WO2023116788A1

WO2023116788A1 - Liver organoid culture chip, liver organoid model, preparation methods therefor, and application

Info

Publication number: WO2023116788A1
Application number: PCT/CN2022/140800
Authority: WO
Inventors: 陈璞; 徐晓冬; 江善青; 谷龙军
Original assignee: 武汉大学
Priority date: 2021-12-24
Filing date: 2022-12-21
Publication date: 2023-06-29
Also published as: CN114276903A

Abstract

Disclosed are a liver organoid culture chip, a liver organoid model, preparation methods therefor, and an application. The preparation method for the chip comprises: obtaining a thin film having a plurality of perforations; coating a material having low cell adhesion on the bottom of a cell culture plate, and then covering the thin film on the bottom of the cell culture plate to obtain a patterned substrate; adding a material having specific cell adhesion into the patterned substrate for coating to obtain a chip; inoculating foregut germ cells in the liver organoid culture chip, removing the thin film having the plurality of perforations after the cells adhere to the wall, suctioning a culture medium in the liver organoid culture chip, and after washing, adding a first culture medium to maintain culture for 3-5 days; and then, sequentially using a second culture medium and a third culture medium to respectively maintain culture, so as to obtain a liver organoid model. The liver organoid model is high in uniformity and can be imaged in situ, and is suitable for optical imaging instruments such as a confocal microscope.

Description

A culture chip of liver organoid, liver organoid model and its preparation method and application

technical field

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

Background technique

The liver is an important organ of the human body and has a variety of important physiological functions, including detoxification, digestion and metabolism. However, related liver diseases such as viral hepatitis, non-alcoholic fatty liver, liver cancer and other diseases have caused a heavy social burden and seriously threatened human life and health. Therefore, in order to reveal the mechanism of the occurrence and development of liver diseases and develop therapeutic methods for liver diseases, it is of great significance to establish a liver research model that is highly relevant to the human body. Liver organoids are an emerging research model of the liver. They have key features of the human liver, such as cell structure and functional characteristics such as albumin synthesis, glycogen synthesis, and lipid accumulation. They have a wide range of applications in basic research, drug development, and other fields. . Compared with traditional animal models, the organoid model has no species difference, and has an apparent metabolic capacity that is highly related to the human body; compared with the two-dimensional culture model, it has a similar cellular microenvironment to the body and is specific for liver tissue Cell populations, the ability to simulate the interaction between parenchymal cells and non-parenchymal cells, etc. At present, liver organoids overcome the limitations of cancer cell lines and PDX models, and can cultivate diseased and healthy tissues from the same patient, providing a personalized drug testing platform that matches the patient, and providing an ideal model for the study of disease mechanisms. It can be seen that the construction of liver organoids in vitro provides an effective model system for the study of liver pathogenesis and the development of therapeutic methods.

At present, the culture of liver organoids is mainly based on the Matrigel droplet culture method. The production process of the glue drop method is to randomly wrap the stem cells in the glue droplet, which leads to the heterogeneity in the glue droplet and between the glue droplet, which is manifested in the shape, size, maturity, and cell population, making it difficult to perform in situ optical imaging. , it is difficult to achieve consistent and reproducible organoid production. Although the existing methods can perform in situ three-dimensional sphere formation, they cannot perform high-throughput in situ imaging. The reason is that the Z-axis height of organoids exceeds the imaging limit of current optical microscopes (such as confocal microscopes and inverted fluorescence microscopes). , so the organoids need to be taken out for sectioning and re-imaging. The process is cumbersome and cannot explain the three-dimensional in situ structure. These problems have seriously affected the commercial application of liver organoids in the biomedical field.

Therefore, in order to overcome the problems of the above-mentioned culture methods, it is urgent to develop a culture chip of liver organoids with high uniformity and in situ imaging, and a new liver organoid model, which is suitable for optical imaging instruments such as confocal microscopes, especially for drugs. Develop the gold standard of high-throughput screening in the industry - high-content fluorescence imaging and cell analysis system, so as to provide an innovative research tool for liver basic research and drug development.

Contents of the invention

The object of the present invention is to provide a liver organoid culture chip, a liver organoid model and its preparation method and application, which have high uniformity and can be imaged in situ, and are suitable for optical imaging instruments such as confocal microscopes.

In order to achieve the above object, the present invention adopts the following technical solutions:

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

obtaining a film with a plurality of perforations;

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

A cell-specific adhesion material is added to the patterned substrate for coating to obtain a culture chip of liver organoids.

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

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

Further, the outer dimension of the film matches the cell culture plate; the cell culture plate includes 384-well plate, 96-well plate, 48-well plate, 24-well plate, 12-well plate, 6-well plate, 3.5cm One of Petri dish, 6cm Petri dish, 10cm Petri dish.

Further, said obtaining a film with multiple perforations includes:

obtaining a male mold with a plurality of micropillar arrays;

Pour the PDMS onto the positive mold, vacuum dry and evacuate, and cover a layer of PMMA on the PDMS, then clamp and fix it with two glass sheets, and dry;

The solidified PDMS layer was taken out and cut into a PDMS membrane adapted to the shape of the cell culture plate to obtain a thin film with multiple perforations.

Further, the height of the micropillar array of the positive membrane is 30 μm˜100 μm.

Further, the perforated film includes a molded porous PDMS/organic silicon film or a porous PDMS/organic silicon film perforated by a puncher.

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

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

Inoculate the foregut embryonic cells in the culture chip of the liver organoid, and after the cells adhere to the wall, remove the film with multiple perforations, and suck off the culture medium in the culture chip of the liver organoid After washing, add the first medium to maintain the culture for 3 to 5 days;

Afterwards, the second culture medium was used to maintain culture for 3 to 5 days and the third culture medium for ≥10 days to obtain a liver organoid model.

Further, the seeding density of the foregut embryo cells is 1×10 ⁵ -9×10 ⁵ (cells/cm ² ).

In the above technical solution, the types of liver organoids include but are not limited to liver organoids derived from PSC or EB, liver organoids derived from adult stem cells, and liver-related tumor organoids.

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

In the fifth aspect of the present invention, the application of the liver organoid model in pharmacology, pharmacodynamics and toxicology 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) A liver organoid model provided by the present invention and its preparation method and application, form a cell-specific adhesion area with a specific pattern on the bottom of the cell culture vessel through patterning technology, and perform low-adhesion or low-adhesion cell adhesion on the non-patterned area Ultra-low adhesion treatment. Stem cells from different sources or different tissue sources can be inoculated on the patterned substrate. After the cells adhere to the wall, the PDMS perforated film is peeled off to obtain cell aggregates with specific edge shapes. Stem cells are induced on the basal to differentiate to the liver lineage, and finally form a new type of liver organoid model with a thickness of 30-200 μm and a specific edge shape, which has high uniformity and can be imaged in situ, and is suitable for optical imaging instruments such as confocal microscopes; this liver type The organ is flattened, resembling a solid domed shape, which is different from the phenotype of previous liver organoids;

(2) The patterned substrate array of the present invention can be designed and customized according to specific needs, and is suitable for various types of organoids or cell culture;

(3) The perforated film of the present invention is suitable for a variety of commercially available cell culture vessels, the preparation process is simple and easy to understand, and the learning cost is low, and it has good advantages for ordinary laboratories and mass production;

(4) The liver organoids in the present invention can be stained and imaged in situ in a well plate without destroying the organoid structure. ) have good compatibility.

(5) The patterned substrate of the present invention is easy to produce in large quantities and standardized, has a wide range of applications, can meet the needs of users, and is very easy to realize commercial production.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings that need to be used in the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.

Fig. 1 is the flow chart and physical map of the patterned substrate preparation of the liver organoid culture chip provided by the embodiment of the present invention; wherein Fig. 1a is the patterned substrate preparation flow chart; Fig. 1b, Fig. 1c and Fig. 1d are the physical maps;

Figure 2 is the culture and characterization of human liver organoids on the patterned substrate of the culture chip of liver organoids provided by the embodiment of the present invention; Figure 2a is a flow chart of patterned liver organoid culture; Figure 2b is a 24-well plate Bright field images of liver organoids; Figure 2c is a bright field image of liver organoids cultured by the traditional glue drop method; Figure 2d is the area representation of patterned liver organoids cultured to different time points; Figure 2e is the patterned liver organoids and glue Comparison of coefficient of variation (CV value) of liver organoid area by drop method;

Figure 3 is the identification of protein levels and transcription levels at various stages of human liver organoid construction; Figure 3a shows the expression of markers at the hiPSC stage and foregut embryo stage; Figure 3b shows the stemness markers at different time points in patterned liver organoids and the expression of bidirectional differentiation potential markers; Figure 3c is the expression of liver lineage markers in patterned liver organoids; Figure 3d is the expression of related genes in patterned liver organoids;

Fig. 4 shows the construction and characterization results of the drug hepatotoxicity evaluation system in the patterned substrate of the liver organoid culture chip provided by the embodiment of the present invention.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly. Those skilled in the art should understand that these specific implementations and examples are used to illustrate the present invention, not to limit the present invention.

Throughout the specification, unless otherwise specified, terms used herein should be understood as commonly used in the art. Therefore, unless otherwise defined, 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. In case of conflict, this specification shall take precedence.

It should be noted that when an element is said to be "fixed" or "set on" another element, it can be directly on another element or indirectly set on another element; when an element is said to be "connected" It may be directly connected to another element or indirectly connected to another element.

It is to be understood that the terms "length", "width", "upper", "lower", "front", "rear", "first", "second", "vertical", "horizontal", "top ", "bottom", "inner", "outer" and other indicated orientations or positional relationships are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the application and simplifying the description, rather than indicating or implying Any device or element must have a specific orientation, be constructed, and operate in a specific orientation, and therefore should not be construed as limiting the application.

In addition, in the description of the present application, the meanings of "plurality" and "several" are two or more, unless otherwise specifically defined.

The general idea of the technical scheme of this application is as follows:

According to a typical embodiment of the present invention, a method for preparing a culture chip of liver organoids is provided, the method comprising:

obtaining a film with a plurality of perforations;

In the above technical scheme, a cell-specific adhesion area with a specific pattern is formed on the bottom of the cell culture vessel through patterning technology, and the non-patterned area is treated with low or ultra-low adhesion of cells, and different sources can be seeded on the patterned substrate. Or stem cells from different tissue sources. After the cells adhere to the wall, the PDMS perforated film is peeled off to obtain cell aggregates with a specific edge shape. On this patterned substrate, stem cells are induced to differentiate into the liver lineage, and finally formed with a thickness of 30- Novel liver organoid model of 200 μm and specific edge shape. High uniformity and in situ imaging, suitable for optical imaging instruments such as confocal microscopes.

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

If the diameter of the circumscribed circle of each perforation is less than 100 μm, there is a disadvantage that organoids are easily lost during liquid exchange, and if the diameter of the circumscribed circle is too large, there is a disadvantage of low flux;

If the distance between two adjacent perforations of the film is less than 0.5 mm, there is a disadvantage that organoids are easy to adhere and cannot grow independently; if the distance is too large, there is a disadvantage of low flux; multiple perforations can wait It can also be arranged at a non-equidistant distance.

As a specific embodiment, the outer dimension of the film matches the cell culture plate; the cell culture plate includes 384-well plate, 96-well plate, 48-well plate, 24-well plate, 12-well plate, 6 One of orifice plate, 3.5cm Petri dish, 6cm Petri dish, 10cm Petri dish. The liver organoid model of the present invention is modified based on the existing cell culture well plate, highly combined with the existing cell culture well plate, and has good compatibility with the existing biological related optical instruments.

As a specific implementation,

The material of the film comprises polydimethylsiloxane;

The low cell adhesion material includes polyethylene glycol; in other embodiments, other low cell adhesion materials can also be used;

The material for cell-specific adhesion includes at least one of Matrigel and Collagen.

As a specific implementation, the obtaining a film with multiple perforations includes:

obtaining a male mold with a plurality of micropillar arrays;

Wherein, the height of the micropillar array of the positive membrane is 30 μm˜100 μm. If the height is too low, the extruded PDMS film is too thin, and it is not easy to unfold; if the height is too high, it is not easy to perforate;

According to another typical implementation of the embodiments of the present invention, a culture chip of liver organoids prepared by the method is provided.

According to another typical implementation of the embodiments of the present invention, a method for preparing a liver organoid model is provided, the method comprising:

Step S1, inoculating foregut embryonic cells in the culture chip of the liver organoid, and after the cells adhere to the wall, remove the film with multiple perforations, and aspirate the inside of the culture chip of the liver organoid After washing, add the first medium to maintain the culture for 3 to 5 days;

The step S1 specifically includes:

S101. Differentiation of stem cells (hESCs or hiPSCs) to endoderm: conventionally cultured hESCs or hiPSCs were digested with Accutase into single cells, and planted in a six-well plate at a density of 1×10 ⁵ cells/cm ² until the cells were confluent. Differentiation begins when it reaches 85%-90%. The culture time is 1 to 3 days, and cultured in D1-D3 medium:

D1 Medium: RPMI medium containing 100ng/mL ActivinA and 50ng/mL BMP4; D2 Medium: RPMI medium containing 100ng/mL ActivinA and 0.2% Knockout serum replacement; D3 Medium: containing 100ng/mL ActivinA and 2% Knockout serum Replacement with RPMI medium.

S102. Differentiation of endoderm cells into foregut embryo cells: culturing with differentiation medium; replacing the medium every day, and routinely culturing the cells in a two-dimensional incubator. On the 6th day of differentiation, obvious three-dimensional structure can be seen.

The differentiation medium is D4-6Medium according to the embodiment of the present invention; the formulation of the differentiation medium is Advanced DMEM/F12 with a final concentration of 450-550ng/mL FGF2, 2-4μM CHIR99021, 1% B27 and 1% N2 Medium, preferably 500ng/mL FGF2 and 3μM CHIR99021;

B27 and N2 of the present invention are serum-free culture additives. "%" in 1%B27 and 1%N2 is the mass fraction.

S103. Digest the foregut embryo cells and inoculate them on the patterned substrate of the liver organoid culture chip, and add the first culture medium to maintain the culture for 3-5 days.

The inoculation density of the foregut germ cells is 1×10 ⁵ -9×10 ⁵ (unit/cm ² ). If the cell seeding density is too low, it is difficult to grow into organoids with a certain thickness; if the cell seeding density is too high, there are too many cells, it is difficult to separate from the PDMS perforated film, and it is impossible to form patterned organoids.

The formulation of the first medium is Advanced DMEM/F12 medium with a final concentration of 75-85ng/mL FGF2, 2-4μM CHIR99021, 1% B27 and 1%N2, preferably 80ng/mL FGF2 and 3μM CHIR99021;

In step S2, the liver organoid model is obtained by sequentially adopting the second medium for maintaining culture for 3-5 days and the third medium for maintaining culture for ≥10 days.

In the step S2,

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

The formula of the third culture medium is the Hepatocyte Culture Medium medium that final concentration is 8～12ng/mL HGF, 0.05～0.2mM Dexamethasone, 18～22ng/mL OSM, preferably 10ng/mL HGF, 0.1mM Dexamethasone, 20ng/mL OSM's Hepatocyte Culture Medium;

According to another typical embodiment of the present invention, a liver organoid model obtained by the method is provided. As a specific implementation of the embodiment of the present invention, the obtained liver organoid is flat, similar to a solid round arch (or similar expression), which is different from the previous liver organoid phenotype; due to the organoid One side is adhered to the bottom of the plate, and the organoid also has a certain thickness; according to the results of the fluorescence image, the surrounding of the organoid is clearly visible and the middle part has poor light transmission, so the thickness in the middle should be thicker than the surrounding , indicating that it is a solid circular arch.

The specific structure of the "solid circular arch" is: the middle part of the liver organoid is arched, and the middle part forms a circular arc with the periphery of the liver organoid; the arch is a three-dimensional concept, and the semicircle is a planar concept. The shape described by the "solid circular arch" can also be expressed in other ways, as long as it can characterize the liver organoid obtained in the present invention.

According to another typical embodiment of the present invention, applications of the liver organoid model in pharmacology, pharmacodynamics, and toxicology analysis are provided.

The preparation method of the novel liver organoid model includes inducing stem cells into foregut embryo cells, planting the foregut embryo cells on a patterned substrate at a certain density, differentiating from the foregut embryo cells to the liver lineage, and finally forming an adhering culture vessel. Arch-like liver organoids with both a certain thickness at the bottom and a specific edge shape, containing cell types including hepatoid cells differentiated from stem cells, cholangioid cells, progenitor cells, and mesenchymal cells; the novel liver organoids The model can be applied to the study of liver development and disease mechanism, drug screening, drug hepatotoxicity evaluation, etc. Compared with the traditional glue drop method and in situ sphere forming method, the present invention provides a new liver organoid model with high throughput, high uniformity and in situ imaging, and provides an innovative research tool for related liver research.

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

Embodiment 1, a culture chip of liver organoid and its preparation method

1. Design of patterned substrate array mask

Use AutoCAD 2018 software to design a patterned array mask. The array units are circular with a diameter of 0.5 mm and the pitch is 1.5 mm. The array units are evenly spaced. The patterned array mask design is shown in Figure 1.

Second, the production of patterned substrate

1. Prepare the SU-8 anode film by using soft lithography technology. The SU-8 anode film is a micro-column array with a pitch of 1.5 mm, the height of the micro-column is 80 μm, and the diameter of the bottom area is 500 μm.

2. Mix PDMS prepolymer (A glue) and crosslinking agent (B glue) at a mass ratio of 10:1, stir evenly, and use a vacuum dryer to remove air bubbles. Pour the PDMS onto the SU-8 positive film, smooth it with a clean pipette tip, make it evenly cover the area of the microcolumn array, and vacuum it with a vacuum desiccator until there are no bubbles on the surface. Cover the surface with a layer of polymethyl methacrylate (PMMA) with a thickness of 0.2mm, fix it with two glass sheets, clamp it in a vise, and dry it in an oven at 80°C for at least 120min.

3. Take out the solidified PDMS perforated film from the vise, and use a 15mm diameter circular punch to punch out a circular perforated film.

4. Configure PEG mixture (PEG1000: 37.5 mg, PEG400: 450 μL, isopropanol 3637.5 μL, pure water 112.5 μL), shake and mix for 3 minutes, add photoinitiator 10 mg, shake and mix again for 3 minutes, and store in the dark. After the orifice plate was treated with Plasma (operating voltage 550V) for 1 min, 150 μL PEG mixture was added to each well and allowed to stand for 5 min. Expose the well plate with PEG mixture in the well to ultraviolet exposure for 1 min. After the exposed orifice plate was washed three times with 70% alcohol, add 1mL of 70% alcohol to each well, spread the circular PDMS perforated film on the surface of the liquid, press it to the bottom of the orifice plate with tweezers, suck out the excess liquid, and place the orifice plate on the surface of the liquid. Dry in an oven at 80°C for about 20 minutes.

5. Put the dried orifice plate into the plasma cleaning machine for Plasma (working voltage 700V), wash three times in total, 4 minutes each time, 3 minutes between each cleaning, and do not take out the orifice plate during the interval.

6. Sterilize the prepared orifice plate by ultraviolet light for 60 minutes, add 1mL 1×DPBS to each well, and pipette repeatedly until there are no air bubbles in the orifice plate. Aspirate 1×DPBS, add 200 μL to each well and add 1% Matrigel:Advanced DMEM/F12, incubate at 37°C for 1 hour for later use, and obtain the culture chip of liver organoids.

3. Culture of human liver organoids

1. The patterned substrates of the various examples and comparative examples were cultured as liver organoids, the method is as follows:

(1) Differentiation of hESCs or hiPSCs to endoderm: conventionally cultured hESCs or hiPSCs were digested into single cells with Accutase, and planted in a six-well plate at a density of 1×10 ⁵ cells/cm ² until the cell confluence reached 85%. Differentiation begins at -90%.

(2) D1 Medium: RPMI medium containing 100ng/mL ActivinA and 50ng/mL BMP4; D2 Medium: RPMI medium containing 100ng/mL ActivinA and 0.2% Knockout serum replacement; D3 Medium: containing 100ng/mL ActivinA and 2 % RPMI medium for Knockout serum replacement.

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

(3) Differentiation of endoderm cells into foregut germ cells: D4-6 Medium: Advanced DMEM/F12 medium containing 500ng/mL of FGF2, 3μM CHIR99021, 1% B27 and 1% N2, the medium was changed every day, and the cells were cultured Conventional two-dimensional culture in the box. On the 6th day of differentiation, obvious three-dimensional structure can be seen.

(4) Inoculation of foregut germ cells in the patterned substrate: differentiated to day 6, digested the foregut germ cells into single cells with Accutase, and inoculated them into well plates at a density of 3×10 ⁵ cells/cm ² , each well 500 μL of Advanced DMEM/F12 medium containing 80 ng/mL FGF2, 3 μM CHIR99021, 1% B27 and 1% N2.

(5) At least 4 hours after inoculating the cells, when the cells are attached to the wall, use tweezers to gently peel off the PDMS film from the bottom edge of the well plate. Aspirate the medium in the well plate, wash the cells gently with 1×DPBS, add 500 μL per well of Advanced DMEM/F12 medium containing 80ng/mL FGF2, 3μM CHIR99021, 1% B27 and 1% N2 to maintain the culture 4 day, and change the solution every other day.

(6) Culture of liver organoids in the patterned substrate: on the 10th to 14th day, maintain the culture with Advanced DMEM/F12 containing 2mM RA, 1% B27 and 1% N2 for 4 days, and change the medium every other day; on the 14th to 24th day The Hepatocyte Culture Medium medium containing 10ng/mL HGF, 0.1mM Dexamethasone, and 20ng/mL OSM was used to maintain the culture for ten days, and the medium was changed every other day.

Example 2

In the embodiment of the present invention, the PDMS perforated film was punched into a circle with a diameter of 10 mm by a circular punch, and spread in a 48-well plate. Other structures and steps were the same as in Embodiment 1.

Example 3

In the embodiment of the present invention, the pattern unit in the PDMS perforated film is circular, the diameter of the circular pattern is 500 μm, the pattern spacing is 1.5 mm, the cell seeding density on the patterned substrate is 1×10 ⁵ cells/cm ² , other structures and steps All with embodiment 1.

Example 4

In the embodiment of the present invention, the pattern unit in the PDMS perforated film is circular, the diameter of the circular pattern is 500 μm, the pattern spacing is 1.5 mm, the cell seeding density on the patterned substrate is 9×10 ⁵ cells/cm ² , other structures and steps All with embodiment 1.

Example 5

In the embodiment of the present invention, the pattern unit in the PDMS perforated film is circular, the diameter of the circular pattern is 100 μm, and the pattern spacing is 1 mm. Other structures and steps are the same as in Example 1.

Example 6

In the embodiment of the present invention, the pattern unit in the PDMS perforated film is circular, the diameter of the circular pattern is 200 μm, and the pattern pitch is 1.2 mm. Other structures and steps are the same as in Embodiment 1.

Example 7

In the embodiment of the present invention, the pattern unit in the PDMS perforated film is circular, the diameter of the circular pattern is 1000 μm, and the pattern pitch is 2 mm. Other structures and steps are the same as in Embodiment 1.

Example 8

In the embodiment of the present invention, the pattern units in the PDMS perforated film are circular, and the circular patterns are arranged in a regular hexagon (including the center point), and the pattern spacing is 1mm. Other structures and steps are the same as in the first embodiment.

Example 9

In the embodiment of the present invention, the pattern units in the PDMS perforated film are circular, and the circular patterns are arranged in a regular hexagon (including the central point), and the pattern spacing is 1.5 mm. Other structures and steps are the same as in the first embodiment.

Example 10

In the embodiment of the present invention, the pattern units in the PDMS perforated film are circular, and the circular patterns are arranged in a regular hexagon (including the central point), and the pattern spacing is 2mm. Other structures and steps are the same as in the first embodiment.

Example 11

In the embodiment of the present invention, the shape of the pattern unit in the PDMS perforated film is an equilateral triangle, the side length is 1 mm, and the pattern spacing is 2 mm. Other structures and steps are the same as in the embodiment 1.

Example 12

In the embodiment of the present invention, the shape of the pattern unit in the PDMS perforated film is an equilateral triangle, the side length is 0.5 mm, and the pattern spacing is 1.5 mm. Other structures and steps are the same as in embodiment 1.

Example 13

In the embodiment of the present invention, the shape of the pattern unit in the PDMS perforated film is rectangular, the length of the long side is 0.5 mm, the length of the short side is 0.15 mm, and the pattern spacing is 1.5 mm. Other structures and steps are the same as in embodiment 1.

Example 14

In the embodiment of the present invention, the shape of the pattern unit in the PDMS perforated film is rectangular, the length of the long side is 1 mm, the length of the short side is 0.3 mm, and the pattern spacing is 2 mm. Other structures and steps are the same as in embodiment 1.

Comparative example 1

The comparative example is conventional Matrigel.

Comparative example 2

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

Comparative example 3

In this comparative example, the seeding density of cells on the patterned substrate is 0.9×10 ⁵ (unit/cm ² ), which is less than 1×10 ⁵ to 9×10 ⁵ (unit/cm ² ) in the embodiment of the present invention. Other structures and steps All with embodiment 1.

Comparative example 4

In this comparative example, the seeding density of cells on the patterned substrate is 9.1×10 ⁵ (cells/cm ² ), which is higher than that of the embodiment of the present invention (1×10 ⁵ -9×10 ⁵ (cells/cm ² ) in other structures and steps. With embodiment 1.

Comparative example 5

In this comparative example, the pattern pitch is 0.4 mm, and other structures and steps are the same as those in Example 1.

Comparative example 6

In this comparative example, the patterning pitch is 3.2 mm, and other structures and steps are the same as those in Embodiment 1.

Experimental example 1

The bases of the above-mentioned Examples 1-14 and Comparative Examples 1-6 were used to carry out statistics on the culture effect of liver organoids, as shown in Table 1, wherein the calculation method of the coefficient of variation of the standard deviation of the area is: coefficient of variation C·V=(standard deviation SD/average Mean)×100%;

Table 1

It can be seen from the data in Table 1 that:

In Comparative Example 1, the traditional Matrigel culture method has the disadvantages of poor organoid uniformity and difficulty in 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 100-1000 μm in the embodiment of the present invention, and there is a disadvantage that organoids are easily lost during liquid replacement.

In Comparative Example 3, the cell seeding density was 0.9×10 ⁵ cells/cm ² , which was lower than the range of 1×10 ⁵ to 9×10 ⁵ (cells/cm ² ) in this example, and it was difficult to grow into organoids with a certain thickness. Shortcomings.

In Comparative Example 4, the cell seeding density was 9.1×10 ⁵ cells/cm ² , which was greater than the range of 1×10 ⁵ to 9×10 ⁵ (cells/cm ² ) in this example, and there were too many cells, which made it difficult to combine with the PDMS perforated film. Disadvantages of isolation and inability to form patterned distributions of organoids.

In Comparative Example 5, the pattern unit spacing of the patterned substrate is 0.4 mm, which is smaller than the range of 0.5-3 mm in this embodiment, and there is a disadvantage that organoids are easy to adhere and cannot grow independently.

In Comparative Example 6, the pattern unit pitch of the patterned substrate is 3.2 mm, which is greater than the range of 0.5-3 mm in this embodiment, and there is a disadvantage that high throughput cannot be achieved.

However, in Example 1-Example 11 of the present invention, the growth is normal, and the coefficient of variation is ≤40%. Compared with the traditional glue drop method and in situ sphere forming method, the present invention provides a new liver organoid model with high throughput, high uniformity and in situ imaging, and provides an innovative research tool for related liver research.

Experimental example 2. Detection of characteristic markers of human liver organoids

1. Use immunofluorescence to detect the characteristic markers of differentiation to each stage: detect the expression of markers OCT3/4 and Nanog at the stage of hESCs or hiPSCs; detect the expression of markers CDX2 and EpCAM at the foregut stage; detect the expression of markers ALB at the stage of liver organoids , HNF4-α, AFP, EpCAM expression.

2. Construction and characterization of the drug hepatotoxicity evaluation system on the patterned substrate of Example 1: On the 24th day, the liver organoids were treated with 0mM, 10mM, 20mM, and 40mM acetaminophen (APAP) for 48 hours. Perform cell death assays.

The results are shown in Fig. 3-Fig. 4, indicating that the embodiment of the present invention successfully obtained human-derived liver organoids and established a drug hepatotoxicity evaluation system.

3. Carry out the culture of human-derived organoids on the patterned substrate of Example 1, and use the traditional glue drop method to culture liver organoids as a control; where Figure 2a is a flow chart of patterned liver organoid culture; Figure 2b is a 24-well plate Bright field image of liver organoids in the middle; Figure 2c is a bright field image of liver organoids cultured by the traditional glue drop method; Figure 2d is the area representation of patterned liver organoids cultured to different time points; Figure 2e is the patterned liver organoids and Comparison of coefficient of variation (CV value) of liver organoid area by glue drop method;

It can be seen from Figure 2e that the coefficient of variation of the area of patterned liver organoids is significantly lower than that of the gel drop method; by comparing the bright field images of Figure 2a and Figure 2c, it can also be seen intuitively that the patterned liver organoids limit the size of the organoids. The growing regions have a consistent shape and size, which are not controllable in droplet-grown organoids.

Finally, it should also be noted that the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, but also Other elements not expressly listed, or inherent to the process, method, article, or apparatus are also included.

While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

A method for preparing a culture chip of liver organoids, characterized in that the method comprises:

obtaining a film with a plurality of perforations;

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

A cell-specific adhesion material is added to the patterned substrate for coating to obtain a culture chip of liver organoids.
The method for preparing a liver organoid culture chip according to claim 1, wherein the shape of each perforated hole includes a circle, an ellipse, a semicircle, a fan, a triangle, a quadrangle, and a pentagon. One of shape, hexagon and arbitrary polygon; the diameter of the circumscribed circle of each perforation is 100 μm-1000 μm, and the distance between two adjacent perforations of the film is 0.5 mm-3 mm.
The method for preparing a liver organoid culture chip according to claim 1, wherein the material of the film comprises polydimethylsiloxane; the material with low cell adhesion comprises polyethylene glycol; The material for cell-specific adhesion includes at least one of Matrigel and Collagen.
The method for preparing a culture chip of a liver organoid according to claim 1, wherein said obtaining a film with multiple perforations comprises:

obtaining a male mold with a plurality of micropillar arrays;

Pour the PDMS onto the positive mold, vacuum dry and evacuate, and cover a layer of PMMA on the PDMS, then clamp and fix it with two glass sheets, and dry;

The solidified PDMS layer was taken out and cut into a PDMS membrane adapted to the shape of the cell culture plate to obtain a thin film with multiple perforations.
The method for preparing a liver organoid culture chip according to claim 4, characterized in that the height of the microcolumn array of the positive membrane is 30 μm˜100 μm.
A culture chip of liver organoids prepared by the method according to any one of claims 1-5.
A method for preparing a liver organoid model, characterized in that the method comprises:

Inoculate the foregut embryonic cells in the culture chip of the liver organoid according to claim 6, and after the cells adhere to the wall, remove the film with multiple perforations, and suck out the inside of the culture chip of the liver organoid. After washing, add the first medium to maintain the culture for 3 to 5 days;

Afterwards, the second culture medium was used to maintain culture for 3 to 5 days and the third culture medium for ≥10 days to obtain a liver organoid model.
The preparation method of a liver organoid model according to claim 7, characterized in that the inoculation density of the foregut germ cells is 1×10 5 -9×10 5 (cells/cm 2 ).
A liver organoid model obtained by the method according to any one of claims 1-8.
An application of the liver organoid model according to claim 9 in pharmacology, pharmacodynamics and toxicology analysis.
The method for preparing a culture chip of a liver organoid according to claim 4, wherein the perforated film comprises a molded porous PDMS/organic silicon film or a porous PDMS/organic silicon film perforated by a puncher .