Preparation method and application of large-size human liver organoids

Abstract

The application discloses a preparation method and application of large-size human liver organoids, and belongs to the technical field of cell culture. In the application, 2D culture mature human primary liver parenchymal cells with different cell amounts are resuspended by using a hepatocyte maturation medium HIM, and are added into a U-shaped bottom 96-hole plate for low-oxygen static culture. After the cells are aggregated, the cells are cultured in normal oxygen and are shaken, so that large-size human liver organoids with different cell contents are prepared. The method does not need biological materials, and can induce human primary liver parenchymal cells to self-assemble and construct functional liver organoids with high uniformity in scale through specific culture conditions. The method solves the problems of large batch difference, high heterogeneity and dedifferentiation in the prior art, and can be widely applied to drug hepatotoxicity testing, in-vitro modeling of liver diseases and development of a bioartificial liver system.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically to a method for preparing and applying a large-sized human liver organoid. Background Technology

[0002] Human primary hepatocytes (PHHs) are the "gold standard" cell model for in vitro studies of liver physiology, pathology, and drug metabolism responses, as they fully preserve the complex functional system of hepatocytes in vivo. However, PHHs are extremely fragile in vitro, rapidly losing their typical morphology and key functions, which constitutes a major technical bottleneck in liver-related in vitro research. Existing major culture strategies all have significant shortcomings, specifically as follows:

[0003] Traditional two-dimensional monolayer culture: This method seeds hepatocytes (PHHs) onto a rigid, planar substrate (such as a plastic culture dish). This two-dimensional environment violates the polarity and three-dimensional clustering characteristics of hepatocytes in vivo, inducing anodic apoptosis and forcing cells to undergo abnormal morphological flattening. This process is accompanied by cytoskeleton rearrangement and nuclear deformation, leading to transcriptional dysregulation at the genome-wide level. As a result, PHHs typically undergo rapid and irreversible dedifferentiation within 24 to 72 hours, with a sharp decline in core liver functions such as cytochrome P450 enzyme (CYP450) activity, transport protein function, and albumin synthesis, failing to meet the needs of any research requiring medium- to long-term stable function.

[0004] Three-dimensional culture based on biological scaffolds: This method uses natural matrix materials such as Matrigel and collagen to provide a three-dimensional growth environment for cells. While it partially improves cell morphology and function maintenance, it has inherent drawbacks: First, natural matrix materials, especially Matrigel, exhibit significant batch-to-batch variability, introducing substantial experimental variability and severely impacting the reproducibility of results. Second, these matrices are complex in composition, often containing growth factors or impurities of unknown origin, interfering with the precise analysis of cell autonomous behavior. Furthermore, with cells embedded within the matrix, the exchange of substances between them and the culture environment (including nutrient uptake, metabolic waste removal, and drug penetration) primarily relies on passive diffusion, easily leading to the formation of nutrient / oxygen gradients at the center of cell aggregates, potentially causing cell necrosis in the central region.

[0005] Emerging hepatocyte organoid technology: This technology typically involves adding complex and costly combinations of growth factors and signaling pathway modulators to mimic liver development or regeneration signals, inducing cells to self-organize into three-dimensional structures. However, its culture cycle is lengthy (often requiring several weeks) and technically challenging. The resulting organoids exhibit significant heterogeneity in size, internal cellular composition (often mixed with hepatic progenitor cells or bile duct cells), and structural polarity. This batch-to-batch variation severely impacts the reliability of data such as drug response and the accuracy of quantitative analysis, making standardized application difficult.

[0006] Hepatocyte spheroids formed by hanging drop or forced aggregation methods: These methods use physical means (such as hanging drop plates or low-attachment plates) to induce rapid aggregation of cells into spheroids. Although the operation is relatively simple, it is difficult to precisely control the final size of each aggregate, resulting in "uniformity" in the size of the generated spheroids. The difference in size directly causes inconsistency in the internal microenvironment: larger spheroids are more prone to central necrosis due to diffusion restriction; smaller spheroids may be functionally immature due to insufficient intercellular interactions. Therefore, the functional state of the spheroid model obtained by this method has "poor reproducibility" and cannot provide a stable and homogeneous experimental system.

[0007] In summary, existing in vitro culture techniques for PHHs generally face systemic bottlenecks such as rapid dedifferentiation, high experimental variability, technical complexity, high cost, poor model uniformity and reproducibility, difficulty in standardization and large-scale application.

[0008] Therefore, there is an urgent need in this field to develop a three-dimensional PHHs culture method that is easy to operate, cost-controllable, highly reproducible, and can quickly construct and maintain a high functional state for a long time. Summary of the Invention

[0009] In view of this, the present invention provides a method for preparing large-sized human liver organoids and its application, which successfully solves the problems of large batch differences, high organoid heterogeneity, and rapid dedifferentiation of hepatocytes in the prior art, and obtains organoids with highly uniform size and mature liver function, simplifies the operation process, and effectively reduces costs.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] The primary objective of this application is to provide a method for preparing a large-sized human liver organoid, comprising the following steps:

[0012] (1) 2D amplification and induced maturation: Resuscitated human primary hepatocytes were seeded in culture dishes coated with type I collagen. After adherent culture, amplification culture and induced maturation culture were performed to complete the 2D-level induced maturation of cells.

[0013] (2) Formation of three-dimensional organoids: The human primary hepatocytes induced to mature in step (1) were digested and resuspended into a single-cell suspension. Human primary hepatocytes of different cell amounts were resuspended using hepatocyte maturation medium HIM and then added to a U-shaped bottom 96-well plate. The cells were cultured statically under hypoxic conditions for 2-5 hours and then transferred to normoxic conditions for shaking culture for 7 days to prepare human liver organoids.

[0014] As a preferred technical solution, the method further includes step (3), the specific process of which is as follows: based on the demand for human liver organoids of different scales, the human liver organoids prepared in multiple steps (2) can be mixed and cultured in a shaker to prepare large-scale human liver organoids.

[0015] As a preferred technical solution, the culture medium for adherent culture in step (1) is as follows: DMEM + 10% FBS + 1% PS; the conditions for adherent culture are as follows: cultured in a hypoxic incubator for 24 hours, with the hypoxic incubator conditions being 37℃, 0.5-2% O2, and 4.5-5.5% CO2.

[0016] As a preferred technical solution, in step (1), the culture medium for amplification culture is HM culture medium, and the conditions for amplification culture are 37℃, 0.5-2% O2, 4.5-5.5% CO2; the amplification culture time is 2-6 days.

[0017] As a preferred technical solution, in step (1), the culture medium for inducing maturation is HIM medium, and the culture conditions for inducing maturation are 37℃, 0.5-2% O2, 4.5-5.5% CO2; the culture time for inducing maturation is 7-10 days.

[0018] As a preferred technical solution, in step (2), the number of cells is 10,000 to 1,000,000.

[0019] As a more preferred technical solution, the number of cells in step (2) is 10,000.

[0020] As a preferred technical solution, in step (2), the low oxygen conditions are: 37℃, 0.5-2% O2, 4.5-5.5% CO2; the normal oxygen conditions are: 37℃, 18.5-20.9% O2, 4.5-5.5% CO2; and the shaking culture speed is 50-60 rpm.

[0021] Another object of this application is to provide: a large-sized human liver organoid prepared by the preparation method.

[0022] Another object of this application is to provide: the application of the aforementioned large-sized human liver organoid in the preparation of in vitro models for drug hepatotoxicity screening, drug metabolism assessment or liver disease modeling.

[0023] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Good standardization and uniformity: It solves the problem of high organoid heterogeneity caused by batch differences and random aggregation of biological materials in traditional 3D culture, and obtains organoids with highly uniform size, which greatly improves the reproducibility of experiments and the reliability of data. It eliminates the need to handle complex, expensive and unstable biological materials (such as Matrigel), simplifies the operation process and effectively reduces costs.

[0025] (2) Mini-organ liver function maturation: Due to the absence of a dense exogenous biological scaffold, the diffusion of nutrients and metabolites inside and outside the organoid is more efficient, which is conducive to the survival and functional maintenance of organoid cells and solves the problem of "limited material exchange". It can maintain a high level of liver-specific functions (such as CYP450 enzyme activity, urea synthesis, and albumin secretion) stably for a long time (up to P16 generation), effectively overcoming the problem of rapid dedifferentiation of hepatocytes in 2D culture, and providing a more reliable platform for drug testing and disease modeling.

[0026] (3) Scalable production: This method can achieve large-scale preparation, meeting the needs of high-throughput drug screening and bioartificial liver systems for a large number of functional hepatocytes in industrial and clinical applications. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 The results are as follows: Experimental results of the optimization process of liver organoid preparation method parameters; where a) is the morphology of human liver organoids cultured in large-area culture dishes; b) is the morphology of human liver organoids cultured in flat-bottomed 96-well plates; c) is the morphology of human liver organoids cultured in U-shaped low-adsorption 96-well plates; and d) is the effect of quiescence time on mini-organ formation.

[0029] Figure 2 Here is a flowchart of the mini-organ preparation method.

[0030] Figure 3The images are: bright-field images of mini-organs with different cell numbers in Example 1; where a is a camera image; b is an image of the mini-organs taken with an upright microscope at 5 hours, day 1, and day 5 with different cell numbers.

[0031] Figure 4 Here are: Schematic diagrams of the internal fine structure of the mini-organ in Example 1 at different time points.

[0032] Figure 5 For example: Expression levels of liver maturation-related genes in mini-organs with different cell masses in Example 1 and

[0033] Example 1: Mini-organ albumin secretion and urea synthesis at different cell sizes.

[0034] Figure 6 The images show: the appearance of the mini-organs prepared in Example 2 and their diameters at different time points; where a is a bright-field image of the mini-organs taken with an upright microscope; b is an image of the uniformly sized mini-organs prepared on a large scale taken with a camera; and c is a statistical chart of the diameters of various organoids.

[0035] Figure 7 The results are as follows: functional assay results of the mini-organ prepared in Example 2; where a is the HE staining image of the mini-organ; b is the immunofluorescence staining image of ALB; c is the immunofluorescence staining image of HNF4A; d is the Dil uptake assay of the mini-organ; and e is the ICG uptake and release assay of the mini-organ.

[0036] Figure 8 The results are as follows: Screening results of hepatotoxic drugs for the mini-organ prepared in Example 2; where a is the IC50 plot of the mini-organ against different drugs; b is the bright field plot of the mini-organ after treatment with different drugs; and c is the PI staining plot of the mini-organ under different drug treatments.

[0037] Figure 9 The image shows the result of fusing mini-organ into macro-organ in Example 3; where a is an image taken with an upright microscope; and b is an image taken with a stereomicroscope.

[0038] Figure 10The images are: macro-organ mesenteric transplantation in Example 3; where a is an appearance image taken by a camera of macro-organ; b is a transplantation image of macro-organ mesenteric transplantation into NSG mice; c is an appearance image of macro-organ taken by a camera one month after transplantation in NSG mice; d is an appearance image of macro-organ taken by a stereoscope one month after transplantation in NSG mice. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The specific composition of the culture medium used in the embodiments of this application is as follows:

[0041] Human primary hepatocyte (PHH) cell expansion medium HM composition table:

[0042] Basic DMEM / F-12 medium (1X, Thermo Fisher, C11330500BT).

[0043] N2 supplement (100x concentrated solution) (1X, Life Technologies, 17502-048).

[0044] B27 supplement (50x concentrated solution), does not contain vitamin A (1X, Life Technologies, 12587-010).

[0045] N-acetylcysteine ​​(1mM, Sigma-Aldrich, A9165-100G).

[0046] Recombinant human epidermal growth factor (40 ng / ml, Peprotech, AF-100-15).

[0047] Recombinant human hepatocyte growth factor (25 ng / ml, Peprotech, 100-39).

[0048] Human [Leu15]-gastrin-I (10 nM, Sigma-Aldrich, G9145).

[0049] A 83-01 (5 μM, Tocris Bioscience, 2939).

[0050] Rho kinase inhibitor Y-27632 (10 μM, Selleck, s1049).

[0051] GSK-3β inhibitor CHIR99021 (3μM, Selleck, S1263).

[0052] Glutamax (1X, Gibco, 35050061, 100mL).

[0053] Sodium pyruvate (1mM, National Pharmaceutical Reagent Shanghai Test, 30169134, 25g).

[0054] Sphingosine 1-phosphate (0.5-2 μM, MCE, HY-108496, 5 mg).

[0055] Oleoyl lysophosphatidyl acid (3-6 μM, MCE, HY-107614, 10 mg).

[0056] NIBR-LTSi (3-6μM, MCE, HY-160769, 10mg).

[0057] LDN193189 (0.3-1μM, MCE, HY-12071, 5mg).

[0058] Penicillin-streptomycin (double antibiotic solution) (1%, Solarbio, P1400).

[0059] Human primary hepatocyte (PHH) maturation medium HIM composition table:

[0060] Basic DMEM / F-12 medium (1X, Thermo Fisher, C11330500BT).

[0061] N2 supplement (100x concentrated solution) (1X, Life Technologies, 17502-048).

[0062] B27 supplement (50x concentrated solution), does not contain vitamin A (1X, Life Technologies, 12587-010).

[0063] N-acetylcysteine ​​(1mM, Sigma-Aldrich, A9165-100G).

[0064] Recombinant human epidermal growth factor (40 ng / ml, Peprotech, AF-100-15).

[0065] Recombinant human hepatocyte growth factor (25 ng / ml, Peprotech, 100-39).

[0066] Human [Leu15]-gastrin-I (10 nM, Sigma-Aldrich, G9145).

[0067] A 83-01 (5 μM, Tocris Bioscience, 2939).

[0068] Rho kinase inhibitor Y-27632 (10 μM, Selleck, s1049).

[0069] GSK-3β inhibitor CHIR99021 (3μM, Selleck, S1263).

[0070] Glutamax (1X, Gibco, 35050061, 100mL).

[0071] Sodium pyruvate (1mM, National Pharmaceutical Reagent Shanghai Test, 30169134, 25g).

[0072] Sphingosine 1-phosphate (0.5-2 μM, MCE, HY-108496, 5 mg).

[0073] Oleoyl lysophosphatidyl acid (3-6 μM, MCE, HY-107614, 10 mg).

[0074] LDN193189 (0.3-1μM, MCE, HY-12071, 5mg).

[0075] Penicillin-streptomycin (double antibiotic solution) (1%, Solarbio, P1400).

[0076] Forsocrine (3-6μM, MCE, HY-15371, 10mg).

[0077] Insulin (0.5-1.5μM, MCE, HY-P73243, 10ug).

[0078] Norepinephrine (0.5-1.5μM, MCE, HY-13715, 100mg).

[0079] Dexamethasone (5-15μM, MCE, HY-14648, 100mg).

[0080] The HM and HIM culture media mentioned above are culture media that we developed and screened ourselves.

[0081] Example 1

[0082] A method for preparing and applying a large-sized human liver organoid

[0083] (1) 2D expansion culture of human primary hepatocytes

[0084] Cryopreserved primary human hepatocytes were purchased from Livo Biotechnology. After thawing, the cryopreserved cells were seeded into cell culture dishes coated with type I rat tail collagen (Corning® type I collagen, high concentration, rat tail, 100 mg; the coating solution was diluted with sterile water to a final concentration of 20 μg / ml). The cells were cultured in DMEM + 10% FBS + 1% PS in a hypoxic incubator (37℃, 0.5% O2, 4.5% CO2). After 24 hours, the culture was replaced with HM medium for expanding primary human hepatocytes. The HM medium contained the lowest possible concentrations of small molecules within the specified range, namely sphingosine 1-phosphate (0.5 μM), oleoyllysophosphatidylcholine (3 μM), NIBR-LTSi (3 μM), and LDN193189 (0.3 μM). The culture was maintained at 37℃, 0.5% O2, and 4.5% CO2. Under CO2 conditions, the culture was passaged and expanded. Two days before constructing organoids, the medium was replaced with the maturation medium HIM. The HIM medium contained the lowest possible concentrations of small molecules within the specified range: sphingosine 1-phosphate (0.5 μM), oleoyllysinic acid (3 μM), LDN193189 (0.3 μM), forsocolin (3 μM), insulin (0.5 μM), norepinephrine (0.5 μM), and dexamethasone (5 μM). Induction maturation was performed at 37°C, 0.5% O2, and 4.5% CO2 for 2 days. Through this culture process, primary human hepatocytes were initially induced to mature at the 2D level.

[0085] (2) Parameter optimization of liver organoid preparation method

[0086] To construct human primary liver parenchyma organoids with uniform scale and high reproducibility, we compared different liver organoid construction methods, as detailed below:

[0087] 1) Experimental results are as follows Figure 1 As shown in Figure a: First, we explored the formation of organoids in culture dishes with different bottom areas. The results showed that human liver organoids cultured in large-bottom-area culture dishes formed multiple liver organoids of varying sizes, and could not form a single organoid of uniform size. Therefore, we subsequently chose 96-well plates with smaller bottom areas for subsequent experiments.

[0088] 2) Next, we compared flat-bottomed 96-well plates, ultra-low adsorption U-shaped bottom 96-well plates, and U-shaped bottom 96-well plates (3DSphaero™ Cell and Tissue Culture Plate, BIOFIL, TCP130096). We found that although the flat-bottomed 96-well plates could aggregate into clusters, some cells adhered and exhibited a 2D cultured cell morphology, failing to form mini-organs (results are shown in Figure 1). Figure 1 (As shown in b) Furthermore, the 96-well plate at the bottom of the U-shaped plate with ultra-low adsorption forms individual, scattered organoids, failing to form single, uniformly sized organoids (results as shown in b). Figure 1 As shown in c), we ultimately chose a U-shaped bottom 96-well plate (3DSphaero™ Cell and Tissue Culture Plate, BIOFIL, TCP130096) for subsequent experiments.

[0089] 3) In addition, we explored the effect of the duration of rest on mini-organ formation. The experimental results are as follows: Figure 1 As shown in d, prolonged resting time can lead to more cell debris appearing at the edges of the formed mini-organs. Therefore, in order to improve hepatocyte activity, the resting time should ideally be controlled within 2-5 hours.

[0090] (3) Preparation of mini-organ

[0091] The 2D human primary hepatocytes induced to mature in step (1) were washed with PBS, digested with 0.05% trypsin at 37°C for 3 minutes, and digested with DMEM + 10% FBS to terminate the digestion. The cells were dispersed into single-cell suspensions, and after counting, 10,000, 70,000, 150,000, 200,000, and 250,000 cell suspensions were aspirated and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cell pellets were resuspended with 100 μL of HIM maturation medium and added to 96-well U-shaped bottom plates (3DSphaero™ Cell and Tissue Culture Plate, BIOFIL, TCP130096). The HIM medium contained the lowest concentration of small molecules in the range, namely sphingosine 1-phosphate (0.5 μM), oleoyllysophosphatidic acid (3 μM), LDN193189 (0.3 μM), forsocolin (3 μM), insulin (0.5 μM), norepinephrine (0.5 μM), and dexamethasone (5 μM). 100 μL of cell suspension was seeded into 96-well plates with a U-shaped bottom and incubated in a hypoxic incubator (37℃, 0.5% O2, 4.5% CO2) for 2-5 hours to allow the cells to aggregate into spheres under gravity. Then, the cells were placed on a shaker and cultured under normal oxygen for 7 days to further induce maturation (37℃, 18.5% O2, 4.5% CO2; 50 rpm) to prepare mini-organs with different cell volumes.

[0092] Experimental flowchart as follows Figure 2 As shown. Mini-organ bright-field plots with different cell volumes are shown below. Figure 3 As shown, Figure 3 In the image, 'a' represents a picture taken by the camera. Figure 3 Image b shows the appearance of the mini-organs taken under an upright microscope at 5 hours, day 1, and day 5 with different cell numbers. The results indicate that the mini-organs constructed by this method have a high cell loading rate, and different cell numbers can self-aggregate into spheres.

[0093] Furthermore, this embodiment also observes the internal fine structure of the mini-organ prepared above, and the experimental results are as follows: Figure 4 As shown.

[0094] The results show that, Figure 4 (With a cell count of 1 million), it can be seen that as the culture time increases, connections are gradually established between cells inside the mini-organ, forming a dense internal structure.

[0095] Furthermore, to verify the function of mini-organs with different cell numbers, their liver maturation-related genes were identified. The specific process is as follows:

[0096] RNA was extracted from mini-organs of varying cell types, and different liver maturation function genes were identified by qPCR. Simultaneously, 2D human primary hepatocytes induced to mature in step (1) were used as the basis for the experimental results, as shown below. Figure 5 As shown, PHH refers to primary hepatocytes, i.e., positive control.

[0097] The results showed that, compared with 2D cultured PHH (cell culture dishes with adherent growth on type I rat tail collagen), mini-organs expressed higher levels of liver maturation function genes, indicating that the 3D cultured mini-organs constructed in this embodiment can induce more mature hepatocyte function. In addition, the expression levels of genes such as ALB, CYP3A4, HNF4A, FGB, ASS1, OTC, and FXR in mini-organs with a cell mass of 10,000 cells were higher than those in mini-organs with other cell mass, indicating more mature function.

[0098] Furthermore, to verify the functions of mini-organ albumin secretion and urea synthesis in different cell masses, the following experiments were conducted:

[0099] After culturing the induced mature mini-organs for 24 hours, the culture supernatant was collected. Albumin secretion and urea synthesis capabilities were then assessed according to the instructions of the human albumin (ALB) ELISA reagent (elabscience) and the blood urea nitrogen (urea) content assay kit (Solepro). The results are as follows: Figure 5 As shown.

[0100] The results showed that mini-organ cells with a cell mass of 10,000 exhibited better albumin secretion and urea synthesis capabilities, indicating more mature function. Therefore, a cell mass of 10,000 mini-organ cells was deemed more appropriate for subsequent experiments.

[0101] Example 2

[0102] (1) Large-scale preparation of uniformly sized mini-organs

[0103] 1) 2D expansion culture of human primary hepatocytes

[0104] Cryopreserved primary human hepatocytes were purchased from Livo Biotechnology. After thawing, the cryopreserved cells were seeded into cell culture dishes coated with type I rat tail collagen (Corning® type I collagen, high concentration, rat tail, 100 mg; the coating solution was diluted with sterile water to a final concentration of 20 μg / ml). The cells were cultured in a hypoxic incubator (37℃, 2% O2, 5.5% CO2) using DMEM + 10% FBS + 1% PS. After 24 hours, the culture was replaced with HM medium for expanding primary human hepatocytes. The HM medium contained the highest concentration of small molecules within its range, namely sphingosine 1-phosphate (2 μM), oleoyllysophosphatidylcholine (6 μM), NIBR-LTSi (6 μM), and LDN193189 (1 μM). The culture was maintained at 37℃, 2% O2, and 5.5% CO2. Under CO2 conditions, the culture was passaged and expanded. Two days before constructing organoids, the medium was replaced with a maturation medium, HIM, containing the highest concentrations of small molecules within the specified range: sphingosine 1-phosphate (2 μM), oleoyllysinic acid (6 μM), LDN193189 (1 μM), forsocolin (6 μM), insulin (1.5 μM), norepinephrine (1.5 μM), and dexamethasone (15 μM). Induction maturation was performed at 37°C, 2% O2, and 5.5% CO2 for 2 days. Through this culture process, primary human hepatocytes were initially induced to mature at the 2D level.

[0105] 2) Preparation of mini-organs

[0106] The 2D human primary hepatocytes induced to mature in step (1) were washed with PBS, digested with 0.05% trypsin at 37°C for 3 minutes, and digested with DMEM + 10% FBS to terminate the digestion. The cells were then dispersed into a single-cell suspension and diluted with the maturation medium HIM to 100,000 cells / mL. The HIM medium contained the highest concentration of small molecules in the range of concentrations, namely sphingosine 1-phosphate (2 μM), oleoyllysin phosphatidic acid (6 μM), LDN193189 (1 μM), forsocolin (6 μM), insulin (1.5 μM), norepinephrine (1.5 μM), and dexamethasone (15 μM). 100 μL of cell suspension was seeded into 96-well round-bottom plates and incubated statically in a hypoxic incubator (37℃, 2% O2, 5.5% CO2) for 5 hours, then transferred to a shaker for normoxic incubation (37℃, 20.9% O2, 5.5% CO2; 60 rpm) for 7 days. The diameter was measured on day 0, day 2, day 5, and day 7. Figure 6 Image a shows a bright-field image of a mini-organ taken with an upright microscope. Figure 6 Image b in the image represents a large-scale image of uniformly sized mini-organs taken with a camera. Figure 6 In the middle, c represents a statistical chart of the diameters of various organ types.

[0107] The results showed that the constructed mini-organs, with a cell count of 10,000, were uniform in size and approximately 735 μM in diameter. The experimental procedure was simple and could be prepared on a large scale.

[0108] (2) Functional experiments of mini-organs

[0109] The mini-organs prepared above were collected, fixed in 4% paraformaldehyde for 24 hours, dehydrated, embedded, sectioned, and stained. The experimental results are as follows: Figure 7 As shown, Figure 7 In the image, 'a' represents the HE staining pattern of a mini-organ. Figure 7 Image b in the image is an immunofluorescence staining pattern of ALB. Figure 7 In the middle, c represents the immunofluorescence staining image of HNF4A. Figure 7 In the experiment, d represents the Dil uptake experiment of mini-organs. Figure 7 In the middle, 'e' represents the ICG uptake and release experiment of the mini-organ.

[0110] The results showed that the internal structure of the HE surface mini-organ was dense, with tight junctions between cells. Immunofluorescence indicated that the mini-organ highly expressed ALB and HNF4A. Furthermore, the mini-organ could actively take up Dil and actively take up and release ICG, all of which indicate that the mini-organ exhibits mature liver function.

[0111] (3) Mini-organ for screening hepatotoxic drugs

[0112] Traditional human liver organoid culture systems commonly use matrix gels (such as Matrigel), but Matrigel exhibits significant batch-to-batch variability and animal-derived issues. Furthermore, in traditional batch cultures, the number and size of organoids are highly variable, leading to significant differences in data wells for drug screening. Our constructed mini-organs are uniform in size, simple to operate, can be prepared on a large scale, and have high reproducibility, making them suitable for screening hepatotoxic drugs. On day seven of induction maturation, mini-organs were supplemented with different concentrations of hepatotoxic drugs (APAP concentrations of 0, 0.1 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM; amiodarone concentrations of 0, 1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 100 μM, 150 μM, and 200 μM). Ketoconazole was administered at concentrations of 0, 0.1 μM, 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, 400 μM, 800 μM, and 1000 μM; tacrine was administered at concentrations of 0, 1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 100 μM, 150 μM, and 200 μM. Seven days after drug administration, the cell viability of mini-organs was measured using an ATP assay kit (CellTiter-Lumi™ luminescent 3D cell viability assay kit; Beyotime; C0065M). The response of mini-organs to different concentrations of hepatotoxic drugs such as APAP, amiodarone, ketoconazole, and tacrine was tested. The results are as follows: Figure 8 As shown, Figure 8 In the figure, 'a' represents the IC50 values ​​of the mini-organ for different drugs. Figure 8 In the middle, b is the bright-field plot of the mini-organ after treatment with different drugs. Figure 8 In the middle, c represents the PI staining pattern of the mini-organ under different drug treatments.

[0113] The results showed that the mini-organ can metabolize drugs into different metabolites through different CYP enzymes, further demonstrating that our in vitro cultured mini-organ highly expresses different CYP enzymes, mimicking the in vivo metabolic process, and can be used as an in vitro drug screening platform.

[0114] Example 3

[0115] (1) Mini-organ merges into macro-organ

[0116] To obtain large-scale human liver organoids, we further seeded multiple mini-organs (10,000 cells each) prepared in Example 2 into 15 mL centrifuge tubes and cultured them in a shaker using mature culture medium HIM, according to the required macro-organ cell quantity. The experimental results are as follows. Figure 9 As shown, Figure 9 Image a is taken with an upright microscope. Figure 9 Image b in the middle is taken with a stereoscope.

[0117] The results showed that multiple mini-organs began to come into contact and fuse with each other on the first day, and formed a large-scale macro-organ on the fourth day.

[0118] (2) Macro-organ mesenteric transplantation

[0119] Macro-organ was transplanted into the mesentery of severely immunodeficient mice (NSG, purchased from Vital River), and changes in macro-organ in vivo at different time points were observed. The results are as follows: Figure 10 As shown, Figure 10 Image 'a' in the middle is an image of the exterior taken with a macro-organ camera. Figure 10 Image b shows a macro-organ mesenteric transplantation image in NSG mice. Figure 10 Image c shows the appearance of the macro-organ taken from an NSG mouse one month after transplantation. Figure 10 Image d shows the appearance of the macro-organ taken with a stereoscope one month after transplantation in an NSG mouse.

[0120] The results showed that the macro-organ remained structurally dense in vivo even after one month, and microvessels began to form inside the macro-organ as the transplantation time increased, indicating that the macro-organ can survive in vivo for a long time and could provide a new treatment alternative for the current shortage of liver transplant donors.

[0121] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0122] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a large-sized human liver organoid, characterized in that, Includes the following steps: (1) 2D amplification and induced maturation: Resuscitated human primary hepatocytes were seeded in culture dishes coated with type I collagen. After adherent culture, they were passaged for amplification culture and induced maturation culture to complete the 2D level induced maturation of cells. (2) Formation of three-dimensional organoids: The human primary hepatocytes induced to mature in step (1) were digested and resuspended into a single-cell suspension. Different amounts of human primary hepatocytes were resuspended using hepatocyte maturation medium HIM and then added to a U-shaped bottom 96-well plate. The plate was cultured statically under hypoxia for 2-5 hours and then transferred to normoxic conditions for shaking culture for 7 days to prepare human liver organoids. In step (2), the low-oxygen conditions are: 37℃, 0.5-2% O2, 4.5-5.5% CO2; the normoxic conditions are: 37℃, 18.5-20.9% O2, 4.5-5.5% CO2; and the shaking incubation speed is 50-60 rpm. The U-shaped bottom 96-well plate is a 3DSphaero™ Cell and Tissue Culture Plate, BIOFIL, TCP130096; The components of the hepatocyte maturation culture medium HIM are as follows: Basic DMEM / F-12 medium, N2 supplement, B27 supplement, N-acetylcysteine, recombinant human epidermal growth factor, recombinant human hepatocyte growth factor, human [Leu15]-gastrin-I, A83-01, Rho kinase inhibitor Y-27632, GSK-3β inhibitor CHIR99021, Glutamax, sodium pyruvate, sphingosine 1-phosphate, oleoyl lysophosphatidic acid, LDN193189, penicillin-streptomycin solution, forsocolin, insulin, norepinephrine, dexamethasone.

2. The preparation method according to claim 1, characterized in that, The method further includes step (3), the specific process of which is as follows: based on the demand for human liver organoids of different scales, multiple human liver organoids prepared in step (2) of claim 1 can be mixed and cultured in a shaker to prepare large-scale human liver organoids.

3. The preparation method according to claim 1 or 2, characterized in that, The culture medium for adherent culture in step (1) is as follows: DMEM + 10% FBS + 1% PS; the conditions for adherent culture are as follows: cultured in a hypoxic incubator for 24 hours, with the hypoxic incubator conditions being 37℃, 0.5-2% O2, and 4.5-5.5% CO2.

4. The preparation method according to claim 1 or 2, characterized in that, In step (1), the culture medium for the subculture amplification culture is HM medium, and the amplification culture conditions are 37℃, 0.5-2% O2, and 4.5-5.5% CO2. The components of the HM culture medium are as follows: Basic DMEM / F-12 medium, N2 supplement, B27 supplement, free of vitamin A, N-acetylcysteine, recombinant human epidermal growth factor, recombinant human hepatocyte growth factor, human [Leu15]-gastrin-I, A83-01, Rho kinase inhibitor Y-27632, GSK-3β inhibitor CHIR99021, Glutamax, sodium pyruvate, sphingosine 1-phosphate, oleoyl lysophosphatidic acid, NIBR-LTSi, LDN193189, and penicillin-streptomycin dual antibiotic solution.

5. The preparation method according to claim 1 or 2, characterized in that, In step (1), the culture medium for the induction maturation culture is HIM medium, and the induction maturation culture conditions are 37℃, 0.5-2% O2, and 4.5-5.5% CO2; the induction maturation culture time is 2 days.

6. The preparation method according to claim 1 or 2, characterized in that, In step (2), the number of cells is 10,000 to 1,000,000.

7. The preparation method according to claim 6, characterized in that, The number of cells mentioned in step (2) is 10,000.

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