In-vitro construction method of hepatic lobule with multi-stage vascular cavity and product of hepatic lobule with multi-stage vascular cavity

Abstract

The invention discloses an in-vitro construction method of a hepatic lobule with a multistage vascular cavity and a product of the hepatic lobule. The method comprises the following steps: firstly, designing and manufacturing a culture container with an imitated hepatic lobule structure to prepare co-cultured hepatic cell spheres with a layered structure, and then forming an imitated hepatic lobule structure pattern in hydrogel by taking the culture container loaded with the hepatic cell spheres as a template through a hydrogel patterning technology to obtain the hepatic lobule with a lumen. And finally, sowing vascular endothelial cells to grow and spread in the lumen to prepare the hepatic lobule with the vascular lumen. Through double-path vascularization of'outer vascular endothelial cell budding-channel vascular endothelial cell attachment ', a'hundred-micron-level main vascular cavity-micron-level capillary' multi-level network is formed, the diffusion efficiency of nutrient substances, oxygen and metabolites is remarkably improved, and the defects of cell dispersion and insufficient vascularization in a traditional 3D model are overcome.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering, and in particular relates to an in vitro construction method of a liver lobule with multi-level vascular cavities and a product thereof. Background Art

[0002] The liver is a crucial metabolic organ in the human body, and drug-induced hepatotoxicity is a major challenge in the development of many new drugs. Currently, drug hepatotoxicity assessment experiments are primarily based on 2D hepatocyte culture models and animal experiments. However, 2D culture models lack the in vivo three-dimensional liver microenvironment and cannot accurately predict drug toxic side effects on the human liver. Animal models also exhibit racial differences from humans, and approximately 50% of drugs found to cause liver damage in clinical trials do not cause any liver damage in animal experiments. The development of a 3D liver tissue model will facilitate reliable in vitro assessment of drug hepatotoxicity.

[0003] The liver is a highly vascularized organ, comprising millions of hepatic lobules arranged in an array. The hepatic lobule is the liver's fundamental structural and functional unit. Each lobule is a regular hexagonal prism, approximately 1 mm wide and 2 mm high. It contains a complex structure, including a central vein, radially arranged hepatic plates, and sinusoids between the plates. Sinusoids are capillary spaces located between adjacent plates and play a vital role in the exchange of nutrients and other molecules between hepatocytes and the bloodstream. With the advancement of tissue engineering, 3D bioprinting and patterning techniques have been widely used to construct hepatic lobule tissue in vitro. However, most current studies have only been able to construct hexagonal structures resembling the appearance of hepatic lobules, primarily composed of discrete individual hepatocytes. This not only requires long culture times to promote hepatocyte proliferation, but also results in dispersed hepatocytes within the hydrogel, with hepatocytes and endothelial cells interlaced within the hydrogel material, lacking a substantial vascular lumen. Constructing in vitro hepatic lobule models with both hundred-micrometer-scale vascular lumens and capillary spaces remains a significant challenge. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for constructing a hepatic lobule with multiple vascular cavities in vitro and a product thereof.

[0005] The present invention is implemented as follows. On the one hand, the present invention provides an in vitro method for constructing a hepatic lobule with multi-level vascular cavities, the method comprising:

[0006] Hepatocytes, non-parenchymal hepatocytes, and vascular endothelial cells are seeded into culture containers at different time points for culture to form pre-vascularized co-cultured hepatocyte spheres with a layered structure; the culture container has a liver lobule-like structure and a vascular-like lumen wall;

[0007] The culture container containing the hepatocyte spheroids is transferred into a hydrogel solution, allowing the hydrogel solution to enter the culture container and submerge the hepatocyte spheroids, thereby solidifying the hydrogel. The culture container is then peeled off from the hydrogel to obtain a patterned hydrogel. Simultaneously, the hepatocyte spheroids further proliferate and differentiate in the hydrogel, forming capillary branches.

[0008] A suspension of vascular endothelial cells is added to the patterned hydrogel. Under the action of gravity, the vascular endothelial cells fall into the grooves caused by the imitation blood vessel cavity wall. After culturing for a certain period of time, the vascular endothelial cells are prompted to adhere to and extend on the inner wall of the groove, forming a vascular cavity with an endothelial cell layer; at the same time, the capillary branches are connected with the vascular endothelial cells in the vascular cavity to form a multi-level vascular cavity, and finally a liver lobule with a multi-level vascular cavity is obtained.

[0009] Preferably, the construction process of the hierarchical pre-vascularized co-cultured hepatocyte spheres is:

[0010] The hepatocytes were mixed evenly with the culture medium and seeded into the culture container to form hepatocyte spheres;

[0011] In the culture container, hepatic non-parenchymal cells are continuously seeded to form core-shell cell spheres in which the hepatic non-parenchymal cells encapsulate the hepatocyte spheres;

[0012] In the culture container, vascular endothelial cells are seeded to form pre-vascularized co-cultured hepatocyte spheres with a core-shell sphere outer layer surrounded by vascular endothelial cells.

[0013] Preferably, the seeding cell numbers of hepatic parenchymal cells, hepatic non-parenchymal cells, and vascular endothelial cells in the pre-vascularized co-cultured hepatocyte spheres are (3-12):1:1; the seeding cell numbers of vascular endothelial cells in the pre-vascularized co-cultured hepatocyte spheres and vascular endothelial cells in the vascular cavity are 1:1.

[0014] Preferably, during the construction of the pre-vascularized co-cultured hepatocyte spheres, the culture time of the hepatocyte parenchymal cells is 1 day, the culture time of the hepatocyte non-parenchymal cells is 1 day, and the culture time of the vascular endothelial cells is 1 day; and the culture time after the vascular endothelial cells are added to the patterned hydrogel is 1 day.

[0015] Preferably, the hepatocytes include at least one of liver cell lines, liver progenitor cells, primary hepatocytes, hepatoma cells, embryonic stem cells, embryonic stem cell-derived hepatocytes, HepaRG, HepG2, C3A, Huh7, and hepatocytes derived from iPS cell-induced differentiation.

[0016] Preferably, the liver non-parenchymal cells include at least one of hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, bile duct epithelial cells, fibroblasts, and mesenchymal cells.

[0017] Preferably, the culture container comprises a plurality of units, each unit is composed of a central area and a plurality of sub-areas, the plurality of sub-areas are equally divided radially from the central area, and there is a simulated blood vessel wall between adjacent sub-areas.

[0018] Preferably, the culture container is made of polydimethylsiloxane.

[0019] Preferably, the hydrogel comprises at least one of methacrylated gelatin, alginate, hyaluronic acid, collagen, and fibrinogen, and the hydrogel curing method comprises at least one of photocrosslinking, ionic crosslinking, and enzyme crosslinking.

[0020] On the other hand, the present invention provides a liver lobule model with multi-level vascular cavities, which is constructed using the above method.

[0021] The present invention integrates structural biomimetic, layered cell co-culture, and dynamic vascularization technologies to construct an in vitro model that closely resembles a real liver lobule in terms of function and morphology. First, a culture vessel imitating the liver lobule structure is designed and fabricated to prepare co-cultured hepatocyte spheres with a layered structure. Then, using hydrogel patterning technology, the culture vessel containing the hepatocyte spheres is used as a template to form a hepatocyte lobule-like structural pattern in the hydrogel, resulting in a hepatic lobule with a lumen. Finally, vascular endothelial cells are seeded and grown and spread within the lumen to prepare a hepatic lobule with a vascular lumen. Specifically:

[0022] (1) The present invention seeded hepatocytes, non-parenchymal hepatocytes and vascular endothelial cells in a time-sequential manner to form a layered pre-vascularized hepatocyte sphere with a three-dimensional core-shell structure of "hepatocyte core-non-parenchymal hepatocyte middle layer-vascular endothelial cell outer layer". The tight connection between cells was used to form rich cell-cell and cell-ECM information exchanges to regulate the function of hepatocytes, so that the prepared liver lobules showed enhanced cell viability and albumin secretion function.

[0023] (2) The present invention uses a culture container that mimics the structure of liver lobule, combined with hydrogel patterning technology, to replicate the radial channel network of liver lobule in the gel. Gravity drives vascular endothelial cells to fill the channels and adhere to grow, forming a main blood vessel cavity of hundreds of microns; at the same time, the outer vascular endothelial cells of the pre-vascularized hepatocyte spheres spontaneously extend capillary branches in the hydrogel and anastomose with the main blood vessel cavity. Through the dual-pathway vascularization of "outer vascular endothelial cells sprouting-channel endothelial cells attachment", a multi-level vascular network of "hundreds of microns main blood vessel cavity-micron-level capillaries" is formed. This structure simulates the physiological distribution of hepatic sinusoids, significantly improves the diffusion efficiency of nutrients, oxygen and metabolic products, and solves the defects of cell dispersion and insufficient vascularization in traditional 3D models.

[0024] (3) The open channel design of the multi-level vascular cavity of the present invention allows drugs to be delivered directly to the hepatocyte spheres through the vascular network, achieving controllable diffusion of drug molecules in three-dimensional space, and can more realistically simulate the drug metabolic pathway in the body, making the drug screening results more sensitive and achieving precise control down to single cell spheres.

[0025] (4) The culture time for each stage of the present invention can be controlled within about 1 day. It only takes about 5 days from cell seeding to the construction of mature liver lobule. Compared with the traditional 3D culture model, the culture time is significantly shortened and the efficiency is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is a flow chart of the in vitro liver lobule construction method provided by an embodiment of the present invention.

[0028] Figure 2 Schematic diagram of the PDMS chip structure and the effect diagram of liver cell sphere preparation based on the PDMS chip provided in an embodiment of the present invention; wherein a is the liver lobule structure, b is the PDMS chip, c is the result diagram of liver cell sphere culture after 1 and 3 days at different cell seeding doses, and d is the comparison result of liver cell sphere diameter at different cell seeding doses.

[0029] Figure 3 This is a schematic diagram of a multi-type cell co-culture spheroid with a layered structure cultured on a PDMS chip provided in Example 2 of the present invention and a diagram of experimental results; wherein a is a schematic diagram of a cell spheroid seeded with three types of cells in steps, and b is a diagram of the experimental results of a cell spheroid seeded with three types of cells in steps.

[0030] Figure 4 This is an evaluation of liver function in co-cultured spheroids of different cell types provided in the examples of the present invention. (a) shows the expression of ALB, CK19, and CD31 proteins in the hierarchical pre-vascularized co-cultured hepatocyte spheroids of the present invention and the control group (mixed co-cultured hepatocyte spheroids). (b) shows the quantitative expression of ALB and CK19 in the hierarchical pre-vascularized co-cultured hepatocyte spheroids of the present invention (Structure group) and the control groups (Random group, 2D group, Mono group). Random group: Hepatocyte spheroids prepared by random mixing of cells. 2D group: Hepatocyte flat culture group. Mono group: Single-component hepatocyte spheroid group.

[0031] Figure 5This is a diagram showing the results of preparing a liver lobule with a simulated vascular cavity using the hydrogel patterning technology provided in an embodiment of the present invention; wherein a is the process of peeling the culture container from the hydrogel to obtain the hydrogel pattern, b is the patterned hydrogel structure, and c is the results on the 1st and 4th days after hydrogel imprinting.

[0032] Figure 6 3 is a diagram showing the result of constructing a multi-level vascular cavity hepatic lobule according to an embodiment of the present invention; a is the process of seeding vascular endothelial cells in the channel, b is the result of optical microscopy observation, and c is the result of laser confocal microscopy observation. DETAILED DESCRIPTION

[0033] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0034] It should be noted that:

[0035] In the present invention, unless otherwise specified, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution.

[0036] In the present invention, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0037] In the present invention, unless otherwise specified, percentages (%) or parts refer to percentages by weight or parts by weight relative to the composition.

[0038] In the present invention, unless otherwise specified, the components involved or their preferred components can be combined with each other to form a new technical solution.

[0039] In the present invention, unless otherwise stated, the "range" disclosed in the present invention is in the form of lower limit and upper limit, which can be one or more lower limits, and one or more upper limits respectively.

[0040] In the present invention, unless otherwise specified, each reaction or operation step can be carried out sequentially or in accordance with the order. Preferably, the reaction method herein is carried out sequentially.

[0041] Unless otherwise indicated, the professional and scientific terms used herein are the same as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0042] At least one embodiment provides a liver lobule model with multi-level vascular cavity and an in vitro construction method thereof, see the attached Figure 1 The method comprises:

[0043] Step S1, seeding hepatocytes, non-parenchymal hepatocytes, and vascular endothelial cells into culture containers at different time points for culture, to form pre-vascularized co-cultured hepatocyte spheres with uniform size and spatial stratification of various cell types; the culture container has a liver lobule-like structure and a vascular-like lumen wall;

[0044] Step S2: transferring the culture container containing the hepatocyte spheroids into the hydrogel solution so that the hydrogel solution enters the culture container and submerges the hepatocyte spheroids, thereby solidifying the hydrogel; peeling the culture container from the hydrogel, thereby imprinting a structure in the hydrogel that is opposite to the pattern of the culture container, thereby obtaining a patterned hydrogel;

[0045] At the same time, the hepatocyte spheres further proliferated and differentiated in the hydrogel, forming capillary branches;

[0046] Step S3: Add a suspension of vascular endothelial cells to the patterned hydrogel. Under the action of gravity, the vascular endothelial cells fall into the grooves caused by the imitation blood vessel cavity wall. After culturing for a certain period of time, the vascular endothelial cells are prompted to adhere to and extend on the inner wall of the groove to form a vascular cavity with an endothelial cell layer; at the same time, the capillary branches are connected with the vascular endothelial cells in the vascular cavity to form a multi-level vascular cavity, and finally a functionally mature liver lobule with a multi-level vascular cavity is obtained.

[0047] In a preferred embodiment, the process of constructing the hierarchical pre-vascularized co-cultured hepatocyte spheres in step S1 is:

[0048] The hepatocytes were mixed evenly with the culture medium and seeded into the culture container to form hepatocyte spheres;

[0049] In the culture container, hepatic non-parenchymal cells are continuously seeded to form core-shell cell spheres in which the hepatic non-parenchymal cells encapsulate the hepatocyte spheres;

[0050] In the culture container, vascular endothelial cells are seeded to form pre-vascularized co-cultured hepatocyte spheres with a core-shell sphere outer layer surrounded by vascular endothelial cells.

[0051] For example, the seeding cell numbers of hepatocytes, non-hepatic cells, and vascular endothelial cells in the pre-vascularized co-cultured hepatocyte spheres are (3-12):1:1, preferably 8:1:1.

[0052] For example, in the process of constructing the pre-vascularized co-cultured hepatocyte spheres, the hepatocytes are cultured for 1 day, the hepatocyte non-parenchymal cells are cultured for 1 day, and the vascular endothelial cells are cultured for 1 day.

[0053] For example, the hepatocytes include at least one of liver cell lines, liver progenitor cells, primary hepatocytes, liver cancer cells, embryonic stem cells, embryonic stem cell-derived hepatocytes, HepaRG, HepG2, C3A, Huh7, and hepatocytes derived from iPS cell differentiation.

[0054] For example, the liver non-parenchymal cells include at least one of hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, bile duct epithelial cells, fibroblasts, and mesenchymal cells.

[0055] The culture container includes a plurality of units, each of which is composed of a central area and a plurality of sub-areas, wherein the plurality of sub-areas are divided equally radially from the central area to the surrounding areas, and there is an imitation blood vessel wall between adjacent sub-areas. For example, the culture container can be made of polydimethylsiloxane. The polydimethylsiloxane (PDMS) chip structure is circular in shape and comprises a plurality of regular hexagonal units with a width of 1 mm. The interior of the regular hexagon is composed of a raised circular middle area and 6 recessed triangular sub-areas. The 6 recessed triangular sub-areas are configured to be divided equally radially from the periphery of the circular middle area. There is a raised imitation blood vessel wall between adjacent triangular sub-areas. Adjacent regular hexagonal units can share part of the triangular sub-areas. The PDMS chip is made by casting PDMS polymer on a mold, and the mold can be prepared by traditional photolithography or digital photopolymerization (DLP) printing. The diameter of the chip can be designed according to the hole diameter of the adapted standardized cell culture well plate.

[0056] The microstructure of the triangular sub-regions of the above-mentioned culture container can limit cell migration and encourage liver cells to form cell spheres with uniform diameter within 24 hours, solving the problem of uneven cell sphere size in traditional culture. At the same time, the raised cavity walls between adjacent sub-regions form grooves after hydrogel imprinting, providing physical support for subsequent vascular endothelial cell attachment, avoiding the randomness of the vascular cavity structure in template-free culture.

[0057] In a preferred embodiment, in step S2, a structure opposite to the pattern of the culture container is embossed in the hydrogel. If the culture container uses a PDMS chip, the patterned hydrogel is composed of a regular hexagonal array with a width of 1 mm, each regular hexagon contains 6 triangular protrusion structures loaded with hepatocyte spheres, and there is a circular groove in the center of the regular hexagon. There are channels between adjacent triangular protrusion structures, and each channel points to the circular groove and is connected to it.

[0058] After being peeled off from the culture container, the hydrogel forms a liver lobule-mimicking structure of "central groove-radial channels-triangular liver plate area". A single chip can produce hundreds of uniform units to meet the needs of high-throughput drug screening; the porous properties of the hydrogel allow the diffusion of nutrients and oxygen, while limiting the disordered migration of cells and maintaining the functional division of the liver plate area and the vascular cavity.

[0059] For example, the hydrogel includes at least one of methacrylated gelatin (GelMA), alginate, hyaluronic acid, collagen, and fibrinogen, and the hydrogel curing method includes at least one of photocrosslinking, ionic crosslinking, and enzyme crosslinking.

[0060] In a preferred embodiment, in step S3, the outermost layer of the pre-vascularized hepatocyte spheres is vascular endothelial cells. After the hydrogel patterning operation, the outer layer of vascular endothelial cells of the pre-vascularized hepatocyte spheres grow and sprout to form capillaries, and connect with the vascular endothelial cells in the channels, forming a multi-level vascular cavity of channel vessels and hepatocyte sphere sprouting capillaries, which is conducive to the transmission and diffusion of nutrients, oxygen, and metabolites between the channels and the hepatocyte spheres.

[0061] Because both ends of the channel are open, that is, the inner port of the channel is connected to the circular grooves of each regular hexagon in the patterned hydrogel, during drug screening, the drug can be delivered to the hepatocyte spheres through the multi-level vascular cavity between the channel and the hepatocyte spheres, making the drug screening results more sensitive.

[0062] For example, vascular endothelial cells are added to the patterned hydrogel and cultured for 1 day.

[0063] For example, the seeding cell number of the vascular endothelial cells in the pre-vascularized co-cultured hepatocyte spheres and the vascular endothelial cells in the vascular lumen is 1:1.

[0064] It should be understood that the contents not described in detail in the description of the above preparation method or the obtained product are all common parameters that are easily thought of by those skilled in the art and can be adjusted by those skilled in the art according to actual conditions, so detailed description thereof can be omitted. For the understanding of those skilled in the art, the technical solution of the present invention is further described in detail below in conjunction with specific examples and comparative examples.

[0065] Example 1: Preparation of a PDMS microstructure chip mimicking a liver lobule structure and its application in hepatocyte spheroid culture

[0066] First, the positive mold pattern is drawn using antoCAD software, and the positive mold structure is made on the silicon wafer using standard photolithography technology. The structure opposite to the positive mold is obtained by PDMS casting through soft photolithography technology, namely the PDMS chip ( Figure 2(b)). The specific steps are: mix the A glue and B glue of the PDMS prepolymer in a ratio of 10:1, then cast them on the positive mold and degas, then place them in an 80℃ oven for 2h to cure, and finally peel off the cured PDMS from the positive mold. According to the hole diameter of the standard cell culture plate (48-well plate), the PDMS chip is cut into a circle with a diameter of 10mm, and 75% alcohol immersion-ultraviolet irradiation is used to disinfect the chip. Finally, it is placed in a 48-well plate and F-127 solution (1%, 500uL) is added for surface anti-adsorption treatment. Digest the HepaRG cells that have converged to 70-80% to obtain a cell suspension. According to 3×10 5 cells / chip, 8×10 5 cells / chip, 12×10 5 The cells were seeded at a seeding rate of 10 cells / cell per chip, and then the culture plate was placed in a cell culture incubator for culture. The results were observed under a bright field microscope. Figure 2 c, On the first day of culture, hepatocytes converged and fused in the triangular microstructure area of the chip. As the culture time increased, the hepatocytes gradually formed a cell sphere structure with uniform size and regular morphology ( Figure 2 d) The optimal cell seeding ratio was calculated based on the cell sphere diameter formed by different seeding doses, which occupied 70-80% of the triangular area of the PDMS chip. In this example, 8×10 5 The seeding amount of cells per chip should be determined for subsequent experiments.

[0067] Example 2: Preparation of multi-type cell co-culture spheroids with layered structure

[0068] Based on the PDMS chip designed by the present invention, co-cultured hepatocyte spheres with a spatial layered structure can be prepared by seeding different types of cells step by step. Figure 3 a, the specific steps are as follows: first, according to 8×10 5 The cell seeding amount of HepaRG cell suspension was seeded into the sterile PDMS chip and placed in an incubator for 1 day. After the HepaRG cells formed cell spheres, the hepatic stellate cell (LX-2) suspension obtained by enzymatic digestion was continued to be seeded into the above chip, so that the seeding amount of LX-2 was 1×10 5 Cells / chip were cultured for 1 day to obtain LX-2-coated hepatocyte spheres. Finally, the vascular endothelial cell (HUVEC) suspension obtained by enzymatic digestion was seeded onto the chip to a seeding density of 1×10 5 cells / chip and continue culturing for 1 day to obtain multi-type cell co-culture spheroids with a spatial layered structure. Figure 3 b shows the experimental results of preparing multi-type cell spheres by layered encapsulation at different stages.

[0069] Liver function was assessed in hepatocyte spheroids with a layered structure. Immunofluorescence staining was used to detect the expression of albumin ALB (a marker of liver synthesis function), CK19 protein (a marker of bile ducts), and CD31 protein (a marker of blood vessels) in hepatocyte spheroids. Figure 4 As shown in Figure a, compared with the hepatocyte spheres prepared by random mixing of cells (Random group), the layered hepatocyte spheres (Structure group) of this example have stronger expression of ALB, CK19 and CD31 proteins. The quantitative expression of ALB and CK19 was analyzed by fluorescence quantitative PCR technology. The results are shown in Figure 4. Figure 4 b- Figure 4 c, The lamellar hepatosphere experimental groups showed higher expression levels.

[0070] Example 3: Preparation of patterned hydrogel structures

[0071] Experimental steps such as Figure 5 As shown in Figure a, hepatocyte spheroids from Example 2 were first cultured on a PDMS chip with a liver lobule-like microstructure. This chip was then used as an imprinting mold to imprint the inverse microstructure onto a GelMA hydrogel. Simultaneously, the hepatocyte spheroids were transferred to the corresponding triangular regions within the hydrogel, resulting in a hydrogel structure that mimicked a liver lobule. Finally, vascular endothelial cells were seeded and cultured on this hydrogel structure to obtain liver lobule microtissues with vascular lumens.

[0072] In order to visualize the hydrogel microstructure after imprinting, the hydrogel solution was dyed with calcein dye and then imprinted. The specific steps are: 1) Place a circular chamber with an inner diameter of 11mm in a 6-well cell culture plate; 2) Pipette 120μL of the prepared GelMA solution (containing a photoinitiator LAP concentration of 0.5%) into the circular chamber, and gently shake it to make the GelMA solution evenly spread in the chamber; 3) Place the PDMS chip containing the hepatocyte spheres flat on the surface of the GelMA solution, and ensure that the microstructured side of the PDMS chip is in contact with the hydrogel solution; 4) Use a 405nm light source for irradiation for 40s to cause the GelMA solution to solidify and form; 5) Gently peel off the PDMS chip to obtain a patterned hydrogel structure ( Figure 5 b).

[0073] After obtaining the patterned hydrogel structure, the tissue structure was cultured. The growth of hepatocyte spheres was observed using optical microscopy and laser confocal microscopy on the first and fourth days after imprinting, respectively. Figure 5As shown in c, the bright field image shows that the hepatocyte spheres further proliferate in the hydrogel, and the cell spheres grow outward with microfilament structures. Combined with the actin staining results, the hepatocyte spheres undergo a morphological transformation, and the extended cell microfilaments are colored by actin (green). It can be observed that the hepatocyte spheres gradually fill the triangular area structure.

[0074] Example 4: Preparation of liver lobule with vascular lumen

[0075] After the patterned hydrogel structure was prepared, vascular endothelial cells were seeded and cultured to obtain liver lobules with vascular cavities. Specifically, the endothelial cells obtained by digestion were prepared into a cell suspension at a rate of 1×10 5 The amount of cells was seeded into the hydrogel structure prepared in Example 3. The effect 15 minutes after seeding is shown in the figure. Figure 6 As shown in a, endothelial cells are evenly filled into the channels of the hydrogel structure. After one day of culture, the endothelial cells grow and extend along the inner wall of the channel under optical microscope observation ( Figure 6 b). To visualize vascular endothelial cells and hepatocyte spheroids, cell tracer DiI dye (red) was used to label vascular endothelial cells, and DiO dye (green) was used to label hepatocyte spheroids. After 3 days of culture, liver lobules were imaged using laser confocal microscopy. The results showed that the liver lobule structure formed a vascular lumen with endothelial cells attached, and the hepatocyte spheroids were evenly distributed in the triangular area ( Figure 6 c).

[0076] The conventional techniques in the above embodiments are well known to those skilled in the art and will not be described in detail here. The specific embodiments described herein are merely illustrative of the spirit of the present invention. Those skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope defined by the appended claims.

[0077] Although the present invention has been described in detail and certain specific embodiments have been cited, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

[0078] Although the above specific embodiments have shown, described and pointed out the novel features applied to various embodiments, it should be understood that various omissions, substitutions and changes may be made to the form and details of the described devices or methods without departing from the spirit of the present disclosure. In addition, the various features and methods described above can be used independently of each other or can be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the above embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it should be understood by those skilled in the art that the present invention can extend beyond the specifically disclosed embodiments to other alternative embodiments and / or applications and their obvious modifications and equivalents. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein. Matters not covered in the present invention are all known technologies.

Claims

1. A method for constructing a hepatic lobule with multi-level vascular cavities in vitro, characterized in that: The method comprises: Hepatocytes, non-parenchymal hepatocytes, and vascular endothelial cells are seeded into culture containers at different time points for culture to form pre-vascularized co-cultured hepatocyte spheres with a layered structure; the culture container has a liver lobule-like structure and a vascular-like lumen wall; The culture container containing the hepatocyte spheroids is transferred into a hydrogel solution, allowing the hydrogel solution to enter the culture container and submerge the hepatocyte spheroids, thereby solidifying the hydrogel. The culture container is then peeled off from the hydrogel to obtain a patterned hydrogel. Simultaneously, the hepatocyte spheroids further proliferate and differentiate in the hydrogel, forming capillary branches. A suspension of vascular endothelial cells is added to the patterned hydrogel. Under the action of gravity, the vascular endothelial cells fall into the grooves caused by the imitation blood vessel cavity wall. After culturing for a certain period of time, the vascular endothelial cells are prompted to adhere to and extend on the inner wall of the groove, forming a vascular cavity with an endothelial cell layer; at the same time, the capillary branches are connected with the vascular endothelial cells in the vascular cavity to form a multi-level vascular cavity, and finally a liver lobule with a multi-level vascular cavity is obtained.

2. The method according to claim 1, characterized in that The construction process of the hierarchical pre-vascularized co-cultured hepatocyte spheres is as follows: The hepatocytes were mixed evenly with the culture medium and seeded into the culture container to form hepatocyte spheres; In the culture container, hepatic non-parenchymal cells are continuously seeded to form core-shell cell spheres in which the hepatic non-parenchymal cells encapsulate the hepatocyte spheres; In the culture container, vascular endothelial cells are seeded to form pre-vascularized co-cultured hepatocyte spheres with a core-shell sphere outer layer surrounded by vascular endothelial cells.

3. The method according to claim 2, characterized in that The seeding cell numbers of hepatic parenchymal cells, hepatic non-parenchymal cells, and vascular endothelial cells in the pre-vascularized co-cultured hepatocyte spheres are (3-12):1:1; the seeding cell numbers of vascular endothelial cells in the pre-vascularized co-cultured hepatocyte spheres and vascular endothelial cells in the vascular cavity are 1:

1.

4. The method according to claim 2, characterized in that During the construction of the pre-vascularized co-cultured hepatocyte spheres, the hepatocytes were cultured for 1 day, the non-parenchymal hepatocytes were cultured for 1 day, and the endothelial cells were cultured for 1 day. After the endothelial cells were added to the patterned hydrogel, the culture time was 1 day.

5. The method according to claim 1, characterized in that: The hepatocytes include at least one of liver cell lines, liver progenitor cells, primary hepatocytes, liver cancer cells, embryonic stem cells, embryonic stem cell-derived hepatocytes, HepaRG, HepG2, C3A, Huh7, and hepatocytes derived from iPS cell differentiation.

6. The method according to claim 1, characterized in that The liver non-parenchymal cells include at least one of hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, bile duct epithelial cells, fibroblasts, and mesenchymal cells.

7. The method according to claim 1, characterized in that: The culture container includes multiple units, each unit consists of a central area and multiple sub-areas, the multiple sub-areas are equally divided radially from the central area, and there is a simulated blood vessel wall between adjacent sub-areas.

8. The method according to claim 1 or 7, characterized in that The culture container is made of polydimethylsiloxane.

9. The method according to claim 1, characterized in that: The hydrogel comprises at least one of methacrylated gelatin, alginate, hyaluronic acid, collagen, and fibrinogen, and the hydrogel curing method comprises at least one of photocrosslinking, ion crosslinking, and enzyme crosslinking.

10. A liver lobule model with multi-level vascular cavities, constructed using the method according to any one of claims 1 to 9.

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