A method for rapid spheroidization and culture of 3D liver spheroids and its application

Abstract

This invention relates to the field of cell culture technology, specifically to a method for rapidly forming 3D liver spheroids and its applications. The method uses a spheroidizing medium to culture a mixture of primary hepatocytes and non-parenchymal liver cells to obtain dense 3D liver spheroids. The spheroidizing medium includes matrix gel, FBS, antibiotics, GlutaMAX, and basal medium, with the matrix gel concentration ranging from 1 to 100 μg / mL. This method rapidly forms spheroids from primary hepatocytes and non-parenchymal liver cells in 15-24 hours, yielding superior 3D liver spheroids. It has lower requirements for cell viability, shorter spheroidizing time, and higher spheroidizing efficiency. The spheroids more closely resemble physiological sizes, are easily standardized, and allow for high throughput. It can be used to establish various miniature liver models, providing excellent models and treatment strategies for related research and disease treatment.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically, to a 3D liver spheroid, a rapid spheroidization culture method, and its application. Background Technology

[0002] 3D liver spheres (Mini-liver) are created by mixing primary hepatocytes and non-parenchymal liver cells in a certain ratio, seeding them onto specially treated consumables, and then forming them into three-dimensional spheres through certain methods.

[0003] Compared to 2D cell culture, 3D liver spheroid culture allows for longer cell culture and maintenance of differentiation. 3D liver spheroid culture better simulates the liver microenvironment, which is highly correlated with cell growth and signal transduction. It is simple to use and allows for the co-culture of various cell types, reproducing cell-cell communication between hepatocytes and non-parenchymal cells, thus more closely resembling the physiological state of humans or animals. It allows for long-term culture and functional maintenance, while maintaining higher albumin secretion levels and hepatic drug-metabolizing enzyme activity. Furthermore, it enables high-throughput and standardized drug screening.

[0004] Studies have shown that after ex vivo culture, hepatocytes undergo rapid dedifferentiation and lose relevant physiological function indicators within 2-5 days of 2D culture, with the culture time typically not exceeding 2 weeks (see: DOI:10.1016 / j.jviromet.2014.06.015.). However, when hepatocytes are co-cultured with non-parenchymal cells in 2D culture, the in vitro culture time can be significantly extended to 4 months, while maintaining the differentiation state and physiological function of hepatocytes (see: doi.org / 10.1016 / j.jviromet.2013.10.010). Therefore, the shorter the spheroidization time, the better the restoration of intercellular communication between hepatocytes and non-parenchymal cells, and the better the maintenance of hepatocyte differentiation state and physiological function.

[0005] Currently, commonly used 3D culture methods include organ-on-a-chip (OIA) and spherical culture. OIA involves seeding different cell types into different channels of an organ-on-a-chip, enabling communication between these cells using microfluidic technology and porous membranes. This method requires microfluidic equipment, organ-on-a-chip components, and sophisticated detection equipment, making it complex, costly, and unable to achieve high throughput.

[0006] The 3D liver spheroid culture method specifically includes the following methods: (1) Hanging drop spheroidization method: By mixing cells in the appropriate culture medium and inverting the culture, the cells self-assemble into spheres due to the factors of liquid surface tension and gravity. This method has high operational risks and the formation of spheres takes a long time, usually more than 3-5 days.

[0007] (2) Agarose sphere formation method: By constructing cell culture wells using agarose, cells will not adhere to the wells and will assemble into spheres due to gravity. This method is cumbersome and the formation of spheres takes a long time, usually more than 3-5 days.

[0008] (3) Ultra-low adsorption spheroidization method: Using consumables treated with ultra-low adsorption, primary cells are mixed and seeded in culture wells, and the cells self-assemble into spheres under gravity. This method is highly dependent on freshly isolated cells and cell viability, and the time required for spheroid formation is long, usually 3 days (compare with Patent 1: A three-dimensional liver microsphere model for co-culture of primary hepatocytes and its preparation method and application, application number: CN202310390858.2). Hepatocytes undergo significant dedifferentiation and loss of function 2-5 days after ex vivo (see: DOI:10.1016 / j.jviromet.2014.06.015.), which is not conducive to the differentiation state and physiological function maintenance of hepatocytes.

[0009] (4) Nucleic acid glue spheroidization method: Cells are treated separately with nucleic acid glues of different properties, and the cells are assembled into spheres through molecular interactions between the glues. This method involves incubating different types of cells separately, which is cumbersome, time-consuming, and prone to causing a decrease in cell activity during incubation. It also requires high cell viability. More importantly, the nucleic acid glue is a non-liver-derived substance, which may affect the metabolic function of hepatocytes and downstream gene detection (see: Patent: Method and application of constructing 3D liver bud organoids based on framework nucleic acid materials, application number: CN202111030909.8).

[0010] The method for rapid formation of 3D liver spheroids described in this invention patent uses natural liver matrix gel as a bio-glue, without introducing non-liver components. It features low cell viability requirements, fast spheroid formation time (less than 1 day, 15-24 hours), high spheroid formation efficiency (greater than 98%), simple operation, low cost, long-term culture capability, spheroids that are closer to physiological levels, easy standardization, and high throughput. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a 3D liver spheroid, a rapid spheroidization culture method and its application.

[0012] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a rapid spheroidization culture method for 3D liver spheroids. The method involves plating a mixture of primary hepatocytes and non-parenchymal liver cells using a spheroidization culture medium to obtain 3D liver spheroids. The spheroidization culture medium includes a matrix gel, which is one of the following: liver matrix gel, matrixgel, kidney matrix gel, small intestinal matrix gel, heart matrix gel, lung matrix gel, collagen combination group, or synthetic matrix gel. The concentration of the liver matrix gel is 0.1-100 μg / mL.

[0013] Based on the above technical solution, the present invention can be further improved as follows.

[0014] Furthermore, the matrix gel composition includes collagen, laminin, adhesion proteins, and fibrils. Liver matrix gel is the most effective and is the main component of the liver, meaning no exogenous components are added; however, it may be possible to use Matrixgel, renal matrix gel, small intestinal matrix gel, cardiac matrix gel, lung matrix gel, collagen combination groups, synthetic matrix gels, etc., as alternatives in certain areas.

[0015] Furthermore, the ratio of primary hepatocytes to non-parenchymal hepatocytes is 3-9:1.

[0016] Furthermore, the primary hepatocytes are either freshly isolated primary hepatocytes or cryopreserved and revived primary hepatocytes.

[0017] Furthermore, the non-parenchymal liver cells include at least one of Kupffer cells, hepatic sinusoidal endothelial cells, and hepatic stellate cells.

[0018] Furthermore, the viability of both the primary hepatocytes and the non-parenchymal hepatocytes is greater than or equal to 70%.

[0019] Furthermore, the culture method includes the following steps: mixing the primary hepatocytes and the non-parenchymal liver cells, then seeding the mixed cells into each well of an ultra-low adsorption 96U plate at a density of 500-5000 cells / well, supplementing with the spheroidizing medium, and culturing at 37°C and 5% CO2 for 15-24 hours.

[0020] Furthermore, the components of the pelleting culture medium also include basal culture medium, antibiotics, serum or serum substitutes, glutamine and its analogues.

[0021] The present invention also provides a 3D liver spheroid obtained by culturing using the method described above.

[0022] The present invention also provides an application of the above-mentioned 3D liver sphere, which can be used to establish experimental models, including one or more of physiological models, disease models, viral infection models, and drug testing models.

[0023] The beneficial effects of this invention are as follows: (1) The rapid spheroidization culture method for 3D liver spheroids of the present invention uses a spheroidization culture medium containing a very low concentration of matrix gel for plate culture, so that primary hepatocytes and non-parenchymal cells can be rapidly spheroidized, and 3D liver spheroids with dense cell spheres and round appearance can be obtained in 15-24 hours, which reproduces the interaction between hepatocytes and non-parenchymal cells, highly simulates the liver microenvironment, and preserves the differentiation state and physiological function of hepatocytes to the maximum extent. (2) The rapid spheroidization culture method for 3D liver spheroids of the present invention has low requirements for cell viability, wide range of cell sources, high spheroidization efficiency (greater than 98%), simple operation, low cost, can be cultured for a long time, the spheroids are closer to the physiological level, easy to standardize and high throughput; (3) The 3D liver spheres of the present invention have good morphology and can be cultured for a long time for 1-2 months without disintegration. Large molecules such as viruses can be infected normally. The free uptake effect of small nucleic acid drugs is much higher than that of 2D cultured hepatocytes. Inclisiran can be delivered to the center of the sphere, and the drug can function normally. (4) The 3D liver sphere of the present invention can be used to establish various experimental models, including physiological models, viral infection models, fatty liver models, liver fibrosis models, drug metabolism models, drug toxicity models, and drug efficacy evaluation models, providing a good model basis for related research and disease treatment. Attached Figure Description

[0024] Figure 1 This is a microscope image taken after 24 hours of cultivation in Example 1 of the present invention. Figure 1 Image A represents a 3D spherical liver containing liver matrix gel. Figure 1 Image B shows a 3D spherical liver shape without liver matrix gel. Figure 2 These are microscope images taken under various viability conditions in Example 2 of the present invention. Figure 2 Image A represents a 3D liver spheroid resulting from a co-culture of human hepatocytes with a viability of 61.5% and human non-parenchymal cells with a viability of 70%. Figure 2 Image B represents a 3D liver spheroid resulting from a co-culture of human hepatocytes with a viability of 70.1% and human non-parenchymal cells with a viability of 70%. Figure 2 C represents a 3D liver spheroid resulting from a co-culture of human hepatocytes with 82.3% viability and human non-parenchymal cells with 70% viability. Figure 2 The image in section D represents a 3D liver spheroid resulting from a co-culture of human hepatocytes with a viability of 92.4% and human non-parenchymal cells with a viability of 70%. Figure 3 This is a 3D microscopic image of a liver spherical after culturing with liver matrix gel at various concentrations, as shown in Example 3 of the present invention. Figure 4This is a 3D microscopic image of a liver spherical body after culturing various primary cell sources, as shown in Example 4 of the present invention. Figure 5 This is a 3D microscopic image of a liver spherical shape after culturing various combinations of non-parenchymal cells in Example 5 of the present invention. Figure 6 This is a 3D microscopic image of a liver spherical after culturing hepatocytes of different species, as shown in Example 6 of the present invention. Figure 7 In Embodiment 7 of the present invention, the infection of lentivirus on 3D liver spheres is shown. The left image is a fluorescence channel diagram, and the right image is an overlay of fluorescence and white light. Figure 8 This is a phase-contrast microscope image of a 3D liver spherical physiological model cultured for different number of days, as shown in Example 8 of the present invention. Figure 9 The image shows the immunofluorescence of the cellular composition of the 3D liver spheroid physiological model in Example 8 of the present invention. The cell spheres are composed of primary hepatocytes that specifically express albumin, Kupffer cells that specifically express CD68, hepatic sinusoidal endothelial cells that specifically express LYVE-1, and hepatic stellate cells that express Desimin. Figure 10 These are the experimental results of the 3D spherical liver physiological model in Example 8 of the present invention. Figure 10 A represents albumin secretion. Figure 10 B represents urea release. Figure 10 C represents the NTCP receptor mRNA level; Figure 11 This is an image of HE staining and Oil Red O staining of a 3D liver spherical physiological model in Embodiment 8 of the present invention. Figure 11 Image A in the image is an HE staining image. Figure 11 In the middle, B is the Oil Red O staining image; Figure 12 The staining images of the 3D liver spherical non-alcoholic fatty liver model in Example 8 of the present invention are, in order, HE staining, Oil Red staining, and Nile Red immunofluorescence. Figure 13 This is a Sirius staining image of a 3D liver spherical liver fibrosis model in Embodiment 8 of the present invention; Figure 14 This is a fluorescence image of the free uptake of the small nucleic acid drug Incrisiran in Example 9 of the present invention. Figure 14 In section A, 2D primary hepatocytes can freely take up their own cells. Figure 14 In the middle section, B represents the free uptake of 3D liver spheroids; Figure 15 This is a laser confocal scanning result of the free uptake of the small nucleic acid drug Incrisiran in Example 9 of the present invention; Figure 16This is a comparison chart of the effect of the small nucleic acid drug Incrisiran in knocking down PSCK9 in Example 9 of the present invention; Figure 17 In Example 9 of the present invention, the effect of Incrisiran drug treatment on lipid droplet formation is described. Figure 17 In the middle section, A represents Oil Red O staining without Incrisiran. Figure 17 In the middle B, 10 pmol of Incrisiran was stained with Oil Red O. Figure 18 This is a comparison of positive drug induction levels in Example 10 of the present invention; Figure 19 In Embodiment 11 of the present invention, Figure 19 Drug A is a hepatotoxic drug. Figure 19 B is a positive drug with no liver toxicity. Detailed Implementation

[0025] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0026] The present invention discloses a rapid spheroidization culture method for 3D liver spheroids, which uses a spheroidization culture medium to plate a mixture of primary hepatocytes and non-parenchymal liver cells to obtain 3D liver spheroids. The primary hepatocyte spheroidization culture medium includes a matrix gel, which is one of liver matrix gel, matrixgel, kidney matrix gel, small intestinal matrix gel, heart matrix gel, lung matrix gel, collagen combination group, and artificial synthetic matrix gel; the concentration of the matrix gel is 40-80 μg / mL.

[0027] The rapid spheroidization culture method for 3D liver spheroids of the present invention uses a culture medium containing a very low concentration of matrix gel for plate culture, which enables primary hepatocytes and non-parenchymal liver cells to rapidly form spheroids, resulting in 3D liver spheroids with good performance, making them suitable for establishing various models.

[0028] Preferably, the matrix gel is liver matrix gel.

[0029] Liver matrix gel is a type of extracellular matrix (ECM) hydrogel, whose specific components include collagen, laminin, adhesion proteins, fibrils, and other extracellular matrix components. It is prepared through gentle decellularization techniques, grinding, and digestion. Its core function is to simulate the natural basement membrane environment of cells in vitro, helping liver cells to quickly adhere to the culture vessel and maintain liver cell differentiation and function during the culture process.

[0030] Since liver matrix gel is derived from the liver, its biological components are closer to those of the organism itself, meaning there is no introduction of exogenous components. Therefore, using liver matrix gel for culture yields the best results and is more conducive to cell aggregation into spheres.

[0031] This invention utilizes the cross-linking properties of liver matrix gel hydrogel. By controlling the amount added, it enables rapid cell connection, achieving effective cell spheroidization culture. The operation is simple, low-cost, highly efficient, and produces good spheroidization results.

[0032] Preferably, the ratio of primary hepatocytes to non-parenchymal hepatocytes is 3-9:1, which is closer to the actual situation of the organism and the experimental requirements.

[0033] Preferably, the primary hepatocytes are one of the following: freshly isolated primary hepatocytes, cryopreserved and revived primary hepatocytes, or cultured and digested primary hepatocytes; the culture method of the present invention is widely applicable and can be used to culture primary hepatocytes in any state.

[0034] Preferably, the primary hepatocytes can be derived from humans, monkeys, pigs, cattle, sheep, donkeys, dogs, cats, rabbits, rats, mice, chickens, ducks, geese, tree shrews, and fish, making the culture method of the present invention widely applicable.

[0035] Preferably, the non-parenchymal cells of the liver include at least one of Kupffer cells, hepatic sinusoidal endothelial cells, and hepatic stellate cells.

[0036] Preferably, the viability of both primary hepatocytes and non-parenchymal liver cells is greater than or equal to 75%. The spheroidization culture method of the present invention has low requirements for the viability of primary hepatocytes and non-parenchymal liver cells, thus making it easier to culture 3D liver spheroids.

[0037] Preferably, the culture method includes the following steps: mixing primary hepatocytes and non-parenchymal liver cells, then seeding the mixed cells into each well of an ultra-low adsorption 96U plate at a density of 1000-3000 cells / well, supplementing with primary hepatocyte spheroidization medium, and culturing at 37°C and CO2 for 18-24 hours; the above-mentioned low-density cell seeding method can better cooperate with the addition of matrix gel and effectively improve the culture effect of 3D liver spheroids.

[0038] Preferably, the primary hepatocyte spheroidizing culture medium also includes basal culture medium, antibiotics, serum or serum substitutes, glutamine and its analogues.

[0039] Further preferably, the composition and mass percentage of the pelleting medium are as follows: 0.1%-20% FBS or serum substitute or platelet lysate, 100 U / mL penicillin-100µg / mL streptomycin, 0.1%-2% GlutaMAX or Glutamin, 0.1-100μg / mL matrix gel, with the remainder being basal medium. The basal medium is one of RPMI-1640, DMEM-F12, William's E, DMEM, MEM, or Leibovitz's L-15 Medium.

[0040] Antibiotics can also be administered in various conventional regimens, such as 50 µg / mL gentamicin, 0.25 - 2.5 µg / mL amphotericin B, Primocin, etc.

[0041] The mass percentages of each component are as follows: 1%-10% FBS, 1x penicillin-streptomycin, 1x glutamine, 10-100 μg / mL matrix gel, and the remainder is basal culture medium; the basal culture medium is one of 1640, DMEM-F12, or William's E.

[0042] The 3D liver spheroids of the present invention are cultured using the method described above. These 3D liver spheroids have good morphology, do not disintegrate during long-term culture, can be normally infected by viruses, and have a much higher free uptake effect of small nucleic acid drugs (Inclisiran positive drugs) than 2D cultured hepatocytes. Furthermore, Inclisiran can be delivered to the center of the spheroid, allowing the drug to function normally.

[0043] The 3D liver sphere of this invention can be used to establish experimental models, including one or more of physiological models, disease models, viral infection models, small nucleic acid drug testing models, and drug testing models, providing a good model foundation for related research.

[0044] The following examples illustrate the concepts. Unless otherwise specified, the actual equipment and instruments used in each example are conventional selections.

[0045] Example 1: Liver matrix colloid as a key adhesive to promote cell spheroidization Human primary hepatocytes were resuscitated using resuscitation medium (LV-Rec001), and then resuspended in PHH plating medium (LV-WEP001). Cell viability and total number of viable cells were determined by trypan blue staining.

[0046] Human liver non-parenchymal cells were resuscitated using complete RPMI 1640 medium (LV-RPMI001), and the cells were resuspended in complete RPMI 1640 medium. Cell viability and total number of viable cells were determined by trypan blue staining.

[0047] PHH plating medium containing 50 μg / mL liver matrix gel (LV-6833-10mL) was prepared. The liver matrix gel in this example contains extracellular matrix components such as collagen, laminin, adhesion proteins, and fibrils. The resuscitation medium, PHH plating medium, RPMI 1640 medium, and liver matrix gel were commercially available from Livo Biotechnology, with catalog numbers LV-Rec001, LV-RPMI001, LV-WEP001, and LV-6833-10mL, respectively.

[0048] Human hepatocytes and non-parenchymal liver cells were mixed at a ratio of 2400 cells to 600 cells and seeded into 96U ultra-low adsorption plates (purchased from Livo Biotechnology, LV-ULA002-96UW, or from Corning, catalog number: 7007). The plates were supplemented with either liver-containing or matrix gel-free plating medium, 100 μL per well. The 96U plates were incubated at 37°C in a 5% CO2 cell culture incubator for 24 h and observed under a phase-contrast microscope.

[0049] Testing showed that the viability of human hepatocytes was 84.77%, and the viability of non-parenchymal liver cells was 90%. Spherical formation was achieved after 24 hours of culture in ultra-low adsorption 96U culture plates with either a final concentration of 50 μg / mL liver matrix gel or without liver matrix gel. The 3D spherical morphology of the liver after 24 hours of culture is as follows. Figure 1 As shown in the figure, Figure A shows the morphology with matrix gel, and Figure B shows the morphology without matrix gel. It is known that 50 μg / mL liver matrix gel can rapidly separate hepatocytes from non-parenchymal cells. Example 2 Cell viability condition test Human primary hepatocytes were resuscitated using resuscitation medium (LV-Rec001), and the cells were resuspended in PHH plating medium (LV-WEP001). Cell viability and total number of viable cells were determined by trypan blue staining.

[0050] Human liver non-parenchymal cells were resuscitated using complete RPMI 1640 medium (LV-RPMI001), and the cells were resuspended in complete RPMI 1640 medium. Cell viability and total number of viable cells were determined by trypan blue staining.

[0051] Human hepatocytes with varying viability and non-parenchymal liver cells were mixed in different combinations at a ratio of 2400 cells to 600 cells per group and seeded into 96-well ultra-low adsorption plates. Each well was supplemented with 50 μg / mL liver matrix gel medium to a final volume of 100 μL. The 96-well plates were incubated at 37°C in a 5% CO2 cell culture incubator for 24 hours and observed under a phase-contrast microscope.

[0052] The cell viability of each group in this embodiment is shown in Table 1, and the morphology under a microscope is shown in Table 1. Figure 2 As shown.

[0053] Table 1. Viability of each cell type before and after purification The above experimental results show that in the 3D liver spheroidization method of the present invention, spheroidization is achieved when the viability of hepatocytes and non-parenchymal liver cells is higher than 70%, while groups with a viability of lower than 70% exhibit severe cell scattering. From the perspective of the refractive index of the cell spheres, a lower refractive index indicates that the cell spheres are compact, indicating a better spheroidization effect; preferably, a viability of 80% or higher, and more preferably 90% or higher.

[0054] Example 3: Test of matrix adhesive concentration and shortest ball formation time Human primary hepatocytes were resuscitated using resuscitation medium (LV-Rec001), centrifuged, and purified using live cell purification medium. Cell viability and total number of live cells were determined by trypan blue staining.

[0055] Human liver non-parenchymal cells were resuscitated using complete RPMI 1640 medium (LV-RPMI001), and the cells were resuspended in complete RPMI 1640 medium. Cell viability and total number of viable cells were determined by trypan blue staining.

[0056] Prepare PHH plate culture media containing 0 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL of liver matrix gel (LV-6833-10 mL).

[0057] Human hepatocytes and non-parenchymal liver cells were mixed at a ratio of 2400 cells to 600 cells and seeded into 96U ultra-low adsorption plates. The medium containing liver matrix gel was added to each well to a final volume of 100 μL. The 96U plates were incubated at 37°C in a CO2 cell culture incubator for 15–24 h and observed under a phase-contrast microscope.

[0058] The culture results using the above-mentioned concentrations of liver matrix gel are as follows: Figure 3 As shown, according to Figure 3 The 3D spherical morphology shows that, except for the 0 μg / mL and 0.1 μg / mL groups, all concentrations can form spherical bodies after 24 hours of incubation, but the optimal concentration is 40-60 μg / mL, more preferably 50 μg / mL, with the fastest spherical formation time being 15 hours. When the liver matrix gel concentration is below 1 μg / mL, spherical bodies cannot form rapidly after 24 hours; when the matrix gel concentration is above 100 μg / mL, the matrix gel will solidify at 37°C, preventing spherical formation.

[0059] Example 4 Primary cell source test Freshly isolated cell group: Freshly isolated primary monkey hepatocytes and non-parenchymal liver cells were mixed at a ratio of 2400 cells to 600 cells, supplemented with PMonH plating medium (LV-WEP007) containing 50 μg / mL liver matrix gel, and seeded into ultra-low adsorption 96U culture plates. The 96U culture plates were placed in a 37℃, 5% CO2 cell culture incubator for 15-24 h and observed under a phase contrast microscope.

[0060] Cryopreserved cell isolation group: Cryopreserved primary monkey hepatocytes were thawed using resuscitation medium (LV-Rec001), centrifuged, and purified using live cell purification medium. Cell viability and total number of viable cells were determined by trypan blue staining. Cryopreserved non-parenchymal hepatocytes were thawed using complete RPMI 1640 medium (LV-RPMI001), resuspended in complete RPMI 1640 medium, and cell viability and total number of viable cells were determined by trypan blue staining. The above hepatocytes and non-parenchymal hepatocytes were mixed at a ratio of 2400 cells to 600 cells and seeded into 96-well ultra-low adsorption plates. PMonH plating medium containing 50 μg / mL liver matrix gel was added to each well to a final volume of 100 μL. The 96-well plates were incubated at 37°C in a 5% CO2 cell culture incubator for 15–24 h and observed under a phase-contrast microscope.

[0061] Cell resuscitation group: Primary monkey hepatocytes were resuscitated and cultured for 2-3 days. Cells were then digested with trypsin, and cell viability and total viable cell count were determined by trypsin blue staining. Non-parenchymal hepatocytes were also resuscitated and cultured for 2-3 days. Cell viability and total viable cell count were determined by trypsin blue staining. The resuscitated hepatocytes and non-parenchymal hepatocytes were mixed at a ratio of 2400 cells:600 cells and seeded into 96-well ultra-low adsorption plates. PMonH plating medium containing 50 μg / mL liver matrix gel was added to each well to a final volume of 100 μL. The 96-well plates were incubated at 37°C in a 5% CO2 cell culture incubator for 15-24 hours and observed under a phase-contrast microscope.

[0062] The 3D liver spheroids cultured from the different cell sources mentioned above are as follows: Figure 4 As shown. According to Figure 4 It can be seen that freshly isolated cells, frozen and then revived cells, and cells that have been cultured for a period of time after revival and then digested, can all form spheres within 24 hours as long as the viability rate is up to standard (above 70%).

[0063] Example 5: Hepatic nonparenchymal cell type and combination test Human primary hepatocytes were resuscitated using resuscitation medium (LV-Rec001), centrifuged, and purified using live cell purification medium. Cell viability and total number of live cells were determined by trypan blue staining.

[0064] Human liver non-parenchymal cells, namely KC, LSEC, and HSC cells, were resuscitated using complete RPMI 1640 medium (LV-RPMI001). Cells were then resuspended in complete RPMI 1640 medium, and cell viability and total number of viable cells were determined by trypan blue staining.

[0065] The above-mentioned hepatocytes and non-parenchymal liver cells were mixed at a ratio of 2400 cells to 600 cells. The specific number of non-parenchymal liver cells in each experimental group was as follows: Experimental group 1: KC 600 cell; Second experimental group: LSEC 600 cells; Third experimental group: HSC 600 cells; Fourth experimental group: KC 300 cells, LSEC 600 cells; Fifth experimental group: KC 300 cells, HSC 600 cells; Experimental group 6: LSEC 600 cell, HSC 600 cell.

[0066] Hepatocytes and non-parenchymal liver cells from each experimental group were seeded into 96U ultra-low adsorption plates, and PMonH plating medium containing 50 μg / mL liver matrix gel was added to each well to 100 μL. The 96U culture plates were incubated at 37℃ in a 5% CO2 cell culture incubator for 15–24 h and observed under a phase-contrast microscope.

[0067] The 3D spherical morphology of the livers after culture in each experimental group is as follows: Figure 5 As shown. According to Figure 5 It can be seen that 2400 hepatocytes mixed with various types of non-parenchymal liver cells, whether using any one type of non-parenchymal liver cell, a mixture of two types of non-parenchymal liver cells, or a mixture of the above-mentioned non-parenchymal liver cells, can form spheres after culturing for 15-24 hours using this sphere-forming method.

[0068] Example 6: Hepatocyte-Nonparanoid Cell Spheroidization Test of Different Species Human, monkey, pig, cattle, sheep, donkey, dog, cat, rabbit, rat, mouse, chicken, duck, goose, tree shrew, and fish primary hepatocytes were revived using resuscitation medium (LV-Rec001). After centrifugation, the cells were purified using live cell purification medium, and cell viability and total number of live cells were counted by trypan blue staining.

[0069] Liver cells from humans, monkeys, pigs, cattle, sheep, donkeys, dogs, cats, rabbits, rats, mice, chickens, ducks, geese, tree shrews, and fish were resuscitated using complete RPMI 1640 medium (LV-RPMI001). Cells were then resuspended in complete RPMI 1640 medium, and cell viability and total number of viable cells were determined by trypan blue staining.

[0070] The aforementioned hepatocytes and non-parenchymal liver cells were mixed at a ratio of 2400 cells to 600 cells and seeded into 96U ultra-low adsorption plates. The appropriate plating medium containing 50 μg / mL liver matrix gel was added to each well to a final volume of 100 μL. The 96U plates were incubated at 37°C in a 5% CO2 cell culture incubator for 15–24 h and observed under a phase-contrast microscope.

[0071] The 3D spherical morphology of the liver obtained after culture in each experimental group is as follows: Figure 6 As shown, according to Figure 6 It can be seen that hepatocytes and non-parenchymal liver cells of different animals can be spherized within 15-24 hours according to the spherization method of the present invention.

[0072] Example 7 Lentiviral Infection Test To answer whether viral particles can enter the center of a 3D liver spheroid, we used lentiviruses (90-120 nanometers in size), a common vector in gene therapy, for research and testing.

[0073] Resuscitate human primary hepatocytes and non-parenchymal liver cells, and spheroidize them according to the method of this invention.

[0074] Lentiviral virus with a green fluorescent tag (purchased from GKG) was used to infect 3D liver spheroids. Fluorescence was observed after 3 days, and the results were as follows: Figure 7 As shown in the image. The left image is the fluorescence channel map, and the right image is the fluorescence and white light overlay map. The results show that lentivirus can normally infect the center of the 3D liver spheroid. Theoretically, viruses smaller than lentivirus, such as hepatitis B virus (42 nm) and adeno-associated virus (20 nm), can successfully infect the center of the spheroid.

[0075] Example 8: Construction and testing of normal physiological models and disease models (fatty liver, liver fibrosis) of 3D liver spheroids. Resuscitate human primary hepatocytes and non-parenchymal liver cells, and spheroidize them according to the method of this invention.

[0076] Normal physiological model group: After spheroidization, 3D liver spheroids were cultured normally using spheroid culture medium. Subsequent procedures included photography, pathological sectioning, HE staining, Oil Red O staining, immunofluorescence, ALB detection, urea detection, and NTCP expression detection. Results are as follows: Figure 8 and Figure 9 , Figure 10 , Figure 11As shown.

[0077] according to Figure 8 , Figure 9 , Figure 10 , Figure 11 As can be seen, the 3D liver spheroids in the physiological model have normal morphology and show no disintegration after 42 days of long-term culture. The cell spheroids are composed of primary hepatocytes specifically expressing albumin, Kupffer cells specifically expressing CD68, sinusoidal endothelial cells specifically expressing LYVE-1, and hepatic stellate cells expressing Desimin. HE staining results show that the structure is similar to that of the liver, and no lipid droplets stained brownish-red were observed in Oil Red O staining. ALB protein secretion continuously increased with prolonged culture and was higher than the lowest albumin level in vivo, while the albumin level of 2D cultured hepatocytes was low and showed a decreasing trend. Urea release was higher than the lowest uric acid level in vivo, while that of 2D cultured hepatocytes was much lower than that of 3D liver spheroids. NTCP expression was stable and closer to physiological levels, while NTCP decreased rapidly under 2D conditions. In conclusion, 3D spheroids can be cultured for a long time, highly simulate the liver's in vivo microenvironment, and maintain the differentiation state and physiological function of hepatocytes.

[0078] In the liver disease model group, after spheroidization, different types of liver disease models were induced by adding high concentrations of glucose, oleic acid, linoleic acid, or TGF-β. After 10 days, pathological sections were prepared, and HE staining, Oil Red O staining, and Sirius Red staining were performed to detect the fat and collagen content in the 3D liver spheroids. The results are as follows: Figure 11 , 12 As shown in Figure 13.

[0079] according to Figure 11 and Figure 12 As can be seen, the 3D liver of the non-alcoholic fatty liver model has a normal spherical morphology and does not disintegrate after long-term culture. HE staining results show fatty degeneration and lipid accumulation. Oil Red O staining shows a large number of lipid droplets stained brownish-red, indicating that the model was successfully constructed.

[0080] according to Figure 13 As can be seen, the 3D liver spherical morphology of the liver fibrosis model is normal. Sirius red staining results show that a large amount of red collagen is aggregated on the outside of the cell spheres, indicating that the fibrosis model was successfully constructed.

[0081] Example 9: Small Nucleic Acid Drug Free Uptake Test Human primary hepatocytes were resuscitated, plated, and cultured for 2 days in medium containing 0, 2.76 pmol, 5.53 pmol, and 10 pmol of inclisiran (green fluorescent, purchased from Suzhou Beixin Biotechnology Co., Ltd.). Fluorescence imaging was performed after 3 days, and the results are as follows: Figure 14 As shown in Figure A.

[0082] Human primary hepatocytes and non-parenchymal hepatocytes were resuscitated and cultured into 3D spheroids according to the method of this invention. After spheroidization, 0, 2.76 pmol, 5.53 pmol, and 10 pmol of inclisiran (green fluorescence) were added to each group, respectively. Fluorescence photography was performed after 3 days, and the results are as follows. Figure 14 As shown in Figure B. Simultaneously, laser confocal scanning was performed to detect the drug uptake effect, and the results are as follows. Figure 15 As shown.

[0083] Furthermore, the expression levels of the PCSK9 gene, a target gene of Inclisiran, were detected by qPCR. Figure 16 As shown.

[0084] Figure 14-16 The results showed that under 2D conditions, the free uptake of inclisiran by hepatocytes was relatively weak ( Figure 14 Under 3D conditions, hepatocytes exhibit strong fluorescence signal absorption of Inclisiran, and the drug can penetrate to the center of the spheroid. Figure 15 Meanwhile, the levels of Inclisiran knockdown of target genes are stronger. Figure 16 This indicates that 3D liver spheroids facilitate the free uptake and evaluation of small nucleic acid drugs.

[0085] Furthermore, inclisiran reduces the expression of PCSK9 protein (a proprotein convertase, subtilisin, and serine protease), thereby inhibiting lysosomal degradation of LDL receptors, increasing the number of LDL receptors reaching the hepatocyte membrane, and reducing circulating LDL-C (low-density lipoprotein cholesterol) levels. Therefore, inclisiran can alleviate lipid droplet accumulation on hepatocytes. On day 14 of the above tests, Oil Red O was used to stain 0 pmol and 10 pmol cell spheres, and the results are as follows... Figure 17 As shown.

[0086] Example 10 Drug metabolism induction test Group 2D (2D PHH): Human primary hepatocytes were resuscitated and cultured in 2D according to standard procedures.

[0087] 3D liver spheroids (mini-liver): Resuscitated human primary hepatocytes and non-parenchymal liver cells were spheroidized according to the method of this invention.

[0088] Different concentrations of positive control substrates (100 μM Omeprazole, 1 μM CITCO, 50 μM Rifampicin) were added to the 2D and 3D liver spheroid groups, respectively. After 48 hours, the mRNA levels of CYP1A2, CYP2B6, and CYP3A4 genes were detected by qPCR. The results are as follows: Figure 18 As shown.

[0089] Comparative analysis showed that, under 2D conditions, the drug-induced levels of CYP2B6 and CYP3A4 in human primary hepatocytes did not reach more than 6 times the principle requirement for drug-induced M12, while all metabolic enzymes in 3D liver mini-livers met the standards.

[0090] Example 11 Drug Toxicity Evaluation Test Resuscitate human primary hepatocytes and non-parenchymal liver cells, and spheroidize them according to the method of this invention.

[0091] After spheroid formation, the control group cultured spheroids in normal culture medium, while the experimental group cultured spheroids in normal culture medium containing drug concentrations of 1, 10, 100, and 300 Cmax (the highest concentration of the drug that can be achieved in plasma or serum after a single dose). Supernatant was collected after 6 days, and albumin was detected using an ELISA kit (Proteintech: KE00076). The results are as follows: Figure 19 As shown.

[0092] The 3D liver spheroid test successfully detected drug A, which is known to be toxic to the liver, and drug B, which is not toxic to the liver.

[0093] As can be seen from the above embodiments, the method of the present invention does not require the addition of exogenous liver components, has a short spheroidization time (15-24 hours), high spheroidization efficiency, low cell usage, low cell viability requirements, simple operation, low cost, can be cultured for a long time, produces spheroids that are closer to physiological levels, is easy to standardize and has high throughput, and can be effectively applied to physiological research, disease research, viral infection, drug efficacy testing, drug metabolism, drug toxicity testing, etc.

[0094] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for rapid spheroidization and culture of 3D liver spheroids, characterized in that, Primary hepatocytes and a mixture of non-parenchymal liver cells were plated and cultured using a spheroidizing medium to obtain 3D liver spheroids. The primary hepatocyte spheroidizing medium included a matrix gel, which was selected from liver matrix gel, matrixgel, kidney matrix gel, small intestinal matrix gel, heart matrix gel, lung matrix gel, collagen combination group, and synthetic matrix gel. The concentration of the matrix gel was 1-100 μg / mL.

2. The method for rapid spheroidization and culture of 3D liver spheroids according to claim 1, characterized in that, The liver matrix gel is a natural extracellular matrix protein composition extracted from the liver, and its components include collagen, laminin, adhesion protein, fibrin, etc.

3. The rapid spheroidization and culture method for 3D liver spheroids according to claim 1, characterized in that, The ratio of primary hepatocytes to non-parenchymal hepatocytes is 3-9:

1.

4. The rapid spheroidization and culture method for 3D liver spheroids according to claim 3, characterized in that, The primary hepatocytes are one of the following: freshly isolated primary hepatocytes, cryopreserved and revived primary hepatocytes, or cultured and digested primary hepatocytes.

5. The rapid spheroidization and culture method for 3D liver spheroids according to claim 3, characterized in that, The non-parenchymal liver cells include at least one of Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells.

6. The method for rapid spheroidization and culture of 3D liver spheroids according to claim 3, characterized in that, The viability of both the primary hepatocytes and the non-parenchymal liver cells before culture was greater than or equal to 70%.

7. A method for rapid spheroidization and culture of 3D liver spheroids according to any one of claims 1-6, characterized in that, The culture method includes the following steps: mixing the primary hepatocytes and the non-parenchymal liver cells, and then seeding the mixed cells at a density of 500-5000 cells / well in ultra-low adsorption plates, bottles, or dishes. Ultra-low adsorption 96U, 96V, 384U, honeycomb culture plates, etc. are commonly used for standardized culture. After supplementing the spheroidizing medium, the cells are cultured at 37℃ and 5% CO2 for 15-24 hours.

8. The method for rapid spheroidization and culture of 3D liver spheroids according to claim 7, characterized in that, The pelleting medium also includes basal medium, antibiotics, serum or serum substitutes, glutamine and its analogues.

9. A 3D liver sphere, characterized in that, It is cultured using the method described in any one of claims 1-8.

10. An application of the 3D liver sphere as described in claim 9, characterized in that, The 3D liver sphere can be used to establish experimental models, including one or more of physiological models, disease models, drug efficacy testing models, drug metabolism testing models, and drug toxicity testing models, for use in basic research and clinical treatment of various diseases.

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