a lower microchannel h and a circular lower chamber i;

The lower microchannel h includes a linear lower microchannel 1h-1 in the microorganism-intestinal symbiosis module I, a linear lower microchannel 2h-2, and a blood-brain barrier (BBB) module II that are connected in sequence through one end. The linear lower microchannel 3h-3 and the linear lower microchannel 4h-4 located in the blood-brain barrier (BBB) module II, wherein the lower microchannel h has a width of 1 mm and a thickness of 0.3 mm; The length of linear lower layer microchannel 1h-1 is 4cm, the length of described linear lower layer microchannel 2h-2 is 3cm, the length of described linear lower layer microchannel 3h-3 is 2.5cm, and the length of described linear lower layer microchannel 3h-3 is 2.5cm. The length of the channel 4h-4 is 5mm, and the outer end angle formed between the linear lower microchannel 4h-4 and the linear lower microchannel 3h-3 is 135°;

The circular lower chamber i is located in the brain organoid module III of the lower layer chip, and the circular lower chamber i is connected with the linear inlet channel j of the lower chamber and the linear inlet channel j of the lower chamber which are consistent with the linear direction in the horizontal direction. exit channel k;

The circular lower chamber i has a diameter of 1.5 cm and a thickness of 3 mm;

The linear inlet channel j of the circular lower chamber i has a length of 1.3 cm and a thickness of 2 mm, and the linear outlet channel k of the circular lower chamber i has a length of 1.5 cm and a thickness of 2 mm;

The inlets and outlets of the microchannels connecting the microorganism-intestinal symbiosis module I and the blood-brain barrier (BBB) module II in the lower chip are ① and ⑦ respectively, the entrance of the lower chamber of the brain organoid module III is ⑩, and the lower layer of the brain organoid module III The chamber outlet is

Further preferably, the linear inlet channel f of the upper chamber, the linear outlet channel g of the upper chamber, the linear inlet channel j of the lower chamber, and the linear outlet channel k of the lower chamber are evenly distributed around the circle.

2. The preparation method of the above-mentioned microorganism-gut-brain axis multi-organ chip, said method comprising the steps of:

(1) Preparing the positive film: using 3D printing technology to prepare the upper chip positive mold and the lower chip positive mold of the multi-organ chip;

(2) Preparation of porous membrane: first, the porous membrane used in the microorganism-intestinal symbiosis module I was prepared by photolithography processing silicon wafers with cylindrical microarrays, and then peeled off from the 6-hole Transwell chamber with a thickness of 20 μm and a vertical pore size of 0.4 μm , a porous membrane with a porosity of 4× ¹⁰ pore/cm ² is used as the porous membrane of the blood-brain barrier (BBB) module II, and the substrate of the Transwell chamber is a transparent polyethylene terephthalate (PET) material, and then A nuclear pore membrane with a thickness of 20 μm, a pore diameter of 8 μm, and a porosity of 1×10 ⁵ pores/cm ² was used as the porous membrane for preparing the brain organoid module III;

(3) Prepare the upper chip and the lower chip: place the upper chip male mold and the lower chip male mold on the mold (acrylic plate) respectively, pour with PDMS prepolymer, degas at -80kPa until all air bubbles are removed, and Curing at 60°C for at least 4 hours, after demoulding, punch holes in the corresponding positions to obtain the upper chip and the lower chip;

(4) Combine the porous membrane prepared in step (2) with the upper chip and the lower chip prepared in step (3), and fix it with a clamp to obtain a microbe-gut-brain axis multi-organ chip.

Preferably, in step (2), the photolithography process specifically includes: mixing polydimethylsiloxane (PDMS) and curing agent at a mass ratio of 9 to 10:1 to obtain a prepolymer, and casting it on On the microarray of the silicon wafer, use the cured PDMS layer as a support layer to cover the uncured prepolymer; then under the pressure of 3kg material, cure and polymerize at 60°C for 12h, peel off the PDMS porous membrane from the silicon wafer;

The size of the microarray is 10 μm in diameter, 30 μm in height, and 25 μm in pitch.

Preferably, in step (3), the PDMS prepolymer is composed of polydimethylsiloxane (PDMS) and curing agent in a mass ratio of 9-10:1.

The beneficial effect of the present invention is that: the present invention discloses a microbe-gut-brain axis multi-organ chip, comprising an upper layer chip (composed of microbe-gut symbiosis module I, blood-brain barrier (BBB) module II, brain organoid module III, liquid mixing module IV) and the lower layer chip (composed of microbiota-gut symbiosis module I, blood-brain barrier (BBB) module II, brain organoid module III), for in vitro modeling and physiology of the microbial gut-brain-axis pathological research. The microbe-gut-brain axis multi-organ chip can be experimented with human-derived cells or organoids, and the experimental results have a good correlation with humans, and there is no interspecies difference between animal experiments and humans; at the same time, the microbe-gut - Brain axis multi-organ chip adopts microfluidic chip technology to continuously perfuse fluid in microchannels and chambers to cultivate cells and tissues, which can significantly speed up the experiment process and shorten the experiment time; in addition, the microorganism-gut-brain axis of the present invention Multi-organ chips combined with microfluidic technology and fluid mechanics use PDMS-processed multi-organ chips instead of animal experiments, which can greatly save experimental costs, and there is no controversy about experimental ethics, so that it can be expanded to large-scale standardized processing and production for High-throughput, large-scale experimental research.

Other advantages, objects and features of the present invention will be set forth in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the investigation and research below, or can be obtained from It is taught in the practice of the present invention. The objects and other advantages of the invention may be realized and attained by the following specification.

Description of drawings

In order to make the purpose of the present invention, technical solutions and advantages clearer, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

Fig. 1 is the plane structural diagram of upper chip;

Fig. 2 is the plane structural diagram of lower layer chip;

Fig. 3 is the plane structural diagram of fixture;

Figure 4 is a structural diagram of the microorganism-gut-brain axis multi-organ chip prepared in Example 1;

Figure 5 is the result of immunofluorescent staining of the tight junction protein ZO-I and DAPI of the intestinal chip module in the microbe-gut-brain axis multi-organ chip of the integrated chip;

Figure 6 shows the results of immunofluorescent staining of intestinal epithelial cells and Bifidobacterium bifidum after intestinal bacteria co-cultured on the intestinal chip module in the microbe-gut-brain axis multi-organ chip after integration;

Figure 7 shows the test results of the transepithelial resistance value of the blood-brain barrier module in the integrated microbe-gut-brain axis multi-organ chip;

Figure 8 shows that the brain organoids in the microbe-gut-brain axis multi-organ chip after integration were subjected to 16-day immunofluorescence staining (a), 45-day immunofluorescence staining (b) and the expression of Alzheimer's disease symptoms (c )the result of.

Detailed ways

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in the following embodiments are only schematically illustrating the basic concept of the present invention, and the following embodiments and the features in the embodiments can be combined with each other in the case of no conflict.

Example 1

Prepare a microbe-gut-brain axis multi-organ chip, the specific method is as follows:

1. Preparation of the positive film: use 3D printing technology to prepare the upper chip positive mold and the lower chip positive mold of the multi-organ chip according to the structure of the upper chip and the lower chip;

直线形出口通道g连接有上层腔室的出口

)、液体混匀模块Ⅳ)(包括折线形微通道d(宽为1㎜、厚为0.5㎜，折线部分的长为1.5㎝)，而折线形微通道d的一端与上层微通道c的另一端相连，在折线形微通道d与上层微通道c的连接处连接有脑类器官培养基进口⑧，折线形微通道d的另一端连接有脑类器官培养基出口⑨)。The upper layer chip includes microorganism-intestinal symbiosis module I (including the linear upper layer microchannel a) (including the linear upper layer microchannel 1a-1, the linear upper layer microchannel 2a-2 and the linear upper layer microchannel connected sequentially through one end 3a-3, wherein the outer end angle formed between the linear upper layer microchannel 2a-2 and the linear upper layer microchannel 1a-1, and the linear upper layer microchannel 3a-3 is 135 °, the linear upper layer microchannel 2a -2 has a length of 2cm, a width of 1mm, and a thickness of 0.5mm, and the linear upper microchannel 1a-1 and the linear upper microchannel 3a-3 have a length of 5mm, a width of 1mm, and a thickness of 0.5mm ) and two linear upper vacuum chambers b (1.5cm in length, 1mm in width, and 0.5mm in thickness) located on both sides of the upper microchannel a respectively. The inlets and outlets ② and ⑤, the two upper vacuum chambers b are respectively connected with the connection ports ③ and ④), the blood-brain barrier (BBB) module II (including the linear upper microchannel c (2cm in length, 1mm in width, 0.5㎜ thick), and one end of the upper microchannel c is connected to the upper microchannel inlet⑥), brain organoid module III (contains a circular upper chamber e (1.5cm in diameter, 5㎜ in thickness), and a circular The upper chamber e is connected with the linear inlet channel f (1.3 cm in length, 1 mm in width, and 2 mm in thickness) of the upper chamber and the linear outlet channel g (length 1.3cm, 1㎜ wide, 2㎜ thick), and the linear inlet channel (f) is connected to the entrance of the upper chamber

The linear outlet channel g is connected to the outlet of the upper chamber

), liquid mixing module Ⅳ) (including zigzag microchannel d (1mm in width, 0.5mm in thickness, and 1.5cm in length of the zigzag part), and one end of zigzag microchannel d is connected to the other end of microchannel c in the upper layer One end is connected, the brain organoid medium inlet ⑧ is connected at the junction of the zigzag microchannel d and the upper microchannel c, and the other end of the zigzag microchannel d is connected to the brain organoid medium outlet ⑨).

The lower chip includes the microbe-intestinal symbiosis module I, the blood-brain barrier (BBB) module II, and the brain organoid module III. The lower chip includes the lower microchannel h and the circular lower chamber i; the lower microchannel h is distributed in the lower layer in a straight line In the microbe-intestinal symbiosis module I and the blood-brain barrier (BBB) module II of the chip (the lower microchannel h includes the linear lower microchannel 1h-1 in the microbe-gut symbiosis module I, the linear The lower microchannel 2h-2, the linear lower microchannel 3h-3 located in the blood-brain barrier (BBB) module II, and the linear lower microchannel 4h-4 located in the blood-brain barrier (BBB) module II, wherein the lower microchannel The channel h has a width of 1mm and a thickness of 0.3mm; the length of the linear lower microchannel 1h-1 is 4cm, the length of the linear lower microchannel 2h-2 is 3cm, and the linear lower microchannel 3h-3 The length of the linear lower layer microchannel 4h-4 is 2.5cm, the length of the linear lower layer microchannel 4h-4 is 5mm, and the outer end angle formed between the linear lower layer microchannel 4h-4 and the linear lower layer microchannel 3h-3 is 135°) ;The circular lower chamber i is located in the brain organoid module III of the lower layer chip, and the circular lower chamber i is connected with the linear inlet channel j of the lower chamber and the linear outlet of the lower chamber in the same horizontal linear direction Channel k, wherein the circular lower chamber i has a diameter of 1.5 cm and a thickness of 3 mm; the linear inlet channel j of the circular lower chamber i has a length of 1.3 cm and a thickness of 2 mm, and the circular lower chamber i The straight outlet channel k of i has a length of 1.5 cm and a thickness of 2 mm.

The linear inlet channel f of the upper chamber, the linear outlet channel g of the upper chamber, the linear inlet channel j of the lower chamber, and the linear outlet channel k of the lower chamber are evenly distributed around the circle.

2. Preparation of porous membrane:

(1) First process by photolithography (the specific method is: mix polydimethylsiloxane (PDMS) and curing agent with a mass ratio of 10:1 to obtain a prepolymer, and cast it on the microarray of silicon wafer (The diameter of the microarray is 10 μm, the height is 30 μm, and the pitch is 25 μm), cover the uncured prepolymer with the cured PDMS layer as a support layer, so that it can be kept flat when peeling off the porous membrane; then under the pressure of 3kg material , Cured and polymerized at 60°C for 12h, peeled off the PDMS porous membrane from the silicon wafer, and cut it into a size of 2cm x 2mm wide with a scalpel to support the growth of intestinal epithelial cells and vascular endothelial cells) with a cylindrical shape Microarray silicon wafers are used to prepare the porous membrane used in the microorganism-intestinal symbiosis module I, which is used to support the growth of intestinal epithelial cells and vascular endothelial cells;

(2) Secondly, peel off the 6-hole Transwell cell (the substrate of the Transwell cell is transparent polyethylene terephthalate (PET) material) with a thickness of 20 μm, a vertical pore of 0.4 μm, and a porosity of 4×10 ⁶ Pore/cm ² porous membrane is used as the porous membrane of blood-brain barrier (BBB) module II to support the growth of vascular endothelial cells and astrocytes;

(3) Then, the nuclear pore membrane with a thickness of 20 μm, a pore diameter of 8 μm, and a porosity of 1×10 ⁵ pores/cm ² was used as the porous membrane for preparing the brain organoid module III to support the brain organoid so that it would not will sink to the bottom of the lower chamber.

直线形进口通道f连接有上层腔室的入口

下层芯片中连接微生物-肠共生模块Ⅰ和血脑屏障(BBB)模块Ⅱ微通道的进出口分别为①和⑦，脑类器官模块Ⅲ的下层腔室入口为⑩，脑类器官模块Ⅲ的下层腔室出口为

即可得到上层芯片(其平面结构图如图1所示)和下层芯片(其平面结构图如图2所示)。3. Prepare the upper chip and the lower chip: place the upper chip male mold and the lower chip male mold on the mold (acrylic plate) respectively, use PDMS prepolymer (polydimethylsiloxane with a mass ratio of 9 to 10:1) oxane (PDMS) and curing agent) for pouring, degassing at -80kPa to remove all air bubbles, and curing at 60°C for at least 4 hours, after demoulding, punch holes in the corresponding positions (the two ends of the upper microchannel a are respectively connected There are inlets and outlets ② and ⑤ of the upper microchannel, the two upper vacuum chambers b are respectively connected with connection ports ③ and ④, one end of the upper microchannel c is connected with the inlet ⑥ of the upper microchannel, and between the zigzag microchannel d and the upper microchannel The junction of channel c is connected to the inlet of brain organoid culture medium ⑧, the other end of zigzag microchannel d is connected to the outlet of brain organoid culture medium ⑨, and the linear outlet channel g is connected to the outlet of the upper chamber

The linear inlet channel f is connected to the inlet of the upper chamber

The inlets and outlets of the microchannels connecting the microorganism-intestinal symbiosis module I and the blood-brain barrier (BBB) module II in the lower chip are ① and ⑦ respectively, the entrance of the lower chamber of the brain organoid module III is ⑩, and the lower layer of the brain organoid module III The chamber outlet is

The upper chip (the plan view of which is shown in FIG. 1 ) and the lower chip (the plan view of which is shown in FIG. 2 ) can be obtained.

4. Combine the porous membrane prepared in step (2) with the upper chip and the lower chip prepared in step (3), and fix it with a clamp (the planar structure of the clamp is shown in Figure 3) to obtain the microorganism-gut- Brain axis multi-organ chip.

The structure diagram of the microorganism-gut-brain axis multi-organ chip prepared in Example 1 is shown in FIG. 4 .

The microorganism-gut-brain axis multi-organ chip prepared in Example 1 is applied, and the specific method is as follows:

1. Cell and organoid culture

(1) Human intestinal epithelial cells Caco-2, human endothelial cells, human astrocytes, and human pluripotent stem cells were cultured according to standard procedures.

(2) Cultivate Bifidobacterium bifidum according to standard procedures.

(3) Human pluripotent stem cells maintained and cultured in mTeSR1 ^TM to maturity were inoculated into 96-well ultra-low adhesion culture plates at 9000 cells/well, and the inoculation medium was embryoid bodies supplemented with 10 μM Rho-kinase inhibitor Form the culture medium, after 5 days of stable culture, transfer it to a 24-well ultra-low adhesion culture plate containing induction medium to induce the differentiation of neuroepithelial cells, use liquid Matrigel to coat the differentiated embryoid bodies after 2 days, and add The expansion medium induces and expands the neuroepithelial structure. From the 10th day, the organoid enters the mature stage, and the mature medium is used for culture. The multi-well plate containing the organoid can be continuously cultured on an orbital shaker. The most current research It can be cultivated for 180 days.

(4) On-chip culture of brain organoids: After the upper and lower layers of the organ chip and the porous membrane are fully sterilized, place the microchannels and chamber openings on the upper layer of the chip facing upward. Carefully remove brain organoids at a specific stage of maturation with a trimmed pipette tip, place in the chamber of the brain organoid section on the upper chip, and add approximately 1 mL of maturation medium. Place the corresponding PET porous membrane and nuclear pore membrane over the blood-brain barrier channel and the brain chamber in turn, carefully align the lower chip combined with the PDMS porous membrane with the channel and chamber of the upper chip, and then quickly place the chip with a clamp fixed.

号口和

号口，以及下层进出口⑩号口和

号口的开口连接不锈钢弯针和Tygon管道，分别以一定的流速经由

号口和⑩号口对上下腔室通入脑类器官成熟培养基。组装芯片到通入培养基的过程尽量控制在10min之内，以保证脑类器官最佳状态和活性。After flipping the chip over, the entrance and exit on the upper layer corresponding to the brain chamber

mouth and

number port, and the entrance and exit of the lower layer ⑩ number port and

The opening of the bell mouth is connected with the stainless steel curved needle and the Tygon pipe, which respectively pass through at a certain flow rate.

Port No. 10 and No. ⑩ port pass through the upper and lower chambers to the brain organoid maturation medium. The process from assembling the chip to introducing the culture medium should be controlled within 10 minutes as much as possible to ensure the best state and activity of the brain organoids.

(5) Porous membrane coating: Use rat tail collagen I and Matrigel to configure extracellular matrix (ECM) solution, take 200 μL from port ① and port ② into microchannels on the upper and lower layers of the microbe-intestine module, and take 200 μL respectively Add the upper and lower microchannels of the blood-brain barrier module from ports ⑥ and ⑦. After standing in the incubator for 1 hour, suck out the extracellular matrix gel solution.

(6) On-chip culture of intestinal and blood-brain barrier modules:

(A) Inject the dissociated Caco-2 cell suspension into the upper microchannel from port ②, and make the cells adhere to the ECM-coated porous membrane at 37°C in a humidified carbon dioxide incubator. The base does not flow. After allowing cells to adhere for 1 hr, culture medium was continuously perfused through the upper channel to establish an intact intestinal epithelial cell monolayer.

(B) At the same time as step (A), inject the dissociated astrocyte suspension into the upper microchannel from port ⑥. Incubate in the same manner for 1 hour, and continuously perfuse the culture medium through the upper channel.

(C), turn the chip upside down, take the dissociated vascular endothelial cell suspension and add it into the lower microchannel of the microbe-intestine module and the blood-brain barrier module from port ① and port ⑦. There is no microchannel in the lower microchannel. Incubate for 1 hour in the incubator with the medium flowing to allow the cells to attach. Then flip the chip to the normal state, and perfuse the endothelial cell culture medium from port ① to the microchannel on the lower layer.

(D) Simultaneous application of vacuum-driven cyclic mechanical strain to the hollow side chamber through port ③ and port ④ to promote the formation of microphysiological structures of the intestinal epithelium and endothelium.

(E) At this stage, port ⑨ is closed, and port ⑧ is the outlet of the vascular access.

2.5 Creating a Physiological Oxygen Concentration Gradient

24 hours before the microorganisms were inoculated into the intestinal epithelial monolayer, the culture medium of the upper and lower microchannels of the microorganism-gut chip module was replaced with a culture medium without adding antibiotics. Enterocyte culture medium was deoxygenated. The microchannels on the module are filled with hypoxic and antibiotic-free medium, and the lower microchannels are filled with oxygenated and antibiotic-free medium. The microbial suspension was inoculated in the upper microchannel. Fluid flow and cyclic stress were suspended in the upper microchannel, and the microfluidic culture was resumed 1 hour after the microorganisms had attached to the apical epithelial surface.

2.6 Microbes are connected to the brain

Open port ⑨ and connect port ⑨ to port ⑩, and inject brain organoid maturation medium from port ⑧, the medium carrying microbial metabolic factors and selectively permeable through the blood-brain barrier and brain organoids. The organ maturation medium is mixed and flows into the lower chamber of the brain organoid chip to exert influence on the brain organoids.

Example 2

Integrating Caco-2 cells, endothelial cells, and Bifidobacterium bifidum into the intestinal chip module in the large microorganism-gut-brain axis multi-organ chip prepared in Example 1 through the inlet and outlet of the microfluidic channel, the endothelial cells , Astrocytes are integrated in the blood-brain barrier module, and finally realize the connection of the gut-brain axis.

Figure 5 is the result of immunofluorescence staining of the tight junction protein ZO-I and DAPI of the intestinal chip module in the microbe-gut-brain axis multi-organ chip of the integrated chip, from which it can be seen that the tight junction protein ZO-1 Well expressed. Figure 6 shows the results of immunofluorescent staining of intestinal epithelial cells and Bifidobacterium bifidum after intestinal bacteria co-cultured on the intestinal chip module in the integrated microbe-gut-brain axis multi-organ chip, from which it can be seen , the large microorganism-gut-brain axis multi-organ chip prepared in Example 1 can successfully construct an aerobic and anaerobic interface to ensure the survival of Bifidobacterium bifidum.

Figure 7 shows the test results of the transepithelial resistance value of the blood-brain barrier module in the microbe-gut-brain axis multi-organ chip after integration. value increases, and vice versa. The TEER value of the blood-brain barrier chip increased from .35Ω*cm2 on the second day to 704.5Ω*cm2, which proved that a blood-brain barrier with good performance had been formed at this time.

Figure 8 shows that the brain organoids in the microbe-gut-brain axis multi-organ chip after integration were subjected to 16-day immunofluorescence staining (a), 45-day immunofluorescence staining (b) and the expression of Alzheimer's disease symptoms (c )the result of. As can be seen, sectioning and immunofluorescent staining of brain organoids from a BrainChip module on a multi-organ chip. The results showed that the 16-day brain organoids expressed the hindbrain marker PAX2 and the forebrain apical progenitor cell marker PAX6, and the 45-day brain organoids expressed the neural progenitor cell marker SOX2 and the neuron marker TUJ1, proving that at this time The brain organoids have differentiated into forebrain and hindbrain brain regions, and nerve cells have begun to differentiate. After the post-induction treatment, the expression of neuron marker MAP2 in brain organoids was reduced, the expression of synaptic marker SYN1 was lost, the expression of Aβ amyloid protein was increased, and the secretion of inflammatory factors was increased, showing the pathogenesis of Alzheimer's disease. symptom. After the gut-brain axis connection, the three different tissues could achieve stable co-culture for more than 48 hours.

The above experimental results prove that in this gut-brain axis multi-organ chip, not only different modules can be stably cultured individually, but also co-culture can be realized. The treatment of Bifidobacterium bifidum decreased the secretion of inflammatory factors.

In summary, the present invention uses organoid and organ chip technology, combined with the application of microfluidic technology and fluid mechanics, to design and manufacture a multi-organ chip for in vitro modeling of microbial gut-brain-axis and physiological pathology Research. The multi-organ chip should be able to create a bionic oxygen concentration gradient to realize the symbiosis of intestinal anaerobic microorganisms and intestinal epithelial cells; the chip should be more in line with the physiological structural characteristics of the human body by optimizing the microchannel design; the chip should be more in line with the size and structure The design makes the flow state of the medium in the chamber for culturing brain organoids laminar, and the state of the fluid can be adjusted to simulate the microenvironment of the brain in vivo with low shear force, so that the brain organoids can obtain sufficient oxygen and nutrients Supply; the chip should be an integrated multi-organ chip with structural design and optimization, which can realize the interconnection, communication and long-term co-cultivation between different organs.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements, without departing from the spirit and scope of the technical solution, should be included in the scope of the claims of the present invention.

Claims (10)

1. A microbe-gut-brain axis multi-organ chip, characterized in that the chip comprises an upper chip and a lower chip from top to bottom; The upper chip includes a microorganism-intestinal symbiosis module (I), a blood-brain barrier module (II), a brain organoid module (III), and a liquid mixing module (IV), and the lower chip includes a microorganism-intestinal symbiosis module (I ), blood-brain barrier module (Ⅱ), brain organoid module (Ⅲ); Among them, the brain organoid module (Ⅲ) is surrounded by the microbe-gut symbiosis module (I), the liquid mixing module (IV) and the blood-brain barrier module (II) in turn. 2. The microbe-gut-brain axis multi-organ chip according to claim 1, characterized in that, the microbe-gut symbiosis module (I) of the upper layer chip comprises an upper layer microchannel (a) and two upper layer microchannels respectively. The linear upper vacuum chamber (b) on both sides of the channel (a), the two ends of the upper microchannel (a) are respectively connected with the inlet and outlet (②) and (⑤) of the upper microchannel, and the two upper vacuum chambers (b ) are respectively connected with connection ports (③) and (④); The upper microchannel (a) comprises a linear upper microchannel 1 (a-1), a linear upper microchannel 2 (a-2) and a linear upper microchannel 3 (a-3) connected successively by one end thereof , wherein the outer end angle formed between the linear upper layer microchannel 2 (a-2), the linear upper layer microchannel 1 (a-1), and the linear upper layer microchannel 3 (a-3) is 135°, so The linear upper layer microchannel 2 (a-2) has a length of 2cm, a width of 1mm, and a thickness of 0.5mm. The linear upper layer microchannel 1 (a-1) and the linear upper layer microchannel 3 (a -3) The length is 5 mm, the width is 1 mm, and the thickness is 0.5 mm; The dimensions of the upper vacuum chamber (b) are 1.5cm in length, 1mm in width and 0.5mm in thickness. 3. The microorganism-gut-brain axis multi-organ chip according to claim 1, wherein the blood-brain barrier module (II) of the upper chip comprises a linear upper microchannel (c), and the upper microchannel One end of (c) is connected with upper floor microchannel inlet (⑥); The linear upper microchannel (c) has a medium length of 2cm, a width of 1mm and a thickness of 0.5mm. 4. The microorganism-gut-brain axis multi-organ chip according to claim 1, characterized in that, the liquid mixing module (IV) of the upper layer chip includes a zigzag microchannel (d), and the zigzag microchannel One end of the channel (d) is connected to the other end of the upper microchannel (c), and the brain organoid culture medium inlet (⑧) is connected at the junction of the zigzag microchannel (d) and the upper microchannel (c), The other end of the zigzag microchannel (d) is connected with a brain organoid culture medium outlet (⑨); The zigzag microchannel (d) has a width of 1mm, a thickness of 0.5mm, and a length of the zigzag part of 1.5cm. 5. The microorganism-gut-brain axis multi-organ chip according to claim 1, wherein the brain organoid module (Ⅲ) of the upper chip contains a circular upper chamber (e) and a circular upper chamber The chamber (e) is connected with the linear inlet passage (f) of the upper chamber and the linear outlet passage (g) of the upper chamber which are consistent with the linear direction in its horizontal direction, and the linear inlet passage (f) is connected with the upper chamber room entrance

Straight outlet channel (g) connected to the outlet of the upper chamber

The circular upper chamber (e) has a diameter of 1.5 cm and a thickness of 5 mm; The linear inlet channel (f) has a length of 1.5cm, a width of 1mm, and a thickness of 2mm; a linear outlet channel (g) has a length of 1.3cm, a width of 1mm, and a thickness of 2mm. 6. The microorganism-intestine-brain axis multi-organ chip according to claim 1, wherein the lower layer chip comprises a lower layer microchannel (h) and a circular lower layer chamber (i); The lower microchannel (h) includes a linear lower microchannel 1 (h-1), a linear lower microchannel 2 (h-2), The linear lower microchannel 3 (h-3) located in the blood-brain barrier module (II) and the linear lower microchannel 4 (h-4) located in the blood-brain barrier module (II), wherein the lower microchannel (h) has a width of 1mm and a thickness of 0.3mm; the length of the linear lower microchannel 1 (h-1) is 4cm, the length of the linear lower microchannel 2 (h-2) is 3cm, The length of the linear lower layer microchannel 3 (h-3) is 2.5cm, the length of the linear lower layer microchannel 4 (h-4) is 5mm, and the linear lower layer microchannel 4 (h-4) The angle between the outer end and the linear lower microchannel 3 (h-3) is 135°; The circular lower chamber (i) is located in the brain organoid module (III) of the lower chip, and the circular lower chamber (i) is connected with a linear inlet channel (j ) and the linear outlet channel (k) of the lower chamber; The circular lower chamber (i) has a diameter of 1.5 cm and a thickness of 3 mm; The length of the linear inlet passage (j) of the circular lower chamber (i) is 1.3 cm and the thickness is 2 mm, and the length of the linear outlet passage (k) of the circular lower chamber (i) is 1.5 cm. cm, thickness 2mm; The inlets and outlets of the microchannels connecting the microbe-intestinal symbiosis module (I) and the blood-brain barrier module (II) in the lower chip are (①) and (⑦) respectively, and the inlets of the lower chamber of the brain organoid module (Ⅲ) are (⑩ ), the outlet of the lower chamber of the brain organoid module (Ⅲ) is

7. The microorganism-gut-brain axis multi-organ chip according to claim 5 or 6, characterized in that the linear inlet channel (f) of the upper chamber, the linear outlet channel (g) of the upper chamber and the lower chamber The linear inlet channel (j) of the chamber and the linear outlet channel (k) of the lower chamber are evenly distributed around the circle. 8. The preparation method of the microorganism-gut-brain axis multi-organ chip according to any one of claims 1 to 7, characterized in that the method comprises the following steps: (1) Preparing the positive film: using 3D printing technology to prepare the upper chip positive mold and the lower chip positive mold of the multi-organ chip; (2) Preparation of porous membrane: first, the porous membrane used in the microorganism-intestinal symbiosis module (I) was prepared by photolithography processing silicon wafers with cylindrical microarrays, and then peeled off from the 6-hole Transwell chamber with a thickness of 20 μm and a vertical pore size of A porous membrane with a porosity of 0.4 μm and a porosity of 4×10 ⁶ pores/cm ² is used as the porous membrane of the blood-brain barrier module (II), and the substrate of the Transwell cell is a transparent polyethylene terephthalate material, and then the thickness A nuclear pore membrane with a diameter of 20 μm, a pore diameter of 8 μm, and a porosity of 1×10 ⁵ pores/cm ² was used as the porous membrane for preparing the brain organoid module (Ⅲ); (3) Prepare the upper chip and the lower chip: place the upper chip male mold and the lower chip male mold on the mold respectively, pour with PDMS prepolymer, degas at -80kPa until all air bubbles are removed, and heat at 60°C Curing for at least 4 hours, after demoulding, punch holes in the corresponding positions to obtain the upper chip and the lower chip; (4) Combine the porous membrane prepared in step (2) with the upper chip and the lower chip prepared in step (3), and fix it with a clamp to obtain a microbe-gut-brain axis multi-organ chip. 9. The preparation method according to claim 8, characterized in that, in step (2), the photolithography process specifically comprises: mixing polydimethylsiloxane and curing agent in a mass ratio of 9 to 10:1 Mix the prepolymer, cast it on the microarray of the silicon wafer, cover the uncured prepolymer with the cured PDMS layer as the support layer; Peel off the PDMS porous membrane on the silicon wafer; The size of the microarray is 10 μm in diameter, 30 μm in height, and 25 μm in pitch. 10. The preparation method according to claim 8, characterized in that, in step (3), the PDMS prepolymer consists of polydimethylsiloxane and a curing agent in a mass ratio of 9 to 10:1.

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