Hepatic vasculature

Abstract

The present invention relates to a method for generating liver tissue in a cell culture device. In this method, a gel precursor containing at least fibrinogen, hepatic stellate cells, and hepatic endothelial cells is provided in the cell culture device. The cells can form liver tissue resembling native liver tissue. The liver tissue contains a vascular network including hepatic sinusoids and can further contain bile canaliculi-like structures and fenestrations. Also provided herein is a microfluidic culture device containing the liver tissue.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention generally relates to methods for generating liver tissue in a cell culture device, wherein the liver tissue closely resembles native liver tissue. In some embodiments, methods are provided in which liver tissue is obtained that includes a vasculature that may be perfusable. In other embodiments, methods are provided in which liver tissue is obtained that includes such vasculature and sinusoid-like structures. In further embodiments, methods are provided in which liver tissue is obtained that includes vasculature, sinusoid-like structures, and bile canaliculi-like structures. The present invention further relates to a culture device containing liver tissue obtained by any one of the methods described herein, and uses thereof.

[0002] Background of the Invention The Background Discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] In vivo, blood vessels are vascular structures containing endothelial cells that form the inner cell layer of blood vessels (e.g., arteries, veins, and capillaries); these endothelial cells are often supported by one or more other cell types, such as fibroblasts. The liver is a highly vascularized organ, with capillary-like structures supplying venous blood to hepatocytes. Hepatocytes play key roles in metabolism, detoxification, and protein synthesis, activating innate immunity by, for example, secreting innate immune proteins into the bloodstream. In vivo, these capillary-like structures, referred to as sinusoids, are low-pressure vascular passageways that receive blood from terminal branches of the hepatic artery and portal vein at the periphery of the lobule and deliver it to the central vein. A schematic diagram of the liver structure is shown in Figure 1. Hepatic sinusoids are notoriously leaky, aiding the diffusion of molecules and particles from the blood toward the basolateral aspects of polarized hepatocytes through a thin extracellular matrix layer (also called the space of Disse). The vasculature of the hepatic sinusoids is supported by stellate cells, often referred to as hepatic pericytes, which reside in the space of Disse (also referred to as the perisinusoidal space) between the endothelial cells and hepatocytes. Hepatic stellate cells, which reside in the perisinusoidal space between the sinusoidal endothelial cells and hepatocytes, are thought to store vitamin A and regulate sinusoidal circulation.

[0004] Hepatocytes are polarized cells that play a central role in metabolism, detoxification, and protein synthesis. They also activate innate immunity against invading microorganisms by secreting innate immune proteins. Hepatocytes transport molecules (often metabolites of parent compounds originating from a series of enzymatic reactions) back into the blood through their basolateral side or into the space between other hepatocytes through their apical side. Hepatocytes are polarized cells that form a network of bile canaliculi (BCs) with their apical surface, enabling the secretion and transport of bile. These canaliculi ultimately drain into larger bile ducts, which transport this fluid back into the intestine.

[0005] Hepatic sinusoids therefore contain a complex, leaky endothelial network supported by stellate cells and lined by polarized hepatocytes.

[0006] In the field of liver tissue engineering, hepatocytes, endothelial cells, and stellate cells have been co-cultured in multicellular spheroids (e.g., as shown in WO2022101675A1) or contained in separate regions of microfluidic platforms (Moradi et al., (2020), Acta Biomaterialia, Vol. 116, p67-83, and Jang et al., (2019), Science Translational Medicine, Vol. 11, p1-12).

[0007] Cells, such as human umbilical vein cells (HUVECs) and normal human lung fibroblasts (NHLFs), are known to interact and self-organize into vascular networks when seeded as single cells and cultured in biomimetic scaffolds, for example, in microfluidic channels. The cells interact and rearrange to form complex, perfusable microvasculature systems in a process resembling the embryonic development of vasculature, termed "angiogenesis." This has led, for example, to the formation of angiogenic tumors or organoid systems. However, most of these vasculature systems rely on readily available cell sources (e.g., HUVECs, dermal endothelial cells, lung fibroblasts, mesenchymal stem cells, etc.) that do not exhibit organ-specific phenotypes. Thus, a liver model that closely resembles the native liver, preferably one that mimics the spatial organization and functionality of the native liver as closely as possible, has yet to be provided. To date, such a liver model has not been successfully developed. Therefore, there is a need in the art to develop a more defined and predictive method for in vitro culturing and obtaining liver tissue, in which cell proliferation and differentiation, as well as the spatial organization and / or functionality of different cells, closely mimic the in vivo situation. In light of this, new products, compositions, methods, and uses for improved in vitro and / or ex vivo liver tissue models would be highly desirable, but are not yet readily available. In particular, there is a clear need in the art for reliable, efficient, and reproducible products, compositions, methods, and uses that make it possible to provide such liver tissue models. Therefore, the technical problem underlying the present invention can be seen in the provision of such products, compositions, methods, and uses to meet any of the above-mentioned needs. This technical problem is solved by the embodiments characterized in the claims and hereinafter.

[0008] Summary of the Invention As embodied and broadly described herein, the present invention relates to the surprising finding that by providing a gel precursor containing fibrinogen, hepatic stellate cells, and hepatic endothelial cells to a culture device, allowing the gel precursor to gel into a scaffold, and culturing cells in the scaffold, liver tissue is obtained that closely resembles native liver tissue, e.g., spatially organized cells containing hepatic endothelial cells and hepatic stellate cells, and forms structures resembling liver tissue (e.g., hepatic sinusoids and bile canaliculi). Surprisingly, the cells forming the resulting liver tissue were found to express relevant liver markers (e.g., MRP-2, LYVE-1, PLVAP). The resulting liver tissue also appears metabolically competent, mimicking fatty liver, as indicated, for example, by the accumulation of lipid droplets (e.g., as illustrated in Figure 12B).

[0009] Thus, in one aspect, the present invention provides an in vitro method for obtaining liver tissue, particularly liver tissue that closely mimics native human liver tissue. Using the present invention, cells are provided and cultured in a culture device in such a way that liver tissue containing a vasculature can be formed, including structures that closely resemble hepatic blood vessels or hepatic blood vessel-like structures, such as hepatic sinusoids. Thus, in one aspect, liver tissue containing hepatic sinusoid-like structures is formed. Furthermore, liver tissue containing bile ducts, bile canaliculi, or bile canaliculi-like structures can be formed. Using the present invention, hepatic stellate cells and hepatic endothelial cells enable the formation of liver tissue described herein, i.e., liver tissue containing a spatial arrangement of liver structures that closely mimics native liver tissue. Other cells, preferably cells commonly found in native liver tissue, may also be included in the methods of the present invention, including, but not limited to, hepatocytes, Kupffer cells, and immune cells.

[0010] In a next aspect, the present invention provides liver tissue, preferably including vasculature, obtainable by the methods described and embodied herein.

[0011] In a further aspect, the present invention provides a culture device comprising liver tissue as described and embodied herein. Preferably, the culture device is a microfluidic culture device.

[0012] In a further aspect of the invention, circulating cells can be added to tissues through the vasculature, mimicking the behavior of circulating cells, preferably immune cells or circulating tumor cells, in tissues.

[0013] In a further aspect, the present invention provides a method of introducing cells into liver tissue as embodied herein, or into a culture device (containing such liver tissue) as disclosed herein, which comprises adding cells to the resulting liver tissue, preferably by adding the cells to the top of the culture chamber, under conditions that allow the cells to attach to the tissue, migrate, and optionally differentiate or specialize.

[0014] In a final aspect, the present invention provides the use of the methods, liver tissue and / or culture devices as described and embodied herein. [Brief explanation of the drawings]

[0015] Embodiments of the present invention are further described hereinafter with reference to the accompanying drawings as follows: [Figure 1] Figure 1: Schematic diagram of the layout of a hepatic lobule, including bile ducts (also bile canaliculi), sinusoids, and hepatocytes. [Figure 2]Figure 2: Schematic diagram of a microfluidic system (OrganoPlate Graft®, MIMETAS BV, The Netherlands) (not to scale). The OrganoPlate Graft® comprises a three-channel system with a large central channel (200) (implantation chamber, center) and adjacent lateral perfusion lanes (202) (microfluidic channels) (left, right) with a phase guide (205) between them. The implantation chamber may have an opening (201), possibly a circular opening. This opening can function as a capillary pressure barrier, as described, for example, in WO2017216113 A2. Furthermore, the implantation chamber may be connected to an inlet (206). The lateral perfusion lanes may be connected to reservoirs (203, 204) that can be used to replenish cell culture media, e.g., for cells present in the implantation chamber, with the addition of other cells, e.g., endothelial cells, that can form tubule-like structures in at least the perfusion lanes. [Figure 3] Figure 3: Schematic representation (not to scale) of angiogenesis in a microfluidic system (OrganoPlate Graft®) including a transplantation chamber (300), perfusion lanes (302), and openings (301). [Figure 4] Figure 4: Hepatic stromal cell-derived vascular networks in fibrin. (A) Vascular network formed with 5416 cells / µL. (B) Vascular network containing 8125 cells / µL. (C) Vascular network formed with 12188 cells / µL. Staining included CD31 (green) and DAPI (blue) for αSMA (red). [Figure 5] Figure 5: LDEC (liver-derived endothelial cells) monoculture in fibrin using ECGM-2 medium (A) and fibroblast-conditioned medium (B). When cultured alone, LDEC do not appear to be substantially capable of forming an extended vascular network. This appears to be improved upon the addition of fibroblast-conditioned medium. [Figure 6]Figure 6: Vascular networks formed by different ratios of liver-derived endothelial cells (LDEC) and stellate cells (HSC). (A) LDEC:HSC ratio 1:1, (B) LDEC:HSC ratio 2:1, (C) LDEC:HSC ratio 4:1, (D) LDEC:HSC ratio 20:1. Aprotopine may be added to prevent fibrin degradation. The cell concentrations used were 6500 LDEC / µL and 6500 HSC / µL. The delivered volume was 1.4 µL per chip; therefore, 9100 LDEC / chip and 9100 HSC / chip. [Figure 7] Figure 7: Calcein-AM staining revealed viable cultures after 7 days of culture, revealing the presence of hepatocytes surrounded by vascular structures (A, B) and sinusoid-like structure and dimensions (C-E). In the cultured vascular structures (A, B), compact cell populations with clear hepatocyte identity were evident throughout. In close contact with hepatocytes, blood vessels appeared reminiscent of hepatic sinusoids, with an average diameter of approximately 10 μm (micrometers), consistent with reported dimensions of human hepatic sinusoids (C-E). Calcein-AM was used to visualize viable cells and is a substrate for MRP-2, an apically expressed transporter in mature hepatocytes. The hepatic structures contained polarized hepatocytes, and accumulation of calcein-AM was observed in the (apical) spaces between hepatocytes, indicating the presence of bile canaliculi. This means that using the method according to the invention, hepatocytes are polarized, with the basolateral side facing the formed vasculature and the apical side facing other hepatocytes, reminiscent of in vivo liver lobules. [Figure 8] Figure 8: (A) MRP-2 expression in polarized hepatocytes: Staining shows MRP-2 lining the apical side of hepatocytes. (B) Immunofluorescence staining shows the presence of mature hepatocytes expressing albumin (green) and partially enveloping the vascular network (CD31, red). [Figure 9] Figure 9: Immunofluorescence staining detecting CD31 and albumin expression. (A-B) Presence of mature hepatocytes expressing albumin (green) and partially enveloping a vascular network (CD31, red) in inverted (A) or not inverted (B). [Figure 10] Figure 10: Expression of LYVE-1 and CD31 by endothelial cells. (AB) LYVE-1 (green), a marker for hepatic sinusoidal endothelial cells, was expressed by LDECs forming vascular networks. Plates were inverted (A) or not (B). HUVECs seeded in the lateral perfusion lane (microfluidic channel) (AB) appeared to express less LYVE-1 and more CD31 (red) compared to the LDEC network, again suggesting a hepatic phenotype. Furthermore, inversion of the culture device appears to promote (e.g., positively influence) angiogenesis. [Figure 11] Figure 11: Expression of PLVAP in vasculature. Positive expression of PLVAP, an endothelial cell-specific protein for the formation of fenestrations, was observed. The presence of fenestrations indicates a healthy phenotype for hepatic endothelial cells. Detecting the presence of fenestrations was surprising because they are generally absent in standard in vitro cell culture methods due to the rapid loss of healthy hepatic endothelium. [Figure 12] Figure 12: Induction of fatty liver by addition of free fatty acids in culture: (A) Control (0.5% DMSO) (Actin (yellow), Bodipy (green), Collagen I (red)), (B) 500 μM oleic acid + 500 μM palmitic acid (Actin (yellow), Bodipy (green), Collagen I (red)). The liver cultures provided herein appear metabolically competent and mimic fatty liver, as indicated by the accumulation of lipid droplets (presumably triglycerides) after 24 hours of exposure to free fatty acids. [Figure 13] Figure 13: Addition of resident macrophages (Kupffer cells) in a liver model. Markers for macrophages, CD45 (red) (A, B), and for liver sinusoidal endothelial cells, LYVE-1 (green) (B), were expressed. [Figure 14] Figure 14: Addition of cholangiocytes (bile duct cells) in a liver model. A marker for cholangiocytes, CK19 (red) (A, B), and a marker for liver sinusoidal endothelial cells, CD31 (green) (B), were expressed. [Figure 15] Figure 15: Liver structure including polarized hepatocytes, where accumulation of calcein-AM (a substrate for MRP-2, an apically expressed transporter in mature hepatocytes) was observed in the spaces between hepatocytes, indicating the presence of bile canaliculi, and the presence of polarized hepatocytes. [Figure 16] Figure 16: Addition of undifferentiated monocytes (approximately 30,000 cells from PBMC Stemcell Technologies) on top of the resulting liver tissue (A), which migrate from the extracellular matrix (ECM) / medium interface into the liver tissue, where they aggregate and differentiate to express macrophage markers CD16 (red), nuclei (blue), and actin (white), associated with the liver vasculature, LYVE-1 (green) (B). [Figure 17] Figure 17: Addition of 2,000 Kupffer cells / µL (Lonza) in the gel precursor, co-seeded with the rest of the hepatocytes after 2 weeks of culture (CD16 (white), LYVE-1 (red)). Kupffer cells are observed outside the vasculature and have an irregular morphology typical of macrophages. [Figure 18] Figure 18: FITC-dextran introduced to visualize the perfusability of the microvasculature (light green network). Macrophages took up the dextran molecules (bright green cells). The liver cultures provided herein with macrophages incorporated into the cultures (as shown in Figure 17) were functional because they were able to phagocytose the dextran molecules used to perform the perfusion assay to visualize the perfusable microvascular network. [Figure 19] Figure 19: T cells (Stemcell Technologies) were labeled with a fluorescent cell tracker (Celltracker Orange, Thermo Fisher Scientific), introduced into the vasculature (i.e., on top of the obtained liver tissue) and perfused for 5 days. The image shows T cells (bright, round red cells) within the liver culture. [Figure 20]Figure 20: Perfusion assay of liver triple culture in fibrin (10,000 Science II HHSECs / μL, 3250 Science II HHSTeCs / μL, 20,000 iHEPs / μL) at day 11 of culture. Image A includes Lonza HUVEC tubules in the perfusion channel, while image B does not. The perfusion assay was performed with 150 kDa FITC-dextran added to the upper left perfusion channel of the OrganoPlate Graft® with the resulting liver tissue. From these images, it can be inferred that the addition of endothelial cells (e.g., Lonza HUVECs) is advantageous for perfusing these chips from left to right. Therefore, to generate a perfusable liver vasculature using the method / system of the present invention, an endothelial network within the gel is preferred, forming connections with the adjacent microfluidic channels. [Figure 21] Figure 21: Vascular liver tissue obtained using the methods of the present invention performed using a unidirectional flow microfluidic system, plate, or device (A) or a bidirectional flow system (B). Vascular network (CD31, red) and cell nuclei (blue). With the use of a unidirectional flow microfluidic device, vascular morphology reflecting a more homeostatic phenotype can be visualized with directed vessels, wider diameters, and more pruning. [Figure 22] Figure 22: Phrodo E. coli particles (white) observed within cells in liver cultures, specifically within endothelial cells and macrophages after one day of perfusion of the particles through the vasculature. Phrodo particles are E. coli particles fused with a pH-sensitive dye. Once inside the acidic environment of cells and lysosomes, they are fluorescent. These data demonstrate that phagocytosis of E. coli particles can be specifically observed in the liver cultures provided herein. LSECs are known to have critical scavenging functions in vivo. In contrast to some previous systems, the scavenging functions of both LSECs and macrophages are maintained in these tissues. [Figure 23]Figure 23: mCherry expression (red) after exposure to AAV (AAV9, factor builder) from a blood vessel. The arrow indicates the direction of flow. A gradient is noted from high to low signal following the direction of flow. This image shows the emergence of a gradient in biological response or cellular phenotype across the gel (i.e., liver tissue) following the direction of flow. Without being bound by any theory, this may indicate the existence of a first-pass effect of cells with the fluid, where the first cells along the vascular connection experience a different environment compared to the last cells, providing a direction or method for modeling liver segmentation. Liver segmentation is a well-known process in which hepatocytes have different, and sometimes opposing, functions depending on where they are positioned along the vascular connection between the portal vein and central vein. [Figure 24] Figure 24: FITC-dextran (green) incorporation by macrophages after delivery by the resulting blood vessels. The arrows indicate the direction of dextran flow. A gradient is noted from high to low signal following the direction of flow. Without being bound by any theory, it can be hypothesized that the fact that FITC-dextran is barely absorbed in downstream parts of the tissue may indicate very effective uptake of the particles by the tissue, mimicking the first-pass effect in vivo. If the tissue is dense and metabolically active enough to process the perfusate, more downstream parts of the tissue may be exposed only to secondary metabolites, recapitulating the sequential nature and complexity of liver metabolism more completely than traditional culture systems. [Figure 25] Figure 25: aSMA expression (green, mainly by astrocytes) is endogenously expressed within the culture medium. A gradient of expression is noted from high to low signal following the direction of flow. [Figure 26]Figure 26: Calcein AM (Thermo Fisher) (top, red) and FITC-dextran (bottom, green) are introduced through the microvasculature of the resulting liver tissue from the left and perfused to the right of the image. While dextran is visible and uniformly perfused throughout the vasculature, the calcein signal appears to exhibit a highly significant gradient. Calcein AM is cell-permeable, but when inside cells, it is converted to fluorescent, cell-impermeant calcein. We speculate (and indirectly observed in other examples) that cells have a high capacity to incorporate calcein AM, leading us to demonstrate that it is indeed possible to achieve a first-pass effect in such a system of the present invention, where cells at the end of the network have fewer (or more) specific molecules than those in the vasculature. [Figure 27] Figure 27: Fluorescence image of GFP-expressing HCT-116 colorectal carcinoma (ATCC) cells within liver tissue (A), and an overlay image (B) showing how the GFP-expressing cancer cells are apparently integrated within a healthy cell population. Cancer cells were introduced into the seeding mixture. The cancer cells grew and integrated into healthy, developing liver tissue. [Figure 28] Figure 28: GFP-labeled HCT-116 colorectal cancer cells were introduced into the vasculature as single cells, allowing them to perfuse through the resulting liver tissue. To this end, HCT-116 cells were mixed with culture medium and introduced into the first channel of a unidirectional flow microfluidic plate / device (see Figure 32 for a schematic diagram of an example of such a device and method for inducing unidirectional flow). Tilting the device induces flow, resulting in the cancer cells perfusing into the resulting liver tissue vasculature. Fluorescence microscopy images were taken 5 days after introducing the cells. This figure illustrates that with the use of a unidirectional flow microfluidic plate, it is possible to introduce circulating tumor cells and model metastasis in liver tissue obtained according to the method / system of the present invention. [Figure 29]Figure 29: mCherry-positive cells (primarily hepatocytes) in four replicating chips (i.e., OrganoPlate Graft®) as a result of gene delivery by adeno-associated virus (AAV) exposure. AAV9 was used. The method / system of the present invention enables a useful liver model for testing AAV transduction. [Figure 30] Figure 30: Immunostaining of liver triplicate cultures (3,250 Science II HHSECs / μL, 812 Science II HHSTeCs / μL, 20,000 iCell iHEPs / μL) exposed to 300,000 AAV9 mCherry genome copies / cell / mL for 72 hours on day 4. Cultures were then maintained for another 4 days (until day 11), after which they were fixed and stained for nuclei (blue), albumin (white), and CD31 (green). (A) The red signal shown for AAV+ cells is from the mCherry payload of AAV9 used in this experiment. Cell morphology resembles hepatocytes (round cells) rather than HHStECs and HHSECs (elongated cells). (B) Overlapping mCherry and albumin signals is evident on the second panel. This figure makes it possible to see that primarily hepatocytes are transfected first, followed by stellate cells and, to a lesser extent, endothelial cells, which is consistent with AAV9 manipulation, which should primarily target receptors on hepatocytes. [Figure 31]Figure 31: Confocal imaging of mCherry+ cells at day 11 in liver triple cultures (3,250 Sciencell HHSECs / μL, 812 Sciencell HHSTeCs / μL, 20,000 iCell iHEPs / μL) exposed to 400,000 AAV9 mCherry genome copies / cell / mL for 72 hours on day 4. Transduction efficiency was assessed every 24 hours starting on day 7. Assays were performed on derived liver tissue with (+monocytes) or without (-monocytes) monocytes added on top of the liver explant, as shown in Figure 16. When cultures include resident immune cells, we observe a decrease in transduction efficiency, consistent with known similar effects observed in vivo. In (A), a graph with the number of AAV+ cells over time is shown. In (B), an image showing the fluorescence of mCherry+ cells is provided. [Figure 32] Figure 32: Top-down schematic of an example of a unidirectional flow microfluidic device useful in the methods of the invention. In (A), the fluid flow path (arrows) from a first reservoir flows through a first channel to a second reservoir through a cell culture chamber. In (B), the fluid flow path from a second reservoir flows through a second channel to the first reservoir.

[0016] explanation: definition A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction). The copyright owner has no objection to the copying or reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights whatsoever.

[0017] Various terms relating to the methods, compositions, uses, and other aspects of the present invention are used throughout the specification and claims. These terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein.

[0018] For purposes of the present invention, the following terms are defined below.

[0019] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like. For example, a method for culturing cells includes culturing a plurality of cells (e.g., tens, hundreds, thousands, tens of thousands, hundreds of thousands, millions, or more).

[0020] As used herein, "about" and "approximately," when referring to measurable values, such as amounts, temporal durations, etc., are meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for practicing the disclosed invention. Unless otherwise clear from the context, all numerical values ​​provided herein include the numerical value modified by the term "about."

[0021] As used herein, "and / or" refers to a situation in which one or more of the stated cases may occur alone or in combination with at least one of the stated cases, up to all of the stated cases.

[0022] As used herein, "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ..., etc. As used herein, the term "up to" a particular value means that particular value or less. For example, "up to 5" is understood to be the same as "5 or less," i.e., 5, 4, 3, ..., -10, -11, etc.

[0023] As used herein, "comprising" or "comprise" is to be interpreted as inclusive and open-ended, not exclusive. Specifically, this term and variations thereof mean that the specified features, steps, or components are included. These terms should not be interpreted to exclude the presence of other features, steps, or components. It also encompasses the more restrictive "consisting of."

[0024] As used herein, "prior art" or "methods known to those skilled in the art" refers to situations in which it would be apparent to one of ordinary skill in the art how to carry out the prior art used in the methods of the present invention. Prior art practices in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology, and related fields are well known to those of skill in the art and are discussed in various handbooks and reference works.

[0025] As used herein, "exemplary" or "for example" means "serving as an example, instance, or illustration" and should not be construed as excluding other configurations, including those disclosed herein.

[0026] As used herein, the term "fibrinogen" refers to a protein, particularly a soluble glycoprotein, often approximately 340 kDa in size, also referred to as clotting factor I. Fibrinogen is generally made and secreted by the liver. Fibrinogen is well described in the art and is known to be formed into fibrin by enzymatic conversion, for example, by thrombin-mediated proteolysis (Pieters et al. Res Pract Thromb Haemost. 2019;3:161-172).

[0027] As used herein, "in vivo" refers to events that take place within a subject's body, while "in vitro" and "ex vivo" refer to events that take place outside of a subject's body. For example, an in vitro assay or method includes any assay or method that is performed outside of a subject. An in vitro assay or method includes cell-based assays using live or dead cells. An in vitro assay also includes cell-free assays that do not use intact cells. The terms "in vivo," "in vitro," and "ex vivo" are well known in the art.

[0028] As used herein, the term "perfusable" describes an object that can be perfused, e.g., that allows fluids, such as (but not limited to) aqueous solutions, suspensions, etc., to flow into, over, and / or through the object.

[0029] As used herein, the term "scaffold" refers to a material or a structure formed by a gel precursor, for example, including fibrinogen, that can gel and form a gel suitable for culturing cells. Within the context of the present invention, a scaffold may provide a structure to allow cell adhesion, cell migration, cell delivery and / or retention, delivery and / or retention of biochemical agents, drug diffusion, etc. A scaffold may be bioactive, for example, by interacting with cells. Certain scaffolds may be used to mimic the extracellular matrix of cells, for example, of natural tissues. Scaffolds have been well described in the art and are commonly used in organ and / or tissue engineering (Chan BP et al. Eur Spine J. 2008;17 Suppl 4(Suppl 4):467-479).

[0030] As used herein, the term "vasculature" refers to a structure, at least a portion of which resembles a blood vessel or a blood vessel-like structure, e.g., comprises an aggregate of cells that resembles a blood vessel or a blood vessel-like structure in vivo. Vasculature, as used herein, may be found in tissue provided using the methods of the present invention. Vasculature may include a network of blood vessels and / or blood vessel-like structures in such tissue.

[0031] Detailed Description The present invention is defined herein, particularly in the appended claims. Subject matter not encompassed by the claims does not form a part of the invention as claimed.

[0032] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use, or composition described herein. Embodiments discussed in the context of a method, use, and / or composition of the invention can be used with respect to any other method, use, or composition described herein. Thus, embodiments with respect to one method, use, or composition can also be applied to other methods, uses, and compositions of the invention.

[0033] As embodied and described herein, the present invention is directed to the surprising finding that by providing hepatic stromal cells, preferably hepatic stellate cells and liver-derived endothelial cells, in a culture device, preferably a gel precursor containing fibrinogen (which gel precursor can be gelled), an in vitro liver tissue is obtained that closely resembles in vivo liver tissue, function, and organization. Surprisingly, the inventors discovered that the obtained liver tissue contains a vasculature. In other words, the liver tissue contained structures resembling blood vessels or vasculature-like structures, e.g., vasculature or vascular network. Furthermore, surprisingly, the obtained liver tissue was found to contain structures resembling hepatic sinusoids formed by hepatic stellate cells and liver-derived endothelial cells. The development of a vasculature appears to be strongly correlated with the presence of stellate cells in the culture. Furthermore, stellate cells are believed to positively influence blood vessel formation (ie, angiogenesis), the formation of bile canaliculi (like structures), and / or the formation of fenestrations by cells, preferably liver (derived) endothelial cells.

[0034] It has been found in the methods of the present invention that cells mixed with a fibrinogen-based gel precursor (e.g., that forms a fibrin gel upon gelation in the presence of thrombin) can form a vascular bed and, as disclosed herein, can connect to blood vessels, e.g., blood vessels formed in a microfluidic channel fluidly connected to a culture chamber containing a gel containing a vascular bed. Enabling such angiogenesis and formation by cells mixed with a fibrinogen-based gel precursor or a fibrin-based gel, particularly the de novo formation of a vascular bed in the gel, is an important aspect of the methods of the present invention. In the present invention, the resulting vascular bed is a vascular bed that forms during the culture of the cells in the gel, and not a vascular bed that has been preformed prior to mixing with the gel precursor. In a preferred embodiment, the cells comprised in the gel (forming the scaffold) are mixed with the gel precursor as substantially single cells and / or as small aggregates of cells, for example, 2 to 200 cells, preferably 10 to 100 single cells, or are provided to the gel (forming the scaffold) as substantially single cells and / or as small aggregates of cells, for example, 2 to 200 cells, preferably 10 to 100 single cells. In a particularly preferred embodiment, cells that will form the vasculature during culturing according to the present invention are mixed with the gel precursor as substantially single cells or are provided to the gel (forming the scaffold) as substantially single cells.

[0035] In a preferred embodiment, when the cells contained in the gel (forming the scaffold) are mixed with or provided to the gel (forming the scaffold), the cells are not in a form resembling a vascular bed or vascular structure. In an embodiment of the method of the present invention, the liver tissue obtained using the method of the present invention, or a part thereof, particularly the vasculature, is obtainable during the culturing step defined herein, and it will be understood by those skilled in the art that this preferably does not require the provision of a pre-existing functional tissue structure, such as a pre-formed or isolated vascular bed, pre-formed or isolated blood vessels, or any other typical tissue structure of the liver, such as those disclosed herein. In a preferred embodiment, the cells provided in the culture chamber, i.e., at least hepatic endothelial cells and hepatic stellate cells, have previously been separately prepared, obtained, or processed before being provided to the culture chamber in the gel precursor or to the scaffold. Those skilled in the art will understand that this does not exclude the cells from being first mixed before being provided to the gel precursor or scaffold. Those skilled in the art will understand that in a preferred embodiment, the liver tissue obtained using the method of the present invention is liver tissue formed in vitro. The liver tissue obtained using the method of the present invention is produced during the culturing method of the present invention.

[0036] Furthermore, it has surprisingly been found that the formation of liver tissue strongly benefits from the presence of a gel precursor containing fibrinogen (and / or a gel containing fibrin). For example, a fibrin-based gel matrix or scaffold formed by gelation of a gel precursor containing fibrinogen is a matrix that is highly permissive for the appearance of vasculature in the resulting liver tissue, thereby leading to liver tissue that is significantly similar to native liver tissue. Embedding the cells disclosed herein, preferably in a scaffold and / or gel precursor containing fibrin and / or fibrinogen, has been found to be highly advantageous for angiogenesis.

[0037] In contrast, when using an extracellular matrix that does not contain fibrin or that contains only a limited amount of fibrin (i.e., in which the gel precursor does not contain fibrinogen or that contains only a limited amount of fibrinogen) in the methods of the present invention, it has been found that obtaining stable cultures that can be formed from liver tissue as disclosed herein is difficult, if not impossible. Thus, the present invention is based in part on the intended use of the fibrinogen-based gel precursor / fibrin-based gel disclosed herein. For example, when using a liver extracellular matrix instead of the fibrinogen precursor / fibrin gel disclosed herein (e.g., as described by Willemse et al. (Biomaterials (2022) Volume 284, 121473; doi.org / 10.1016 / j.biomaterials.2022.121473)), it has been found that obtaining stable cultures is difficult, if not impossible, and the liver structure disclosed herein (obtainable using the methods of the present invention using fibrinogen-based gel precursor / fibrin-based gel) cannot be exhibited (data not shown). The liver extracellular matrix described by Willemse is rich in collagen and does not mention the presence of any (minimal) levels of fibrinogen / fibrin. Thus, the present inventors have surprisingly discovered that in the method of the present invention, fibrinogen-based gel precursors / fibrin-based gels provide liver tissue that more closely resembles in vivo liver tissue structure and function, even compared to more stable liver cell cultures and / or liver-based extracellular matrices.

[0038] In further experiments conducted by the present inventors, it was found that scaffolds of Matrigel or BME (with its abundant protein, collagen) resulted in the contraction of stellate cells present in culture. Thus, in some embodiments, collagen in the gel precursor or gel comprises 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1% or less of the total protein of the gel precursor or gel. Thus, in some embodiments, fibronectin in the gel precursor or gel comprises 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1% or less of the total protein of the gel precursor or gel. Thus, in some embodiments, laminin in the gel precursor or gel comprises 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1% or less of the total protein of the gel precursor or gel. In some embodiments, the precursor gel and / or gel does not comprise collagen. In some embodiments, the precursor gel and / or gel does not comprise fibronectin. In some embodiments, the precursor gel and / or gel does not contain laminin. Those skilled in the art will appreciate that in some embodiments, Matrigel or BME (basement membrane extract) may be mixed with fibrinogen / fibrin to obtain a gel precursor or scaffold (gel) in accordance with the present disclosure. For example, the weight ratio of the protein contained in the BME or Matrigel to fibrinogen (or fibrin) may be between 5:1 and 1:5, e.g., about 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5. Based on the present disclosure, those skilled in the art will understand how to provide such a gel precursor / scaffold, thereby enabling the in vitro methods for obtaining liver tissue disclosed herein.

[0039] In addition, it has surprisingly been found that angiogenesis can also occur in the presence of additional cells, such as tumor-derived cells, e.g., HUVECs, stem cells, umbilical cord blood cells, etc. Preferably, the additional cells include (liver) cells, e.g., hepatocytes, Kupffer cells, cholangiocytes, (other) hepatic stromal cells, etc. These additional cells can be provided in the gel precursor and / or in the side tubes / channels of the microfluidic device, preferably, the additional cells, e.g., HUVECs, etc., are provided in the side tubes / channels. Thus, surprisingly, the present invention also provides liver tissue containing additional (liver) cells, e.g., hepatocytes, Kupffer cells, cholangiocytes, etc. This finding means that liver tissue can be obtained that even more closely resembles native liver tissue. For example, it has been found that the addition of additional liver cells, e.g., hepatocytes, etc., results in the formation of bile canaliculi or bile canaliculus-like structures.

[0040] In addition, it has been surprisingly found that the resulting liver tissue appears to be metabolically competent, meaning that the liver tissue can mimic the metabolism of native liver tissue. For example, it has been surprisingly found that the resulting liver tissue can mimic fatty liver (i.e., excessive fat accumulation by the liver) after exposure to free fatty acids.

[0041] Additionally, it has surprisingly been found that cells added to the tissue, preferably by addition at the top of the tissue culture chamber, can be incorporated into the tissue and differentiated towards more functional cells, for example, PBMCs can be added to the top of the tissue and differentiated towards Kupffer-like cells.

[0042] Thus, in some embodiments, the present invention provides an in vitro method for obtaining liver tissue, wherein the method comprises: a) providing a culture device, wherein the culture device includes at least a culture chamber; b) providing a culture chamber with a gel precursor, wherein the gel precursor comprises at least fibrinogen, and wherein the gel precursor further comprises hepatic endothelial cells and hepatic stellate cells; c) gelling the gel precursor to obtain a scaffold, wherein the scaffold comprises the cells of step (b); d) contacting the scaffold of step (c) with a cell culture medium; and e) culturing the cells in the scaffold to obtain liver tissue, preferably wherein the liver tissue is present in the scaffold.

[0043] Alternatively, in some embodiments, the method comprises: A) providing a culture device, wherein the culture device comprises at least a culture chamber, and wherein the culture chamber comprises a gel precursor comprising fibrinogen, hepatic endothelial cells, and hepatic stellate cells; B) gelling the gel precursor to obtain a scaffold, wherein the scaffold contains the cells of step (A); C) contacting the scaffold of step (B) with a cell culture medium; and D) culturing cells in the scaffold to obtain liver tissue, preferably wherein the liver tissue is present in the scaffold; Includes.

[0044] Alternatively, a culture device may be provided in which a gel precursor is gelled to obtain a scaffold and / or in which fibrinogen has been converted to fibrin, for example by enzymatically converting the fibrinogen, for example by thrombin-mediated proteolysis. In other words, in an alternative embodiment, an in vitro method for obtaining liver tissue is provided, comprising the steps of: AA) providing a culture device, wherein the culture device comprises at least a culture chamber, and wherein the culture chamber comprises a scaffold, preferably a scaffold comprising fibrin and / or fibrinogen, hepatic endothelial cells and hepatic stellate cells; B-B) contacting the scaffold of step (A) with a cell culture medium; and CC) Culturing cells in a scaffold to obtain liver tissue, preferably wherein the liver tissue is present in the scaffold.

[0045] While in the specification and claims, reference is made to the first method described above (e.g., involving steps (a)-(e)), those skilled in the art will understand that any method, use, or composition described herein can similarly be practiced with respect to a method presented using alternative language (e.g., involving steps (A)-(D) or (AA)-(CC)). Those skilled in the art will also understand that when reference is made to cells contained in a gel precursor or contained in a scaffold, these cells may, in some embodiments, be provided via the gel precursor and / or, in some embodiments, be provided in the gel. Those skilled in the art will also understand that in embodiments of the method according to the present invention, certain cells may be initially provided by being contained in the gel precursor, while the same or different types of cells may be provided after the gel precursor has gelled, e.g., during the culture of the cells to obtain liver tissue.

[0046] In the method according to the present invention, in step (a), a culture device is provided that includes a culture chamber (which may also be referred to as an "implantation chamber" or "culture chamber"). The term "culture device" as used herein includes any cell culture vessel known and described in the art. As provided herein, the culture device includes at least a culture chamber, in other words, a vessel, channel, or plate, in which cells can be held and cultured, such as a Petri dish, culture flask, or well. Preferably, the culture chamber provides an (at least partially) sealed chamber for culturing cells. The culture chamber (also referred to as an implantation chamber) may have an opening, preferably a circular opening, which can be used for introducing cells, gels, aqueous solutions, suspensions, and / or dispersions, etc., into the culture chamber. The opening in the culture chamber does not necessarily have to be circular, but may include any shape suitable for providing an (additional) inlet to the implantation chamber. This opening can also function as a capillary pressure barrier, as described, for example, in WO2017216113 A2. The opening can be partially or completely closed by a commonly used method or tool for closing an opening in a culture chamber, such as a film, a lid, etc. Preferably, the opening is located at the top of the culture chamber; however, the opening can also be located on any other side of the culture chamber, e.g., laterally (e.g., on the left or right side), as long as it allows the introduction of cells, gels, aqueous solutions, suspensions, and / or dispersions, etc., into the culture chamber. Multiple openings that allow the introduction of cells, gels, aqueous solutions, suspensions, and / or dispersions, etc., into the culture chamber are also envisioned and encompassed herein. Alternatively, the culture chamber can include one or more channels connected to openings suitable for allowing the introduction of substances. In such embodiments, the culture chamber itself can be closed except for the openings provided via one or more channels.In other embodiments, the culture chamber is accessible through a closable opening in addition to one or more channels, for example, where the opening is located on the upper side of the culture chamber. Various methods for introducing substances into the culture chamber can be combined in a culture device. In the art, systems use standard culture plates and various barrier inserts (e.g., Transwell® permeable supports) in an attempt to culture cells that more closely represent their in vivo characteristics. It is contemplated herein that any culture plate may be used for the methods of the present invention. Alternatively, bioreactors or other devices suitable for culturing cells and including at least a culture chamber, e.g., vessel-like devices, may also be used in the methods of the present invention. However, in a preferred embodiment, the culture device is a microfluidic device that includes at least a culture chamber, preferably including at least one channel fluidly connected to the culture chamber. It has been found that the amount and volume of cells used in the microfluidic devices disclosed herein enable cells to be cultured in a manner that closely resembles native liver tissue.

[0047] Furthermore, in step (b), a gel precursor is provided to the culture chamber, and then in step (c), the gel precursor is gelled (ie, solidified, eg, polymerized) in the culture device.

[0048] Gels and gel precursors for use in tissue engineering, e.g., for providing scaffolds for obtaining tissues or organs, are well known in the art. For example, the gel precursor may be a hydrogel precursor, typically an extracellular matrix (ECM) gel precursor. The ECM may include, for example, collagen, fibronectin, fibrinogen, and / or a basement membrane extract, such as Matrigel or a synthetic gel. As provided herein, the gel precursor includes at least fibrinogen. For example, the gel precursor may be provided (e.g., introduced) into the culture device using a pipette (typically a repeating pipette, e.g., an Eppendorf Multipette® M4 (Eppendorf AG, Germany, catalog number 4982 000.012), in combination with an Eppendorf Combitips advanced® (Eppendorf AG, Germany, catalog number 0030 089.405)). Preferably, the gel precursor comprises at least fibrinogen, wherein preferably the fibrinogen is present in the gel precursor at a concentration sufficient to allow the formation of a fibrin scaffold, for example, between 1 mg / mL and 80 mg / mL, 1 mg / mL and 50 mg / mL, 1 mg / mL and 25 mg / mL, or 1 mg / mL and 10 mg / mL, preferably at a final concentration of 3, 4, 5, 6, 7, 8 mg / mL, more preferably 5 mg / mL, and may further comprise a basement membrane extract, an extracellular matrix component, collagen, collagen I, collagen IV, fibronectin, laminin, vitronectin, D-lysine, entactin, heparan sulfide proteoglycan, or a combination thereof.

[0049] In a preferred embodiment, in the gel precursor, fibrinogen is the most abundant protein (by weight) present in the gel precursor. In an embodiment, in the gel, fibrin is the most abundant protein (by weight) present in the gel material (i.e., excluding any proteins contained in cells contained in the gel / gel precursor).

[0050] In another embodiment, in the gel precursor, fibrinogen constitutes at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the protein in the gel precursor by weight. In an embodiment, in the gel, fibrin constitutes at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more (e.g., 100%) of the protein in the gel material by weight. In some embodiments, the gel precursor comprises a mixture of Matrigel and / or BME and fibrinogen, as described herein. In some embodiments, the gel (scaffold) comprises a mixture of Matrigel and / or BME and fibrin, as discussed herein.

[0051] In embodiments, the gel precursors comprise more fibrinogen (by weight) than collagen. In embodiments, the gel precursors comprise more fibrinogen (by weight) than elastin. In embodiments, the gel precursors comprise more fibrinogen (by weight) than fibronectin. In embodiments, the gel precursors comprise more fibrinogen (by weight) than laminin.

[0052] The gel precursor provided in the method of the present invention is intended to contain at least fibrinogen, and gelling the gel precursor results in obtaining a scaffold containing at least fibrinogen and / or fibrin. Preferably, the scaffold may contain fibrin, such as a fibrin gel or fibrin gel matrix (e.g., obtainable by enzymatically converting fibrinogen). Fibrin-based scaffolds have been found to be beneficial for the formation of vasculature by cells in the method of the present invention. Therefore, it is intended that gel precursors containing fibrinogen will result in liver tissue with improved vasculature compared to methods that do not use fibrinogen (and / or fibrin) in the gel precursor / gel. The use of a fibrin scaffold is further preferred because such a scaffold has a relatively low stiffness (e.g., a shear modulus of less than 150 Pa, preferably about 100 Pa or less) compared to other scaffolds. It has been found that the low stiffness makes the fibrin gel suitable for inducing and testing angiogenesis in liver tissue in vitro. Similarly, the use of fibrinogen and / or fibrin in the gel precursor / gelling gel is believed to facilitate obtaining liver tissue in accordance with the present invention, providing liver tissue that more closely resembles in vivo liver tissue, e.g., in terms of (spatial) organization and / or functionality.

[0053] The gel precursor provided in step (b) further comprises hepatic endothelial cells and hepatic stellate cells. The hepatic endothelial cells and / or hepatic stellate cells are preferably primary culture cells, i.e., primary hepatic stellate cells and / or primary liver-derived endothelial cells, and the cells are preferably derived from the liver, meaning that they have been isolated from an organ or tissue, e.g., for in vitro use. It is contemplated that primary cell lines are advantageous in the methods of the present invention, e.g., are beneficial for angiogenesis, and are therefore preferred over (immortalized) cell lines or cells derived from pluripotent stem cells. In some embodiments, the hepatic stellate cells in step (b) do not comprise LX-2 cells. Both the hepatic endothelial cells and the hepatic stellate cells can be passaged (e.g., cultured) one or more times before providing the cells in the gel precursor. In other words, the cells do not need to be derived directly from the liver, e.g., fresh. The cells may, for example, be frozen according to common cell processing protocols and may need to be thawed prior to the method of the present invention.

[0054] It is understood that the gel precursor containing fibrinogen, hepatic endothelial cells, and / or hepatic stellate cells can be provided to the culture chamber as a mixture or suspension, for example, in the form of an aqueous solution containing at least fibrinogen, hepatic endothelial cells, and / or hepatic stellate cells. Alternatively, the gel precursor, fibrinogen, hepatic endothelial cells, and / or hepatic stellate cells can be provided to the culture chamber separately and mixed or suspended when in the culture chamber. However, it is highly preferred to introduce the gel precursor containing fibrinogen and the cells together. After the gel precursor is allowed to gel (form a gel), preferably at least a portion, preferably most or all (e.g., at least 5%, 10%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the number of cells) of the provided hepatic endothelial cells and / or hepatic stellate cells are embedded in a scaffold, thereby allowing the cells to be cultured in the scaffold (and the subsequent formation of, for example, vasculature, bile canaliculi, and other liver tissue structures disclosed herein in the gel (scaffold)). It is contemplated that adding such a cell suspension and forming a scaffold will ultimately result in liver tissue that closely mimics a natural liver. However, in some embodiments, it is contemplated that a portion of the cells (e.g., a portion of stellate cells, or a portion of hepatic endothelial cells, or both, or a portion of any other cells included as part of the method of the present invention) may be added during and / or after obtaining the scaffold (i.e., during or after the gel precursor forms a gel) and / or on top of the gel (precursor) once it has been introduced. However, without being bound by theory, adding (a portion of) the cells subsequent to the gel precursor, while possible, may result in less uniform distribution of the cells throughout the scaffold. This may adversely affect gelation, for example, in the case of a gel precursor containing fibrinogen, where thrombin must diffuse into the gel precursor and cells. Therefore, adding a suspension / dispersion of one or more gel precursors containing fibrinogen and cells is preferred. Thus, in some embodiments, the cells are more or less uniformly distributed throughout the gel after gelation. In some embodiments, the cells are not uniformly distributed throughout the gel after gelation.

[0055] Mixing of the gel precursor containing fibrinogen and / or cells is typically performed before the gel precursor at least partially, preferably completely, gels for the (gel) scaffold in step I. Preferably, a cell suspension, i.e., in which the gel precursor contains at least some hepatic endothelial cells or hepatic stellate cells, is introduced into the cell chamber, thus enabling cell embedding in the scaffold. In some embodiments, the gel precursor (containing hepatic endothelial cells and hepatic stellate cells) is provided as a whole to the culture chamber. In some embodiments, a first portion of the gel precursor (e.g., containing hepatic endothelial cells and / or hepatic stellate cells or no cells) is provided to the culture chamber and preferably allowed to gel, before a subsequent portion of the gel precursor (e.g., containing hepatic endothelial cells and / or hepatic stellate cells or no cells) is introduced into the cell culture chamber. In such embodiments, a different spatial arrangement of cells, including hepatic endothelial cells and / or hepatic stellate cells, throughout the scaffold may be obtained before culturing the cells therein.

[0056] In some embodiments, the cells are not provided as a viscous body of cells in step b) of the methods of the present invention. In some embodiments, the methods according to the present invention do not include a step of mixing the cells disclosed herein with collagen and heparin before performing step a) of the methods disclosed herein. In some preferred embodiments, the present invention does not include a step of centrifuging a mixture comprising the cells disclosed herein, preferably the cells, collagen, and heparin, to obtain, for example, a viscous body of cells, before and / or during performing the methods disclosed herein.

[0057] One skilled in the art can identify that hepatic endothelial cells and / or hepatic stellate cells are being used in the methods of the invention by identifying cell markers, such as (but not limited to) LYVE-1 and / or PLVAP for hepatic endothelial cells, and DESMIN, αSMA, and / or PDGFRβ for hepatic stellate cells. Alternatively, one skilled in the art can identify the type of cells used by (single cell) sequencing techniques known in the art.

[0058] The gel precursor can be introduced into a culture device, e.g., a microfluidic culture device, by allowing the gel precursor to enter and fill at least (part of) the culture chamber and, optionally, other microfluidic channel networks that may be present (see below). In embodiments where the culture device is a microfluidic device, it may be possible to (partially) fill selected regions of the microfluidic device, such as at least one of the microfluidic channels that may be present, with the aid of commonly used patterning techniques, e.g., photolithographic patterning, and potentially patterning involving gravity-assisted capillary pressure techniques. For example, it is possible to stop the gel precursor from filling a specific portion of the culture device, preferably a microfluidic device, using a capillary pressure barrier, e.g., a phase guide (Vulto et al. (2011), Lab on a chip. 11. 1596-602), or similar suitable methods for stopping the transport of the gel precursor. The capillary pressure barrier should not be understood as a wall or cavity consisting of elements that are filled with the gel precursor but that ensure that the gel precursor does not spread openly due to surface tension. This concept is referred to as meniscus pinning (Vulto et al. (2011), Lab on a Chip. 11, 1596-602). As such, stable confinement of a fluid-fluid meniscus, e.g., a gas-liquid meniscus, consisting of the gel precursor is achieved in microfluidic devices. As an example, the capillary pressure barrier can be a series of materials, e.g., in the form of edges, grooves, holes, or combinations thereof, that prevent the gel precursor from flowing beyond the capillary pressure barrier, e.g., due to meniscus pinning. In another embodiment, the capillary pressure barrier can be made of pillars at selected intervals that line the area occupied by the gel. Particularly advantageous capillary pressure barriers have been previously described by the applicant, for example in WO2017216113 A2 and WO2014038943 A1.

[0059] Further, in step (c), the gel precursor described herein is gelled into a scaffold. The gel precursor is allowed to gel (solidify) in a specific region of the culture device, thereby occupying at least a portion of the device. It is understood that the gel precursor at least partially gels in the culture chamber, thereby occupying at least a portion of the culture chamber in the form of a scaffold. Following gelling, it is preferred that at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or at least 99% of the volume of the culture chamber is occupied by the scaffold. With regard to the term "occupying" at least a portion of the culture device and at least the culture chamber, it will be understood by those skilled in the art that the scaffold is not required to be present throughout the culture device, but preferably occupies a specific area, leaving selected regions accessible for introducing additional gel precursor, fluid, or growth medium, for example, for perfusion flow. It will also be understood that the scaffold should not block the passage of growth medium through the device. The gel precursor may be solidified into a scaffold using, for example, a photosensitive crosslinker, such that the gel precursor gels upon exposure to, for example, UV light. Alternatively, some gel precursors can self-assemble into a scaffold, for example, by adding an enzyme that enables their conversion into a solid gel. Thus, in a preferred embodiment, a fibrinogen-containing gel precursor is solidified into a scaffold by adding the enzyme thrombin, which cleaves fibrinogen into fibrin monomers and enables self-assembly into a scaffold (Janmey et al. JR Soc. Interface (2009) 6, 1-10). It is known in the art that the gelation of a fibrinogen-containing gel precursor can be tuned to change the gelation time and / or mechanical properties of the fibrin scaffold. For example, a culture device containing the gel precursor can be placed in a humidified incubator for a period of time. Gelation (eg, polymerization or solidification) of the gel precursor into the scaffold preferably takes 5 to 30 minutes, preferably 15 to 20 minutes, after inducing gelation, for example, by enzymatic conversion.The time to gelation may depend on the type and / or concentration of ECM, conditions, and / or the concentration of enzymes, such as thrombin. Preferably, gelation results in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or at least 99% w / w gelation of the gel precursor into a scaffold. In other words, it is preferred that at least a portion of the gel precursor gels into a solid scaffold, although some of the gel precursor may still not solidify. After the gel precursor is provided, gelation is initiated, preferably before the introduction of additional fluids, aqueous solutions, or culture media. Several techniques for determining the mechanical properties of hydrogels have been described in the art (Handbook - Topics in Tissue Engineering, Vol. 4 (2008)). Whether the gel precursor has partially or completely gelled into a gel / scaffold can be determined by methods known to those skilled in the art, such as simple inversion of the culture device.

[0060] Furthermore, in step (d) of the method according to the present invention, the scaffold obtained in step (c) and containing the cells of step (b) is contacted with a cell culture medium. Those skilled in the art will understand that multiple cell culture medium compositions can be contacted with the cell-containing scaffold, for example, contacting the scaffold with a first medium suitable for supporting cell growth, followed by a second medium, and then a third or fourth medium with a similar or different composition (as long as it allows the cells to grow and survive). In other words, the cell culture medium can be replaced one or more times during cell culture. Typically, the cell culture medium is an aqueous medium. Preferably, the cell culture medium is a growth medium that can sufficiently deliver sufficient nutrients and other compounds essential for cell growth, differentiation, and / or proliferation, but preferably does not contain compounds / drugs that are potentially harmful to cell growth, differentiation, and / or proliferation.

[0061] Furthermore, it is contemplated herein that, depending on the cells used in the method according to the present invention, the culture medium can be adjusted by those skilled in the art to provide an appropriate growth medium for the cells in step (b). If desired, the growth medium can be supplemented with additional growth factors. For example, hepatic endothelial cells and / or stellate cells benefit from a scaffold contacted with EGM-2 / EGCM-2 medium, e.g., EGM-2 (Lonza™), and / or hepatocytes benefit from a scaffold contacted with iHEP plating medium (Cellular Dynamins, Fujifilm). An appropriate culture medium may promote, i.e., positively influence, the formation of the vasculature in the method according to the present invention. Other suitable media known to those skilled in the art are also possible and, therefore, are fully encompassed by the present invention. Therefore, selecting an appropriate, e.g., cell-specific, medium for culturing, differentiating, and / or expanding the cells is preferred, but not necessary. When performing step (d), the growth medium can be provided in a flow to contact the scaffold, for example, by allowing a fluid stream to pass through the scaffold through microfluidic channels and any vasculature-like structures formed by the cells in the scaffold. In the case of a flow, the growth medium can also be used to remove or dilute waste metabolites produced by the cells. Alternatively, the growth medium can be provided without the use of a flow to the scaffold, for example, by providing a predetermined volume of medium directly to the scaffold through holes in the culture chamber.

[0062] Further, in step e) of the method, the cells are cultured in the scaffold to obtain liver tissue. In other words, the cells of step (b) at least partially embedded in the scaffold can be cultured and / or proliferated and / or differentiated until at least a portion of liver tissue is formed.

[0063] It is contemplated that the methods of the present invention allow hepatic stellate cells and hepatic endothelial cells to interact and proliferate, leading to the formation of distinct morphological patterns of liver tissue that closely resemble native liver tissue in terms of spatial organization and / or function, and that include at least liver structures that include hepatic stellate cells and hepatic endothelium.

[0064] Regarding the formation of liver tissue in the scaffold, this includes the presence of a network of cells that interact with liver tissue, for example, physically and / or through signal molecules, which is preferably confluent or at least partially confluent. Such a confluent network includes, for example, about 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the cells present in the scaffold, for example, by physically interacting with each other, i.e., by forming one or more (parts of) cells that are in direct contact with each other and spatially organized in the form of vasculature, ducts, bile canaliculi-like structures, fenestrations, bile ducts, and / or cell clusters, and thus forming liver tissue. 100% confluency is preferred, but not required.

[0065] Preferably, the liver tissue is embedded in the scaffold, e.g., at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90, or 100 percent of the cells forming the liver tissue. Preferably, the liver tissue embedded in the scaffold resembles an arrangement (e.g., spatial organization) of cells that closely resembles in vivo tissue. For example, the liver tissue includes an arrangement of cells, such as (but not limited to) endothelial cells, hepatocytes, Kupffer cells, stellate cells, etc., which is highly vascularized and includes hepatic sinusoids, bile canaliculi, etc.

[0066] It has been found that improved liver tissue can be obtained by the method according to the invention by including hepatocytes in addition to hepatic stellate cells and hepatic endothelial cells in the gel precursor in step (b). In particular, it has been surprisingly found that the addition of hepatocytes causes the liver tissue to resemble even more closely native liver tissue in vivo, for example by exhibiting a similar spatial arrangement as native liver tissue.

[0067] In particular, it has been found that the inclusion of hepatocytes in the method results in liver tissue comprising functionally (polarized) and spatially organized hepatocytes, preferably in which at least a portion of the hepatocytes are in direct contact with and / or interact with hepatic stellate cells and / or hepatic endothelial cells through signaling molecules. Thus, in some embodiments, methods according to the present invention are provided, in which the gel precursor of step (b) further comprises hepatocytes.

[0068] As will be understood by those skilled in the art, when hepatocytes are added to the gel precursor in step (b), it is preferable to (also) use a cell culture medium suitable for culturing (e.g., maintaining), differentiating, and / or expanding the hepatocytes in step (d). In embodiments, the hepatocytes provided herein are derived from or differentiated from induced pluripotent stem cells and / or are (primary) human-derived hepatocytes. Although not preferred, it has been found that hepatic endothelial cells and hepatocytes can form structures that share some similarities with native liver tissue, even without the presence of hepatic stellate cells. While structures consisting solely of hepatic endothelial cells and hepatocytes do not resemble native tissue, they are similarly robust and / or lack the advantages, at least not the combination of hepatic stellate cells and hepatic endothelial cells shown herein.

[0069] For example, the present inventors have found that the use of at least hepatic stellate cells and hepatic endothelial cells enables angiogenesis in culture. In other words, it has been found that it is highly preferable for hepatic endothelial cells to be supported by stellate cells and form liver tissue containing a vasculature in a scaffold containing at least fibrin (e.g., a fibrin scaffold).

[0070] In some embodiments, a method according to the present invention is provided, in which the culturing in step (e) provides liver tissue comprising a vasculature formed by at least hepatic endothelial cells and hepatic stellate cells. In some embodiments, at least hepatic endothelial cells and hepatic stellate cells can be cultured and allowed to proliferate and / or differentiate until liver tissue comprising at least a vasculature is formed. Optionally, during the method of the present invention, a (bio)chemical gradient is provided, growth factors are provided and / or removed, the culture medium (components) are exchanged, etc. Preferably, the cells are provided at the total number of cells contained in the gel precursor provided in step (b), which is at least 1,000 cells per microliter of gel precursor. Furthermore, preferably, the hepatic endothelial cells and hepatic stellate cells are provided at a ratio of hepatic endothelial cells:hepatic stellate cells equal to or greater than 20:1. Also preferably, the cells are cultured for at least 3 days after steps (a) to (c), preferably after initiating step (d) of the method according to the present invention.

[0071] In some embodiments, the vasculature formed herein may extend outward from the resulting liver tissue. For example, (a portion of) the vasculature may extend outward from a first group of cells forming (a portion of) the liver tissue and extend toward a second group of cells forming (a portion of) the liver tissue, or may extend toward (a portion of) the vasculature extending outward from the second group of cells forming (a portion of) the liver tissue. The vasculature may extend toward (the vasculature of) a plurality (e.g., 3, 4, 5, 10, 100, 1,000, 10,000, etc.) of such additional groups of cells forming (a portion of) the liver tissue. In other words, the cells that (partially) form the vasculature may form a vascular network (as part of the liver tissue) by directly (e.g., physically) and / or indirectly interacting with additional cells in the scaffold. In some embodiments, the vasculature may extend outside of the scaffold and / or resulting liver tissue, for example, the vasculature may extend into other tissue structures, such as tubule-like structures formed by HUVECs in one or more microfluidic channels, or other tissue structures, such as tubule-like structures formed by HUVECs (and / or other endothelial cells), may extend into the scaffold and / or liver tissue, thereby interacting with cells contained within the scaffold / liver tissue.

[0072] Preferably, the vasculature also comprises the formation of at least some tubules and / or tubule-like structures, which preferably allow perfusion of fluids through the tubule(-like) structures, e.g., through a portion of the vasculature.

[0073] Preferably, the vasculature comprises at least one tubule-like structure having an average diameter between 1 and 100 μm, e.g., 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90, 100 μm, preferably between 1 and 50 μm, more preferably between 5 and 50 μm, or even between 5 and 30 μm. Surprisingly, it has been found that the vasculature of the liver tissue described herein exhibits a close similarity to the average diameter of the vasculature in native liver tissue.

[0074] In some embodiments, a method according to the present invention is provided, wherein the culturing in step (e) provides liver tissue comprising one or more sinusoidal structures formed by at least hepatic endothelial cells and hepatic stellate cells. Preferably, at least hepatic endothelial cells and hepatic stellate cells are cultured until liver tissue comprising a vasculature, preferably with one or more sinusoidal structures, is formed.

[0075] In some embodiments, methods according to the present invention are provided in which hepatocytes are included in addition to stellate cells and endothelial cells. Hepatocytes are mixed with stellate cells and endothelial cells in a gel precursor and provided in a culture chamber. In some embodiments, hepatocytes can be grown in a scaffold. Sinusoidal or sinusoid-like structures have been found to be at least partially surrounded by hepatocytes, as illustrated, for example, in Figures 7A-C. It is contemplated that liver tissue containing vasculature and one or more sinusoidal structures can include any other cell types commonly found in native liver tissue, such as other parenchymal and / or non-parenchymal cells, e.g., Kupffer cells, immune cells, bile duct cells, etc. Those skilled in the art will recognize additional suitable cell types.

[0076] Sinusoidal structures are capillaries typically found in native liver tissue. Sinusoidal structures have been described in the art, for example, by Brunt et al. (Brunt, EM et al. (2014) Histopathology 64, 907-920). In in vivo liver sinusoids, the endothelium is continuous, but some endothelial cells are loosely attached to each other, allowing fluids and proteins to leak from one side of the endothelium to the other side through intercellular junctions. Further leakage can be enabled by the formation of fenestrations in the liver endothelium. Liver tissue can be obtained using the method of the present invention, and it has been found to contain sinusoidal and / or sinusoid-like structures by providing at least hepatic stellate cells and hepatic endothelial cells in step (b) of the method of the present invention. Preferably, the liver tissue contains a vasculature including sinusoidal and / or sinusoid-like structures.

[0077] When cells are cultured, differentiated, and / or expanded in the methods according to the present invention, hepatic endothelial cells preferably contain vascular structures resembling hepatic sinusoidal structures, and preferably also form vasculature in scaffolds containing fenestrations. The discovery of fenestrations was surprising because the presence of fenestrations indicates a healthy phenotype for hepatic endothelial cells, and particularly because such a healthy phenotype is rapidly lost when hepatic endothelial cells, preferably liver-derived endothelial cells, are cultured using standard cell culture methods. Thus, the methods according to the present invention surprisingly enable the in vitro formation of liver tissue, which contains fenestrations and resembles the presence of such fenestrations in healthy in vivo liver tissue.

[0078] Fenestrations are known in the art and can be described as transcellular holes, small openings, or pores commonly found in aggregates of cells, particularly endothelial cells. In in vivo situations, fenestrations are often found in the endothelium of organs, e.g., tissues, such as the liver, pancreas, kidney, small intestine, and endocrine glands, where a higher rate of exchange between the intravascular and extravascular compartments is required. Generally, fenestrations allow the movement of large molecules from or into the blood and / or extravascular compartments. For example, fenestrations can be found in hepatic endothelial cells, allowing the transfer of substrates (e.g., lipoproteins) between the blood and hepatocytes. Typically, a single fenestration is approximately 50-300 nm or even 80-100 nm in diameter. In vivo, the majority of fenestrations are arranged in groups of 10-100, referred to as the "liver sieve plate."

[0079] In a preferred embodiment, the average diameter of the sinusoidal structures obtainable using the method of the present invention is between 1 and 50 μm, preferably between 5 and 30 μm, and more preferably about 10 μm, when human cells are used. Thus, it has been found that the average diameter of the sinusoidal structures provided by the method of the present invention is about 2, 5, 10, 15, 20, 25, 27.5, 30, 35, 40, 45, or 50 μm. Those skilled in the art will understand that the sinusoidal structures formed by cells are naturally occurring structures and therefore do not have the same diameter throughout the entire sinusoidal structure. In other words, the diameter of the sinusoidal structures is not 10 μm throughout the entire sinusoidal structure. Conversely, the diameter can be 60 μm in one portion of the sinusoidal structure, 1 μm in another portion of the sinusoidal structure, and 20 μm in a further portion of the sinusoidal structure. The mean diameter is therefore considered herein as the average (estimated) diameter throughout the sinusoid(-like) structure, determined, for example, by measuring the diameter at different positions in the sinusoid(-like) structure and determining the mean diameter thereon using methods known to those skilled in the art.

[0080] In a preferred embodiment, the cells contained in the scaffold are cultured for at least 2 days, at least 4 days, 5 days, 6 days, preferably about 7 days, and liver tissue containing vasculature with sinusoidal and / or sinusoid-like structures forms in the scaffold.

[0081] In some embodiments, methods are provided according to the present invention, wherein the liver tissue further comprises polarized hepatocytes, wherein the polarized hepatocytes align to form bile canaliculi or bile canaliculi-like structures. As used herein, the term "align" means that the hepatocytes, when polarized, are arranged in a line, e.g., a (mono)layer / sheet / group opposite at least one other line, e.g., a further (mono)layer / sheet / group of hepatocytes, preferably with a bile canaliculus or bile canaliculus-like structure separating both or more lines of polarized hepatocytes. Thus, in some embodiments, methods are provided according to the present invention, wherein the polarized hepatocytes form a layer / sheet / group of cells, wherein the layer / sheet / group is preferably formed in a scaffold.

[0082] Upon providing a gel precursor in step (b) further comprising hepatocytes, the hepatocytes in step (e) were found to form structures in which the basolateral sides of the hepatocytes are aligned and oriented around or toward the vasculature, and / or in which the apical sides of the hepatocytes face the apical sides of other hepatocytes. Surprisingly, it was found that the apical sides of in vitro cultured hepatocytes form structures resembling bile canaliculi. As such, hepatocytes form bile canaliculi-like structures, such as bile canaliculi or bile canaliculi-like structures. These bile canaliculi-like structures were identified by providing calcein-AM (which is a substrate for the apically expressed transporter MRP-2) in the culture device and detecting the accumulation of calcein-AM in the intercellular space between polarized hepatocytes (apical side). Thus, in some embodiments, there is provided a method according to the invention, wherein in the liver tissue, polarized hepatocytes are aligned such that the basal side of the polarized hepatocytes is adjacent to (or oriented towards) the vasculature and / or the apical side of the polarized hepatocytes is adjacent to or oriented towards (i.e., together with the apical domain of other polarized hepatocytes, forms) a bile canalicular or bile canalicular-like structure. Preferably, at least one bile canalicular or bile canalicular-like structure is obtained in the liver tissue obtained using the method according to the invention.

[0083] One skilled in the art can distinguish the apical side from the basolateral side by using methods known in the art, for example, by observing marker molecules that indicate the apical or basolateral side of a cell, e.g., a hepatocyte.

[0084] A suitable marker substance for establishing the presence of polarized hepatocytes is, for example, calcein-AM, which is a substrate for MRP-2, which is expressed and transported apically in (mature) hepatocytes. In the present invention, it has been found that detection of MRP-2 expression (or calcein-AM accumulation) can be used to identify the apical alignment of polarized hepatocytes and to identify bile canaliculi or bile canaliculi-like structures in the obtained liver tissue. Those skilled in the art may also use other marker substances indicative of hepatocytes, such as mature hepatocytes, to identify hepatocytes in the scaffolds provided herein. For example, albumin expression is used to identify mature hepatocytes.

[0085] Also provided is that the hepatocytes, preferably polarized hepatocytes, are capable of forming layers / clusters / sheets of cells, wherein the layers / clusters / sheets of cells comprise an apical side and a basal side, the basal side facing towards the vasculature and / or the apical side facing towards bile canaliculus-like structures.

[0086] The use of perfusion flow, for example, applying perfusion flow through a microfluidic device, has also been found to be beneficial in forming blood vessels containing polarized hepatocytes.

[0087] In the present invention, the basolateral membrane of a hepatocyte is the membrane facing a vasculature-like structure, preferably a sinusoid-like structure. It is contemplated herein that at least a portion of the polarized hepatocytes in the scaffold comprise a basal side adjacent to or oriented toward the vasculature and / or an apical side adjacent to or oriented toward the bile canaliculi.

[0088] Also provided is that the hepatocytes are positioned with respect to the bile canaliculi and / or bile canaliculi such that the bile canaliculi and / or bile canaliculi are at least partially positioned between the apical sides of two or more layers / groups / sheets of hepatocytes. It is contemplated that the bile canaliculi and / or bile canaliculi preferably have a morphology or shape such that more than two apical layers / groups / sheets of polarized hepatocytes face the bile canaliculi and / or bile canaliculi.

[0089] In some embodiments, the method provides for obtaining liver tissue having as a characteristic feature the formation of a vascular network, wherein the vascular network comprises a (transparent) lumen, further comprises one or more hepatic sinusoids, and further comprises (a layer of) (polarized) hepatocytes, the basolateral sides of which are adjacent to or oriented towards the vasculature, and the apical sides of the hepatocytes form bile canaliculi.

[0090] In some embodiments of the method, the gel precursor of step (b) further comprises one or more cells selected from the group consisting of immune cells, preferably liver-resident immune cells, and tumor cells.

[0091] More specifically, the immune cells are selected from neutrophils; eosinophils; basophils; lymphocytes, more specifically T cells, B cells and natural killer cells, monocytes, macrophages; mixtures comprising peripheral blood mononuclear cells (PBMCs); and combinations thereof.

[0092] In a particular embodiment of the liver resident immune cells, Kupffer cells, are included in the gel precursor of step (b).

[0093] In certain embodiments, the tumor cells are cells derived from a liver tumor or from other types of tumors, preferably circulating tumor cells.

[0094] The gel precursor containing tumor cells allows for the testing of cancer models in the obtained liver tissue, for example, for testing potential therapeutic agents. It also provides a model for studying the morphology and preferred tissue sites affected by tumors. It also allows for characterizing and monitoring the development of tumors and their tumor (micro)environment over time and / or in response to (immune) treatment. Thus, in some embodiments, liver tissue obtainable using the methods of the present invention is provided, which further comprises tumor cells and / or tumors.

[0095] In some embodiments of the method for obtaining liver tissue, it further comprises adding one or more of undifferentiated cells, immune cells, infectious agents, nucleic acid carriers, preferably gene therapy vectors, organoids, tumor cells and / or test compounds after step (e) of culturing cells in the scaffold to obtain liver tissue.

[0096] In a more specific embodiment, the undifferentiated cells are differentiated in the obtained liver tissue. A specific embodiment of the undifferentiated cells includes a source of monocytes, preferably added as one of the cell types in PBMCs. As illustrated in the figure (Figure 16), the addition of undifferentiated monocytes to the previously obtained liver tissue allowed the monocytes to adhere (i.e., attach) to the formed tissue (i.e., in the formed vasculature) and differentiate into macrophages (cells expressing the CD16 marker). In the same manner, other undifferentiated cells may be added and differentiated in situ under the (culture) conditions required to induce such differentiation, as known to those skilled in the art.

[0097] In another specific embodiment of the method, other cell types may be added after step (e) of the method, i.e., selected from immune cells, preferably neutrophils; eosinophils; basophils; lymphocytes, more specifically T cells, B cells, and natural killer cells, monocytes, and macrophages; a mixture comprising peripheral blood mononuclear cells (PBMCs); and combinations thereof. Preferred immune cell types added after step (e) of the method are T cells and macrophages.

[0098] In another, and specific, embodiment of the method, after step (e), the method comprises the addition of one or more infectious agents (i.e., unicellular or multicellular, or disease-causing agents or organisms) to the previously obtained liver tissue, preferably selected from the group consisting of bacteria, viruses, fungi, protozoa, and helminths.

[0099] In another specific embodiment of the method, after step (e), one or more nucleic acid carriers are added to the obtained liver tissue, which are preferably selected from viral vectors, preferably adeno-associated viral vectors. The addition of these nucleic acid carriers, preferably AAV, allows for the testing of gene therapy. In more specific embodiments, the AAV is of a capsid serotype specific for hepatocytes, preferably AAV1, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, nucleic acids are added to the obtained liver tissue, and in some embodiments, the nucleic acid is DNA, and in some embodiments, the nucleic acid is RNA. In some embodiments, the RNA or DNA is encapsulated in a delivery vehicle, such as a liposome or nanoparticle. In some embodiments, the RNA or DNA is in the form of a vaccine.

[0100] In another and particular embodiment of this method, after step (e), one or more organoids are preferably added to the top of culture device to obtain transplanted organoids.Organoids can be selected from any origin and / or mimic any organ at any stage of development.Preferably, organoids are liver organoids.

[0101] In another specific embodiment of the method, after step (e), one or more tumor cells (cancer cells) are added. These may be, in particular, liver tumor cells or alternative non-liver tumor cells. The addition of liver tumor cells allows for a model of liver cancer. The addition of non-liver tumor cells allows for a model of liver metastasis.

[0102] In other, and certain, embodiments of the method, candidate test compounds are added, for example, during or after steps b, c, d, or e. These compounds may be of any nature and may be used, for example, to screen for compounds that modulate liver development or provide a prophylactic or therapeutic effect in a liver model of disease. Thus, and in some embodiments, screening methods are provided that include testing the compounds to be screened in liver tissue obtained using the methods of the invention, or in the methods for obtaining liver tissue disclosed herein.

[0103] Several approaches are possible for the addition of any of these cells, drugs, or compounds, e.g., after step (e). In one approach, these cells and / or drugs and / or compounds can be added as components of the culture medium, e.g., to the top of the cell culture chamber. In an alternative, they can be added to any side tube / channel of the culture device, with or without the vasculature of endothelial cells (e.g., HUVECs).

[0104] Also provided is that in some embodiments, the culture device including the culture chamber is a microfluidic culture device. Microfluidic cell culture is an increasingly important technology. This technology finds its applications in drug screening, tissue culture, toxicity screening, and biological research. A major advantage of microfluidic cell culture is that it can add aspects such as perfusion flow, enhanced co-culture, and stable gradients to traditional cell culture, which can provide higher quality data, reduced reagent consumption, and lower costs. The microfluidic culture device preferably includes at least one microfluidic channel, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., connected to the culture chamber. In the case of a microfluidic device, the culture chamber can be in the form of a channel, preferably a channel that is the central channel of two other channels. The microfluidic channel serving as the culture chamber can be larger in size (e.g., include a larger diameter) than adjacent channels. An exemplary microfluidic device is shown (not to scale) in FIG. 2 of the present disclosure. As provided herein, one or more microfluidic channels are preferably positioned to the side of the culture chamber. More preferably, the channels are positioned substantially parallel to the culture chamber.

[0105] When the culture chamber is in the form of a channel, it preferably includes at least an inlet.

[0106] It is contemplated that the channels, preferably channels that are not culture chambers, can be used to benefit the culture chambers, for example, by adding additional cells, preferably endothelial cells, such as HUVECs, hepatic endothelial cells, etc. (or a mixture thereof), to the culture device, thereby enabling the formation of additional tubule-like structures in the channels, preferably in channels that are not culture chambers, for example, in channels positioned laterally relative to the culture chambers (as a non-limiting example, in FIG. 2, two channels are positioned on either side of the central culture chamber, and in FIG. 3, the channels positioned laterally relative to the culture chambers (named "transplantation chambers" in the examples) are referred to as "perfusion channels"). The tubule-like structures can further be used, for example, to replenish the culture in the channels and / or in the culture chambers with additional aqueous culture medium, nutrients, fluids, etc., and / or to remove and / or dilute waste metabolites produced by the cells.

[0107] Advantageously, in the context of the present invention, the microfluidic channel is used to enable perfusion or flow, for example, to enable flow (e.g., of culture medium) through at least a portion of the vasculature. In a preferred embodiment, liver endothelial cells are added to the microfluidic device by using a channel, preferably at least two channels, to allow the formation of tubule-like structures in the two channels. The channel or channels are positioned laterally relative to the culture chamber. For example, in the case of two channels, one channel can be positioned on one side of the culture chamber and another channel can be positioned on the opposite side of the culture chamber. The advantage of allowing endothelial cells to form tubule-like structures in the channels is that this can be advantageous in the formation of vasculature in the scaffold. For example, endothelial cells in one or more of the lateral channels can interact with cells in the scaffold, e.g., endothelial cells, for example, by direct and / or indirect contact, thereby connecting the endothelial cells in the channel to the cells in the scaffold, thereby allowing the formation of vasculature that can extend from the channel into and through the scaffold. In such embodiments, for example, blood vessels in the microfluidic channels are fluidly connected to a vascular bed in the culture chamber, allowing perfusion to both. The application of flow or perfusion, for example, with aqueous solutions or culture media, can further benefit the formation of vasculature in the scaffold.

[0108] In another preferred embodiment, one or more, preferably at least two, microfluidic channels are fluidly connected. It is further preferred that the microfluidic channel includes an inlet and an outlet. The network formed by one or more microfluidic channels may be referred to as a microfluidic channel network. In some embodiments, a method according to the present invention is provided, wherein the culture device is a microfluidic culture device including a culture chamber, further including at least one or at least two microfluidic channels fluidly connected to the culture chamber, and wherein endothelial cells, preferably hepatic endothelial cells, are cultured in at least one or at least two microfluidic channels. The endothelial cells may be provided in a gel precursor that forms into an endothelial cell-containing scaffold upon settling into the microfluidic channels. The (additional) endothelial cells are preferably hepatic endothelial cells. Other (liver) cells may be provided together with the (liver) endothelial cells. It is further contemplated that the tubule-like structures formed by the endothelial cells in one or more of the microfluidic channels may already be present in the culture device provided in step (a) of the method. Also contemplated are additional hepatic endothelial cells capable of forming tubule-like structures in the microfluidic channel before, during (any step), or after performing steps (a)-(e) according to the methods provided herein. Preferably, the tubule-like structures are formed during step (e) and thereby form an integral part of the resulting liver tissue.

[0109] As described above, tubules and / or tubule-like structures are preferably obtained in a microfluidic culture device comprising at least one or at least two microfluidic channels fluidly connected to a culture chamber, wherein the tubule-like structures are formed by cells, preferably endothelial cells.

[0110] In a preferred embodiment, the tubules and / or tubule-like structures have an average diameter similar to that of native liver tissue. The average diameter has been found to be between 1 μm and 100 μm, e.g., 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90, or 100 μm, preferably between 1 and 50 μm, more preferably between 5 and 30 μm, or even 5 to 15 μm. Preferably, the average diameter closely resembles that of the blood vessels of native liver tissue.

[0111] Within the context of the present invention, a microfluidic channel network is a hollow volume defined by two sidewalls (surfaces), a bottom substrate, and a top substrate enclosing the channel network. Both sidewalls, the top substrate, and the bottom substrate can be referred to as walls when they are in contact with the microfluidic channel network. The channel network is further connected to an inlet, typically a hole in the top substrate, which is used to fill the network from the outside world. The channel network can include one microfluidic channel or multiple microfluidic channels connected to each other. The microfluidic channel network can also be connected to further inlets or outlets.

[0112] The microfluidic channel network, into which cells may be further introduced, is characterized by the presence of a first portion constructed to provide a fluid passage to the cells, and / or a second portion constructed to provide a fluid passage from the cells, preferably hepatic stellate cells and hepatic endothelial cells, to and from the culture chamber containing the cells. This allows, for example, a flow of growth medium through the channels in the culture chamber and along the cells present in the channels. Thus, in some embodiments, a method according to the present invention is provided, in which the culture device is a microfluidic culture device comprising a culture chamber and at least two microfluidic channels, in which the at least two microfluidic channels are each independently fluidly connected to the culture chamber, and in which the culture chamber connects the two microfluidic channels to each other, and in which the liver tissue obtained in step (e) is perfusable, thereby allowing fluid to flow from one microfluidic channel to another through the scaffold containing the cells, preferably through the obtained liver tissue provided by the method of the present invention.

[0113] Adjacent to the scaffold is a further microfluidic channel network, which is in contact with the scaffold but in which the channel is not in direct contact with a further microfluidic channel (not a culture chamber) containing cells, e.g., endothelial cells. For example, if the scaffold is present in a channel adjacent to the channel into which the cells are introduced, the scaffold thus forms part of the wall of this channel. On the other side of the scaffold, there may be a further channel, which may be used, for example, to provide nutrients or compounds to the scaffold or to collect substances secreted by the cells. Alternatively, the scaffold may be present on both sides of a channel used for perfusion, e.g., a perfusion channel. This embodiment has the advantage that the maximum scaffold surface area is oriented and therefore exposed towards the tubule-like structures formed in the microfluidic channel.

[0114] Also provided is a method, wherein the culture device is a microfluidic culture device including a culture chamber and further including at least one or at least two microfluidic channels fluidly connected to the culture chamber, and wherein after step (e) of culturing cells in the scaffold to obtain liver tissue, one or more of undifferentiated cells, immune cells, infectious agents, nucleic acid carriers, tumor cells, and test compounds are added to at least one microfluidic channel.

[0115] Also provided is a method in which a culture chamber is at least partially aligned with and / or connected to a first reservoir, and in which the culture chamber is connected, preferably fluidly connected, to a second reservoir by at least one microfluidic channel. The channel may have at least one, two, or more reservoirs at one or both ends of the channel. The reservoirs may serve different purposes, but preferably serve the purpose of supplying nutrients, fluids, drugs, culture medium, cells, etc. to / from the cell culture in the culture device, and / or withdrawing waste products, cells, fluids, drug metabolites, culture medium, etc.

[0116] The reservoirs may have the form of wells (optionally microtiter plates), bottles, tubes, flacons, etc. The reservoirs may have any volume appropriate for the situation in which the microfluidic device is used. The first and second reservoirs may comprise different volumes. By way of example, the first reservoir may have a volume of about 15 μL, and the second reservoir may have a volume of about 100 μL. Preferably, the reservoirs are fixed to allow for the exchange of liquid in the reservoirs, e.g., culture medium.

[0117] Also provided is a method in which the liver tissue extends from the first reservoir to the second reservoir. Also provided is a method in which the culture chamber is further connected, preferably fluidly connected, to a third reservoir by at least one microfluidic channel, and in which a fluid flow is introduced between the second and third reservoirs. For example, a fluid inlet can be provided somewhere between the second and third reservoirs. The fluid flow is preferably introduced by passive leveling, but alternative ways of providing a fluid flow are not excluded and can be appropriately applied by those skilled in the art. Preferably, the liver tissue extends to the third reservoir. Furthermore, the liver tissue may extend to any additional reservoir forming part of the microfluidic channel network and / or microfluidic device.

[0118] Alternatively, cells can be added to any one or more of the reservoirs and can interact directly and / or indirectly with (the cells forming) liver tissue. For example, HUVECs can be added to the reservoirs and then grown on top of the liver tissue and / or migrate from the reservoirs into the liver tissue, e.g., by migrating into a culture chamber.

[0119] Also, in some embodiments of the present methods, the culture device including the culture chamber is a unidirectional flow microfluidic culture device. A unidirectional flow microfluidic culture device is defined as a microfluidic culture device in which the microfluidic network is configured such that, upon tilting of the device, any liquid provided in the reservoirs and / or the microfluidic channels flows in only one direction through the microfluidic channels and cell culture chamber. In this manner, the microfluidic network is configured such that perfusion flow through at least a portion of the microfluidic network and culture chamber flows in a predominantly unidirectional direction during reciprocal tilting of the device to two or more positions and angles. This direction can sometimes be adjusted depending on the operating mode of the microfluidic culture device (i.e., positioning, titer plate angle). This is referred to as unidirectional flow, as opposed to bidirectional flow, in which liquid flow changes direction when the tilt direction of the microfluidic device is changed. Any embodiment relating to the addition of additional cells in a channel is also applicable to methods using a unidirectional flow microfluidic culture device.

[0120] In a preferred embodiment, the culture device comprising the culture chamber is a unidirectional flow microfluidic culture device comprising at least one microfluidic network comprising: (a) a microfluidic layer including a first channel and a second channel; and (b) a reservoir layer disposed on the microfluidic layer and including first and second reservoirs, wherein the first reservoir and the second reservoir have an access port to the first flow path and an access port to the second flow path, respectively, and the first flow path and the second flow path form a fluid circuit with the first reservoir and the second reservoir; Therein, the access ports in the first and second reservoirs are spaced apart, and in use, tilting the device (i.e., titer plate) at a first angle causes the volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir primarily via the first flow path, and adjusting the tilt so that the device is tilted at a second angle causes the volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir primarily via the second flow path.

[0121] In even more specific embodiments of the method in which a unidirectional flow microfluidic culture device is used, the method comprises: (a) tilting the device (i.e., titer plate) at a first angle by rotating it about a first axis to induce a volume of fluid in a first reservoir to flow from the first reservoir to a second reservoir via a first flow path; and (b) adjusting the tilt, and tilting the device (i.e., titer plate) at a second angle by rotating about the first axis, causing the volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir via the second flow path.

[0122] Advantageously, the method of the present invention, i.e., the use of a unidirectional flow microfluidic culture device to apply unidirectional flow, provides tissue grafts, particularly liver tissue grafts, with improved tissue structure, in the sense that the resulting tissue and vasculature closely mimic in vivo tissue and vasculature. As illustrated in the figures, when applying unidirectional flow, tissue morphology highly similar to in vivo tissue is obtained, and even improved tissue morphology can be obtained compared to cultures obtained under bidirectional flow conditions. Surprisingly, the application of unidirectional flow also enables the phenomenon of tissue regionalization, particularly in the case of liver tissue, the phenomenon of sinusoidal regionalization. As explained above, liver regionalization is a well-known process in which liver cells have different, and sometimes contradictory, functions depending on where they are located along the vascular junction between the portal vein and the central vein.

[0123] Another particularly advantageous application of the unidirectional flow microfluidic culture device for carrying out the method of the present invention is the ability to obtain metastasis in a liver tissue cancer model that is highly similar to the actual in vivo situation. This is mainly due to the high degree to which the previously obtained liver tissue mimics the actual sinusoids and liver vasculature. Preferably, metastasis is induced by introducing tumor cells into at least one microfluidic channel and perfusing the tumor cells into the liver tissue, i.e., into the vasculature.

[0124] In the same way, unidirectional flow can be applied while providing analysis or simulation of real infection processes (e.g., bacteria, viruses, etc.) Specific infections with viruses and other vector particles, such as adeno-associated virus particles, further provide for testing of gene therapy in liver and other tissues.

[0125] The application of unidirectional flow also allowed the observation that cell alignment is regulated by the fluid flow involved in the process of obtaining tissue models. Those skilled in the art will recognize that many tissues in the human body exhibit structural arrangements of cells that imply anisotropic, direction-dependent properties. All this is achieved by means of specific directional connections and migration, usually accompanied by a specific spatial distribution of components of the extracellular matrix (ECM).

[0126] Several configurations can be provided to operate a microfluidic system as a unidirectional flow microfluidic device. An example of a preferred device for the acquisition of biological tissue models and other tissue models in plates is depicted schematically in FIG. 32, where the flow of liquids during operation is illustrated using arrows in the microfluidic channels. Here, the device includes one or more microfluidic networks, including: (a) a microfluidic layer including at least a first flow path (1) and a second flow path (2) fluidly connected to a cell culture chamber (3); and (b) a reservoir layer, preferably disposed on the microfluidic layer, including at least a first reservoir (4) and a second reservoir (5), wherein the at least first reservoir and the second reservoir have access ports to the first flow path and the second flow path, respectively, and the first flow path and the second flow path form a flow circuit with the at least first reservoir and the second reservoir.

[0127] Accordingly, there is also disclosed a unidirectional flow microfluidic culture device comprising one or more microfluidic networks, the microfluidic network comprising: (a) a microfluidic layer comprising at least a first flow path (1) and a second flow path (2) fluidly connected to a cell culture chamber (3); and (b) a reservoir layer preferably disposed above the microfluidic layer and adapted to receive a volume of fluid (i.e., liquid), comprising at least a first (4) and a second (5) reservoir, each having an access port to the first flow path and an access port to the second flow path, such that the first flow path and the second flow path form a fluid circuit involving at least two reservoirs (numbering in reference to Figure 32). The access ports in the first and second reservoirs are spaced apart such that, during use, tilting the device (i.e., titer plate) at a first angle induces the volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir primarily via the first flow path, and adjusting the tilt so that the device (i.e., titer plate) is tilted at a second angle induces the volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir primarily via the second flow path.

[0128] In particular, this unidirectional flow microfluidic culture device is used with a tilt system, preferably a rocker, where a first tilt angle is imposed on the device, thereby inducing a volume of fluid in at least a first reservoir to flow from the first reservoir to a second reservoir via a first flow path; and adjusting the tilt to a second tilt angle causes a volume of fluid in at least a second reservoir to flow from the second reservoir to the first reservoir via a second flow path, whereby tilting the device at least at the first angle induces a volume of fluid in the first reservoir to flow from the at least first reservoir through the cell culture chamber to the second reservoir.

[0129] In some embodiments, a method according to the present invention is provided, wherein openings are formed in the scaffold during step (c) and / or after step (e). The openings preferably allow for the introduction of solutions, fluids, and / or drugs. The openings may have any suitable shape for the intended purpose. For example, holes may be created suitable for allowing the pipetting of drugs dissolved in solution directly into the gel. For example, drugs may be added that have an effect on angiogenesis by liver endothelial cells, such as VEGF. As such, the holes may provide access to the luminal side of the vasculature, for example, for fluids.

[0130] In some embodiments, methods according to the present invention are provided in which the hepatic endothelial cells, hepatic stellate cells, and / or hepatocytes are not separated from one another by an artificial membrane. For example, the hepatic stellate cells, hepatic endothelial cells, and / or hepatocytes, and optionally any additional cells, can be in direct contact with one another without the presence of an artificial membrane or support (i.e., a thin membrane or layer (e.g., 5-60 micrometers or less) formed, for example, based on polyester, polycarbonate, or polytetrafluoroethylene, or collagen-coated polytetrafluoroethylene), e.g., a non-natural (e.g., plastic, glass, etc.) and / or exogenously introduced membrane, such as a membrane used in a Transwell system. Furthermore, it is contemplated that no artificial membrane or support is present between the scaffold formed by gelation in the culture chamber and any adjacent (microfluidic) channels, thereby allowing direct contact between the tubule-like structures that may be formed by endothelial cells in the (microfluidic) channels (as described herein) and the liver tissue formed in the culture chamber. In a preferred embodiment, no artificial (porous) membrane is used in the method according to the present invention. Within the context of the present invention, those skilled in the art will understand that the gel precursors, gels, and / or scaffolds used in the present invention are not considered artificial membranes or supports. Within the context of the present invention, an artificial membrane is not a membrane formed during the implementation of the culture method of the present invention. Those skilled in the art will understand that, in embodiments, an artificial membrane is not a non-biological membrane, but a membrane created by humans. Thus, within the context of the present invention, in a preferred embodiment, the cells used in the method according to the present invention are not separated from each other by the presence of a membrane, e.g., an artificial membrane, that would prevent the cells from being in contact with each other, in particular from being in direct cell-cell contact. For example, in a preferred embodiment, a membrane, e.g., an artificial membrane, does not separate the obtained polarized hepatocytes from the obtained vasculature. The present invention is therefore based in part on the absence of any artificial membrane separating cells in the obtained liver tissue, thereby making it possible to obtain a perfusable network, e.g., a perfusable vascular bed and / or blood vessels.

[0131] In some embodiments, a method according to the present invention is provided, wherein the total number of cells contained in the gel precursor provided in step (b) is at least 1,000 cells per microliter. The cell number is preferably up to 100,000 cells per microliter, more preferably 50,000 cells per microliter. The total number of cells will likely be limited by the nutrients and / or oxygen available in the culture chamber. Also provided is a method according to the present invention, wherein the ratio of the number of hepatic endothelial cells to hepatic stellate cells provided in the gel precursor in step (b) is preferably equal to or greater than 20:1. Furthermore, a method is provided, wherein the total number of cells contained in the gel precursor is at least 1,000 cells per microliter, and the ratio of the number of hepatic endothelial cells to hepatic stellate cells is equal to or greater than 20:1. It has been found that this cell number is advantageous for angiogenesis. At least 1,000, e.g., 1,500, 2,000, 2,500, 3,000, 5,000, 7,500, 8,000, 9,500, 10,000, 11,000, 12,500, 20,000, 30,000, 40,000, and / or up to 50,000 cells (and any amount between 1,000 and 50,000 cells) can be provided per microliter, preferably per microliter of gel precursor. In preferred embodiments, the total cell count is at least 5,000 cells per microliter, more preferably at least 7,500 cells per microliter. Preferably, the total cell count is between about 8,000 and 15,000 cells per microliter, preferably up to 30,000 cells per microliter. Furthermore, a specific ratio of the amount of hepatic stellate cells relative to hepatic endothelial cells appears to be highly advantageous for the formation of liver tissue according to the present invention, with stellate cells preferably present at a ratio of at least 1 stellate cell per 20 hepatic endothelial cells, meaning that, for example, when 10,000 hepatic endothelial cells are provided, at least 500 hepatic stellate cells are provided.Still, ratios equal to or greater than 20:1 (hepatic endothelial cells:hepatic stellate cells), for example, with increasing preference, equal to or greater than 15:1, equal to or greater than 10:1, equal to or greater than 4:1, equal to or greater than 1:1, or equal to or greater than 0.5:1, were found to result in beneficial angiogenesis. As non-limiting, illustrative examples, several ratios of hepatic endothelial cells to hepatic stellate cells (1:1, 2:1, 4:1, and 20:1) were tested and are shown in Figure 6 of the present disclosure. While ratios of hepatic endothelial cells to hepatic stellate cells less than 20:1, such as 25:1 or 30:1, may be used in the disclosed methods, these smaller ratios of hepatic endothelial cells:hepatic stellate cells are not preferred for the methods. However, ratios equal to or greater than 20:1 (hepatic endothelial cells:stellate cells) were found to enable robust vascular development. Most preferably, this ratio is about 4:1, meaning that for every four hepatic endothelial cells, there is one hepatic stellate cell in the gel precursor. It is particularly preferred that the cell numbers and / or ratios referred to herein are representative of the number of cells present in step (b) of the method, i.e., before step (c). The cell numbers used to determine the ratio do not include proliferating and / or differentiated cells according to step (e) of the method, nor do they include endothelial cells further introduced into one or more microfluidic channels. The cell numbers provided herein include any additional cells, i.e., hepatocytes, Kupffer cells, immune cells, bile duct cells, etc., added to the gel precursor according to step (b).

[0132] In some embodiments, Kupffer cells can be added in the method. It has been found that adding Kupffer cells results in obtaining liver tissue containing live Kupffer cells. The addition of Kupffer cells is beneficial to inflammatory response in the obtained liver tissue. Therefore, providing Kupffer cells allows the liver tissue obtained in the present invention to more closely resemble natural liver tissue. For example, when providing Kupffer cells in the method according to the present invention, it has been found that the cells are functional when mimicking fatty liver, for example, by providing free fatty acids.

[0133] In some embodiments, Kupffer cells are added (e.g., during step c) and / or step d)) on top of the scaffold formed in step c) and / or on top of the liver tissue obtained in step e) and allowed to migrate into the scaffold and / or liver tissue.

[0134] In some embodiments, cholangiocytes can be added in this method.It has been found that adding cholangiocytes can result in liver tissue containing living cholangiocytes.Therefore, by providing cholangiocytes, the liver tissue obtained in the present invention can more closely resemble natural liver tissue.

[0135] It is contemplated that the ratio need not include an absolute number, e.g., a round number such as 1 in "the ratio is 1:1," but may include, for example, "1,00001:1" when referring to the ratio of the number of hepatic endothelial cells to the number of hepatic stellate cells.

[0136] In some embodiments, methods according to the present invention are provided in which the ratio of the number of hepatocytes to the sum of the number of hepatic endothelial cells and hepatic stellate cells is equal to or greater than 1:1. For example, when hepatocytes are provided in methods according to the present invention, there are at least two hepatocytes for every one hepatic endothelial cell and one hepatic stellate cell (i.e., a ratio equal to 1:1). As such, it is preferred that there are more hepatocytes in the gel precursor than there are hepatic endothelial cells and hepatic stellate cells present in the gel precursor. Optionally, i.e., the number of cells for determining the ratio does not include cells proliferated and / or differentiated according to step (e) of the method, and the number of cells does not include hepatic endothelial cells further introduced into one or more microfluidic channels. For example, there may be about 8,000-10,000 hepatocytes per microliter, about 3,000-3,500 hepatic endothelial cells per microliter, and about 1,000-2,000 hepatic stellate cells per microliter.

[0137] Furthermore, the ratio of the number of hepatocytes to the sum of the number of hepatic endothelial cells and hepatic stellate cells is preferably greater than 2:1. It is contemplated that an increased number of hepatocytes relative to hepatic endothelial cells and hepatic stellate cells is advantageous for the formation of liver-specific structures, such as bile canaliculus-like structures. In a further embodiment, the ratio of the number of hepatocytes to the sum of the number of hepatic endothelial cells and hepatic stellate cells is between (and including) 1:1 and 30:1, for example, 1:1, 2:1, 4:1, 5:1, 6:1, 10:1, 15:1, 20:1, 25:1, 29:1, 29.5:1, or 30:1. Any ratio of the number of hepatocytes to the sum of the number of hepatic endothelial cells and hepatic stellate cells between 1:1 and 30:1 is encompassed by the present invention. Preferably, the ratio is between 1:1 and 10:1, even more preferably between 1:1 and 5:1. When referring to the ratio of the number of hepatocytes to the sum of the number of hepatic endothelial cells and hepatic stellate cells, the ratio does not necessarily include absolute numbers, e.g., round numbers such as 1:1, but can include 1,00001:1.

[0138] For example, when the total number of cells / μL contained in the gel precursor provided in step (b) is about 100,000 cells / μL, the amount of hepatocytes relative to the total amount of hepatic endothelial cells and hepatic stellate cells can be, for example, about 50,000 hepatocytes: about 50,000 hepatic endothelial cells and hepatic stellate cells, but can also be about 80,000 hepatocytes: about 20,000 hepatic endothelial cells and hepatic stellate cells.

[0139] In some preferred embodiments, there is provided a method according to the present invention, wherein the gel precursor of step (b) comprises per microliter: Between 1,000 and 10,000 hepatic endothelial cells; and -Between 100 and 10,000 hepatic stellate cells. The gel precursor of step (b) preferably contains between 1,000 and 10,000 hepatic endothelial cells per microliter. Thus, the gel precursor may contain 1,000, 1,500, 2,500, 5,000, 7,500, 8,000, 9,000, or any other number between 1,000 and 10,000 hepatic endothelial cells per microliter. Fewer hepatic endothelial cells, e.g., 500 cells per microliter, may also be used, and therefore, these lower numbers of hepatic endothelial cells are also contemplated by the present invention. However, an amount of between 1,000 and 10,000 hepatic endothelial cells per microliter is preferred for obtaining liver tissue as disclosed herein, and preferably for vascularization.

[0140] The gel precursor of step (b) preferably contains between 100 and 10,000 hepatic stellate cells per microliter. Therefore, the gel precursor may contain 500, 1,000, 2,500, 5,000, 7,500, 8,000, 9,000, or any other number between 100 and 10,000 hepatic stellate cells per microliter. Fewer hepatic stellate cells, for example, 50 cells per microliter, may also be used and are therefore encompassed by the present invention. However, an amount between 100 and 10,000 hepatic stellate cells per microliter is preferred for liver tissue acquisition and, preferably, for vascularization.

[0141] It is further preferred that the gel precursor of step (b) contains between 5,000 and 80,000 hepatocytes per microliter, preferably between 5,000 and 50,000. Preferably, the gel precursor may contain 5,500, 7,500, 8,000, 9,000, 10,000, 12,500, 25,000, 30,000, 40,000, 45,000, or any other number between 5,000 and 50,000 hepatocytes per microliter. It is contemplated that fewer hepatocytes, e.g., 500 cells per microliter, may be used, and therefore, these fewer numbers of hepatocytes are also encompassed by the present invention. However, an amount of between 5,000 and 50,000 hepatocytes per microliter is preferred for obtaining liver tissue and, preferably, for vascularization and / or bile canalicular (or bile canalicular) formation.

[0142] In one particularly preferred embodiment, there is provided a method according to the invention, wherein the gel precursor of step (b) is - between 1,000 and 10,000 hepatic endothelial cells; Between 100 and 10,000 hepatic stellate cells; and -Contains between 5,000 and 50,000 hepatocytes.

[0143] In some embodiments, a method according to the present invention is provided, wherein the gel precursor in step (b) has a volume between 0.1 μL and 50 μL. The gel precursor in step (b) preferably has a volume between 0.5 μL and 20 μL. The gel precursor in step (b) may have a volume of 0.5, 1, 2, 5, 10, 15, 20, 25, 30, or 40 microliters, or any other volume between 0.1 μL and 50 μL. One skilled in the art can adjust the amount of cells to match the volume of the gel precursor. In an exemplary embodiment, between 0.5 and 5 μL, preferably about 1.5 μL or about 1.35 μL, of gel precursor is provided. As disclosed herein, the volume of gel precursor provided to the culture chamber in step (b) may be provided all at once (as a whole) or in separate subvolumes. In some embodiments, the volume of gel precursor in step (b) is preferably provided in a microfluidic culture device.

[0144] One advantage of using a relatively small volume of gel precursor is that it allows for a high cell density, i.e., a relatively large number of cells in a relatively small volume of gel precursor. For example, the gel precursor provided in step b) herein may preferably have a volume of between 0.5 μL and 20 μL and contain a total cell amount of up to 100,000 cells / μL. Thus, the volume of the gel precursor and the amount of cells provided herein allow for a relatively high cell density to be used in the methods of the present invention.

[0145] In some embodiments, methods according to the present invention are provided, wherein the scaffold comprises a basement membrane matrix gel, an extracellular matrix gel, a collagen gel, and / or a fibrin gel. In some embodiments, methods according to the present invention are provided, wherein the scaffold of step (c) further comprises a collagen scaffold.

[0146] In some embodiments, a method according to the present invention is provided, wherein the gel precursor comprises at least a fibrinogen gel precursor and a mixture of any one or more selected from the group consisting of a basement membrane matrix gel precursor, an extracellular matrix gel precursor, and a collagen gel precursor. For example, the additional gel precursor may comprise a mixture of a collagen gel precursor and a fibrin gel precursor, or may comprise a mixture of a basement membrane matrix gel precursor, a collagen gel precursor, and a fibrin gel precursor.

[0147] In some embodiments, a method according to the present invention is provided, wherein the gel precursor of step (b) comprises fibrinogen at a concentration of at least 1 mg / mL. The gel precursor of step (b) preferably comprises fibrinogen at a concentration of up to 50 mg / mL. The gel precursor of step (b) can therefore comprise between about 1 mg / mL and about 50 mg / mL. Thus, fibrinogen can be present at, for example, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 45 mg / mL, 49 mg / mL, and any other concentration between about 1 mg / mL and about 50 mg / mL, or between any two of the above concentrations. It is even more preferred that the final concentration of fibrinogen in the gel precursor of step (b) is about 5 mg / mL.

[0148] In one non-limiting example, to achieve an appropriate final concentration of fibrinogen, the following mixture (60 μL) may be provided in step (b): 8125 cells / μL stellate cells (final concentration 1,625 cells / μL) in 10 μL DMEM (10% FBS, 1% P / S), 16,250 cells / μL hepatic endothelial cells (final concentration 3,250 cells / μL) in 10 μL DMEM (10% FBS, 1% P / S), 25,000 cells / μL hepatocytes (final concentration 10,000 cells / μL) in 20 μL DMEM (10% FBS, 1% P / S), 5 μL of 50 mg / mL fibrinogen (final concentration 5 mg / mL), and 5 μL of 1 U / mL thrombin (final concentration 0.1 U / mL).

[0149] The concentration of fibrinogen in the gel precursor in step (b) may be achieved by adding a specific volume of a fibrinogen stock solution to the gel precursor, preferably the mixture / suspension / dispersion forming the gel precursor. Although not preferred, it is also possible to achieve the concentration of fibrinogen in the gel precursor by adding a specific volume of fibrinogen in step (b) and a further volume of fibrinogen in step (c), at least as long as the gel precursor has not yet completely gelled into a scaffold, i.e., when at least a portion of the gel precursor has not gelled. Preferably, the gel precursor contains sufficient fibrinogen to allow the gel precursor to gel into a scaffold, for example under the influence of thrombin.

[0150] In some embodiments, a method according to the present invention is provided, wherein the gel precursor of step (b) further comprises thrombin. Preferably, the gel precursor of step (b) comprises thrombin at a concentration of at least 0.01 U / mL or at most 1 U / mL. The concentration of thrombin in the gel precursor of step (b) may be, for example, 0.01 U / mL, 0.05 U / mL, 0.1 U / mL, 0.25 U / mL, 0.5 U / mL, 0.75 U / mL, 0.9 U / mL, 0.99 U / mL, or any other value between 0.01 U / mL and 1 U / mL. It is highly preferred that the gel precursor of step (b) comprises about 0.1 U / mL thrombin. It is contemplated that the concentration of thrombin should be sufficient for enzymatic conversion of the fibrinogen-containing gel precursor to a fibrin-containing scaffold, i.e., thrombin-mediated proteolytic conversion. Additionally, one skilled in the art may wish to adjust the concentration of thrombin depending on the hardness of the gel; for example, for a particular use, a less stiff scaffold may be desirable; in such cases, the concentration of thrombin provided may be reduced, e.g., so that at least a portion of the concentration of fibrinogen in the gel precursor is not converted to fibrin.

[0151] The concentration of thrombin in the gel precursor in step (b) may be achieved by adding a specific volume of thrombin stock solution to the gel precursor, preferably to the mixture / suspension / dispersion that forms the gel precursor. It is also possible to achieve the concentration of fibrinogen in the gel precursor by adding a specific volume of fibrinogen in step (b) and further adding a volume of fibrinogen in step (c), at least as long as the gel precursor has not yet completely gelled into a scaffold, i.e., when at least a portion of the gel precursor has not gelled. Alternatively, it is also possible to achieve the concentration of thrombin in the gel precursor by adding a volume of thrombin in step (c), at least as long as the gel precursor has not yet completely gelled into a scaffold.

[0152] In some embodiments, methods according to the present invention are provided, wherein the culture medium in step (d) has a volume of between 20 and 500 μL, e.g., 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 490 μL, or any other volume between 20 and 500 μL. In some embodiments, methods according to the present invention are provided, wherein the culture chamber has a volume of between 0.1 μL and 50 μL, e.g., 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 20, 25, 27.5, 30, 35, 40, 45, 49.5 μL. Even more preferably, the culture chamber has a volume of between 0.1 μL and 20 μL. In another embodiment, there is provided a method according to the present invention, wherein the culture medium in step (d) has a volume between 20 and 500 μL, and wherein the culture chamber has a volume between 0.1 μL and 50 μL.

[0153] In step (d), and further in step (e), more than one culture medium may be provided, e.g., 2, 3, 4, 5, 6, 7, 8, 10, etc. One culture medium may be provided multiple times in step (d). Different culture media may be provided (and replaced) multiple times. The present invention is not particularly dependent on a particular culture medium for contacting the scaffold containing cells and / or for culturing the cells in step (e), as long as it is appropriate for growing the cells within the context of the present invention. For example, cells may be provided in a gel precursor dispersed / suspended in a first culture medium, e.g., iHEP medium (Cellular Dynamics, Fujifilm), and the cells may then be contacted with a second culture medium, e.g., during steps (b) and / or (c) and / or (d), and optionally with a third or fourth culture medium on day 1. In other words, cells may be contacted with multiple culture medium compositions during the method of the present invention, and optionally with one or more culture medium compositions on day 1. While it is contemplated that the culture medium compositions can be mixed, preferably the different culture medium compositions are provided separately (e.g., at different time points), and optionally after removal of the previous culture medium composition to which the cells were exposed. The scaffold containing the cells may then be contacted again with either the first or subsequent culture medium, or with a different culture medium.

[0154] The culture medium can include, for example, Dulbecco's Modified Eagle Medium (DMEM, Gibco, catalog number 11965-092), which has been supplemented with additional compounds such as 10% FBS (Gibco), penicillin-streptomycin (P / S, Sigma-Aldrich, catalog number P4333-100ML), and sodium pyruvate (Thermo Fisher Scientific); or RPMI medium (Sigma-Aldrich, catalog number R8758-500ML), which has been supplemented with additional compounds such as B27 supplement (Gibco, catalog number 17404-001, lot number 2209534), oncostatin M, dexamethasone (MP Biomed, catalog number 194561), gentamicin (Gibco, catalog number 15750-060), and iCell Hepatocyte 2.0 Medium Supplement (Cellular Dynamics, catalog number M1024, lot number 106063), or may contain Endothelial Cell Growth Basal Medium-2 (EBM-2, Lonza, catalog number CC-3156), which may be supplemented with, for example, additional compounds such as FBS, hydrocortisone, hFGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF, GA-1000, and heparin. Other suitable media are commercially available, such as Hepatocyte Basal Medium (CC-3199), HCM™ SingleQuots™ Kit (CC-4182) (Lonza), Williams E Medium, and the like.

[0155] In some embodiments, at least one of the culture media provided in the methods according to the invention preferably comprises one or more growth factors selected from FGF, VEGF, and / or EGF, preferably at concentrations beneficial to the formation of vasculature.

[0156] In some embodiments, methods are provided according to the invention, wherein the culture medium in step (d) is changed (at least partially) every 24 to 144 hours. In some embodiments, methods are provided according to the invention, wherein the cells are cultured for at least 3, 4, 5, 6, or 7 days with increasing preference. Further, in some embodiments, methods are provided according to the invention, wherein the culture medium in step (d) is changed every 24 to 144 hours, and methods are provided according to the invention, wherein the cells are cultured for at least 3, 4, 5, 6, or 7 days with increasing preference.

[0157] The culture medium provided in step (d) and / or the culture medium in which the cells are cultured in step (e) may be changed at least every 24 hours and / or at most every 144 hours. The culture medium may be changed, for example, every 24, 25, 26, 27, 28, 29, 30, 36, 40, 44, 48, 50, 56, 60, 66, 72, 84, 96, 108, 120, 132, and / or 144 hours. Replacing the culture medium in step (d) at any time between 24 and 144 hours is also encompassed by the present invention. The culture medium may be replaced with the same culture medium or a different culture medium. It is preferable to replace the medium once the nutrients in the culture medium have (for the most part) been absorbed / used by the cells. Optionally, the culture medium is replaced to affect cell differentiation and / or proliferation.

[0158] Preferably, the cells are cultured for at least 3 days. The cells may be cultured for longer periods. After about 6 or 7 days of cell culture, the cells are preferably ready for imaging (e.g., by adding an imaging agent, such as calcein AM, to the culture medium). It has been found that about 3 days after initiating step (d), the cells begin to differentiate and / or proliferate and form liver tissue, preferably liver tissue containing a vasculature as disclosed herein and / or containing sinusoidal and / or bile canalicular structures. Therefore, in some embodiments, it is contemplated that about 7 days after performing steps (a)-(c) of the method is preferably sufficient to obtain liver tissue as disclosed herein. It is also contemplated herein that any one or more of steps (a)-(c) may be terminated, for example, by cryopreserving the culture device containing the cell-containing gel precursor and / or scaffold. In such cases, it is contemplated that after thawing the cryopreserved culture device, liver tissue and / or liver-like tissue can be obtained within 7 days of culture. It is also contemplated that any one of steps (d) to (e) may be terminated, for example, by cryopreserving the culture device containing the cells and / or the obtained liver tissue (portion). Furthermore, the method may include step (f), in which the obtained liver tissue is preserved after step (e), for example, by cryopreservation.

[0159] In some embodiments, a method according to the present invention is provided, wherein the gel precursor of step (b) further comprises hepatocytes; and wherein one or more, preferably all, of the cells are cultured for a period of time sufficient to allow the formation of vasculature, preferably wherein the vasculature is accessible (e.g., fluidly and / or physically) and / or has a diameter of about 5-50 μm; and / or wherein one or more, preferably all, cells are cultured for a period of time sufficient to allow the formation of polarized hepatocytes, preferably layers / sheets / clusters of polarized hepatocytes, wherein the basal side of the polarized hepatocytes is adjacent to the vasculature and / or the apical side of the polarized hepatocytes is adjacent to bile canaliculi and / or bile canaliculus-like structures; wherein one or more, preferably all, of the cells are cultured for a period of time sufficient to allow the formation of bile canaliculi and / or bile canaliculus-like structures; and / or wherein one or more, preferably all, of the cells are cultured for a period sufficient to allow expression of the LYVE-1 marker in hepatic endothelial cells and / or expression of the PLVAP marker in hepatic endothelial cells and / or expression of MRP-2 in hepatocytes; and / or wherein one or more cells are cultured for a period of time sufficient to allow for the formation of fenestrations in liver tissue.

[0160] In some embodiments, a method according to the present invention is provided, wherein the gel precursor in step (b) and / or the culture medium in step (d) contain an antifibrinolytic agent. The antifibrinolytic agent is preferably selected from the group consisting of bovine pancreatic trypsin inhibitor, lysine analogs, coagulation factors (e.g., factor XIII), or serine protease inhibitors, such as aminocaproic acid, aprotinin, epsilon-aminocaproic acid, and tranexamic acid. The agent may be any one or combination of the antifibrinolytic agents mentioned herein. Alternative agents with antifibrinolytic activity may be appropriately selected and used by those skilled in the art. Preferably, the antifibrinolytic agent is aprotinin. Preferably, the agent, and the concentration of the agent, is provided during step (b) and / or step (d) of the method, thereby at least partially inhibiting the degradation of fibrinogen and / or fibrin in the scaffold. Furthermore, the agent preferably does not affect cell growth, differentiation, and / or proliferation and / or liver tissue production.

[0161] As described herein, the concentration of the agent depends on various parameters, such as the volume of the scaffold and the concentrations of fibrinogen and / or thrombin used. Preferably, an amount of aprotinin between about 10 and 1000 kIU (kallikrein inhibitor units) is used and provided in a suspension / solution in culture medium. For example, aprotinin can be provided in about 50 μL of culture medium. Thus, by way of example, aprotinin can be provided at a concentration between about 0.2 kIU / μL and about 20 kIU / μL, e.g., about 2 kIU / μL of aprotinin can be provided in either the culture medium, the gel precursor, or both.

[0162] Also provided is a method, in which the culture medium, preferably the culture medium used in step (d), contains any one or more agents selected from glucose, pyruvate, amino acids, preferably free amino acids, vitamins, salts, insulin, proteins, preferably cytokines and / or growth factors, such as, but not limited to, oncostatin M and / or VEGF. Preferably, the growth factors include at least FGF, EGF, and / or VEGF. The culture medium may be adjusted to be beneficial for cell differentiation and / or proliferation. Those skilled in the art know how to appropriately adjust the culture medium containing any one of the agents mentioned herein, selected from the group consisting of glucose, pyruvate, amino acids, preferably free amino acids, vitamins, salts, insulin, proteins, preferably cytokines and / or growth factors. Furthermore, those skilled in the art can routinely adjust the concentration of any one of the agents depending on the amount and / or cell type of cells maintained in culture in the culture device.

[0163] Furthermore, in some embodiments of the present invention, the culture medium preferably used in step (d) does not contain TGF-β inhibitor, hepatocyte growth factor, activin A, R-spondin protein, fibroblast growth factor (preferably fibroblast growth factor 10), epidermal growth factor, GSK-3 inhibitor, preferably CHIR99021. It is contemplated that any one of these disapproved drugs, for example, when used at a certain concentration in the culture medium, may have a negative impact on the acquisition of liver tissue by carrying out the method of the present invention.

[0164] In some embodiments, a method according to the present invention is provided, wherein the hepatic endothelial cells, hepatic stellate cells, and / or hepatocytes are mammalian cells (e.g., monkey, cat, dog, sheep, mouse, rat, human), preferably human cells, preferably human-derived cells, e.g., primary culture cells derived from a human subject (e.g., iCell Hepatocytes, Cellular Dynamics (Fujifilm)). The cells may be derived from a subject, preferably a human subject, suffering from a disease / disorder. The human-derived cells may be cryopreserved cells.

[0165] In some aspects, the liver tissue described herein is an artificial liver tissue.An artificial liver tissue can refer to a liver tissue that mimics natural tissue, but it is modified (obtained by using tissue engineering methods, for example, methods as provided herein), which is preferably cell-based and may be partially derived from stem cells, and in which at least a portion of the obtained liver tissue comprises a biopolymer matrix, such as a fibrin scaffold, to mimic a portion of natural liver tissue, as contemplated herein.

[0166] In some embodiments, a method according to the present invention is provided, wherein the liver tissue obtained in step (e) allows the accumulation of intracellular lipid droplets when cells, preferably the obtained liver tissue, are contacted with free fatty acids for a period of 12 to 48 hours. Preferably, a sufficient amount of free fatty acids is provided to allow the formation of lipid droplets. In one non-limiting example (FIG. 12B), the obtained liver tissue was exposed to approximately 1000 μM of free fatty acids (500 μM oleic acid and 500 μM palmitic acid). The free fatty acids may be provided together with or mixed into the culture medium. Any period between 12 and 48 hours for exposing the cells to the free fatty acids is contemplated and thus encompassed herein, for example, 12, 16, 20, 24, 26, 30, 32, 36, 40, 42, or 48 hours. Any free fatty acid and / or mixture of free fatty acids is contemplated and thus encompassed herein.

[0167] In an alternative embodiment, specifically when hepatocytes are provided in step (b) of the present method, the hepatocytes may be exposed to free fatty acids before the cells are provided in step (b).Thus, for example, the hepatocytes provided herein in step (b) may contain lipid droplets within the cells.Preferably, due to the accumulation of lipid droplets in the hepatocytes provided in the present method, at least some hepatocytes can still proliferate and / or differentiate, and optionally form into at least one or more structures typically present in healthy native liver tissue.In some uses of the present method, it is preferred that the concentration and / or duration of exposing hepatocytes to free fatty acids is sufficient to induce hepatic steatosis, which is indicated, for example, by the accumulation of lipid droplets in the liver.The accumulation of such lipid droplets in the liver tissue obtained and provided in the present invention will be considered by those skilled in the art to be indicative of metabolically capable liver tissue.

[0168] In some embodiments, methods according to the present invention are provided, further comprising adding a compound to a cell culture to observe its effect on the formation or function of liver tissue and / or any of the structures and / or cells therein (e.g., vasculature, fenestrations, bile ducts, bile canaliculi, polarized hepatocytes, stellate cells, and endothelial cells). It is contemplated that the expression of liver markers in the cells provided herein can be detected via immunofluorescence techniques and / or mRNA (PCR, single-cell RNA analysis). For example, secreted molecules in the supernatant, such as albumin produced by hepatocytes or CTGF produced by stellate cells and / or endothelial cells, may be detected and / or analyzed. Furthermore, compounds may be provided that are parent compounds, e.g., drugs, that are metabolized as a result of liver metabolism. Also, for example, free fatty acids may be added to the culture medium, suitable for testing / monitoring, for example, the accumulation of fat stores. Additionally, fluorescent dyes (eg, CMFDA, calcein-AM, etc.) may be added to observe polarized transport, for example, by adding them to the formed bile canaliculi.

[0169] The present invention further provides liver tissue obtained by the methods disclosed and embodied herein. Preferably, the liver tissue comprises a vasculature. The liver tissue obtained according to the methods of the present invention, more particularly, the liver tissue comprising a vasculature, may comprise any one or more of the following characteristics: a) a viable population of at least endothelial cells and stellate cells, preferably endothelial cells, stellate cells, and hepatocytes; b) a sinusoidal vascular network comprising a detectable lumen, preferably with a diameter between 5 μm and 30 μm, and / or comprising an at least partially fenestrated endothelium; c) dense parenchymal cell layers / sheets / groups, preferably containing epithelial morphology, at least partially located between vascular structures; d) polarized epithelia with transporters expressed either on the basolateral side facing blood vessels or on the apical side facing other parenchymal cells (e.g., the apical transporter MRP2); e) (polarized) bile canaliculus(-like) structures between two layers / sheets of hepatocytes; f) fluidly accessible liver tissue; g) Physically accessible liver tissue.

[0170] Preferably, a viable cell population is formed when at least about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% of the total number of cells in the population are viable. Cell viability may be tested by using methods known and commonly used in the art. A cell population is considered viable when it has at least a certain percentage of live, healthy cells within the population. Those skilled in the art can determine and classify a cell population as viable.

[0171] It is further contemplated that the liver tissue obtained by the method according to the present invention may comprise a sinusoidal vascular network. This vascular network preferably comprises a detectable lumen. The lumen can be detected by common means used in the art, and preferably has a diameter of between 5 and 30 μm, e.g., 10 μm, 15 μm, 20 μm, etc. It is further preferred that the hepatic sinusoidal endothelial cells (i.e., endothelial cells included herein in forming hepatic sinusoidal (like) structures) comprise at least partially fenestrated endothelium (i.e., comprising fenestrations and / or fenestration-like structures).

[0172] It is also contemplated that at least a portion of the parenchymal cells (i.e., primarily hepatocytes) provided herein form at least one, and preferably more, layers / sheets / clusters of hepatocytes. Preferably, the cells form in a manner similar to native liver epithelial morphology, i.e., in compact or densely packed hepatocytes between at least two tubules and / or tubule-like structures.

[0173] Furthermore, the polarized epithelium formed as part of the liver tissue is preferably at least partially, preferably at least 50%, 60%, 70%, 75%, 80%, or 90%, formed by hepatocytes. The polarized epithelium may also contain transporters found at least partially in native liver tissue. Thus, preferably, the transport proteins include one or more of Ntcp, Bsep, MRP-2, Mdr1, Mdr2, AE2, AQP-8, ASBT, and CFTR.

[0174] Preferably, the obtained liver tissue is fluidly and / or physically accessible. This may include, for example, being able to achieve perfusion through the obtained liver tissue, e.g., from a fluid inlet to a fluid outlet. Fluid flow may be achieved, for example, by allowing fluid to flow from one lateral microfluidic channel to another, thereby allowing liquid to flow in / through (the vasculature of) the obtained liver tissue. The obtained liver tissue may also include being able to add and / or extract, for example, soluble factors or drugs, e.g., by mixing them with fluids or culture media. The obtained liver tissue may also be accessible, for example, with specific drugs and / or tools, such as, but not limited to, antibodies, nanobodies, cells, fluorescent stains, nanocapsules, nanosensors, etc., to test the liver tissue and / or use parts of the liver tissue in tests, e.g., drug metabolism screening.

[0175] The present invention further provides a culture device comprising liver tissue obtained by the methods disclosed and embodied herein. The culture device is preferably a microfluidic culture device. Preferably, the microfluidic culture device comprises any one of the features disclosed and described herein. The microfluidic device preferably comprises at least one culture chamber and at least one, preferably two, microfluidic channels adjacent to the culture chamber. An exemplary schematic diagram (not to scale) of a suitable microfluidic device is found in Figure 2.

[0176] Finally, also provided are uses of the methods, liver tissue, and / or culture devices disclosed and embodied herein. The uses may be in an in vitro and / or ex vivo setting, but may be for example, but not limited to, any one of the following uses: testing hepatotoxicity, including drug-induced liver injury, nanoparticle toxicity, environmental exposures, such as alcohol and diet, including exposure to biologics; testing pharmacokinetics, including distribution, secretion, and metabolism of drugs, detoxification of xenobiotics; evaluation of drug-drug interactions; testing for gene therapy and cell therapy; and enzyme replacement therapy for metabolic disorders; testing for oxidative stress; testing the uptake and biological effects of microparticles, lipids, amino acids, and / or carbohydrates. Examining liver function in each cell type, including metabolism, vitamin and mineral storage, and production of macromolecules and micromolecules (e.g., albumin, vLDL, glucose, cholesterol); Examining liver disease as a result of genetic, drug-induced, or environmental factors, such as fatty liver, fibrosis, cirrhosis, hepatocellular carcinoma, cholestasis, hypertension, capillary formation, cholangiocarcinoma, polycystic liver disease, etc.; Examining liver disease as a result of infection, such as hepatitis virus, malaria parasites, bacteria, etc.; Examining whether the liver harbors primary metastatic tumors or serves as a metastatic niche for circulating cancer cells. studying the metastasis of hepatocytes acting as a niece, studying the interaction between hepatocytes and immune components, studying liver infection with infectious agents (e.g., bacteria, viruses, fungi, protozoa), studying the cellular circulation of cells (e.g., immune cells) through the vasculature of liver tissue, studying the transduction and / or transfection of liver tissue cells with nucleic acid carriers, preferably viral vectors (e.g., adeno-associated viruses), studying fibrosis in liver tissue, studying liver segmentation, preferably hepatic sinusoidal segmentation, studying liver regeneration, studying liver development, studying polar transport and bile production and secretion, studying molecules in hepatic bile fractions, studying nutrient storage, studying the effects of fluid flow and shear stress, studying the effects of radiation, studying host-graft interactions, studying biological effects after exposure to patient-derived samples (e.g., serum, plasma, whole blood).

[0177] Preferably the use is one selected from the group consisting of: testing hepatotoxicity, testing pharmacokinetics, testing detoxification of xenobiotics, assessing drug-drug interactions, testing gene and / or cell therapy, testing enzyme replacement therapy, testing oxidative stress, testing uptake and biological effects of (micro)particles, testing liver function, testing liver diseases, testing cancer in the liver, testing metastasis in the liver, testing cell interactions, testing liver infections by infectious agents (e.g. bacteria, viruses, fungi, protozoa), cell circulation of cells through the vasculature of liver tissue (e.g. studying liver tissue cells, studying liver function, studying liver function, studying liver function (immune cells), studying transduction and / or transfection of liver tissue cells with nucleic acid carriers, preferably viral vectors (e.g., adeno-associated viruses), studying fibrosis in liver tissue, studying liver segmentation, preferably hepatic sinusoidal segmentation, studying liver regeneration, studying liver development, studying polar transport and bile production and secretion, studying hepatic bile fractionation, studying nutrient storage, studying the effects of fluid flow and shear stress, studying the effects of radiation, studying host-graft interactions, and studying the effects after exposure to patient-derived samples (e.g., serum, plasma, whole blood).

[0178] Also embodied herein is a method for introducing cells into liver tissue or into a culture device as disclosed in this description, comprising adding the cells to the obtained liver tissue, preferably to the top of the culture chamber, under conditions that allow the cells to attach to the tissue, migrate, and optionally differentiate or specialize. Alternatively, the cells can be introduced in at least one microfluidic channel and perfused into the vascularized liver tissue in the cell culture chamber.

[0179] The manner of adding these cells has been previously disclosed earlier in this description when referring to the embodiment of the method for obtaining liver tissue.

[0180] The foregoing description of specific embodiments fully reveals the general nature of the invention so that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt such specific embodiments for various applications without undue experimentation and without departing from the general concept of the invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0181] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are hereby incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited references. In addition, the entire contents of the references cited within the references cited herein are also hereby incorporated by reference in their entirety.

[0182] It is to be understood that the phraseology or terminology used herein is for the purpose of description and not of limitation, and that the phraseology or terminology used herein should be interpreted by the skilled artisan in light of the teaching and guidance presented herein, in combination with the knowledge of those skilled in the art.

[0183] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of an embodiment of the invention are equally applicable to any other aspect or embodiment of the invention, and therefore it is not necessary to separately recite all such details, embodiments, and preferences for every aspect.

[0184] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limitations of the invention. Further aspects and embodiments will be apparent to those skilled in the art.

[0185] Example material cell [Table 1]

[0186] reagent [Table 2]

[0187] device [Table 3]

[0188] Medium preparation Complete DMEM Dulbecco's modified Eagle's medium (DMEM, Gibco, catalog number 11965-092) was supplemented with 10% FBS (Gibco), 1% penicillin-streptomycin (P / S, Sigma-Aldrich, catalog number P4333-100ML), and 1 mM sodium pyruvate (Thermo Fisher Scientific).

[0189] iHEP plating medium. IHep plating medium was prepared according to the manufacturer's instructions (Cellular Dynamics, Fujifilm). Briefly, 96 mL of RPMI media (Sigma-Aldrich, catalog number R8758-500ML) was supplemented with 2 mL of B27 supplement 50X (Gibco, catalog number 17404-001, lot number 2209534) (final concentration 1X), 0.2 mL of oncostatin M 10 μg / mL (final concentration 20 ng / mL), 2 μL of dexamethasone 5 mM (MP Biomed, catalog number 194561) (final concentration 0.1 μM), 50 μL of gentamicin 50 mg / mL (Gibco, catalog number 15750-060) (final concentration 25 μg / mL), and 2 mL of iCell Hepatocyte 2.0 media supplement (Cellular Dynamics, catalog number M1024, lot number 106063) (final concentration 1X).

[0190] EGM-2 medium Endothelial Cell Growth Medium-2 Bullet Kit (EGM-2, Lonza, catalog number CC-3162) was prepared as follows: Endothelial Cell Growth Basal Medium-2 (EBM-2, Lonza, catalog number CC-3156) was supplemented with Large Vessel Endothelial Cell Growth Medium SingleQuots and growth factors (Lonza, catalog number CC-4146). The kit contained 10 mL of FBS, 0.20 mL of hydrocortisone, 2 mL of hFGF-B, 0.5 mL of VEGF, 0.5 mL of R3-IGF-1, 0.5 mL of ascorbic acid, 0.5 mL of hEGF, 0.5 mL of GA-1000, and 0.5 mL of heparin.

[0191] procedure Cell banking Cell banking for LDEC (liver endothelial cells, ScienCell) was performed as follows: Prior to thawing, T75 orange-cap flasks were coated with fibronectin at 33.3 μg / mL for 2 hours. To obtain a 33.3 μg / mL fibronectin solution, 150 μL of 1 mg / mL fibronectin was diluted in 5 mL of PBS. Using 5 mL of 33.3 μg / mL fibronectin in a T75 flask results in a coating of 1 μg / mL. After 2 hours of incubation at 37°C, the fibronectin solution was removed. This fibronectin solution was routinely stored and used again for coating, after which it was discarded. After removing the fibronectin solution, the flask was washed 1× with PBS. 14 mL of ECM (Sciencell) was then added to the flask, and the flask was then placed back into the incubator to allow the medium to equilibrate. Next, a P1 vial of LDEC (Sciencell) was removed from -150°C storage and immediately placed in a 37°C water bath. After the contents of the vial were thawed, they were transferred directly into a fibronectin-coated T75 flask, which was then placed back into the incubator. 18-24 hours after the thawing procedure, the medium was aspirated and replaced with fresh, prewarmed ECM (Sciencell). Subsequent medium changes were performed every other day. LDEC were maintained in these flasks until D6 (thawing day = day 0). On day 6, cell banking was initiated by aspirating the medium and washing the cells with PBS. A 0.013% trypsin / EDTA solution was then prepared by diluting 0.5 mL of 0.25% trypsin / EDTA in 9.5 mL of PBS. Trypsin solution was added to the cells for 5 minutes at 37°C, after which 5 mL of trypsin neutralizing solution (TNS) was added to the flask, and the total volume of 10 mL was transferred to a 50 mL falcon containing 10 mL of FBS. The flask was then washed with another 5 mL of TNS, which was then transferred to the falcon. Cells were counted from the total volume of 25 mL using a Luna automated cell counter. The cell solution was then centrifuged at 1000 RPM for 5 minutes, after which the supernatant was aspirated.The cells were then resuspended in a chilled freezing solution consisting of 20% FBS, 10% DMSO, and 70% ECM and transferred to cryovials. The cryovials were then placed in Mr. Frosty and placed in a -80°C freezer. After 24 hours, the cryovials were transferred to -150°C. The cell banking procedure for HHStECs was nearly identical. Instead of a fibronectin coating, a poly-l-lysine coating was used at 1 μg / cm. 2 and StECM was used instead of ECM. Additionally, cells were trypsinized using 0.025% trypsin / EDTA instead of 0.013%.

[0192] Cell subculture HHSTeCs (HSCs) (hepatic stellate cells, ScienCell) (passages 2 > 3 or 3 > 4) and LDECs (passages 2 > 3) were thawed in 14 mL of warm StECM and ECM, respectively. The cells were then seeded into T75 orange flasks coated with poly-l-lysine (HHSTeC) or fibronectin.

[0193] HHSteCs and LDECs (passages 2 to 3) were thawed in 14 mL of warm SteCM and ECM, respectively. The cell suspension was transferred into T-75 flasks coated (for 2 h) with 150 μL of poly-L-lysine in 10 mL of MilliQ water (HHSteCs) or 5 mL of fibronectin in PBS (HHSECs, hepatic endothelial cells, ScienCell). Medium was refreshed for 24 h after plating and every 2–3 days for a total of 5 days after seeding.

[0194] Preparation of cell suspension iHep (iPSC-derived hepatocytes, Cellular Dynamics (Fujifilm)) were thawed in a 10 mL tube containing warm iHep plating medium. The iHep vial was thawed in a water bath for exactly 3 minutes, and the contents were then transferred to a tube containing 10 mL of warm iHep plating medium. The tube was centrifuged at 200 g for 3 minutes. The supernatant was discarded, the pellet was resuspended in 2 mL of plating medium, and the cells were counted using the trypan blue exclusion method with a LUNA cell counting device. The iHep were then resuspended in complete DMEM to a final concentration of 25,000 cells / μL.

[0195] HHSteCs were dissociated using 9 mL of PBS + 1 mL of 2.5% trypsin for 3 minutes. LDECs were dissociated using 8 mL of PBS + 2 mL of 2.5% trypsin for 3-5 minutes. The trypsin was then neutralized using 5 mL of TNS. An additional 5 mL of TNS was used to rinse the flask and collect the cells in the same tube. Cells were then counted using the trypan blue exclusion method with a LUNA cell counting device. The cell suspension was centrifuged at 200 g for 5 minutes and resuspended in DMEM (10% FBS, 1% P / S) to a final concentration of 8,125 cells (HHSTeC) / μL and 16,250 cells (LDEC) / μL.

[0196] Seeding and maintenance of OrganoPlates. A mixture of 10 μL of HHSTeC (final concentration: 1625 cells / μL), 10 μL of LDEC (final concentration: 3250 cells / μL), 20 μL of iHep (final concentration: 10000 cells / μL), 5 μL of 50 mg / mL fibrinogen (final concentration: 5 mg / mL), and 5 μL of 1 U / mL thrombin (final concentration: 0.1 U / mL) was prepared in a tube and mixed vigorously. 1.35 μL of this cell suspension was introduced through the transplantation chamber hole in the OrganoPlate Graft plate using a Sartorius P10 pipette with minimal dispensing speed. The plate was then placed in a humidified incubator (37°C, 5% CO2) for 10 minutes. 50 μL of DMEM (10% FBS, 1% P / S) was added to the transplantation chamber. After 1-2 hours, the medium in the transplantation chamber was replaced with iHep plating medium containing 100 kIU aprotinin. 50 μL of EGM-2 was added to each well of the perfusion channel. The plates were kept stationary in a humidified incubator, and a complete medium change was performed every 2-3 days for a total of 7 days.

[0197] Calcein-AM staining. On day 7 of culture, a 1:100 mixture of calcein AM in EGM-2 was prepared and added to each well of some chips. The plates were incubated in a humidified incubator for 20 minutes and imaged using a confocal microscope (10x magnification).

[0198] result The results obtained by the methods provided herein are shown in the accompanying figures and figure legends.

Claims

1. 1. An in vitro method for obtaining liver tissue, comprising: a) providing a culture device, wherein said culture device comprises at least a culture chamber; b) providing a gel precursor to the culture chamber, wherein the gel precursor comprises at least fibrinogen, and wherein the gel precursor further comprises hepatic endothelial cells and hepatic stellate cells; c) gelling the gel precursor to obtain a scaffold, wherein the scaffold comprises the cells of step (b); d) contacting the scaffold of step (c) with cell culture medium; and e) culturing cells in the scaffold to obtain liver tissue, preferably wherein the liver tissue is present in the scaffold; A method comprising:

2. The method of claim 1 , wherein the gel precursor of step (b) further comprises hepatocytes.

3. 3. The method of claim 1 or 2, wherein the gel precursor of step (b) further comprises one or more cells selected from the group consisting of immune cells, preferably liver-resident immune cells, and tumor cells.

4. The method according to any one of claims 1 to 3, wherein the culturing in step (e) provides liver tissue comprising a vasculature formed by at least hepatic endothelial cells and hepatic stellate cells.

5. 5. The method according to any one of claims 1 to 4, wherein the culturing in step (e) provides liver tissue, preferably comprising a vasculature, comprising one or more sinusoidal structures formed by at least hepatic endothelial cells and hepatic stellate cells, preferably in which the sinusoidal structures have an average diameter of between 1 and 50 μm, more preferably between 5 and 30 μm, even more preferably about 10 μm.

6. 6. The method of any one of claims 1 to 5, wherein the liver tissue, preferably comprising vasculature, further comprises polarized hepatocytes, wherein said polarized hepatocytes are aligned to form bile canaliculi or bile canaliculus-like structures.

7. 7. The method of any one of claims 1 to 6, wherein the polarized hepatocytes are aligned such that the basal side of the polarized hepatocytes is adjacent to the vasculature and / or the apical side of the polarized hepatocytes is adjacent to bile canaliculi or bile canaliculus-like structures.

8. The method of any one of claims 1 to 7, wherein the polarized hepatocytes form layers / sheets / clusters of cells.

9. 9. The method of any one of claims 1 to 8, wherein the bile canaliculi or bile canaliculus-like structures are at least partially located between the apical sides of two or more layers / sheets / groups of polarized hepatocytes.

10. 10. The method according to any one of claims 1 to 9, wherein after step (e) of culturing the cells in the scaffold to obtain liver tissue, one or more of undifferentiated cells, immune cells, infectious agents, nucleic acid carriers, organoids, tumor cells, and test compounds are added to the obtained liver tissue.

11. 11. The method according to any one of claims 1 to 10, wherein the culture device comprising the culture chamber is a microfluidic culture device, preferably wherein said microfluidic culture device comprises at least one, preferably at least two microfluidic channels fluidly connected to said culture chamber, preferably wherein said at least one, preferably at least two microfluidic channels comprise an inlet and an outlet.

12. 12. The method of any one of claims 1 to 11, wherein the culture device is a microfluidic culture device comprising a culture chamber and further comprising at least one or at least two microfluidic channels fluidly connected to the culture chamber, and wherein after step (e) of culturing cells in the scaffold to obtain liver tissue, one or more of undifferentiated cells, immune cells, infectious agents, nucleic acid carriers, tumor cells, and test compounds are added to the at least one microfluidic channel.

13. 13. The method according to any one of claims 1 to 12, wherein the culture device is a microfluidic culture device comprising a culture chamber and further comprising at least one or at least two microfluidic channels fluidly connected to said culture chamber, and wherein endothelial cells, preferably hepatic endothelial cells, are cultured in the at least one or at least two microfluidic channels, preferably to obtain tubules or tubule-like structures, more preferably blood vessels and / or blood vessel-like structures.

14. 14. The method according to any one of claims 1 to 13, wherein the culture device is a microfluidic culture device comprising a culture chamber and at least two microfluidic channels, wherein the at least two microfluidic channels are each independently fluidly connected to the culture chamber, and wherein the culture chamber connects the two microfluidic channels to each other, and wherein the liver tissue obtained in step (e) is perfusable, thereby allowing liquid to flow from one microfluidic channel to the other microfluidic channel.

15. The method according to any one of claims 1 to 14, wherein the culture device comprising the culture chamber is a unidirectional flow microfluidic culture device.

16. A culture device comprising a culture chamber is a unidirectional flow microfluidic culture device comprising at least one microfluidic network, comprising: (a) a microfluidic layer comprising a first flow path and a second flow path; and (b) a reservoir layer disposed on the microfluidic layer and comprising first and second reservoirs, wherein the first and second reservoirs have access ports to the first and second flow paths, respectively, and the first and second flow paths form a fluid circuit with the first and second reservoirs; 16. A method according to any preceding claim, wherein the access ports in the first and second reservoirs are spaced apart, and wherein, in use, tilting the device at a first angle induces a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir predominantly via a first flow path, and adjusting the tilt so as to tilt the device at a second angle causes a volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir predominantly via a second flow path.

17. A culture device comprising a culture chamber, the culture device comprising at least one microfluidic network, the method comprising: (a) tilting the device at a first angle by rotating about a first axis to induce a volume of fluid in a first reservoir to flow from the first reservoir to a second reservoir through a first flow path; and 17. The method of any one of claims 1 to 16, comprising: (b) adjusting the tilt such that the device tilts at a second angle by rotating about a first axis, thereby causing a volume of fluid in a second reservoir to flow from the second reservoir to the first reservoir via a second flow path.

18. 18. The method of any one of claims 1 to 17, wherein openings are formed in the scaffold during step (c) and / or after step (e), preferably wherein said openings allow the introduction of solutions, fluids or drugs.

19. The method according to any one of claims 1 to 18, wherein the hepatic endothelial cells, hepatic stellate cells, and / or hepatocytes are not separated from each other by an artificial membrane.

20. 20. The method of any one of claims 1 to 19, wherein the total number of cells contained in the gel precursor is at least 1,000 cells per microliter, preferably at least 5,000 cells per microliter, more preferably at least 7,500 cells per microliter, even more preferably between 8,000 and 15,000 cells per microliter, and / or up to 100,000 cells per microliter, preferably up to 50,000 or 30,000 cells per microliter, and / or the ratio of the number of hepatic endothelial cells to hepatic stellate cells is equal to or greater than 20:1, with increasing preference equal to or greater than 15:1, equal to or greater than 10:1, equal to or greater than 4:1, equal to or greater than 1:1, or equal to or greater than 0.5:

1.

21. 21. The method according to any one of claims 1 to 20, wherein the ratio of the number of hepatocytes to the sum of the number of hepatic endothelial cells and hepatic stellate cells is greater than 1:1, preferably greater than 2:1, preferably between 1:1 and 30:1, preferably between 1:1 and 10:1, or between 1:1 and 5:

1.

22. The gel precursor of step (b) contains, per microliter: Between 1,000 and 10,000 hepatic endothelial cells; and - A method according to any one of claims 1 to 21, comprising between 100 and 10,000 hepatic stellate cells.

23. 23. The method of any one of claims 1 to 22, further comprising between 5,000 and 50,000 hepatocytes per microliter of gel precursor.

24. The method according to any one of the preceding claims, wherein the gel precursor of step (b) has a volume between 0.1 μL and 50 μL, preferably between 0.5 μL and 20 μL.

25. The method according to any one of claims 1 to 24, wherein the gel precursor in step (b) further comprises any one or more selected from the group consisting of a basement membrane matrix gel precursor, an extracellular matrix gel precursor, and a collagen gel precursor; and / or the scaffold in step (c) comprises a collagen scaffold and / or a fibrin scaffold.

26. 26. The method of any one of claims 1 to 25, wherein the gel precursor of step (b) comprises fibrinogen in a concentration of at least 1 mg / mL, preferably up to 50 mg / mL, more preferably about 5 mg / mL.

27. 27. The method of any one of claims 1 to 26, wherein the gel precursor of step (b) further comprises thrombin, preferably wherein the gel precursor of step (b) comprises thrombin at a concentration of at least 0.01 U / mL, preferably at most 1 U / mL, more preferably about 0.1 U / mL.

28. 28. The method according to any one of the preceding claims, wherein the culture medium in step (d) has a volume between 20 μL and 500 μL and / or the culture chamber has a volume between 0.1 μL and 50 μL, preferably between 0.1 μL and 20 μL.

29. 29. The method of any one of claims 1 to 28, wherein the gel precursor of step (b) and / or the culture medium of step (d) comprises an antifibrinolytic agent, preferably wherein the antifibrinolytic agent is selected from the group consisting of bovine pancreatic trypsin inhibitor, a lysine analogue, a coagulation factor (e.g., factor XIII), or a serine protease inhibitor, more preferably wherein the antifibrinolytic agent is aprotinin.

30. The method according to any one of claims 1 to 29, wherein the hepatic endothelial cells, hepatic stellate cells and / or hepatocytes are human cells, preferably cells of human origin.

31. 31. The method of any one of claims 1 to 30, wherein the culture medium in step (d) is changed every 24 to 144 hours and / or the cells are cultured with increasing preference for at least 3, 4, 5, 6, or 7 days.

32. The gel precursor of step (b) further comprises hepatocytes; and wherein one or more, preferably all, of the cells are cultured for a period sufficient to allow the formation of vasculature, preferably wherein said vasculature is accessible and / or has a diameter of about 5-50 μm; and / or wherein one or more, preferably all, cells are cultured for a period sufficient to allow the formation of polarized hepatocytes, preferably layers / sheets / clusters of polarized hepatocytes, wherein the basal side of said polarized hepatocytes is adjacent to the vasculature and / or the apical side of said polarized hepatocytes is adjacent to bile canaliculi and / or bile canaliculus-like structures; wherein one or more, preferably all, of the cells are cultured for a period of time sufficient to allow the formation of bile canaliculi and / or bile canaliculus-like structures; and / or wherein one or more, preferably all, of the cells are cultured for a period sufficient to allow expression of the LYVE-1 marker in hepatic endothelial cells and / or expression of the PLVAP marker in hepatic endothelial cells and / or expression of MRP-2 in hepatocytes; and / or wherein one or more, preferably all, of the cells are cultured for a period of time sufficient to allow the formation of fenestrations in the liver tissue; 32. The method according to any one of claims 1 to 31.

33. 33. The method of any one of claims 1 to 32, wherein the liver tissue obtained in step (e) allows intracellular lipid droplet accumulation when the cells are contacted with free fatty acids for a period of between 12 and 48 hours.

34. 34. The method according to any one of claims 1 to 33, wherein the culture device comprising the culture chamber is a unidirectional flow, preferably a microfluidic culture device, and wherein the liver tissue obtained in step (e) allows for unidirectional flow of culture medium through the liver tissue and results in absorption or metabolism of compounds in the medium that mimics the first pass effect observed in liver tissue in vivo.

35. 35. The method of any one of claims 1 to 34, further comprising adding a compound to the cell culture and observing its effect on the formation or function of liver tissue, preferably including the vasculature.

36. Liver tissue, preferably comprising vasculature, obtainable by the method according to any one of claims 1 to 35.

37. 37. The liver tissue of claim 36, further comprising one or more sinusoidal structures, one or more bile canaliculus-like structures, one or more fenestrations and / or one or more bile duct-like structures.

38. A culture device, preferably a microfluidic culture device, containing liver tissue, preferably containing vasculature, obtained by a method according to any one of claims 1 to 35.

39. 39. The culture device of claim 38, which is a unidirectional flow microfluidic culture device.

40. testing hepatotoxicity, testing pharmacokinetics, testing xenobiotic detoxification, evaluating drug-drug interactions, testing gene and cell therapy, testing enzyme replacement therapy, testing oxidative stress, testing uptake and biological effects of (micro)particles, testing liver function, testing liver diseases, testing cancer in the liver, testing cancer metastasis, preferably testing cancer metastasis in the liver, testing cell interactions, testing liver infections with infectious agents, testing cell circulation of cells through the vasculature of liver tissue, testing transduction and / or transfection of liver tissue cells with nucleic acid carriers, preferably viral vectors. and use of the method of any one of claims 1 to 35, the liver tissue of claim 36 or 37, and / or the culture device of claim 38 or 39 in any one use selected from the group consisting of: studying fibrosis in liver tissue, studying liver segmentation, preferably liver sinusoidal segmentation, studying liver regeneration, studying liver development, studying polar transport and bile production and secretion, studying hepatic bile fractionation, studying nutrient storage, studying the effect of fluid flow and shear stress, studying the effect of radiation, studying host-graft interactions, and studying the effect after exposure to a patient-derived sample (e.g. serum, plasma, whole blood).

41. A method for introducing cells into liver tissue according to claim 36 or 37 or into a culture device as disclosed in claim 38 or 39, comprising adding the cells to the obtained liver tissue, preferably by adding them to the top of the culture chamber, under conditions that allow the cells to attach to the tissue, migrate and, optionally, differentiate or specialize.

42. 42. The method of claim 41, wherein the introduced cells are immune cells, preferably monocytes.

43. 43. The method of claim 41 or 42, which allows the introduced cells to differentiate completely or partially towards a specific cell type, preferably Kupffer cells.

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