A culture chip of liver organoids, a liver organoid model and a preparation method and application thereof

Abstract

The application discloses a liver organoid culture chip, a liver organoid model and a preparation method and application thereof. The preparation method of the chip comprises the following steps: obtaining a film with multiple perforations; coating a cell low-adhesion material on the bottom of a cell culture plate, then covering the film on the bottom of the cell culture plate to obtain a patterned substrate; adding a cell-specific adhesion material to the patterned substrate for coating to obtain the chip; inoculating foregut cells in the liver organoid culture chip, removing the film with multiple perforations after the cells adhere, aspirating the culture medium in the liver organoid culture chip, and adding a first culture medium for maintaining culture for 3-5 days after washing; then, a second culture medium and a third culture medium are used in sequence to maintain culture, and the liver organoid model is obtained. 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.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tissue engineering and organ chip technology, in particular to a liver organoid culture chip, a liver organoid model and a preparation method and application thereof. BACKGROUND

[0002] The liver is an important organ of the human body, which has a variety of important physiological functions, including detoxification, digestion and metabolism, etc. However, related liver diseases such as viral hepatitis, non-alcoholic fatty liver, liver cancer and other diseases have caused heavy social burden, and seriously threaten human life and health. Therefore, in order to reveal the mechanism of liver disease development, develop treatment methods for liver disease, it is of great significance to establish a liver research model highly related to the human body. Liver organoids are a new liver research model, which has key features of human liver, such as cell composition and functions such as albumin synthesis, glycogen synthesis, and lipid accumulation, and has a wide range of applications in basic research, drug development, etc. Compared with traditional animal models, organoid models do not have species differences and have highly related epigenetic metabolism to the human body; compared with two-dimensional culture models, they have similar cell microenvironments to the body, have liver tissue-specific cell populations, and can simulate the interaction between parenchymal cells and non-parenchymal cells. At present, liver organoids overcome the limitations of cancer cell lines and PDX models, can culture diseased and healthy tissues from the same patient, provide a personalized drug testing platform matched to the patient, and at the same time provide an ideal model for disease mechanism research. Therefore, the construction of an in vitro liver organoid provides an effective model system for the study of liver pathogenesis and the development of treatment methods.

[0003] At present, the culture of liver organoids is mainly based on Matrigel droplet culture method. The production process of the droplet method is to randomly wrap stem cells in the droplet, which causes heterogeneity in the droplet and between the droplets, specifically in morphology, size, maturity, and cell population, making it difficult to perform in situ optical imaging and achieve consistency and reproducibility of organoid production. The existing method can perform in situ three-dimensional balling, but cannot perform in situ imaging at high throughput, because the Z-axis height of the organoid exceeds the imaging limit of the current optical microscope (such as confocal microscope, inverted fluorescence microscope), so the organoid needs to be taken out for sectioning and imaging, which is a tedious process and cannot explain the three-dimensional in situ structure, and these problems seriously affect the commercial application of liver organoids in the biomedical field.

[0004] Therefore, in order to overcome the problems existing in the above culture method, it is urgent to develop a liver organoid culture chip with high uniformity and in-situ imaging, a new liver organoid model, which is suitable for optical imaging instruments such as confocal microscopes, especially high-content fluorescence imaging and cell analysis systems, which are the gold standard of high-throughput screening in the drug development industry, so as to provide an innovative research tool for liver basic research and drug development. SUMMARY

[0005] The purpose of the present application is to provide a liver organoid culture chip, a liver organoid model, and a preparation method and application thereof, which have high uniformity and can be imaged in-situ, and are suitable for optical imaging instruments such as confocal microscopes.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] In the first aspect of the present application, a preparation method of a liver organoid culture chip is provided, which comprises:

[0008] obtaining a thin film with a plurality of perforations;

[0009] coating a cell low-adhesion material 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;

[0010] adding a cell-specific adhesion material to the patterned substrate for coating to obtain a liver organoid culture chip.

[0011] Further, the hole shape of each perforation includes one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrilateral, a pentagon, a hexagon, and any polygon; the diameter of the circumscribed circle of each perforation is 100-1000 μm, and the spacing between two adjacent perforations of the thin film is 0.5-3 mm.

[0012] Further, the material of the thin film includes polydimethylsiloxane; the cell low-adhesion material includes polyethylene glycol; and the cell-specific adhesion material includes at least one of Matrigel and Collagen.

[0013] Further, the outer size of the thin film matches the cell culture plate; and the cell culture plate includes 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.

[0014] Further, the obtaining of the thin film with a plurality of perforations comprises:

[0015] obtaining a male mold with a plurality of micro-column arrays;

[0016] Pour PDMS onto the male mold, vacuum dry and evacuate, then cover the PDMS with a layer of PMMA, clamp and fix it with two glass plates, and dry.

[0017] The solidified PDMS layer was removed and cut into PDMS membranes that fit the shape of cell culture plates to obtain a membrane with multiple perforations.

[0018] Furthermore, the height of the micropillar array of the cation membrane is 30 μm to 100 μm.

[0019] In a second aspect of the invention, a culture chip for liver organoids prepared using the method is provided.

[0020] In a third aspect of the present invention, a method for preparing a liver organoid model is provided, the method comprising:

[0021] Foregut germ cells were seeded into the liver organoid culture chip. After the cells adhered, the membrane with multiple perforations was removed, the culture medium in the liver organoid culture chip was aspirated, and after washing, the first culture medium was added to maintain culture for 3 to 5 days.

[0022] The liver organoid model was then obtained by maintaining the culture in the second culture medium for 3-5 days and the third culture medium for ≥10 days.

[0023] Furthermore, the seeding density of the foregut embryonic cells is 1×10⁻⁶. 5 ~9×10 5 (pieces / cm) 2 ).

[0024] In the above technical solutions, 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.

[0025] In a fourth aspect of the invention, a liver organoid model obtained by the method is provided.

[0026] In a fifth aspect of the invention, the application of the liver organoid model described herein in pharmacological, pharmacodynamic, and toxicological analyses is provided.

[0027] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0028] (1) The application 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 through a patterning technology, and a cell low-adhesion or ultra-low-adhesion treatment is performed on a non-patterned area. Different source or different tissue source stem cells can be inoculated on the patterned substrate. After the cells adhere, the PDMS punctured film is removed to obtain a cell aggregate with a specific edge shape. The stem cells are induced to differentiate into a liver lineage on the patterned substrate, and finally a novel liver organoid model with a thickness of 30-200 μm and a specific edge shape is formed. The uniformity is high and can be imaged in situ, and is suitable for optical imaging instruments such as a confocal microscope. The liver organoid is flat and similar to a solid circular arch, which is different from the phenotype of the previous liver organoid;

[0029] (2) The patterned substrate array of the application can be designed and customized according to specific needs, and is suitable for various types of organoids or cell culture;

[0030] (3) The punctured film of the application is suitable for various commercially available cell culture vessels, and the preparation process is simple and easy to understand, with low learning cost. It has good advantages for ordinary laboratories and batch production;

[0031] (4) The liver organoid in the application can be stained and imaged in situ in a well plate without damaging the organoid structure, and has good compatibility with existing biological analysis and imaging instruments (such as high-content instruments) for drug development field.

[0032] (5) The patterned substrate of the application is easy to mass-produce, has a wide range of applications, can meet the needs, and is very easy to realize commercial production. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0034] Figure 1 The patterned substrate preparation process chart and physical map of the culture chip of the liver organoid provided by the embodiments of the application; wherein Figure 1 a is the patterned substrate preparation process chart; Figure 1 b, Figure 1 c and Figure 1 d is the physical map;

[0035] Figure 2The culture and characterization of human liver organoids on the patterned substrate of the culture chip of the liver organoids provided by the embodiments of the present application; wherein Figure 2 a is a patterned liver organoid culture flow chart; Figure 2 b is a bright field image of liver organoids in a 24-well plate; Figure 2 c is a bright field image of liver organoids cultured by a traditional gel drop method; Figure 2 d is an area characterization of the patterned liver organoids cultured to different time points; Figure 2 e is a comparison of the area coefficient of variation (CV value) of the patterned liver organoids and the gel drop method liver organoids;

[0036] Figure 3 The protein level and transcription level identification of each stage of the human liver organoids are constructed; wherein Figure 3 a is the expression of hiPSC stage and foregut embryo stage markers; Figure 3 b is the expression of related stemness markers and bidirectional differentiation potential markers of the patterned liver organoids at different time points; Figure 3 c is the expression of liver lineage markers of the patterned liver organoids; Figure 3 d is the expression of related genes of the patterned liver organoids;

[0037] Figure 4 The construction and characterization results of the drug hepatotoxicity evaluation system in the patterned substrate of the culture chip of the liver organoids provided by the embodiments of the present application. DETAILED DESCRIPTION

[0038] The advantages and various effects of the present application will be more clearly presented from the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0039] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs. If there is a contradiction, the present specification takes precedence.

[0040] It should be noted that when an element is referred to as being "fixed to" or "provided on" another element, it can be directly on the other element or indirectly provided 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 indirectly connected to the other element.

[0041] It should be understood that the terms "length", "width", "upper", "lower", "front", "back", "first", "second", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0042] In addition, in the description of the present application, the meaning of "multiple" or "several" is two or more, unless otherwise explicitly and specifically limited.

[0043] The general idea of the technical solution of the present application is as follows:

[0044] According to a typical embodiment of the present application, a preparation method of a liver organoid culture chip is provided, the method comprising:

[0045] obtaining a thin film having a plurality of perforations;

[0046] coating a cell low-adhesion material on the bottom of a cell culture plate, then covering the thin film on the bottom of the cell culture plate to obtain a patterned substrate;

[0047] adding a cell-specific adhesion material to the patterned substrate for coating to obtain a liver organoid culture chip.

[0048] In the above technical solution, a cell-specific adhesion area with a specific pattern is formed on the bottom of a cell culture vessel by patterning technology, and a cell low-adhesion or ultra-low-adhesion treatment is performed on the non-patterned area. Different source or different tissue source stem cells can be inoculated on this patterned substrate. After the cells adhere, the PDMS perforated film is removed to obtain a cell aggregate with a specific edge shape. Stem cells are induced to differentiate into liver lineage on this patterned substrate, and finally a new liver organoid model with a thickness of 30-200 μm and a specific edge shape is formed. It has high uniformity and can be imaged in situ, and is suitable for optical imaging instruments such as confocal microscopes.

[0049] As a preferred embodiment, each of the perforations has a shape including one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrilateral, a pentagon, a hexagon, and any polygon; and a diameter of a circumscribed circle of each of the perforations is 100 μm to 1000 μm, and a spacing between two adjacent perforations of the thin film is 0.5 mm to 3 mm.

[0050] If the diameter of the circumscribed circle of each of the perforations is less than 100 μm, there is a disadvantage that the organoids are easily lost during liquid change; and if the diameter of the circumscribed circle is too large, there is a disadvantage of low flux.

[0051] If the distance between two adjacent perforations of the film is less than 0.5 mm, there is a disadvantage that the organoids are easy to adhere to each other and cannot grow independently; if the distance is too large, there is a disadvantage of low flux; the plurality of perforations can be arranged equidistantly or non-equidistantly.

[0052] As a specific embodiment, the film has a size matching the cell culture plate; the cell culture plate includes one of 384-well plate, 96-well plate, 48-well plate, 24-well plate, 12-well plate, 6-well plate, 3.5cm culture dish, 6cm culture dish, and 10cm culture dish. The liver organoid model of the present application is based on the existing cell culture well plate and has high compatibility with the existing cell culture well plate and the existing bio-related optical instrument.

[0053] As a specific embodiment,

[0054] The material of the film includes polydimethylsiloxane;

[0055] The cell low-adhesion material includes polyethylene glycol; other cell low-adhesion materials can also be used in other embodiments;

[0056] The cell-specific adhesion material includes at least one of Matrigel and Collagen.

[0057] As a specific embodiment, the film having a plurality of perforations is obtained by:

[0058] A positive mold having a plurality of microcolumn arrays is obtained;

[0059] PDMS is poured into the positive mold, vacuum dried and vacuumed, and a layer of PMMA is covered on the PDMS, and then two glass sheets are clamped and fixed, and dried;

[0060] The solidified PDMS layer is taken out and cut into a PDMS film suitable for the shape of the cell culture plate to obtain a film having a plurality of perforations.

[0061] The height of the microcolumn array of the positive film is 30μm-100μm. If the height is too low, the pressed PDMS film is too thin and not easy to unfold; if the height is too high, it is not easy to perforate;

[0062] According to another typical embodiment of the present application, a culture chip of the liver organoid prepared by the method is provided.

[0063] According to another typical embodiment of the present application, a preparation method of the liver organoid model is provided, which includes:

[0064] Step S1, seeding foregut cells in the liver organoid culture chip, after the cells adhere, removing the membrane with multiple perforations, aspirating the culture medium in the liver organoid culture chip, and adding the first culture medium for maintaining culture for 3-5 days after washing;

[0065] The step S1 specifically comprises:

[0066] S101, inducing stem cells (hESCs or hiPSCs) to differentiate into endoderm: conventionally cultured hESCs or hiPSCs are digested into single cells with Accutase, and are seeded in a six-well plate at a density of 1×10 5 / cm 2 When the cell confluence reaches 85%-90%, the differentiation is started. The culture time is 1-3 days, and D1-D3 medium is used for culture:

[0067] D1 Medium: RPMI medium containing 100 ng / mL ActivinA and 50 ng / mL BMP4; D2 Medium: RPMI medium containing 100 ng / mL ActivinA and 0.2% Knockout serum replacement; and D3 Medium: RPMI medium containing 100 ng / mL ActivinA and 2% Knockout serum replacement.

[0068] S102, differentiating endoderm cells into foregut cells: using a differentiation medium for culture; the medium is replaced every day, and the cells are conventionally cultured in a two-dimensional manner in a culture box. On the 6th day of differentiation, obvious three-dimensional structures can be observed.

[0069] The differentiation medium is D4-6 Medium of the embodiment of the application; the formula of the differentiation medium is Advanced DMEM / F12 medium with a final concentration of 450-550 ng / mL FGF2, 2-4 μM CHIR99021, 1% B27 and 1% N2, preferably 500 ng / mL FGF2 and 3 μM CHIR99021;

[0070] The B27 and N2 of the application are serum-free culture additives. The “%” in 1% B27 and 1% N2 is a mass fraction.

[0071] S103, after the foregut cells are digested, the foregut cells are seeded on the patterned substrate of the liver organoid culture chip, and the first culture medium is added for maintaining culture for 3-5 days.

[0072] The seeding density of the foregut cells is 1×10 5 -9×105 (Individual / cm 2 If the cell inoculation density is too small, it is difficult to grow into organoids with a certain thickness; if the cell inoculation density is too large, there are too many cells, which are difficult to separate from the PDMS perforated film and cannot form patterned organoids.

[0073] The first medium is Advanced DMEM / F12 medium with a final concentration of 75-85 ng / mL FGF2, 2-4 μM CHIR99021, 1% B27 and 1% N2, preferably 80 ng / mL FGF2 and 3 μM CHIR99021;

[0074] In step S2, the second medium is used for maintaining culture for 3-5 days and the third medium is used for maintaining culture for ≥10 days, to obtain the liver organoid model.

[0075] In step S2,

[0076] The second medium is Advanced DMEM / F12 medium with a final concentration of 1-3 mM RA, 1% B27 and 1% N2, preferably containing 2 mM RA;

[0077] The third medium is Hepatocyte Culture Medium medium with a final concentration of 8-12 ng / mL HGF, 0.05-0.2 mM Dexamethasone and 18-22 ng / mL OSM, preferably 10 ng / mL HGF, 0.1 mM Dexamethasone and 20 ng / mL OSM;

[0078] According to another typical embodiment of the present application, the liver organoid model obtained by the method is provided. As a specific embodiment of the present application, the obtained liver organoid is flat, similar to a solid circular arch (or similar expression), which is different from the phenotype of the previous liver organoids; since one side of the organoid is adhered to the bottom of the plate, and the organoid also has a certain thickness; as shown by the fluorescence map, the periphery of the organoid is clearly visible, while the middle part is less transparent, so the middle thickness should be thicker than the periphery, indicating a solid circular arch.

[0079] The "solid circular arch" has the following specific structure: the middle part of the liver organoid is arched, and the middle part and the periphery of the liver organoid form a circular arc; while 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 described by other expressions, as long as it can represent the liver organoid obtained by the present application.

[0080] According to another typical embodiment of the present application, the liver organoid model is applied in pharmacology, pharmacodynamics, and toxicology analysis.

[0081] The preparation method of the novel liver organoid model comprises inducing stem cells into foregut blast cells, seeding the foregut blast cells on a patterned substrate at a certain density, differentiating the foregut blast cells into a liver lineage, and finally forming an arch-like liver organoid adhering to the bottom of a culture vessel and having a certain thickness and specific edge shape, which comprises cell types including liver-like cells, bile duct-like cells, progenitor cells and interstitial cells differentiated from stem cells; the novel liver organoid model can be applied to research liver development and disease mechanism, drug screening, drug hepatotoxicity evaluation, etc. Compared with traditional gel drop method and in-situ sphere formation method, the present application provides a novel liver organoid model with high throughput, high uniformity and in-situ imaging, which provides an innovative research tool for related liver research.

[0082] A liver organoid model and a culture method according to the present application will be described in detail below with reference to the accompanying drawings.

[0083] Embodiment 1, a culture chip of a liver organoid and a preparation method thereof

[0084] I. Design of patterned substrate array mask

[0085] The AutoCAD 2018 software is used to design the patterned array mask, the array unit is a 0.5mm diameter circle, the pitch is 1.5mm, and the array units are uniformly arranged at equal intervals. The design diagram of the patterned array mask is shown in Figure 1 .

[0086] II. Fabrication of patterned substrate

[0087] 1. SU-8 positive film is prepared using soft lithography technology, the SU-8 positive film is a microcolumn array with a pitch of 1.5mm, the microcolumn height is 80μm, and the bottom area diameter is 500μm.

[0088] 2. The PDMS prepolymer (A glue) and the crosslinking agent (B glue) are mixed at a mass ratio of 10:1, stirred uniformly, and then the vacuum dryer is used to remove the bubbles. The PDMS is poured on the SU-8 positive film, and a clean gun head is used to flatten it, so that it uniformly covers the microcolumn array area. Vacuum drying is performed until there are no bubbles on the surface. A layer of 0.2mm thick polymethyl methacrylate (PMMA) is covered on the surface, and after being fixed by two glass sheets, it is clamped and fixed in the bench clamp, and then placed in an 80℃ oven for drying for at least 120min.

[0089] 3. The solidified PDMS punched film is taken out of the bench clamp, and a 15mm diameter circular punch is used to punch a circular punched film.

[0090] 4. Prepare PEG mixture (PEG 1000: 37.5 mg, PEG 400: 450 μL, isopropyl alcohol 3637.5 μL, pure water 112.5 μL), shake well for 3 min, then add 10 mg of photoinitiator, shake well again for 3 min, and store in the dark. After treating the well plate with Plasma (working voltage 550 V) for 1 min, add 150 μL of PEG mixture to each well, and stand for 5 min. Expose the well plate with PEG mixture in the wells to ultraviolet light for 1 min. After washing the exposed well plate with 70% alcohol three times, add 1 mL of 70% alcohol to each well, place the circular PDMS punched film flat on the liquid surface, press it to the bottom of the well plate with tweezers, suck out the excess liquid, and dry the well plate in an 80°C oven for about 20 min.

[0091] 5. Place the dried well plate in a plasma cleaning machine and treat with Plasma (working voltage 700 V) for a total of three times, 4 min each time, with a 3 min interval between each treatment. Do not remove the well plate during the interval.

[0092] 6. Ultraviolet sterilize the prepared well plate for 60 min, add 1 mL of 1x DPBS to each well, and repeatedly blow with a pipette gun until there are no bubbles in the well plate. Suck out the 1x DPBS, add 200 μL of 1% Matrigel: Advanced DMEM / F12 to each well, and incubate at 37°C for 1 h for standby, i.e., obtain the culture chip of liver organoids.

[0093] III. Culture of human liver organoids

[0094] 1. Culture the patterned substrates of each example and comparative example with liver organoids as follows:

[0095] (1) Differentiation of hESCs or hiPSCs into endoderm: conventionally cultured hESCs or hiPSCs were digested into single cells with Accutase, and seeded in a six-well plate at a density of 1x10 5 cells / cm 2 . When the cell confluence reached 85%-90%, differentiation was started.

[0096] (2) D1 Medium: RPMI medium containing 100 ng / mL Activin A and 50 ng / mL BMP4; D2 Medium: RPMI medium containing 100 ng / mL Activin A and 0.2% Knockout serum replacement; D3 Medium: RPMI medium containing 100 ng / mL Activin A and 2% Knockout serum replacement.

[0097] The above D1-D3 medium is used for the first three days of differentiation to induce differentiation of stem cells to endoderm;

[0098] (3) Differentiation of endoderm cells to foregut spheroids: D4-6 Medium: Advanced DMEM / F12 medium containing 500 ng / mL FGF2, 3 mM CHIR99021, 1% B27 and 1% N2, medium is changed every day, cells are routinely cultured in two dimensions in an incubator. On the 6th day of differentiation, obvious three-dimensional structures can be seen.

[0099] (4) Seeding foregut spheroids in patterned substrate: On the 6th day of differentiation, foregut spheroids are digested into single cells with Accutase, and seeded into the well plate at a density of 3 x 10 5 cells / cm 2 , 500 μL of Advanced DMEM / F12 medium containing 80 ng / mL FGF2, 3 mM CHIR99021, 1% B27 and 1% N2 per well.

[0100] (5) After the cells are seeded for at least 4 hours, the cells are attached, and the PDMS film is gently removed from the edge of the well plate with tweezers. The medium in the well plate is aspirated, the cells are gently washed with 1 x DPBS, and 500 μL of Advanced DMEM / F12 medium containing 80 ng / mL FGF2, 3 mM CHIR99021, 1% B27 and 1% N2 per well is added to maintain culture for 4 days, with medium changed every other day.

[0101] (6) Culture of liver organoids in patterned substrate: On the 10th-14th day, culture is maintained for 4 days with Advanced DMEM / F12 containing 2 mM RA, 1% B27 and 1% N2, with medium changed every other day; on the 14th-24th day, culture is maintained for ten days with Hepatocyte Culture Medium containing 10 ng / mL HGF, 0.1 mM Dexamethasone, 20 ng / mL OSM, with medium changed every other day.

[0102] Example 2

[0103] In the embodiment of the present application, the PDMS perforated film is punched into a 10 mm diameter circle by a round punch, and is laid in a 48-well plate. The other structures and steps are the same as those in Example 1.

[0104] Example 3

[0105] In the embodiment of the present application, the pattern unit in the PDMS perforated film is a circle, the diameter of the circular pattern is 500 μm, the pattern spacing is 1.5 mm, and the cell seeding density on the patterned substrate is 1 x 10 5 cells / cm 2 . The other structures and steps are the same as those in Example 1.

[0106] Example 4

[0107] In the embodiment of the present application, the pattern unit in the PDMS perforated film is a circle, the diameter of the circular pattern is 500 μm, the pattern spacing is 1.5 mm, and the cell seeding density on the patterned substrate is 9 x 10 5 cells / cm 2 . The other structures and steps are the same as those in Example 1.

[0108] Example 5

[0109] In the embodiment of the present application, the pattern unit in the PDMS perforated film is a circle, the diameter of the circular pattern is 100 μm, the pattern spacing is 1 mm, and the other structures and steps are the same as those in Example 1.

[0110] Example 6

[0111] In the embodiment of the present application, the pattern unit in the PDMS perforated film is a circle, the diameter of the circular pattern is 200 μm, the pattern spacing is 1.2 mm, and the other structures and steps are the same as those in Example 1.

[0112] Example 7

[0113] In the embodiment of the present application, the pattern unit in the PDMS perforated film is a circle, the diameter of the circular pattern is 1000 μm, the pattern spacing is 2 mm, and the other structures and steps are the same as those in Example 1.

[0114] Example 8

[0115] In the embodiment of the present application, the pattern unit in the PDMS perforated film is a circle, the circular pattern is arranged in a regular hexagon (including the center point), the pattern spacing is 1 mm, and the other structures and steps are the same as those in Example 1.

[0116] Example 9

[0117] In the embodiment of the present application, the pattern unit in the PDMS perforated film is a circle, the circular pattern is arranged in a regular hexagon (including the center point), the pattern spacing is 1.5 mm, and the other structures and steps are the same as those in Example 1.

[0118] Example 10

[0119] In this embodiment of the invention, the pattern unit in the PDMS perforated film is circular, the circular patterns are arranged in a regular hexagonal pattern (including the center point), the pattern spacing is 2mm, and other structures and steps are the same as in Embodiment 1.

[0120] Example 11

[0121] In this embodiment of the invention, the pattern unit shape in the PDMS perforated film is an equilateral triangle with a side length of 1 mm and a pattern spacing of 2 mm. Other structures and steps are the same as in Embodiment 1.

[0122] Example 12

[0123] In this embodiment of the invention, the pattern unit shape in the PDMS perforated film is an equilateral triangle with a side length of 0.5 mm and a pattern spacing of 1.5 mm. Other structures and steps are the same as in Embodiment 1.

[0124] Example 13

[0125] In this embodiment of the invention, the pattern unit shape in the PDMS perforated film is rectangular, with a long side length of 0.5 mm, a short side length of 0.15 mm, and a pattern spacing of 1.5 mm. Other structures and steps are the same as in Embodiment 1.

[0126] Example 14

[0127] In this embodiment of the invention, the pattern unit shape in the PDMS perforated film is rectangular, with a long side length of 1 mm, a short side length of 0.3 mm, and a pattern spacing of 2 mm. Other structures and steps are the same as in Embodiment 1.

[0128] Comparative Example 1

[0129] The comparison ratio is the traditional Matrigel.

[0130] Comparative Example 2

[0131] In this comparative example, the diameter of the circular pattern in the PDMS perforated film is 95 μm, the pattern spacing is 1 mm, and other structures and steps are the same as in Example 1.

[0132] Comparative Example 3

[0133] In this comparative example, the cell seeding density on the patterned substrate was 0.9 × 10⁻⁶. 5 (pieces / cm) 2 (), smaller than 1×10 of the present invention embodiment. 5 ~9×10 5 (pieces / cm) 2 The other structures and steps are the same as in Example 1.

[0134] Comparative Example 4

[0135] In this comparative example, the cell seeding density on the patterned substrate was 9.1 × 10⁻⁶. 5 (pieces / cm) 2 (), greater than 1×10 of the embodiments of the present invention. 5 ~9×10 5 (pieces / cm) 2 The other structures and steps are the same as in Example 1.

[0136] Comparative Example 5

[0137] In this comparative example, the pattern spacing is 0.4 mm, and the other structures and steps are the same as in Example 1.

[0138] Comparative Example 6

[0139] In this comparative example, the patterning spacing is 3.2 mm, and the other structures and steps are the same as in Example 1.

[0140] Experimental Example 1

[0141] The liver organoid culture effect of the substrates of Examples 1-14 and Comparative Examples 1-6 was statistically analyzed, as shown in Table 1. The standard deviation coefficient of variation of the area was calculated as follows: coefficient of variation C·V = (standard deviation SD / mean Mean) × 100%.

[0142] Table 1

[0143]

[0144]

[0145] The data in Table 1 shows that:

[0146] In Comparative Example 1, the traditional Matrigel culture method is used, which has the disadvantages of poor organoid uniformity and difficulty in imaging.

[0147] 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 embodiments of the present invention. This has the disadvantage that the organoids are easily lost during fluid exchange.

[0148] In Comparative Example 3, the cell seeding density was 0.9 × 10⁻⁶. 5 pcs / cm 2 Smaller than 1×10 in this embodiment 5 ~9×10 5 (pieces / cm) 2 Within the range of ), there is a drawback that it is difficult to grow into organoids with a certain thickness.

[0149] In Comparative Example 4, the cell seeding density was 9.1 × 10⁻⁶. 5 pcs / cm 2 Larger than 1×10 in this embodiment 5 ~9×10 5 (pieces / cm) 2 The range of cells is limited, and there are disadvantages such as too many cells, making it difficult to separate them from the PDMS perforated membrane and preventing the formation of patterned organoids.

[0150] In Comparative Example 5, the spacing between pattern units on the patterned substrate is 0.4 mm, which is smaller than the range of 0.5 to 3 mm in this embodiment. This results in the disadvantage that organoids tend to adhere to each other and cannot grow independently.

[0151] In Comparative Example 6, the spacing between pattern units on the patterned substrate is 3.2 mm, which is larger than the range of 0.5 to 3 mm in this embodiment, resulting in the disadvantage that high throughput cannot be achieved.

[0152] In Examples 1-11 of this invention, normal growth was observed with a coefficient of variation ≤40%. Compared with traditional droplet and in-situ spheroidization methods, this invention provides a novel liver organoid model with high throughput, high uniformity, and in-situ imaging capabilities, offering an innovative research tool for related liver studies.

[0153] Experimental Example 2: Detection of Characteristic Biomarkers for Human Liver Organoids

[0154] 1. Immunofluorescence was used to detect characteristic markers at each differentiation stage: the expression of markers OCT3 / 4 and Nanog was detected at the hESCs or hiPSCs stage; the expression of markers CDX2 and EpCAM was detected at the foregut stage; and the expression of markers ALB, HNF4-α, AFP, and EpCAM was detected at the liver organoid stage.

[0155] 2. Construction and characterization of a drug hepatotoxicity evaluation system on a patterned substrate in Example 1: On day 24, liver organoids were treated with 0 mM, 10 mM, 20 mM and 40 mM acetaminophen (APAP) for 48 hours, and cell viability was determined.

[0156] The results are as follows Figures 3-4 This indicates that the embodiments of the present invention have successfully obtained human liver organoids and constructed a drug hepatotoxicity evaluation system.

[0157] 3. Human organoids were cultured on the patterned substrate of Example 1, and liver organoids were cultured using the conventional droplet method as a control; wherein... Figure 2 a is a patterned flowchart of liver organoid culture; Figure 2 b is a bright-field image of a liver organoid in a 24-well plate; Figure 2 c is a bright-field image of liver organoids cultured using the traditional gel drop method;Figure 2 d is the area characterization of the patterned liver organoids cultured at different time points; Figure 2 e is the comparison of the area coefficient of variation (CV value) of the patterned liver organoids and the gel-drop liver organoids;

[0158] From Figure 2 e, it can be seen that the area coefficient of variation of the patterned liver organoids is significantly lower than that of the gel-drop liver organoids; through Figure 2 a and Figure 2 The comparison of the bright field images of a, b and c can also directly show that the patterned liver organoids limit the growth area of the organoids, have consistent shape and size, while the organoids cultured by the gel-drop method are uncontrollable in these aspects.

[0159] Finally, it should be noted that the terms "comprising", "containing" or any other variant thereof are intended to cover non-exclusive inclusions, so that a process, method, article or apparatus that includes a list of elements not only includes those elements, but also includes other elements not explicitly listed, or inherent to such a process, method, article or apparatus.

[0160] Although preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to cover all preferred embodiments and all changes and modifications falling within the scope of the present application.

[0161] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A method for preparing a culture chip for liver organoids, characterized in that, The method includes: Obtain a thin film with multiple perforations; A cell-low adhesion material is coated on the bottom of a cell culture plate, and then the film is covered on the bottom of the cell culture plate to obtain a patterned substrate; A cell-specific adhesion material is added to the patterned substrate for coating to obtain a liver organoid culture chip; The material of the film includes polydimethylsiloxane; the material with low cell adhesion includes polyethylene glycol; and the material with specific cell adhesion includes at least one of Matrigel and Collagen.

2. The method for preparing a liver organoid culture chip according to claim 1, characterized in that, The shape of each perforation includes one of the following: circular, elliptical, semi-circular, fan-shaped, triangular, quadrilateral, pentagonal, hexagonal, and any other polygon; the diameter of the circumscribed circle of each perforation is 100μm to 1000μm, and the distance between two adjacent perforations of the film is 5mm to 3mm.

3. The method for preparing a liver organoid culture chip according to claim 1, characterized in that, The process of obtaining a thin film with multiple perforations includes: Obtain a positive mold with multiple micropillar arrays; Pour PDMS onto the male mold, vacuum dry and evacuate, then cover the PDMS with a layer of PMMA, clamp and fix it with two glass plates, and dry. The solidified PDMS layer was removed and cut into PDMS membranes that fit the shape of cell culture plates to obtain a membrane with multiple perforations.

4. The method for preparing a liver organoid culture chip according to claim 3, characterized in that, The height of the micropillar array in the positive mold is 30μm to 100μm.

5. A culture chip for liver organoids prepared by the method according to any one of claims 1-4.

6. A method for preparing a liver organoid model, characterized in that, The method includes: Foregut germ cells are seeded into the liver organoid culture chip as described in claim 5. After the cells adhere to the wall, the membrane with multiple perforations is removed, the culture medium in the liver organoid culture chip is aspirated, and after washing, the first culture medium is added to maintain culture for 3 to 5 days. The liver organoid model was then obtained by maintaining the culture in the second culture medium for 3-5 days and the third culture medium for ≥10 days.

7. The method for preparing a liver organoid model according to claim 6, characterized in that, The foregut progenitor cells are seeded at a density of 1 x 105 to 9 x 105 cells / cm2 2 .

8. A liver organoid model obtained by the method according to any one of claims 6 to 7.

9. The application of the liver organoid model as described in claim 8 in pharmacological, pharmacodynamic, and toxicological analyses.

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