CN114276903A

CN114276903A - Liver organoid culture chip, liver organoid model, and preparation method and application thereof

Info

Publication number: CN114276903A
Application number: CN202111594737.7A
Authority: CN
Inventors: 陈璞; 徐晓冬; 江善青; 谷龙军
Original assignee: Wuhan University WHU
Current assignee: Hefei Runyin Biotechnology Co ltd
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-04-05
Also published as: WO2023116788A1

Abstract

The invention discloses a liver organoid culture chip, a liver organoid model, a preparation method and application thereof, wherein the preparation method of the chip comprises the following steps: obtaining a film having a plurality of perforations; coating a material with low cell adhesion on the bottom of a cell culture plate, and then covering the film on the bottom of the cell culture plate to obtain a patterned substrate; adding a cell-specific adhesive material into the patterned substrate for coating to obtain a chip; inoculating foregut embryonic cells into the culture chip of the liver organoid, removing the film with the plurality of perforations after the cells adhere to the wall, absorbing the culture medium in the culture chip of the liver organoid, washing, and adding a first culture medium for maintaining and culturing for 3-5 days; and then sequentially adopting a second culture medium and a third culture medium to respectively maintain and culture to obtain the liver organoid model. The uniformity is high, in-situ imaging can be realized, and the method is suitable for optical imaging instruments such as a confocal microscope.

Description

Liver organoid culture chip, liver organoid model, and preparation method and application thereof

Technical Field

The invention relates to the technical field of tissue engineering and organ chips, in particular to a liver organoid culture chip, a liver organoid model, a preparation method and application thereof.

Background

The liver is an important organ of the human body and has a variety of important physiological functions including detoxification, digestion, metabolism and the like. However, related liver diseases such as viral hepatitis, non-alcoholic fatty liver, liver cancer and the like cause a heavy social burden and seriously threaten the life health of human beings. Therefore, it is important to develop a treatment method for liver diseases and establish a liver research model highly related to the human body in order to reveal the mechanism of occurrence and development of liver diseases. Liver organoids are a new liver research model, have key characteristics of human liver, such as cell composition and functional characteristics of albumin synthesis, glycogen synthesis, lipid accumulation and the like, and have wide application in the fields of basic research, drug research and development and the like. Compared with the traditional animal model, the organoid model has no species difference and has the apparent metabolic capacity highly related to the human body; compared with a two-dimensional culture model, the method has the advantages of similar cell microenvironment to that in vivo, liver tissue specific cell population, capability of simulating the interaction between parenchymal cells and non-parenchymal cells and the like. At present, the liver organoid overcomes the limitations of cancer cell lines and PDX models, can culture pathological changes and healthy tissues from the same patient, provides a personalized medicine test platform matched with the patient, and provides an ideal model for disease mechanism research. Therefore, the construction of the in vitro liver organoid provides an effective model system for researching the pathogenesis of the liver and developing a treatment method.

At present, the cultivation of liver organoids is mainly based on Matrigel gel drop culture. The glue drop method has the manufacturing process that stem cells are randomly wrapped in the glue drops, so that heterogeneity exists in the glue drops and among the glue drops, the heterogeneity is particularly represented by the shape, the size, the maturity and the cell population, in-situ optical imaging is difficult to perform, and the consistency and the reproducibility of organoid production are difficult to realize. Although the existing method can carry out in-situ three-dimensional balling, the in-situ imaging cannot be carried out with high flux because the Z-axis height of the organoid exceeds the imaging limit of the current optical microscope (such as a confocal microscope and an inverted fluorescence microscope), so that the organoid needs to be taken out for slicing and then imaging, the process is complicated, the three-dimensional in-situ structure cannot be explained, and the problems seriously influence the commercial application of the liver organoid in the biomedical field.

Therefore, in order to overcome the problems of the above culture methods, it is urgently needed to develop a high-homogeneity in-situ imageable liver organoid culture chip and a novel liver organoid model, which are suitable for confocal microscopes and other optical imaging instruments, especially a high-throughput screening gold standard-high-content fluorescence imaging and cell analysis system in the pharmaceutical development industry, so as to provide an innovative research tool for liver basic research and drug research and development.

Disclosure of Invention

The invention aims to provide a liver organoid culture chip, a liver organoid model, a preparation method and application thereof, which have high uniformity and can be used for in-situ imaging and are suitable for optical imaging instruments such as a confocal microscope and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a method for preparing a culture chip for liver organoids, the method comprising:

obtaining a film having a plurality of perforations;

coating a material with low cell adhesion on the bottom of a cell culture plate, and then covering the film on the bottom of the cell culture plate to obtain a patterned substrate;

and adding a material with cell specificity adhesion into the patterned substrate for coating to obtain the culture chip of the liver organoid.

Further, the hole shape of each of the perforations includes one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrangle, a pentagon, a hexagon, and an arbitrary polygon; the diameter of the circumscribed circle of each perforation is 100-1000 microns, and the distance between two adjacent perforations of the film is 0.5-3 mm.

Further, the material of the film comprises polydimethylsiloxane; the cell-low adhesion material comprises polyethylene glycol; the cell-specifically adhesive material includes at least one of Matrigel and Collagen.

Further, the outer dimensions of the membrane match the cell culture plate; the cell culture plate comprises one of a 384-well plate, a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate, a 3.5-cm culture dish, a 6-cm culture dish and a 10-cm culture dish.

Further, the obtaining a film having a plurality of perforations comprises:

obtaining a male mold having a plurality of micropillar arrays;

pouring PDMS onto the male mold, vacuum drying and vacuumizing, covering a layer of PMMA on the PDMS, clamping and fixing by two glass sheets, and drying;

the solidified PDMS layer was taken out and cut into a PDMS membrane that fit the shape of the cell culture plate, obtaining a thin film with multiple perforations.

Further, the height of the micro-column array of the anode membrane is 30-100 μm.

In a second aspect of the invention, a liver organoid culture chip prepared by the method is provided.

In a third aspect of the invention, there is provided a method of preparing a liver organoid model, the method comprising:

inoculating foregut embryonic cells into the culture chip of the liver organoid, removing the film with the plurality of perforations after the cells adhere to the wall, absorbing the culture medium in the culture chip of the liver organoid, washing, and adding a first culture medium for maintaining and culturing for 3-5 days;

and then, maintaining and culturing for 3-5 days by adopting a second culture medium and maintaining and culturing for more than or equal to 10 days by adopting a third culture medium in sequence to obtain the liver organoid model.

Further, the seeding density of the pre-intestinal germ cells is 1 × 10⁵～9×10⁵(pieces/cm)²)。

In the above embodiments, the liver organoid includes, but is not limited to, PSC or EB derived liver organoids, adult stem cell derived liver organoids, liver-related tumor organoids.

In a fourth aspect of the invention, there is provided a liver organoid model obtained using the method.

In the fifth aspect of the invention, the application of the liver organoid model in pharmacological, pharmacodynamic and toxicological analysis is provided.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

(1) the invention provides a liver organoid model and a preparation method and application thereof.A cell specific adhesion area with a specific pattern is formed at the bottom of a cell culture vessel by a patterning technology, cell low adhesion or ultra-low adhesion treatment is carried out on a non-pattern area, stem cells with different sources or different tissue sources can be inoculated on the patterned substrate, a PDMS (polydimethylsiloxane) perforated film is removed after the cells adhere to the wall to obtain a cell aggregate with a specific edge shape, the stem cells are induced to differentiate towards a liver lineage on the patterned substrate, and a novel liver organoid model with the thickness of 30-200 mu m and the specific edge shape is finally formed, so that the liver organoid model has high uniformity and can be imaged in situ, and is suitable for optical imaging instruments such as a confocal microscope; the liver organoid is flat, resembling a solid dome, which is a phenotype different from previous liver organoids;

(2) the patterned substrate array can be designed and customized according to specific requirements, and is suitable for culturing various types of organoids or cells;

(3) the perforated film is suitable for various commercially available cell culture vessels, the preparation process is simple and easy to understand, the learning cost is low, and the perforated film has good advantages for common laboratories and mass production;

(4) the liver organoid of the present invention can be subjected to in-situ staining and imaging in the well plate without destroying the organoid structure, and has good compatibility with the existing bioanalysis and imaging instruments (such as high content instruments) used in the field of drug development.

(5) The patterned substrate is easy for large-scale standardized production, has wide application range, can meet the demand and is very easy to realize commercial production.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a flow chart and a schematic diagram of a patterned substrate of a liver organoid culture chip according to an embodiment of the present invention; wherein FIG. 1a is a flow chart of a patterned substrate preparation process; FIG. 1b, FIG. 1c and FIG. 1d are pictorial representations;

FIG. 2 is a schematic diagram of the culture and characterization of human liver organoid on the patterned substrate of the liver organoid culture chip according to the embodiment of the present invention; wherein FIG. 2a is a flow chart of a patterned liver organoid culture; FIG. 2b is a bright field diagram of liver organoids in a 24-well plate; FIG. 2c is a bright field diagram of a liver organoid cultured by the conventional gel drop method; FIG. 2d is a plot of area characterization of patterned liver organoids cultured to different time points; FIG. 2e is a comparison of the coefficient of variation (CV value) for the area of patterned liver organoids and of the area of the liver organoids by the gel drop method;

FIG. 3 shows the identification of protein levels and transcription levels at various stages of the establishment of human liver organoids; wherein FIG. 3a is the expression of the hipSC stage and foregut embryo stage markers; FIG. 3b shows the expression of dry markers and bidirectional differentiation potential markers associated with different time points of patterned liver organoids; FIG. 3c is the expression of patterned liver organoid liver lineage markers; FIG. 3d shows the expression of genes associated with patterned liver organoids;

fig. 4 is a diagram illustrating the construction and characterization of a drug hepatotoxicity evaluation system in a patterned substrate of a liver organoid culture chip according to an embodiment of the present invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.

It will be understood that when an element is referred to as being "fixed" or "disposed" on another element, it can be directly on the other element or be indirectly on the other element; when an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element.

It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "first," "second," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, refer to an orientation or positional relationship indicated in the drawings for convenience in describing the present application and to simplify description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be operated in a particular manner, and is not to be construed as limiting the present application.

In addition, in the description of the present application, "a plurality" or "a plurality" means two or more unless specifically defined otherwise.

The technical scheme of the application has the following general idea:

according to an exemplary embodiment of the present invention, there is provided a method of preparing a culture chip for liver organoids, the method including:

obtaining a film having a plurality of perforations;

In the technical scheme, a cell specific adhesion area with a specific pattern is formed at the bottom of a cell culture vessel through a patterning technology, cell low adhesion or ultra-low adhesion treatment is carried out on a non-pattern area, stem cells of different sources or different tissue sources can be inoculated on the patterned substrate, a PDMS perforated film is removed after the cells adhere to the surface, a cell aggregate with a specific edge shape is obtained, the stem cells are induced to differentiate towards a liver lineage on the patterned substrate, and finally, a novel liver organ model with the thickness of 30-200 mu m and the specific edge shape is formed. The uniformity is high, in-situ imaging can be realized, and the method is suitable for optical imaging instruments such as a confocal microscope.

As a preferred embodiment, the hole shape of each of the perforations includes one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrangle, a pentagon, a hexagon, and an arbitrary polygon; the diameter of the circumscribed circle of each perforation is 100-1000 microns, and the distance between two adjacent perforations of the film is 0.5-3 mm.

If the diameter of the circumscribed circle of each perforation is less than 100 mu m, the defect that the organoid is easy to lose during liquid exchange exists, and if the diameter of the circumscribed circle is too large, the defect that the flux is low exists;

if the distance between two adjacent perforations of the film is less than 0.5mm, the defects that the organoids are easy to adhere and cannot grow independently exist; if the distance is too large, the flux is low; the plurality of through holes may be arranged at equal intervals or at non-equal intervals.

As a specific embodiment, the outer dimensions of the membrane match the dimensions of the cell culture plate; the cell culture plate comprises one of a 384-well plate, a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate, a 3.5-cm culture dish, a 6-cm culture dish and a 10-cm culture dish. The liver organoid model is reconstructed based on the existing cell culture pore plate, is highly combined with the existing cell culture pore plate, and has good compatibility with the existing biological related optical instruments.

As a specific embodiment, it is possible to use,

the material of the film comprises polydimethylsiloxane;

the cell-low adhesion material comprises polyethylene glycol; other cell-low adhesion materials may also be used in other embodiments;

the cell-specifically adhesive material includes at least one of Matrigel and Collagen.

As a specific embodiment, the obtaining a film having a plurality of perforations comprises:

obtaining a male mold having a plurality of micropillar arrays;

Wherein the height of the micro-column array of the anode membrane is 30-100 μm. If the height of the PDMS film is too low, the PDMS film is pressed out too thin and is not easy to unfold; the height is too high, so that the hole is not easy to be punched;

according to another exemplary embodiment of the present invention, there is provided a liver organoid culture chip prepared by the method.

According to another exemplary embodiment of the present invention, a method for preparing a liver organoid model is provided, the method comprising:

s1, inoculating foregut embryonic cells in the culture chip of the liver organoid, removing the film with the plurality of perforations after the cells adhere to the wall, absorbing the culture medium in the culture chip of the liver organoid, washing, and adding a first culture medium to maintain for 3-5 days;

the step S1 specifically includes:

s101, induction of stem cell (hESCs or hiPSCs) differentiation into endoderm: routinely cultured hESCs or hipSCs were digested into single cells at 1X 10 with Accutase⁵Per cm²The six-well plate is laid at the density, and the differentiation is started when the cell fusion degree reaches 85-90%. The culture time is 1-3 days, and the culture is carried out by adopting a D1-D3 culture medium:

d1 Medium RPMI Medium containing 100ng/mL ActivinA and 50ng/mL BMP 4; d2 Medium RPMI Medium containing 100ng/mL ActivinA and 0.2% Knockout serum replacement; d3 Medium RPMI Medium containing 100ng/mL ActivinA and 2% Knockout serum replacement.

S102, endoderm cells are differentiated to anterior enterocyte cells: culturing with a differentiation medium; the medium was changed daily and the cells were routinely grown in two dimensions in an incubator. A distinct three-dimensional structure was visible at day 6 of differentiation.

The differentiation Medium is D4-6Medium in the examples of the invention; the formula of the differentiation medium is an Advanced DMEM/F12 medium with the final concentration of 450-550 ng/mL FGF2, 2-4 mu M CHIR99021, 1% B27 and 1% N2, and preferably 500ng/mL FGF2 and 3 mu M CHIR 99021;

the B27 and N2 are serum-free culture additives. The "%" in 1% B27 and 1% N2 is a mass fraction.

S103, digesting the foregut embryonic cells, inoculating the digested foregut embryonic cells onto the patterned substrate of the liver organoid culture chip, and adding a first culture medium to maintain and culture for 3-5 days.

The inoculation density of the foregut embryonic cells is 1 multiplied by 10⁵～9×10⁵(pieces/cm)²). If the cell seeding density is too low, the defect that organoids with certain thickness are difficult to grow exists; if the cell seeding density is too high, there are disadvantages that the number of cells is too large, it is difficult to separate from the PDMS porous membrane, and organoids cannot be formed in a patterned distribution.

The formula of the first culture medium is an Advanced DMEM/F12 culture medium with the final concentration of 75-85 ng/mL FGF2, 2-4 mu M CHIR99021, 1% B27 and 1% N2, and preferably 80ng/mL FGF2 and 3 mu M CHIR 99021;

and S2, sequentially adopting a second culture medium to maintain and culture for 3-5 days and a third culture medium to maintain and culture for more than or equal to 10 days to obtain the liver organoid model.

In the step S2, in the above step,

the formula of the second culture medium is Advanced DMEM/F12 with the final concentration of 1-3 mM RA, 1% B27 and 1% N2, and preferably contains 2mM RA;

the third Culture Medium is a Hepatocyte Culture Medium with the final concentration of 8-12 ng/mL HGF, 0.05-0.2 mM Dexamethane and 18-22 ng/mL OSM, preferably a Hepatocyte Culture Medium with 10ng/mL HGF, 0.1mM Dexamethane and 20ng/mL OSM;

according to another exemplary embodiment of the present invention, a liver organoid model obtained by the method is provided. As a specific implementation of an embodiment of the present invention, the obtained liver organoids are flat, resembling a solid dome (or similar expression), which is a different phenotype than the previous liver organoids; one side of the organoid is adhered to the bottom of the plate, and the organoid also has a certain thickness; from the results of the fluorescence images, the peripheral portions of the organoids are clearly visible and the central portion is less transparent to light, so the central thickness should be thicker than the peripheral portions, indicating a solid dome shape.

The solid circular arch-shaped structure is as follows: the middle part of the liver organoid is arched, and the middle part and the periphery of the liver organoid form an arc shape; the arch is a three-dimensional concept, and the semicircle is a plane concept. The shape described by the "solid dome shape" can also be expressed in other ways as long as the liver organoids obtained by the present invention can be characterized.

According to another exemplary embodiment of the present invention, there is provided a use of the liver organoid model in pharmacological, pharmacodynamic, toxicological analysis.

The preparation method of the novel liver organoid model comprises the steps of inducing stem cells into foregut embryonic cells, planting the foregut embryonic cells on a patterned substrate in a certain density, differentiating the foregut embryonic cells to a liver lineage, and finally forming an arch-shaped liver organoid which is adhered to the bottom of a culture vessel and has a certain thickness and a specific edge shape, wherein the cell type of the liver organoid comprises liver-like cells, bile duct-like cells, progenitor cells and interstitial cells differentiated by the stem cells; the novel liver organoid model can be applied to research of liver development and disease mechanisms, drug screening, drug hepatotoxicity evaluation and the like. Compared with the traditional gum dripping method and the in-situ balling method, the invention provides a novel liver organoid model which has high flux and high uniformity and can be imaged in situ, and provides an innovative research tool for relevant liver research.

A liver organoid model and a culturing method according to the present application will be described in detail below with reference to the accompanying drawings.

Example 1A liver organoid culture chip and method for preparing the same

First, design of patterned substrate array mask

The patterning array mask is designed by using AutoCAD 2018 software, the array units are 0.5mm in diameter and circular, the spacing is 1.5mm, and the array units are uniformly arranged at equal intervals. The patterned array mask layout is shown in FIG. 1.

Second, manufacturing of patterned substrate

1. The SU-8 positive film is prepared by using a soft lithography technology, the SU-8 positive film is a micro-column array with the interval of 1.5mm, the height of the micro-column is 80 mu m, and the diameter of the bottom area is 500 mu m.

2. The PDMS prepolymer (glue A) and the crosslinking agent (glue B) are mixed according to the weight ratio of 10: 1, stirring uniformly, and then pumping out bubbles by using a vacuum drier. And pouring the PDMS on the SU-8 positive membrane, leveling the PDMS by using a clean gun head to uniformly cover the area of the micro-column array, and vacuumizing the micro-column array by using a vacuum drier until no bubbles exist on the surface. Covering 1 layer of polymethyl methacrylate (PMMA) with the thickness of 0.2mm on the surface, fixing the PMMA with two glass sheets, then placing the PMMA in a bench clamp for clamping and fixing, and placing the PMMA in an oven with the temperature of 80 ℃ for drying for at least 120 min.

3. The solidified PDMS punched film was removed from the vise and punched down with a 15mm diameter round punch.

4. Preparing PEG mixture (PEG1000:37.5mg, PEG400:450 μ L, isopropanol 3637.5 μ L, and pure water 112.5 μ L), shaking and mixing for 3min, adding photoinitiator 10mg, shaking and mixing for 3min, and storing in dark place. After the plate was treated with Plasma (working voltage 550V) with the plate being uncapped for 1min, 150. mu.L of PEG mixture was added to each well, and the mixture was allowed to stand for 5 min. The well plate with PEG mix in the well was UV exposed for 1 min. After the exposed orifice plate was washed three times with 70% alcohol, 1mL of 70% alcohol was added to each orifice, the circular PDMS perforated film was spread flat on the liquid surface, pressed to the bottom of the orifice plate with tweezers, excess liquid was aspirated, and the orifice plate was placed in an 80 ℃ oven and dried for about 20 min.

5. And putting the dried pore plate into a Plasma cleaner for Plasma (working voltage of 700V), and cleaning for three times, wherein each time is 4min, the cleaning interval is 3min, and the pore plate is not taken out at intervals.

6. And (3) carrying out ultraviolet sterilization on the prepared pore plate for 60min, adding 1mL of 1 XDPBS into each pore, and repeatedly blowing and beating by using a pipette until no air bubbles exist in the pore plate. The 1 XDPBS was aspirated, 200. mu.L of 1% Matrigel: Advanced DMEM/F12 was added to each well, and the mixture was incubated at 37 ℃ for 1 hour to prepare a liver organoid culture chip.

Culture of human liver organoid

1. The patterned substrates of the examples and comparative examples were subjected to liver organoid culture as follows:

(1) differentiation of hESCs or hiPSCs into endoderm: routinely cultured hESCs or hipSCs were digested into single cells at 1X 10 with Accutase⁵Per cm²The six-well plate is laid at the density, and the differentiation is started when the cell fusion degree reaches 85-90%.

(2) D1 Medium RPMI Medium containing 100ng/mL ActivinA and 50ng/mL BMP 4; d2 Medium RPMI Medium containing 100ng/mL ActivinA and 0.2% Knockout serum replacement; d3 Medium RPMI Medium containing 100ng/mL ActivinA and 2% Knockout serum replacement.

The above D1-D3 medium was used for the first three days of differentiation to induce differentiation of stem cells into the endoderm;

(3) endoderm cells differentiated towards anterior enteroblast cells: d4-6 Medium: advanced DMEM/F12 medium containing 500ng/mL FGF2, 3. mu.M CHIR99021, 1% B27 and 1% N2, the medium was changed daily and the cells were cultured in the incubator in two dimensions as usual. A distinct three-dimensional structure was visible at day 6 of differentiation.

(4) Seeding of pre-gut germ cells in patterned substrates: differentiation to day 6, digestion of foregut blasts into single cells with Accutase at 3X 10⁵Per cm²Was inoculated into a well plate, 500. mu.L per well of Advanced DMEM/F12 medium containing 80ng/mL FGF2, 3. mu.M CHIR99021, 1% B27 and 1% N2.

(5) After the cells are seeded for at least 4 hours, the PDMS film is gently lifted off from the bottom edge of the well plate by forceps after the cells are attached. After aspirating the medium from the well plates and gently washing the cells with 1 XDPBS, 500. mu.L of Advanced DMEM/F12 medium containing 80ng/mL FGF2, 3. mu.M CHIR99021, 1% B27 and 1% N2 per well was added again to maintain the culture for 4 days, with the medium changed every other day.

(6) Culture of liver organoids in patterned substrates: culturing on days 10-14 with Advanced DMEM/F12 containing 2mM RA, 1% B27 and 1% N2 for 4 days, with fluid changes every other day; the Culture is maintained for ten days by using a Hepatocyte Culture Medium containing 10ng/mL HGF, 0.1mM Dexamethane and 20ng/mL OSM on days 14-24, and the Culture Medium is changed every other day.

Example 2

In the embodiment of the invention, a PDMS perforated film is punched into a circle with the diameter of 10mm by a round punch and is paved in a 48-hole plate, and other structures and steps are the same as those in the embodiment 1.

Example 3

In the embodiment of the invention, the pattern unit in the PDMS perforated film is circular, the diameter of the circular pattern is 500 μm, the pattern interval is 1.5mm, and the cell seeding density on the patterned substrate is 1 multiplied by 10⁵Per cm²The other structures and steps are the same as those of embodiment 1.

Example 4

In the embodiment of the invention, the pattern units in the PDMS perforated film are circular, the diameter of the circular pattern is 500 μm, the pattern space is 1.5mm, and the cell seeding on the patterned substrate is denseDegree of 9X 10⁵Per cm²The other structures and steps are the same as those of embodiment 1.

Example 5

In the embodiment of the invention, the pattern units in the PDMS perforated film are circular, the diameter of the circular pattern is 100 μm, the pattern pitch is 1mm, and other structures and steps are the same as those in embodiment 1.

Example 6

In the embodiment of the invention, the pattern units in the PDMS perforated film are circular, the diameter of the circular pattern is 200 μm, the pattern pitch is 1.2mm, and other structures and steps are the same as those in embodiment 1.

Example 7

In the embodiment of the invention, the pattern units in the PDMS perforated film are circular, the diameter of the circular pattern is 1000 μm, the pattern pitch is 2mm, and other structures and steps are the same as those in embodiment 1.

Example 8

In the embodiment of the invention, the pattern units in the PDMS perforated film are circular, the circular patterns are arranged in regular hexagons (including the central point), the pattern spacing is 1mm, and other structures and steps are the same as those in embodiment 1.

Example 9

In the embodiment of the invention, the pattern units in the PDMS perforated film are circular, the circular patterns are arranged in regular hexagons (including the central point), the pattern pitch is 1.5mm, and other structures and steps are the same as those in embodiment 1.

Example 10

In the embodiment of the invention, the pattern units in the PDMS perforated film are circular, the circular patterns are arranged in regular hexagons (including the central point), the pattern pitch is 2mm, and other structures and steps are the same as those in embodiment 1.

Example 11

In the embodiment of the invention, the shape of the pattern units in the PDMS perforated film is equilateral triangle, the side length is 1mm, the pattern interval is 2mm, and other structures and steps are the same as those in the embodiment 1.

Example 12

In the embodiment of the invention, the pattern units in the PDMS perforated film are equilateral triangles, the side length is 0.5mm, the pattern pitch is 1.5mm, and other structures and steps are the same as those in embodiment 1.

Example 13

In the embodiment of the invention, the pattern units in the PDMS perforated film are rectangular, the length of the long side is 0.5mm, the length of the short side is 0.15mm, the pattern interval is 1.5mm, and other structures and steps are the same as those in embodiment 1.

Example 14

In the embodiment of the invention, the pattern units in the PDMS perforated film are rectangular, the length of the long side is 1mm, the length of the short side is 0.3mm, the pattern interval is 2mm, and other structures and steps are the same as those in embodiment 1.

Comparative example 1

The comparative example is a conventional Matrigel.

Comparative example 2

In this comparative example, the diameter of the circular pattern in the PDMS perforated film was 95 μm, the pattern pitch was 1mm, and the other structures and steps were the same as in example 1.

Comparative example 3

In this comparative example, the cell seeding density on the patterned substrate was 0.9X 10⁵(pieces/cm)²) Smaller than 1X 10 in the examples of the present invention⁵～9×10⁵(pieces/cm)²) The other structures and steps are the same as those of embodiment 1.

Comparative example 4

In this comparative example, the cell seeding density on the patterned substrate was 9.1X 10⁵(pieces/cm)²) Greater than 1X 10 in the examples of the invention⁵～9×10⁵(pieces/cm)²) The other structures and steps are the same as those of example 1.

Comparative example 5

In this comparative example, the pattern pitch was 0.4mm, and the other structure and procedure were the same as in example 1.

Comparative example 6

In this comparative example, the patterning pitch was 3.2mm, and the other structure and procedure were the same as in example 1.

Experimental example 1

Liver organoid culture effect statistics were performed on the substrates of the above examples 1 to 14 and comparative examples 1 to 6, as shown in table 1, in which the standard deviation coefficient of variation of the area was calculated by: coefficient of variation C · V ═ (standard deviation SD/Mean) × 100%;

TABLE 1

From the data in table 1, it can be seen that:

in comparative example 1, the conventional Matrigel culture method has the disadvantages of poor organoid homogeneity and difficult imaging;

in comparative example 2, the pattern unit of the patterned substrate is a circle with a diameter of 95 μm, which is smaller than the range of 100 to 1000 μm in the example of the present invention, and there is a disadvantage that the organoid is easily lost when the fluid is changed.

In comparative example 3, the cell seeding density was 0.9X 10⁵Per cm²Smaller than 1X 10 in the present embodiment⁵～9×10⁵(pieces/cm)²) There is a disadvantage that it is difficult to grow organoids having a certain thickness.

In comparative example 4, the cell seeding density was 9.1X 10⁵Per cm²Greater than 1 × 10 in the present embodiment⁵～9×10⁵(pieces/cm)²) The range (2) has the disadvantage that the number of cells is too large, the cells are difficult to separate from the PDMS-made perforated film, and the organoids cannot be formed in a patterned distribution.

In comparative example 5, the pattern unit pitch of the patterned substrate was 0.4mm, which is smaller than the range of 0.5 to 3mm in this example, and there was a disadvantage that the organoids were easily adhered to each other and could not grow independently.

In comparative example 6, the pattern unit pitch of the patterned substrate was 3.2mm, which is larger than the range of 0.5 to 3mm in this example, and there was a disadvantage that high throughput could not be achieved.

In the examples 1 to 11 of the present invention, the growth was normal and the coefficient of variation was 40% or less. Compared with the traditional gum dripping method and the in-situ balling method, the invention provides a novel liver organoid model which has high flux and high uniformity and can be imaged in situ, and provides an innovative research tool for relevant liver research.

Experimental example 2 detection of human liver organoid marker

1. Differentiation to stage signature markers were detected using immunofluorescence: detecting the expression condition of the markers OCT3/4 and Nanog at the stage of hESCs or hipSCs; foregut stage detection markers CDX2, EpCAM expression; detecting the expression conditions of the markers ALB, HNF 4-alpha, AFP and EpCAM in the liver organoid stage.

2. Example 1 construction and characterization of drug hepatotoxicity evaluation system on patterned substrates: on day 24, dead and alive assays were performed 48 hours after treatment of liver organoids with 0mM, 10mM, 20mM, 40mM acetaminophen (APAP), respectively.

The results are shown in fig. 3-4, which show that the embodiment of the invention successfully obtains the human liver organoid and constructs a drug hepatotoxicity evaluation system.

3. Culturing human organoids on the patterned substrate of example 1 and culturing liver organoids using traditional gum drop method as control; wherein FIG. 2a is a flow chart of a patterned liver organoid culture; FIG. 2b is a bright field diagram of liver organoids in a 24-well plate; FIG. 2c is a bright field diagram of a liver organoid cultured by the conventional gel drop method; FIG. 2d is a plot of area characterization of patterned liver organoids cultured to different time points; FIG. 2e is a comparison of the coefficient of variation (CV value) for the area of patterned liver organoids and of the area of the liver organoids by the gel drop method;

as can be seen from fig. 2e, the area coefficient of variation of the patterned liver organoids is significantly lower than that of the liver organoids by the gel drop method; it can also be seen visually from a comparison of the brightfield plots of fig. 2a and 2c that the patterned liver organoids bound the regions of organoid growth, having consistent shape and size, whereas the gum-drop cultured organoids were not controllable in these respects.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method for preparing a culture chip of a liver organoid, the method comprising:

obtaining a film having a plurality of perforations;

2. The method of claim 1, wherein the shape of each of the holes of the plurality of holes includes one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrangle, a pentagon, a hexagon, and an arbitrary polygon; the diameter of the circumscribed circle of each perforation is 100-1000 microns, and the distance between two adjacent perforations of the film is 0.5-3 mm.

3. The method of claim 1, wherein the thin film is made of polydimethylsiloxane; the cell-low adhesion material comprises polyethylene glycol; the cell-specifically adhesive material includes at least one of Matrigel and Collagen.

4. The method for preparing a culture chip of liver organoid according to claim 1, wherein the obtaining of the thin film having a plurality of perforations comprises:

obtaining a male mold having a plurality of micropillar arrays;

5. The method of claim 4, wherein the height of the micro-column array of the anode membrane is 30 μm to 100 μm.

6. A liver organoid culture chip prepared by the method of any one of claims 1 to 5.

7. A method of preparing a liver organoid model, the method comprising:

inoculating foregut embryonic cells into the liver organoid culture chip of claim 6, removing the film with the plurality of perforations after the cells adhere to the wall, sucking off the culture medium in the liver organoid culture chip, washing, and adding a first culture medium for maintaining and culturing for 3-5 days;

8. The method of claim 7, wherein the pre-embryonic intestine is prepared by the method of preparing a liver organoid modelThe seeding density of the cells was 1X 10⁵～9×10⁵(pieces/cm)²)。

9. A liver organoid model obtained by the method of any one of claims 1 to 8.

10. Use of the liver organoid model of claim 9 in pharmacological, pharmacodynamic, toxicological assays.