CN115558601A

CN115558601A - Mini mammal model for detecting drug effect, toxicity and pharmacokinetics and application thereof

Info

Publication number: CN115558601A
Application number: CN202211518830.4A
Authority: CN
Inventors: 张秀莉; 曲玥阳; 王静; 张慧雪; 季珍妮; 杨瑒; 徐曙辉; 罗勇
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2023-01-03
Anticipated expiration: 2042-11-30
Also published as: CN115558601B

Abstract

The invention discloses a mini-mammal model for drug effect, toxicity and pharmacokinetic detection and application thereof, wherein the mini-mammal model is formed by superposing a lower substrate, a middle substrate, an upper substrate and a top plate together; the lower substrate is an organ layer and is provided with a plurality of tissue culture chambers; the middle substrate is an artery and capillary layer, a first micro-channel and a second micro-channel are etched on the lower surface of the middle substrate, and the second micro-channel is connected to the first micro-channel; the upper layer substrate is a vein layer, and a third micro-channel is etched on the vein layer; the middle substrate is provided with a culture solution inlet communicated with the first microchannel, and the top plate is provided with a culture solution outlet communicated with the third microchannel. The mini-mammal model of the invention can simulate intestinal absorption, liver metabolism, kidney excretion and drug distribution, and the time curve, the drug effect and the toxicity of the drug can be measured on the mini-mammal.

Description

Mini mammal model for drug effect, toxicity and drug metabolism detection and application thereof

Technical Field

The invention relates to the technical field of drug screening, in particular to a mini-mammal model for drug effect, toxicity and drug generation detection and application thereof.

Background

At present, the cost for discovering new drugs is high, and one important reason is animal experiments. Because of individual differences of experimental animals, scientists need to perform the same experiment by using a plurality of similar animals and then take an average value in order to collect reliable data, which leads to the rapid increase of the dosage of the experimental animals and further greatly improves the research and development cost of new drugs.

The organ chip is an organ physiological microsystem constructed on a chip with the size of a glass slide, and comprises key elements of organ microenvironments such as living cells, tissue interfaces, biological fluids, mechanical force and the like. The method can simulate the main structural and functional characteristics of different tissues and organs of a human body and the relation between complex organs in vitro, is used for predicting the response of the human body to drugs or external different stimuli, and has wide application prospect in the fields of life science, medical research, new drug research and development, personalized medical treatment, toxicity prediction, biological defense and the like.

Disclosure of Invention

The invention aims to provide a mini-mammal model based on an organ chip technology, which can simulate intestinal absorption, liver metabolism, kidney excretion and drug distribution, and can measure the drug time curve, drug effect and toxicity of drugs on the mini-mammal.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a mini mammal model, which is formed by stacking a lower substrate, a middle substrate, an upper substrate and a top plate from bottom to top in sequence; wherein, the first and the second end of the pipe are connected with each other,

the lower substrate is an organ layer and is provided with a plurality of tissue culture chambers, and the tissue culture chambers are respectively used for culturing different micro tissues to simulate mammal organs;

the middle substrate is an artery and capillary layer, a first micro-channel representing an artery blood vessel and a plurality of second micro-channels representing a capillary are etched on the lower surface of the middle substrate, the first micro-channel and the second micro-channels are respectively positioned above the tissue culture chambers and are communicated with the corresponding tissue culture chambers, and porous membranes are arranged between the tissue culture chambers and the first micro-channel and the second micro-channel; the plurality of second microchannels are each connected to the first microchannel;

the upper substrate is a vein layer, a third microchannel representing vein blood vessels is etched on the vein layer, and the third microchannel is respectively arranged above the first microchannel and the second microchannel; the tail ends of the first microchannel and the third microchannel are provided with corresponding interfaces, and the third microchannel is communicated with the first microchannel and the second microchannel below through the interfaces;

the culture medium circulation device is characterized in that a culture solution inlet communicated with the first micro-channel is formed in the middle substrate, a culture solution outlet communicated with the third micro-channel is formed in the top plate, and the culture solution inlet and the culture solution outlet are communicated with an external culture medium circulation device to realize the internal circulation of a culture solution in the first micro-channel, the second micro-channel, the third micro-channel and the tissue culture chamber.

In the invention, the first microchannel is equivalent to an arterial blood vessel, the second microchannel is equivalent to a capillary blood vessel, and the third microchannel is equivalent to a venous blood vessel.

In the present invention, the organ layer may be formed by stacking a plurality of plates. The micro-tissues cultured in the tissue culture chamber can be mammal primary tissues, cell balls, organoids and the like, and the culture mode can be two-dimensional culture or three-dimensional culture.

Further, the bottom surface of the tissue culture chamber is a low-adhesion super-hydrophobic surface with a micro-nano morphology, and the contact angle of the surface with a water phase solution is more than 120 degrees;

or the bottom surface of the tissue culture chamber is modified with a low-adhesion hydrophilic polymer coating in a micro-nano shape, and the contact angle between the polymer coating and the aqueous phase solution is less than 90 degrees.

In the present invention, the bottom surface of the tissue culture chamber is a modified low adhesion surface so that when the micro-tissue is cultured, the micro-tissue does not adhere to the bottom wall of the chamber and thus disintegrates.

In one embodiment of the present invention, the method for forming the low-adhesion superhydrophobic surface is: mixing nano silicon dioxide particles, normal hexane and chloroform, and then carrying out ultrasonic treatment to disperse the nano silicon dioxide particles in the mixed solution; and then, immersing a polymethyl methacrylate (PMMA) plate into the mixed solution, taking out and airing to obtain the low-adhesion super-hydrophobic surface.

Preferably, the method for forming the low-adhesion superhydrophobic surface is: weighing hydrophobic nano silica particles with the diameter of 2-200 zxft 3252 and 0.25-2 g, and measuring 20-500 zxft 3532 n-hexane and 0.5-50 mL chloroform. The materials are mixed and then subjected to ultrasonic treatment, so that the nano silicon dioxide particles are completely dispersed in the mixed solution. And (3) immersing the PMMA plate into the solution for about 5-300 s, taking out and airing to obtain the PMMA plate with the super-hydrophobic surface.

In another embodiment of the present invention, the method for forming the low-adhesion superhydrophobic surface is: soaking a Polydimethylsiloxane (PDMS) plate into tetraethyl orthosilicate to swell the PDMS plate, taking out the swelled PDMS plate, and placing the PDMS plate in an ethylene diamine aqueous solution; and taking out the PDMS plate, washing and then carrying out heat treatment to obtain the super-hydrophobic surface.

Preferably, the method for forming the low-adhesion superhydrophobic surface is: the PDMS sheet is immersed in tetraethyl orthosilicate at 30-70 ℃ for 10-200 min, and then the swollen PDMS sheet is taken out of the tetraethyl orthosilicate solution and immediately floats in a 5-40% aqueous solution of ethylenediamine. 3-24, h, taking out and washing with deionized water for 3-5 times, and carrying out heat treatment on 0.5-5 h in an oven to obtain the PDMS plate with the super-hydrophobic surface.

In still another embodiment of the present invention, the method of forming the low adhesion superhydrophobic surface comprises the steps of:

a. pouring the pre-polymerization liquid of the elastic resin on a super-hydrophobic template with a micro-nano structure, polymerizing the pre-polymerization liquid of the elastic resin into a first elastic solid, and then stripping the first elastic solid from the template;

b. performing silanization modification on the surface of the first elastic solid, pouring the elastic resin pre-polymerization liquid by taking the silanized and modified first elastic solid as a template, and polymerizing the elastic resin pre-polymerization liquid into a second elastic solid;

c. and (3) peeling the second elastic solid from the silanized and modified first elastic solid template, wherein the surface of the second elastic solid forms the low-adhesion super-hydrophobic surface.

The super-hydrophobic template with the micro-nano structure can be made of natural materials such as cicada's wing, lotus leaf and the like; the coating can also be prepared by a manual method, such as the methods of etching an aluminum plate on the surface, etching a high polymer plate on the surface, processing MEMS (micro-electromechanical systems), enriching silica micro-nano particles on the surface, spraying super-hydrophobic coating on the surface and the like.

Further, the preparation method of the low-adhesion hydrophilic polymer coating comprises the following steps:

s1, mixing glycidyl methacrylate and other polymerization monomers in water, adding tetramethylethylenediamine and an initiator, and carrying out copolymerization reaction; after the reaction is finished, dialyzing the copolymer solution, and removing unreacted monomers and an initiator to obtain a high-molecular coating solution;

s2, pretreating the bottom surface of the tissue culture chamber to enable the bottom surface to generate polar groups, and then placing the pretreated bottom surface in the polymer coating liquid for soaking treatment; then taking out, cleaning the polymer coating liquid on the surface, and drying to form a low-adhesion hydrophilic polymer coating on the bottom surface of the tissue culture chamber;

wherein the other polymerized monomer comprises one or more of 2- (2-methoxyethoxy) ethyl methacrylate, methoxyethyl methacrylate, methyl dimethacrylate, hexafluorobutyl acrylate and methyl methacrylate; the polar group comprises one or more of hydroxyl, amino and carboxyl.

Further, in step S1, the mixing time of glycidyl methacrylate and other polymerized monomers in water is 5-30 min to ensure uniform mixing.

Further, in the step S1, the adding concentration of the tetramethylethylenediamine is 0.1wt% to 3wt%, and the adding concentration of the initiator is 0.1wt% to 3wt%.

Further, in step S1, the initiator may be an initiator commonly used in the art, and is preferably potassium persulfate. The time for the copolymerization reaction is preferably 30 min to 1 d.

Further, in step S1, the dialysis specifically includes: and completely dialyzing the copolymer solution for two days by using a dialysis membrane, and changing the solution every 6-12 h to remove monomers and small molecules which do not have polymerization reaction, wherein the solution in the dialysis bag is the high-molecular coating solution.

In step S2, groups such as hydroxyl, amino, carboxyl and the like are introduced into the bottom surface of the tissue culture chamber, so that the hydrophilic modification liquid can be firmly bonded on the bottom surface of the tissue culture chamber through covalent bonds, and a high polymer coating with a micro-nano morphology on the surface is formed. Preferably, the tissue culture chamber is treated with plasma to introduce polar groups into its bottom surface. For example, oxygen plasma treatment is employed to introduce hydroxyl groups.

Further, in the step S2, the temperature of the soaking treatment is 30-70 ℃, and the time of the soaking treatment is 30 min-5 d; the drying temperature is 0-75 ℃, and the drying time is 30 min-5 d.

Furthermore, a through hole is formed in the bottom surface of the tissue culture chamber, and the through hole is used for enabling a micro stirring paddle or a sensor to extend into the tissue culture chamber. Since the tissue culture chamber is superhydrophobic, the culture solution does not leak out of the through-hole in the bottom.

Further, the mini-mammal model has a similar appearance to a real mammal, thereby facilitating discrimination between species of the simulated mammal.

Further, the position of at least one tissue culture chamber in said mini-mammal model corresponds to the position of the organ it represents in the mammal body and is similar to the shape of the organ it represents, which facilitates accurate placement of the corresponding micro-tissue in the tissue culture chamber.

Further, the tissue culture chamber includes a brain culture chamber, an eye culture chamber, a nose culture chamber, an ear culture chamber, a tongue culture chamber, a trachea culture chamber, a heart culture chamber, a lung culture chamber, a liver-intestine culture chamber, a kidney culture chamber, a stomach culture chamber, a pancreas culture chamber, a spleen culture chamber, a skin culture chamber, a fat culture chamber, a bone marrow culture chamber, a muscle culture chamber, a testis culture chamber, and a tumor culture chamber, but is not limited to the above organ culture chambers.

Further, the culture solution inlet is communicated with the first microchannel above the lung culture chamber, and the culture solution outlet is communicated with the tail end of the third microchannel above the heart culture chamber.

Furthermore, the porous membrane between the tissue culture chamber and the first and second microchannels is used for culturing vascular endothelial cells, and immune cells are placed above the vascular endothelial cells.

Further, an air passage is connected to a side wall of some tissue culture chambers, and the air passage communicates with the outside to supply oxygen to the tissue culture chambers. The airways are preferably also superhydrophobic so that the culture fluid does not flow out through the airways.

Further, the liver-intestine culture chamber comprises a liver tissue culture area and an intestinal cell culture area positioned below the liver tissue culture area, the liver tissue culture area is used for culturing liver micro-tissues and is separated from the first micro-channel and the second micro-channel above the liver micro-tissues by a porous membrane, the upper surface of the porous membrane is used for culturing vascular endothelial cells, and immune cells are cultured on the vascular endothelial cells.

The liver tissue culture area and the intestinal cell culture area are separated by another porous membrane, and the intestinal cells are cultured on the lower surface of the porous membrane; the intestinal cell culture region is communicated with the outside through a separate digestion channel, and the digestion channel is used for simulating the circulation flow of a digestive fluid.

Furthermore, the number of the kidney culture chambers is two, wherein one kidney culture chamber is used for culturing kidney micro tissues and simulating the metabolic function of the kidney; the other kidney culture chamber is provided with a dialysis membrane and is communicated with the outside through a separate elimination channel for simulating the elimination of wastes and medicines in the body.

Further, the mammals include, but are not limited to, commonly used experimental animals, such as mice, rats, monkeys, etc.

In a preferred embodiment, the present invention provides a mini rat model comprising 19 organs, a blood circulation system, an excretory system and a functional immune system, which can survive more than two worship.

The invention also discloses application of the mini mammal model in drug effect detection, toxicity detection or drug-induced detection.

Compared with the prior art, the invention has the beneficial effects that:

1. the mini-mammal model of the invention comprises micro-tissues (primary tissues, organoids or cytospheres) instead of simulating an organ in cells or discrete combinations of cells; the tissue culture zone has a low adhesion surface so that tissue or organ-like functionality is present in the mini animal model in a suspended state for a prolonged period of time; the tissue culture area is connected with a micro-channel system which can provide nutrition and oxygen for the micro-tissues; the mini-mammal has a shape profile and organ distribution similar to those of the corresponding mammal, and has a shape similar to those of the corresponding organ, which is easily distinguishable and has excellent reproducibility. The mini-mammal can simulate ADME process of medicine, namely intestinal absorption, liver metabolism, distribution and kidney elimination process, and can measure time course, drug effect and toxicity of medicine.

2. The mini rat model provided by the invention can survive more than two worship, can easily research the pharmacodynamics, systemic toxicity and pharmacokinetics of the medicine, and opens up an effective way for reducing the individual difference of experimental animals.

3. Particularly, considering that large animals such as monkeys can be divided into hundreds of monkey chips, the invention can reduce the animal consumption of the industry by at least one order of magnitude, thereby practically promoting the practice of the 3R principle in the field of new drug development.

Drawings

FIG. 1 is an electron micrograph of the surface of PDMS after it has been overmolded twice with lotus leaves;

FIG. 2 is a state of a droplet on a superhydrophobic PDMS surface;

FIG. 3 is a bright field photograph of tumor spheres on a PDMS superhydrophobic surface;

FIG. 4 shows the change of contact angle after 12 repeated uses;

FIG. 5 is a surface electron micrograph of a polydimethylsiloxane sheet of silica microbubbles;

FIG. 6 is a brightfield photograph of the surface tumor spheres of a polydimethylsiloxane sheet of silicon dioxide microbubbles;

FIG. 7 is an electron microscope photograph of a superhydrophobic PMMA surface;

FIG. 8 is a bright field photograph of tumor spheres on a superhydrophobic PMMA surface;

FIG. 9 is an atomic force microscope image of the surface of a polymer coating;

FIG. 10 shows the fluorescent protein adsorption of the modified PDMS surface (A) and the unmodified PDMS surface (B);

FIG. 11 shows tumor spheres formed on the surface of the polydimethylsiloxane polymer coating;

FIG. 12 is a disintegration of renal micro-tissue on a normal surface;

FIG. 13 is a pictorial photograph of a mini mouse model;

FIG. 14 is a design drawing of the venous, arterial and capillary layers, organ layers of a mini mouse model;

FIG. 15 shows the type of tissue contained within each chamber;

FIG. 16 is a bright field image of each tissue;

FIG. 17 is an organ structure diagram of a mini mouse model;

FIG. 18 is the mass ratio of the major organs of a real rat;

FIG. 19 is the direction of blood flow in the mini mouse model;

FIG. 20 is a design of a mini mouse model renal elimination unit;

FIG. 21 is a plot of sodium fluorescein elimination at different blood and urine flow rates;

FIG. 22 is a design of a mini mouse model liver and bowel unit;

FIG. 23 is an erlotinib dosing time curve based on the mini mouse model;

FIG. 24 shows the evaluation results of general toxicity and drug effect of doxorubicin based on the mini mouse model;

FIG. 25 is a design drawing of a mini-mannequin;

FIG. 26 is a design drawing of a mini dog model;

FIG. 27 is a design drawing of a mini monkey model;

FIG. 28 is a design drawing of a mini-pig model;

wherein: 1. a tissue culture chamber; 2. an airway; 3. a micro-stirrer; 4. micro-tissue; 5. vascular endothelial cells; 6. an immune cell; 7. eliminating the channel; 8. a porous membrane; 9. liver microtissue; 10. intestinal cells; 11. a digestive channel; 12. a lung; 13. the left atrium and ventricle; 13', the right atrium and ventricle; 14 to 23,14 to 23' and a capillary vessel end interface;

a. a heart culture chamber; b. a liver-intestine culture chamber; c. a spleen culture chamber; d. a lung culture chamber; e. a kidney culture chamber; f. a brain culture chamber; g. an eye culture chamber; h. an ear culture chamber; i. a bone marrow culture chamber; j. a muscle culture chamber k and a trachea culture chamber; l, a fat culture chamber; m, testis culture chamber; n, pancreas culture chamber; o, gastric culture chamber; p, tumor culture chamber; q, a nasal culture chamber; r, tongue culture chamber; s, skin culture chamber.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Example 1: preparing super-hydrophobic surface by using natural material as template

Fixing lotus leaves on a glass plate, pouring liquid polydimethylsiloxane on the surface of the lotus leaves, standing overnight to enable the liquid polydimethylsiloxane to be polymerized into a solid, stripping solidified polydimethylsiloxane sheets from the lotus leaves to serve as a template of the next step, performing silanization modification on the surface of the polydimethylsiloxane template, pouring liquid polydimethylsiloxane on the template, heating to perform polymerization, stripping newly solidified polydimethylsiloxane sheets from the silanization modified polydimethylsiloxane sheet template, wherein an electron microscope picture of the surface of the newly solidified polydimethylsiloxane sheets is shown in figure 1, and the polydimethylsiloxane sheets have a super-hydrophobic micro-nano structure, a contact angle is larger than 120 degrees (figure 2), and the preparation of the super-hydrophobic polydimethylsiloxane plate is finished.

Low adhesion characterization:

the plane is used for tumor cell balling experiment, human embryonic lung fibroblast (MRC-5) and human lung adenocarcinoma cell (NCI-H1792) are cultured in a mixed way in a culture medium RPMI 1640+10% FBS, the culture medium is placed in a hole with the super-hydrophobic surface, the culture ratio of the fibroblast to the cancer cell is 1: 1, the culture environment is 37 ℃, and the culture environment is 5% CO ₂ . Tumor cells did not adhere to the wall after 3 days, but rather spheronized (fig. 3), demonstrating the low adhesion of superhydrophobic surfaces.

And (3) durability characterization:

the test repeatability showed that the wells were filled with various cell culture media for 48h, then poured off, washed, air dried, and the above experiment was repeated 12 times, and the contact angle was again measured and still greater than 120 ° (fig. 4), indicating that the low adhesion surface could be reused at least 12 times for more than 24 days.

Example 2: direct preparation of polydimethylsiloxane superhydrophobic surface

A polydimethylsiloxane plate was immersed in tetraethyl orthosilicate at 50 ℃ for 20 min. Then, the tetraethyl orthosilicate swollen polydimethylsiloxane plate was taken out from the tetraethyl orthosilicate solution, immediately floated in a 10% ethylenediamine aqueous solution, and silica microbubbles were gradually formed on the surface of the polydimethylsiloxane plate. 10 After h, the polydimethylsiloxane plate with the silicon dioxide microbubbles was removed and rinsed 3 times with deionized water. Finally, the polydimethylsiloxane plates with the silica microbubbles were heat treated in an oven at 1 h. An electron micrograph of the prepared superhydrophobic surface is shown in fig. 5.

Low adhesion characterization:

the plane is used for tumor cell balling experiment, human embryonic lung fibroblast (MRC-5) and human lung adenocarcinoma cell (NCI-H1792) are cultured in a mixed way in a culture medium RPMI 1640+10% FBS, the culture medium is placed in a hole with the super-hydrophobic surface, the culture ratio of the fibroblast to the cancer cell is 1: 1, the culture environment is 37 ℃, and the culture environment is 5% CO ₂ . Tumor cells did not adhere to the wall after 3 days, but instead spheronized (fig. 6), demonstrating the low adhesion of superhydrophobic surfaces.

And (3) durability characterization:

the repeatability of the test shows that the wells were filled with cell culture medium (1640 medium +10% fetal bovine serum) for 48h, then poured off, washed, air dried, the above experiment was repeated 12 times, and finally the contact angle was measured as 135.8 °, still greater than 120 °, indicating that the low adhesion surface could be reused at least 12 times for more than 24 days.

Example 3: preparation of a polymethyl methacrylate Superhydrophobic surface

Weighing hydrophobic nano silica particles with the diameter of 20 nm and 0.25 g, weighing 50 mL n-hexane and 2.5 mL chloroform, mixing, performing ultrasonic treatment to completely disperse the nano silica particles in a mixed solution, immersing a polymethyl methacrylate plate into the mixed solution to obtain about 15 s, taking out and airing to obtain the polymethyl methacrylate super-hydrophobic surface (figure 7).

Low adhesion characterization:

the plane is used for tumor cell balling experiments, human embryonic lung fibroblast (MRC-5) and human lung adenocarcinoma cell (NCI-H1792) are cultured in a mixed mode, a culture medium is RPMI 1640+10% FBS, the human embryonic lung fibroblast and human lung adenocarcinoma cell are placed in the hole with the super-hydrophobic surface, the culture ratio of the fibroblast to the cancer cell is 1: 1, the culture environment is 37 ℃, and 5% CO is 5% ₂ . Tumor cells did not adhere to the wall after 3 days, but instead spheronized (fig. 8), demonstrating the low adhesion of superhydrophobic surfaces.

And (3) durability characterization:

the test repeatability shows that the well plate is added with cell culture medium (1640 medium +10% fetal bovine serum) for 48h, then poured off, cleaned, air dried, the above experiment is repeated for 12 times, and finally the contact angle is measured to be 132.6 degrees and still greater than 120 degrees, which indicates that the low-adhesion surface can be repeatedly used for at least 12 times, and the continuous use time is more than 24 days.

Example 4: preparation of polydimethylsiloxane super-hydrophilic surface

Uniformly mixing 2 mL methyl methacrylate, 0.04mL polyglycidyl methacrylate and 38 mL water for 10 min, then adding 0.4 g potassium persulfate and 2 mL water, after fully dissolving, adding 0.04mL TEMED, carrying out copolymerization reaction at 25 ℃ for 15 min, stopping the reaction after the solution color changes from transparent to milky, immediately transferring into a 1.5W dialysis bag for dialysis for two days, changing the solution every 12 h to remove monomers and small molecules which do not generate polymerization reaction, and obtaining the solution in the dialysis bag as the macromolecular coating solution.

Firstly, generating hydroxyl on the surface of polydimethylsiloxane by oxygen plasma, then soaking the surface of 1 h in the macromolecular coating liquid, then washing the macromolecular coating liquid for three times by deionized water, and drying 1 h at 75 ℃.

First, the surface is characterized by atomic force microscopy, and as is apparent from fig. 9, after the surface is modified, a layer of high molecular polymer is formed on the surface, and nano-ravine structures are formed on the high molecular polymer.

Then, protein adsorption prevention characterization is carried out. The fluorescence labeled secondary antibody solutions were injected onto the modified and unmodified surfaces, respectively, and it is evident that the unmodified surface had a high fluorescence intensity, indicating that protein adsorption was severe, whereas on the modified surface, almost no fluorescence was detected, indicating that the coating well prevented protein adsorption (fig. 10).

Finally, anti-cell adhesion characterization was performed. Human embryonic lung fibroblast (MRC-5) and human lung adenocarcinoma cell (NCI-H1792) are cultured in a mixed manner in a culture medium RPMI 1640+10% FBS. The culture ratio of fibroblast to cancer cell is 1: 1, the culture environment is 37 deg.C, and 5% CO ₂ . After 3 days, the cells did not adhere, but formed tumor spheres (fig. 11), confirming the ability of the coating to prevent cell adhesion. The same surface, 5 consecutive tumor balling experiments were successful, demonstrating the durability of the low adhesion surface.

Kidney tissue was placed on a non-low-adhesive surface and cells were found to crawl outward and the tissue disintegrated quickly (fig. 12).

Example 5: mini mouse model

This example provides a mini mouse model, as shown in FIG. 13, which is shaped like a real mouse. As shown in fig. 14, the mini mouse model is formed by laminating an organ layer (PMMA), an artery and capillary layer (PDMS) and a vein layer (PMMA) from bottom to top, and the lower layer (i.e. the organ layer) of the mini mouse model comprises 19 PMMA superhydrophobic chambers: heart culture chamber a, liver-intestine culture chamber b, spleen culture chamber c, lung culture chamber d, kidney culture chamber e, brain culture chamber f, eye culture chamber g, ear culture chamber h, bone marrow culture chamber i, muscle culture chamber j, trachea culture chamber k, fat culture chamber l, testis culture chamber m, pancreas culture chamber n, stomach culture chamber o, tumor culture chamber p, nose culture chamber q, tongue culture chamber r, skin culture chamber s (figure 15), the bottom surface of the super-hydrophobic chamber is a super-hydrophobic surface, and the contact angle of the super-hydrophobic chamber and the aqueous phase solution is more than 120 degrees. The super-hydrophobic modification process comprises the following steps: weighing 0.25 g hydrophobic nano silica particles with the diameter of 20 nm, weighing 50 mL n-hexane and 2.5 mL chloroform, mixing, performing ultrasonic treatment to completely disperse the nano silica particles in a mixed solution, immersing the PMMA plate in the mixed solution for about 15 s, taking out and airing to obtain the PMMA superhydrophobic surface.

Each chamber contained one primary micro-tissue including brain, eye, nose, ear, tongue, trachea, heart, lung, liver, kidney, stomach, pancreas, spleen, skin, fat, bone marrow, muscle, testis, and tumor (fig. 16). The method for obtaining each micro-tissue comprises the following steps: the organs were first cut into pieces with scissors, then masked and finally sieved, leaving tissue particles of 70-100 μm, i.e. the micro-tissues simulating the organs in the chip, the culture medium used for each micro-tissue being shown in table 1 below.

TABLE 1 culture media for various microtissue

In the present invention, the contour of each chamber is similar to the corresponding organ, and the distribution of the chambers is similar to the distribution of the real organ in the body (fig. 15), thereby facilitating the accurate positioning and identification of various micro-tissues.

As shown in FIG. 17, a porous membrane 8 is clamped in the middle of each tissue culture chamber 1, vascular endothelial cells 5 are cultured on the membrane, immune cells 6 are arranged above the vascular endothelial cells 5, and microtissue 4 is arranged below the vascular endothelial cells 5. The bottom of the tissue culture chamber 1 is provided with a through hole, a micro stirrer 3 is extended into the through hole, and culture solution in the chamber is stirred for strengthening mass transfer. Since the tissue culture chamber 1 is superhydrophobic, the culture solution does not leak out of the small hole in the bottom.

An air passage 2 is also arranged in the tissue culture chamber 1 and is communicated with the peripheral air to supplement oxygen for the micro-tissues 4 in the chamber. Airway 2 is also superhydrophobic, and the modification method is as described above, and the cell culture fluid does not leak out. The microtissue 4, vascular endothelial cells 5 and immune cells 6 constitute the organs of the mini mouse model. The mass ratio between the major organs of the mini rat model was consistent with the mass ratio of the corresponding organs in the real rat (fig. 18).

As shown in fig. 14, the arteries and capillaries of the mini mouse model were etched in the middle PDMS slab, and the capillaries were located right above the chamber shown in fig. 17, and the capillary networks of different organs were connected by arterial blood vessels. The veins of the mini mouse model were carved in the upper PMMA plate (fig. 14), which was also connected to an external peristaltic pump and culture medium pot (fig. 19), and the lower side of the upper PMMA plate was tightly attached to the PDMS plate of the arterial and capillary layers.

As shown in FIG. 19, "arterial blood" first enters the lungs 12 from the culture medium reservoir, then enters the left atrium and ventricles 13, and then is shunted through the porous membrane to the organs below the artery and capillary layers, and "arterial blood" becomes "venous blood". Then, the blood vertically enters vein layers (14 to 14',15 to 15',16 to 16',17 to 17',18 to 18',19 to 19',20 to 20',21 to 21',22 to 22',23 to 23 ') through capillary vessel end interfaces 14 to 23', and the blood at the capillary vessel end interfaces 14' to 23' is converged into the right atrium and the ventricle 13', and then the venous blood enters the culture medium tank from the right atrium and the ventricle 13' to become arterial blood again. Fresh "blood" then flows into the lungs 12 again, completing a "blood circulation" process. When the blood circulates, the micro-tissues in the mini mouse model can survive for a long time, and the micro-tissues have lives similar to the simulated mice.

Referring to fig. 15, the mini mouse model has two kidneys. Wherein, the kidney at one side of the abdomen is provided with a kidney microtissue for detecting the drug nephrotoxicity; while the kidney, which is close to the back, is used to excrete body wastes and drugs, and a separate elimination channel 7 is provided below it (fig. 20). A typical elimination curve for fluorescein sodium (molecular weight 320) is shown in FIG. 21.

Referring to fig. 22, in the mini mouse model, the intestine is designed below the hepatic microtissue 9, separated by the porous membrane 8, and the intestinal cells 10 are cultured on the lower surface of the porous membrane 8 and form a dense intestinal cell membrane. The intestinal cell culture section is also in communication with the outside through a separate digestive channel 11 to allow circulation of the digestive fluid stream. The drug is added from the digestive tract 11 and may simulate oral administration, where the drug is first absorbed by the intestine, then metabolized by the liver, and finally enters the simulated blood.

In this example, an adult rat was killed, various tissues were extracted, parallel 3 mini-mouse models each having a length of 10 cm were prepared, and the drug-time curve (dose 1.08 mg) of erlotinib was measured using the mini-mouse models. As can be seen from fig. 23, the blood concentration of the drug tends to increase first and then decrease, similar to the real time curve, and the error of each data point is small.

In addition, systemic toxicity and drug effect of doxorubicin were also measured using the mini mouse model, and it was found that doxorubicin had toxicity and drug effect that differed for each organ (fig. 24).

Example 6: mini human model, mini dog model, mini monkey model and mini pig model

FIGS. 25-28 show the design of these mini-mammal models, including the arterial and capillary layers, the venous layer and the organ layer, and the corresponding species of micro-tissue placed in the chamber, which can be substituted for the corresponding real mammal for drug evaluation.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. A mini mammal model is characterized in that the model is formed by stacking a lower substrate, a middle substrate, an upper substrate and a top plate from bottom to top in sequence; wherein the content of the first and second substances,

the lower substrate is an organ layer, and a plurality of tissue culture chambers are formed on the lower substrate and are respectively used for culturing different micro tissues to simulate each organ of a mammal;

the middle substrate is an artery and capillary layer, a first micro-channel representing an artery blood vessel and a plurality of second micro-channels representing a capillary are etched on the lower surface of the middle substrate, the first micro-channel and the second micro-channels are positioned above the tissue culture chambers and are communicated with the corresponding tissue culture chambers, and porous membranes are arranged among the tissue culture chambers, the first micro-channel and the second micro-channels; the plurality of second microchannels are each connected to the first microchannel;

2. The mini-mammal model of claim 1, wherein the bottom surface of the tissue culture chamber is a low adhesion super-hydrophobic surface with micro-nano topography, and the contact angle with the aqueous solution is more than 120 °;

or the bottom surface of the tissue culture chamber is modified with a low-adhesion hydrophilic polymer coating in a micro-nano shape, and the contact angle between the polymer coating and the water-phase solution is less than 90 degrees.

3. The mini-mammal model of claim 2, wherein the method of forming the low adhesion superhydrophobic surface is: mixing nano silicon dioxide particles, normal hexane and chloroform, and then carrying out ultrasonic treatment to disperse the nano silicon dioxide particles in the mixed solution; and then, soaking the PMMA board into the mixed solution, taking out and airing to obtain the low-adhesion super-hydrophobic surface.

4. The mini-mammal model of claim 2, wherein the method of forming the low adhesion superhydrophobic surface is: soaking the PDMS plate into tetraethyl orthosilicate to swell the tetraethyl orthosilicate, taking out the swelled PDMS plate, and placing the swelled PDMS plate in an ethylene diamine aqueous solution; and taking out the PDMS plate, washing and then carrying out heat treatment to obtain the low-adhesion super-hydrophobic surface.

5. The mini-mammal model of claim 2, wherein the method of forming the low adhesion superhydrophobic surface comprises the steps of:

a. pouring the pre-polymerization liquid of the elastic resin on a super-hydrophobic template with a micro-nano structure on the surface, polymerizing the pre-polymerization liquid of the elastic resin into a first elastic solid, and then stripping the first elastic solid from the template;

c. and peeling the second elastic solid from the silanized and modified first elastic solid template to form the low-adhesion super-hydrophobic surface on the surface of the second elastic solid.

6. The mini-mammal model of claim 2, wherein the method for preparing the low adhesion hydrophilic polymer coating comprises the following steps:

s2, pretreating the bottom surface of the tissue culture chamber to enable the bottom surface to generate polar groups, and then placing the pretreated bottom surface in the polymer coating liquid for soaking treatment; then taking out, washing off the polymer coating liquid on the surface, and drying to form a low-adhesion hydrophilic polymer coating on the bottom surface of the tissue culture chamber;

7. The mini-mammal model as claimed in claim 1, wherein the tissue culture chamber has a through hole formed in a bottom surface thereof, the through hole being used for inserting a micro paddle or a sensor into the tissue culture chamber.

8. The mini-mammal model of claim 1, wherein the mini-mammal model has a similar appearance to a real mammal;

at least one tissue culture chamber is positioned in the mini-mammal model at a location corresponding to the location of the organ it represents within the mammal body and similar in shape to the organ it represents.

9. The mini-mammal model of claim 1, wherein the tissue culture chamber comprises a brain culture chamber, an eye culture chamber, a nose culture chamber, an ear culture chamber, a tongue culture chamber, a trachea culture chamber, a heart culture chamber, a lung culture chamber, a liver-intestine culture chamber, a kidney culture chamber, a stomach culture chamber, a pancreas culture chamber, a spleen culture chamber, a skin culture chamber, a fat culture chamber, a bone marrow culture chamber, a muscle culture chamber, a testis culture chamber, and a tumor culture chamber.

10. A mini-mammal model according to claim 9, wherein the culture fluid inlet communicates with the first microchannel above the lung culture chamber and the culture fluid outlet communicates with the end of the third microchannel above the heart culture chamber.

11. The mini-mammal model of claim 1, wherein the porous membrane between the tissue culture chamber and the first and second microchannels is used to culture vascular endothelial cells, and immune cells are placed on the vascular endothelial cells.

12. The mini-mammal model of claim 1, wherein an airway is provided in a sidewall of the tissue culture chamber, the airway communicating with the outside to provide oxygen to the tissue culture chamber.

13. The mini-mammal model of claim 9, wherein the liver-intestine culture chamber comprises a liver tissue culture region for culturing liver micro-tissues separated from the first and second micro-channels by a porous membrane, and an intestinal cell culture region located below the liver tissue culture region, and the upper surface of the porous membrane is used for culturing vascular endothelial cells on which immune cells are cultured;

14. The mini-mammal model of claim 9, wherein there are two kidney culture chambers, one of the kidney culture chambers being used to culture kidney micro-tissues to simulate kidney metabolic function; the other kidney culture chamber was equipped with a dialysis membrane and was in communication with the outside through a separate elimination channel for simulating the elimination of waste and drugs in the body.

15. Use of the mini-mammal model of any one of claims 1 to 14 in a pharmacodynamic, toxicity, or pharmacokinetic assay.