Liver vasculature

Abstract

The present invention relates to a method for generating a liver tissue in a cell culture device. In the method a gel precursor at least comprising fibrinogen, liver stellate cells and liver endothelial cells is provided in the cell culture device. The cells may form a liver tissue that resembles native liver tissue. The liver tissue may comprise a vascular network comprising liver sinusoids and further can comprise bile canaliculi(-like) structures and fenestrations. Also provided herein is a microfluidic culturing device comprising said liver tissue.

Description

[0001] LIVER VASCULATURE

[0002] FIELD OF THE INVENTION

[0003]

[0001] This invention pertains in general to a method for generating a liver tissue in a cell culture device, wherein the liver tissue closely resembles native liver tissue. In some embodiments methods are provided wherein liver tissue comprising vasculature, that may be perfusable, is obtained. In other embodiments methods are provided wherein liver tissue comprising such vasculature and sinusoidal-like structures are obtained. In further embodiments methods are provided wherein liver tissue comprising vasculature, sinusoidal-like structures and bile canaliculus-like structures are obtained. The invention further pertains to a culturing device comprising the liver tissue as obtained by any one of the methods described herein, as well as to the use thereof.

[0004] BACKGROUND OF THE INVENTION

[0005]

[0002] The background description 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.

[0006]

[0003] In vivo blood vessels are vascular structures comprising endothelial cells forming the inner cellular lining of the blood vessels (e.g., arteries, veins, and capillaries), which 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 a major role in metabolism, detoxification, protein synthesis and activate innate immunity, e.g., by secreting innate immunity proteins into the bloodstream. In vivo, these capillary-like structures referred to as sinusoids are low pressure vascular channels that receive blood from terminal branches of the hepatic artery and portal vein at the periphery of lobules and deliver it into central veins. A schematic example of a liver structure is shown in Figure 1. Hepatic sinusoids are notoriously leaky, aiding the diffusion of molecules and particles from the blood through a thin extracellular matrix layer (also called Space of Disse) towards the basolateral side of polarized hepatocytes. The liver sinusoidal vasculature is supported by stellate cells (residing in the Space of Disse (also referred to as perisinusoidal space), between the endothelial cells and hepatocytes) which are often referred to as liver pericytes. Hepatic stellate cells residing in the perisinusoidal space, between sinusoidal endothelial cells and hepatocytes, store vitamin A, and are believed to regulate sinusoidal circulation.

[0007]

[0004] Hepatocytes are polarized cells and play pivotal roles in metabolism, detoxification, and protein synthesis. Hepatocytes also activate innate immunity against invading microorganisms by secreting innate immunity proteins. Hepatocytes transport molecules (often metabolites of parent compounds originating after a series of enzymatic reactions) either back into the blood through their basolateral side or through their apical side into the space between other hepatocytes. Hepatocytes are polarized cells that form a network of bile canaliculi (BC) with their apical surfaces, enabling secretion and transport of bile. Such bile canaliculi eventually drain into larger bile ducts transporting this fluid back into the intestinal track.

[0008]

[0005] Liver sinusoids comprise therefore an intricate, leaky endothelial network supported by stellate cells and lined by polarized hepatocytes.

[0009]

[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 included in separated 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).

[0010]

[0007] It is known that cells such as human umbilical vein cells (HLIVECs) and normal human lung fibroblasts (NHLFs), when seeded as single cells and cultured in a biomimetic scaffold interact and self-organize into vascular networks, for example in a microfluidic channel. The cells interact and rearrange, creating an intricate and perfusable microvascular system in a process similar to the embryonic emergence of vascular structures called “vasculogenesis.” This has led, for example, to the formation of vascularized tumour or organoid systems. However, the majority of these vascular systems rely on readily available cell sources (such as HLIVECs, dermal endothelium, lung fibroblasts, mesenchymal stem cells) which do not represent an organ-specific phenotype. Thus, there yet has to be provided for a liver model that closely resembles a native liver, preferably one that mimics the spatial organization and functionality of a native liver as close as possible. As of yet no such liver model has been developed successfully. So, there is a need in the field to develop a more defined and predictive method for in vitro culturing and obtaining a liver tissue, in which the proliferation and the differentiation of cells, and the spatial organisation and / or functionality of the different cells is closely mimicking 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 allow to provide such a liver tissue model. Accordingly, the technical problem underlying the present invention can been seen in the provision of such products, compositions, methods and uses for complying with any of the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

[0011] SUMMARY OF THE INVENTION

[0012]

[0008] As embodied and broadly described herein, the present invention is directed to the surprising finding that by providing a gel precursor comprising fibrinogen, liver stellate cells and liver endothelial cells to a culturing device, allowing the gel precursor to gelate into a scaffold and culturing the cells in the scaffold a liver tissue is obtained that closely resembles native liver tissue, for example the spatial organization cells, including liver endothelial cells and liver stellate, and the forming of structures that resemble liver tissue(-like) structures such as, at least, liver sinusoids and bile canaliculi. In addition, it was surprisingly found that the cells forming the obtained liver tissue express relevant hepatic markers (e.g., MRP-2, LYVE-1 , PLVAP). In addition, the obtained liver tissue appears to be metabolically competent, e.g., as indicated by the accumulation of lipid droplets (e.g., examplified in Fig. 12B), mimicking hepatic steatosis.

[0013]

[0009] Therefore, in an aspect, the invention provides for an in vitro method for obtaining a liver tissue, particular a liver tissue that closely mimics native human liver tissue. With the invention cells are provided and cultured in a culturing device in such a way that a liver tissue comprising vasculature may be formed, comprising structures that closely resemble liver blood vessels or liver blood vessel-like structures, such as liver sinusoids. Therefore, in one aspect, a liver tissue comprising liver sinusoidal- like) structures is formed. Moreover, a liver tissue comprising bile ducts, bile canaliculi or bile canaliculi(-like) structures may be formed. With the invention liver stellate cells and liver endothelial cells allow for the formation of a liver tissue as described herein, i.e. , liver tissue comprising spatial organization of liver structures that closely mimics native liver tissue. Other cells, preferably cells commonly found in native liver tissue, may be included in the method of the invention as well, such as, but not limited to hepatocytes, Kupffer cells, immune cells and the like.

[0014]

[0010] In a next aspect the invention provides for a liver tissue, preferably comprising a vasculature, obtained by the method as described and embodied herein.

[0015]

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

[0016]

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

[0017]

[0013] In a further aspect the invention provides for a method of introducing cells to a liver tissue as embodied herein, or to a culturing device as disclosed herein (comprising such liver tissue), comprising adding the cells to the obtained liver tissue, preferably by adding the cells on top of the culturing chamber, under conditions to allow the cells to attach to the tissue, migrate and optionally differentiate or to specialize.

[0018]

[0014] In a last aspect the invention provides for uses of the method, the liver tissue and / or the culturing device as described and embodied herein.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

[0015] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0021]

[0016] Figure 1 : Schematic representation of the lay-out of a liver lobule, comprising a bile ductule (also bile canaliculus), a sinusoid and hepatocytes.

[0022]

[0017] Figure 2: Schematic drawing of the microfluidic system (OrganoPlate Graft®, MIMETAS B.V., the Netherlands) (not to scale). The OrganoPlate Graft® comprises a three-channel system, with phaseguides (205) between the large middle channel (200) (graft chamber, middle) and the adjacent lateral perfusion lanes (202) (microfluidic channels) (left, right). The graft chamber may have an opening (201), optionally a circular one. This opening can function as a capillary pressure barrier e.g., as described in WO2017216113 A2. Further the graft chamber may be connected to an inlet (206). The lateral perfusion lanes may be connected to reservoirs (203, 204) and can be used to add other cells, such as endothelial cells, that may form tubule(-like) structures, at least in the perfusion lane, to supplement the cell culture, for example the cells residing in the graft chamber, with medium.

[0023]

[0018] Figure 3: Schematic drawings of vasculogenesis in the microfluidic system (OrganoPlate Graft®) (not to scale) comprising a graft chamber (300), perfusion lanes (302) and an opening (301).

[0024]

[0019] Figure 4: Liver stromal cell-derived vascular network in fibrin (A) vascular network formed by 5416 cells I pl. (B) vascular network comprising 8125 cells I pl. (C) vascular network formed by 12188 cells I pl. The staining comprises CD31 (green) for aSMA (red), and DAPI (blue).

[0025]

[0020] Figure 5: LDECs (liver-derived endothelial cells) monoculture in fibrin with ECGM-2 medium (A) and fibroblast-conditioned medium (B). When cultured alone, LDECs do not appear to be substantially capable of generating an extended vascular network. This appears to be improved upon addition of fibroblast-conditioned medium.

[0021] Figure 6: Vascular networks formed by different ratios of liver-derived endothelial cells (LDEC) and stellate cells (HSC) (A) ratio LDEC : HSC 1 :1 , (B) ratio LDEC : HSC 2:1 , (C) ratio LDEC : HSC 4:1 , (D), ratio LDEC : HSC 20:1. Aprotopin may be added to prevent fibrin degradation. The concentration of cells used was 6500 LDECs / pl and 6500 HSCs / pl. The provided volume was 1.4pl / per chip; so, 9100 LDECs / chip and 9100 HSCs / chip.

[0026]

[0022] Figure 7: Calcein-AM staining, after 7 days of culturing, reveals live cultures revealing the presence of hepatocytes surrounded by vascular structures (A, B) and sinusoidal-like architecture and dimensions (C - E). In the culture (A, B) vascular structures where evident throughout a compact population of cells, with clear hepatocyte identity. The vessels, in close contact to hepatocytes, appear to possess an architecture reminiscent of liver sinusoids, with an average diameter of about 10 pm (micrometre) which matches with the reports of human hepatic sinusoid dimension (C - E). Calcein-AM, used to visualize live cells, is also a substrate for MRP-2, an apically-expressed transporter in mature hepatocytes. Hepatic structures contained polarized hepatocytes and an accumulation of Calcein-AM was observed in the (apical) spaces between hepatocytes indicating the presence of bile canaliculi. This indicates that, using the method according to the invention, the hepatocytes are polarized, with a basolateral side facing the formed vasculature and an apical side facing other hepatocytes which is reminiscent of in vivo liver lobules.

[0027]

[0023] Figure 8: (A) MRP-2 expression in polarized hepatocytes; the staining indicates MRP-2 lining the apical side of hepatocytes (B) Immunofluorescence staining indicate the presence of mature hepatocytes expressing albumin (in green) surrounded and partially enveloping a vascular network (CD31 , red).

[0028]

[0024] Figure 9: Immunofluorescence staining detecting the expression of CD31 and albumin. (A - B) the presence of mature hepatocytes expressing albumin (in green) surrounded and partially enveloping a vascular network (CD31 , red). Plates were inverted (A), or not inverted (B).

[0029]

[0025] Figure 10: Expression of LYVE-1 and CD31 by endothelial cells. (A - B) LYVE- 1 (green), a marker for liver sinusoidal endothelial cells, was expressed by the LDECs forming the vascular network. Plates were inverted (A), or not inverted (B). HLIVECs seeded in the lateral perfusion lanes (microfluidic channels) (A - B) appear to be expressing less LYVE-1 and more CD31 (red) compared to the LDEC network, again suggesting the hepatic phenotype of the latter. Further, it seems inversion of the culturing device may promote, e.g., positively affect, vasculature formation.

[0030]

[0026] Figure 11 : Expression of PLVAP in vascular structures. A positive expression of PLVAP, which is an endothelial cell-specific protein for the formation of fenestrations, was observed. Presence of fenestrations indicates a healthy phenotype for liver endothelial cells. It was surprising to detect the presence of fenestrations because these are generally absent in in vitro standard cell culture methods due to the rapid loss of healthy liver endothelium.

[0031]

[0027] Figure 12: Induction of hepatic steatosis by addition of free fatty acids in culture medium (A) Control (0.5% DMSO) (Actin (yellow), Bodipy (green), Collagen I (red)), (B) 500pM oleic acid + 500pM palmitic acid (Actin (yellow), Bodipy (green), Collagen I (red)). The herein provided liver culture appears to be metabolically competent, as indicated by the accumulation of lipid droplets (probably triglycerides) after 24h exposure to free fatty acids, mimicking hepatic steatosis.

[0032]

[0028] Figure 13: Addition of resident macrophages (Kupffer cells) in the liver model. CD45 (red) (A, B), a marker for macrophages and LYVE-1 (green) (B), a marker for liver sinusoidal endothelial cells was expressed.

[0029] Figure 14: Addition of biliary cells (cholangiocytes) in the liver model. CK19 (red) (A, B), a marker for biliary cells and CD31 (green) (B), a marker for liver sinusoidal endothelial cells was expressed.

[0033]

[0030] Figure 15: Hepatic structures containing polarized hepatocytes where an accumulation of Calcein-AM (a substrate for MRP-2, an apically-expressed transporter in mature hepatocytes) is observed in the spaces between hepatocytes indicating the presence of bile canaliculi and indicating presence of polarized hepatocytes.

[0034]

[0031] Figure 16: Addition of undifferentiated monocytes (around 30.000 cells, from PBMCs Stemcell Technologies) added on top of the obtained liver tissue (A), migrate from an extracellular matrix (ECM) / medium interface into the liver tissue, populating it, and differentiate to express macrophage markers CD16 (in red) nuclei in blue and actine in white, associated with liver vasculature, LYVE-1 (green) (B).

[0035]

[0032] Figure 17: Addition of Kupffer cells 2.000 cells I pl (Lonza) in the gel precursor co-seeded with the rest of the liver cells after two weeks culture (CD16 in white, LYVE- 1 in red). Kupffer cells are observed outside the vasculature and have the typical irregular morphology of macrophages.

[0036]

[0033] Figure 18: FITC dextran introduced to visualize the perfusability of the microvasculature (faint green network). Macrophages have taken up the dextran molecules (bright green cells). The herein provided liver culture with the macrophages integrated within the culture (as shown in Figure 17) were functional as they were capable of phagocytosis of the dextran molecules used to perform perfusion assays to visualize perfusable microvascular network.

[0037]

[0034] Figure 19: T-cells (Stemcell Technologies) have been labelled with a fluorescent cell tracker (Celltracker Orange, Thermo Fisher) and introduced in the vasculature (i.e. , on top of the obtained liver tissue and perfused for 5 days. Image shows T-cells (red bright circular cells) within the liver culture.

[0038]

[0035] Figure 20: Perfusion assay of a Liver Triculture (10.000 Sciencell HHSECs I pl, 3250 Sciencell HHSTeCs / pl, 20.000 iHEPs / pl) in fibrin on day 11 of culture. Image A comprises a Lonza HUVEC tubule in the perfusion channel, whereas image B does not. Perfusion assay was performed with 150kDa FITC-Dextran that was added to the top left perfusion channel of the OrganoPlate Graft® with the obtained liver tissue. From these images, it is deducible that the addition of endothelial cells (e.g., Lonza HUVECs) is advantageous to perfuse these chips from left to right. Thus, to generate perfusable liver vasculature with the method / system of the invention, the endothelial network within the gel is preferably to form connection with the microfluidic channels next to it.

[0039]

[0036] Figure 21 : Vascular liver tissue obtained with the method of the invention carried out with a unidirectional flow microfluidic system, plate or device (A), or with a bidirectional flow system (B). Vascular network (CD31 , red) and nuclei of cells in blue. With the use of a unidirectional flow microfluidic device, a vascular morphology reflecting a more homeostatic phenotype is visualizable by directional vessels, wider diameter, and more pruning.

[0040]

[0037] Figure 22: Phrodo E-coli particles seen (white) within cells in the liver culture, particularly in endothelial cells and macrophages after 1 day perfusion of the particle through the vasculature. Phrodo particles are E.coli particles fused with a pH sensitive dye. Once within a cell and inside the acidic environment of lysosomes they become fluorescent. These data illustrate that in the herein provided liver culture the phagocytosis of E.coli particles particularly can be observed. LSECs are known to possess a very important scavenger function in vivo. In contrast with some conventional systems, the scavenging function of both LSECs and macrophages is maintained in these tissues.

[0041]

[0038] Figure 23: mCherry expression (red) after exposure to AAV (AAV9, Factor builders) from the blood vessels. Arrow indicates direction of flow. A gradient is noticeable from high to low signal following the direction of the flow. This image shows the emergence of gradients of biological responses or cellular phenotype across the gel (i.e. , liver tissue), following the direction of the flow. Without being bound to any theory, this might indicate the existence of a first-pass effect of the cells on the fluid, where the first cells along the vascular connection experience a different environment compared to the last ones and provides a way or method to model liver zonation. Liver zonation is a well-known process in which liver cells have different, and sometimes opposing, functions depending on where they are located along the vascular connection between portal to central vein.

[0042]

[0039] Figure 24: FITC dextran (green) incorporation by macrophages after being delivered by the obtained blood vessels. Arrow indicates direction of flow of dextran. A gradient is noticeable from high to low signal following the direction of the flow. Without being bound to any theory, it could be hypothesized that the fact that almost no FITC dextran is absorbed in the downstream part of the tissue could indicate a very effective uptake of particles by the tissue mimicking the in vivo first pass effect. If the tissues are dense and metabolically active enough to process the perfusate fully, more downstream parts of the tissue could be exposed to secondary metabolites only, recapitulating the sequential nature and complexity of liver metabolism more completely than conventional culture systems.

[0043]

[0040] Figure 25: aSMA expression (in green, mostly by stellate cells) is expressed endogenously within the culture. A gradient of expression is noticeable from high to low signal following the direction of the flow.

[0044]

[0041] Figure 26: Calcein AM (Thermo Fisher) (top red) and FITC dextran (bottom green) are introduced via the microvasculature of the obtained liver tissue from the left and perfuses to the right side of the image. While dextran perfuses and is visible through the vasculature uniformly, calcein signal seems to show a very significant gradient. Calcein AM is cell permeable but transformed into the fluorescent and cell impermeable Calcein when inside a cell. We speculate that cells have a high capacity of incorporating calcein AM (also seen indirectly in other instances) leading us to show that indeed it is possible to achieve a first pass effect in such a system of the invention, where cells at the end of the network have less (or more) of certain molecules in the vasculature

[0045]

[0042] Figure 27: Fluorescent imaging of GFP-expressing HCT-116 colorectal (ATCC) cancer cells within the liver tissue (A) and overlay image showing how GFP-expressing cancer cells are seemingly integrated within the healthy cell population (B). Cancer cells were introduced in the seeding mixture. Cancer cells grow and are integrated within the healthy developing liver tissue.

[0046]

[0043] Figure 28: GFP-labelled HCT-116 colorectal cancer cells introduced as single cells in the vasculature allowing them to perfuse through the obtained liver tissue. To this end, HCT-116 cells were mixed with the medium and introduced in the first flow channel of a unidirectional flow microfluidic plate / device (see Figure 32 for schematic 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 obtained liver tissue vasculature. Fluorescent microscopy image was taken 5 days after introducing the cells. This figure illustrated that with the use of a unidirectional flow microfluidic plate it is possible to introduce circulating tumor cells to model metastasis in the liver tissue obtained according to the method / system of the invention.

[0047]

[0044] Figure 29: mCherry positive cells (mainly hepatocytes) in four replicate 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 invention allows for a liver model useful to study AAV transduction.

[0048]

[0045] Figure 30. Immunostaining of a liver triculture (3.250 Sciencell HHSECs / pl, 812 Sciencell HHSTeCs / pl, 20.000 iCell iHEPs / pl) that was exposed to 300.000 AAV9 mCherry genome copies / cell / ml for 72 hours on day 4. The culture was subsequently maintained for another 4 days (until day 11), whereafter they were fixed and stained for Nuclei (Blue), Albumin (White), CD31 (green). (A) The displayed red signal for AAV+ cells is from the mCherry payload of the AAV9 used in this experiment. The morphology of the cells is akin to hepatocytes (rounded cells), rather than the HHStECs and HHSECs (elongated cells). (B) Overlap of the mCherry signal and the albumin signal is clear on the second panel. This Figure allows to affirm that it appears to be mainly the hepatocytes to be transfected at first, followed by stellates and to a lesser extent endothelial cells. This is in line with the engineering of AAV9 which should target predominantly receptors on hepatocytes.

[0049]

[0046] Figure 31 : Day 11 confocal imaging of mCherry+ cells in a liver triculture (3.250 Sciencell HHSECs / pl, 812 Sciencell HHSTeCs / pl, 20.000 iCell iHEPs / pl) that was 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. The assay was performed in obtained liver tissue with monocytes (+ Monocytes) or without monocytes (- Monocytes) added on top of the liver grafts as indicated in Figure 16. If the culture contains resident immune cells, we observe a reduction in the transduction efficiency, in line with known similar effects observed in vivo. In (A) a graph with the number of AVV+ cells over time is shown. In (B) an image showing fluorescence of mCherry+ cells is provided.

[0050]

[0047] Figure 32: Top schematic view of an example of a unidirectional flow microfluidic device useful in the method of the invention. In (A), the path of a liquid flow (arrow lines) from a first reservoir flows through a first channel to a second reservoir passing through a cell culturing chamber. In (B), the path of a liquid flow from the second reservoir flows through a second channel to the first reservoir. DESCRIPTION:

[0051] Definitions

[0052]

[0048] 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 facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0053]

[0049] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such 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 definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0050] For purposes of the present invention, the following terms are defined below.

[0054]

[0051] As used herein, the singular form terms “a,” “an,” and “the” include plural referents unless the content 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 a cell includes the culturing of a plurality of cells (e.g., 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more).

[0055]

[0052] As used herein, “about” and “approximately", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed invention. Unless otherwise clear from context, all numerical values provided herein include numerical values modified by the term “about.”

[0056]

[0053] As used herein, “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0057]

[0054] 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 "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e. , 5, 4, 3, ... .-10, -11 , etc.

[0058]

[0055] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps, or components. It also encompasses the more limiting “to consist of.”

[0059]

[0056] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of conducting the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques 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 literature references.

[0060]

[0057] 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.

[0061]

[0058] As used herein the term “fibrinogen” refers to a protein, specifically a soluble glycoprotein, often being about 340 kDa in size, also referred to as coagulation factor I. Fibrinogen is commonly made and secreted by the liver. Fibrinogen is well described in the art and known to be formed into fibrin by enzymatical conversion, e.g., by thrombin-mediated proteolysis (Pieters et al. Res Pract Thromb Haemost. 2019;3:161- 172).

[0062]

[0059] As used herein, "in vivo" refers to an event that takes place in a subject's body; "in vitro" and “ex vivo" refer to an event that takes places outside of a subject's body”. For example, an in vitro assay or method encompasses any assay or method conducted outside of a subject. In vitro assays or methods encompass cell-based assays in which cells, alive or dead, are employed. In vitro assays also encompass a cell-free assay in which no intact cells are employed. The terms "in vivo", “in vitro" and “ex vivo" are well known in the art.

[0063]

[0060] As used herein, the term “perfusable” describes subject matter that can be perfused, e.g., that allows for a fluid, such as (but not limited to) an aqueous solution, suspension and the like, to flow in, over and / or through the subject matter.

[0061] As used herein, the term “scaffold” refers to a material, or a structure formed by a material, for example by a gel precursor comprising fibrinogen that is allowed to gelate to form a gel suitable for culturing cells. Scaffolds, within the context of the current invention, may provide an architecture for allowing cell attachment, cell migration, delivery and / or retaining of cells, delivery and / or retaining of biochemical agents, enabling diffusion of agents and the like. Scaffolds may have bioactivity, e.g., by interacting with cells. Certain scaffolds may be used to mimic the extracellular matrices of cells, e.g., of native tissue. Scaffolds have been duly 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).

[0064]

[0062] As used herein, the term “vasculature” refers to a structure wherein at least a part of the structure comprises an assembly of cells that resemble a blood vessel or a blood vessel-like structure, e.g., that resemble a blood vessel or a blood vessel-like structure in vivo. It may be that the vasculature as used herein is found in a tissue as provided with the method of the invention. It may be that the vasculature comprises a network of blood vessels and / or a blood vessel-like structures in such tissue.

[0065] Detailed description

[0066]

[0063] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0067]

[0064] 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 methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0068]

[0065] As embodied and described herein, the present invention is directed to the surprising finding that by providing liver stromal cells, preferably wherein the liver stromal cells are, or at least comprise, liver stellate cells and liver(-derived) endothelial cells, in a culturing device, preferably in a gel precursor comprising fibrinogen (which gel precursor is allowed to gelate), an in vitro liver tissue is obtained and that closely resemble in vivo liver tissue, function and organization. It was surprisingly found by the inventors that the obtained liver tissue comprises vasculature, in other words, that the liver tissue comprised a structure that resembles blood vessels or blood-vessel like structures, e.g., a vascular structure or vascular network. Moreover, it was surprisingly found that the obtained liver tissue comprised structures resembling liver sinusoidal structures, formed by liver stellate cells and liver(-derived) endothelial cells. It is believed that the vasculature development strongly correlates with the presence of stellate cells in a culture. Moreover, it is believed that stellate cells positively affect the vascular formation (i.e., vasculogenesis), formation of bile canaliculi(-like structures) and / or formation of fenestrations by cells, preferably by liver(-derived) endothelial cells.

[0069]

[0066] In the method of the invention, the cells that are mixed with the fibrinogen- based gel precursor (forming a fibrin gel upon gelating, for example in the presence of thrombin) where found to be able to form a vascular bed and that, in the method of the invention, is able to connect to blood vessels, for example, blood vessels that are formed in a microfluidic channel that is fluidically connected to the culture chamber comprising the gel comprising the vascular bed, as disclosed herein. Allowing such vasculogenesis by the cells that are mixed with the fibrinogen-based gel precursor or fibrin-based gel, and the formation, in particular the de novo formation of the vascular bed in the gel is an important aspect of the method of the current invention. In the invention, the vascular bed that is obtained is a vascular bed that is formed during the culturing of the cells in the gel and is not a vascular bed that has been preformed before mixing with the gel precursor. In a preferred embodiment, the cells that are comprised in the gel (forming the scaffold) are mixed with the gel precursor substantially as single cells and / or as small aggregates of cells, for example cell aggregates (or clumps) of 2 - 200 cells, preferably 10 -100 cells single cells, or are provided to the gel (forming the scaffold) substantially as single cells and / or as small aggregates of cells, for example cell aggregates (or clumps) of 2 - 200 cells, preferably 10 -100 cells single cells. In a particular preferred embodiment, in particular the cells that will form the vasculature during the culturing according to the invention are mixed with the gel precursor substantially as single cells or are provided to the gel (forming the scaffold) substantially as single cells.

[0070]

[0067] In a preferred embodiment, when the cells that are comprised in the gel (forming the scaffold) are mixed with the gel precursor or are provided to the gel (forming the scaffold), the cells are not in a form resembling a vascular bed or vascular structure. It is understood by the skilled person that in embodiments of the method of the invention, the liver tissue obtained with the method of the invention, or parts thereof, in particular the vasculature is obtainable during the culturing steps defined herein, and preferably does not require the provision of an existing functional tissue structure, such as a preformed or isolated vascular bed or a preformed or isolated blood vessel, or any other typical tissue structure of a liver, such as those disclosed herein. In a preferred embodiments, the cell that are provided to the culture chamber, i.e. the at least liver endothelial cells and liver stellate cells have be prepared, obtained or treated before individually before being provided to the culture chamber in a gel precursor, or to the scaffold. The skilled person understands this does not exclude that the cells are first mixed before being provided to the gel precursor or scaffold. The skilled person will understand that in a preferred embodiment, the liver tissue obtained with the method of the invention is an in vitro formed liver tissue. The liver tissue that is obtained with the method of the invention is created during the culturing method of the invention.

[0071]

[0068] Further, it was surprisingly found that the forming of a liver tissue strongly benefits from the presence of a gel precursor comprising fibrinogen (and / or of a gel comprising fibrin). For example, it was found that a fibrin-based gel matrix or scaffold formed by gelation of a gel precursor comprising fibrinogen is a very permissive matrix for the emergence of vasculature in the obtained liver tissue, thereby leading to a liver tissue that strikingly resembles a native liver tissue. It was found to be highly advantageous for vasculogenesis to embed the herein disclosed cells in a scaffold and / or gel precursor, preferably comprising fibrin and / or fibrinogen.

[0072]

[0069] In contrast, it was found that if in the method of the invention an extracellular matrix is used that does not, or only to a limited extent, comprises fibrin (i.e. wherein the gel precursor does not, or only to a limited extent, comprises fibrinogen), obtaining stable cultures that are able to form for a liver tissue as disclosed herein, is difficult, if not impossible, to achieve. Therefore, the invention is in part based on the purposely use of a fibrinogen-based gel precursor / fibrin-based gel as disclosed herein. For example, it was found (data not shown) that in case, instead of a fibrinogen precursor / fibrin gel as disclosed herein, a liver extracellular matrix was used (for example as described by Willemse et al (Biomaterials (2022) Volume 284, 121473; doi.org / 10.1016 / j. biomaterials.2022.121473), it was difficult, if not impossible to obtain a stable culture and shows liver structures as disclosed herein (obtainable with the method of the invention wherein a fibrinogen based gel precursor / fibrin based gel is used). The liver extracellular matrix described by Willemse is rich in collagen, with no mention of the presence of any (minimal levels of) fibrinogen / fibrin. Therefore, the inventors have surprisingly found that in the method of the invention, a fibrinogen- based gel precursor / fibrin-based gel provides for a more stable liver cell culture, and / or liver tissue more resembling in vivo liver tissue structure and function, even in comparison to a liver-based extracellular matrix.

[0073]

[0070] In yet other experiments performed by the inventors it was found that a Matrigel or BME (with collagen being an abundant protein comprised therein) scaffold results in contraction of stellate cells present in the culture. Therefore, in some embodiments, in the gel precursor or gel, collagen constitutes no more than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or less of total protein of the gel precursor or gel. Therefore, in some embodiments, in the gel precursor or gel, fibronectin constitutes no more than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or less of total protein of the gel precursor or gel. Therefore, in some embodiments, in the gel precursor or gel, laminin constitutes no more than 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1 % or less of 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 comprise laminin. The skilled person understood that in some embodiments, Matrigel, or BME (basement membrane extract) may be mixed with fibrinogen / fibrin, to thus obtain a gel precursor or scaffold (gel) in accordance with he disclosure herein. For example, in a ratio such that the weight ratio of proteins comprised in BME or Matrigel to fibrinogen (or fibrin) is between 5: 1 and 1 :5, for example about 4:1 , 3: 1 , 2:1 , 1 :1 , 1 :2, 1 :3, 1 :4 or 1 :5. Based on the current disclosure, the skilled person will understand how to provide for such gel precursor / scaffold, and that allows for the in vitro method for obtaining a liver tissue as disclosed herein.

[0074]

[0071] Additionally, it was surprisingly found that vasculogenesis may also occur in the presence of further cells, e.g., tumor(-derived) cells, such as HLIVECs, stem cells, umbilical cord blood cells and the like. Preferably the further cells comprise (liver) cells such as hepatocytes, Kupffer cells, biliary cells, (other) liver stromal cells and the like. These further cells can be provided in the gel precursor and / or in the lateral tubes / channels of the microfluidic device, preferably further cells, such as HLIVECs, are provided in the lateral tubes / channels. Hence, surprisingly, the invention also results in the obtaining of a liver tissue comprising further (liver) cells, such as hepatocytes, Kupffer cells, biliary cells and the like. This finding means that a liver tissue can be obtained that even more closely resembles a native liver tissue. For example, it was found that the addition of further liver cells, such as hepatocytes, resulted in the forming of bile canaliculi or bile-canaliculi-like structures.

[0075]

[0072] Additionally, it was surprisingly found that the obtained liver tissue appears to be metabolically competent, meaning that the liver tissue can mimic the metabolism of a native liver tissue. For example, it was surprisingly found that the obtained liver tissue can mimic hepatic steatosis (i.e. (excessive) fat accumulation by the liver) after exposure to free fatty acids.

[0076]

[0073] Additionally, it was surprisingly found that cells added to the tissue, preferably by addition on top of the tissue culture chamber, could be incorporated in the tissue and could differentiate towards more functional cells, e.g., PBMCs could be added on top of the tissue and be differentiated towards Kupffer like cells.

[0077]

[0074] Therefore, in some embodiments, the invention provides for an in vitro method for obtaining a liver tissue, wherein the method comprises: a) providing a culturing device wherein the culturing device at least comprises a culture chamber; b) providing the culture chamber with a gel precursor, wherein the gel precursor at least comprises fibrinogen, and wherein the gel precursor further comprises liver endothelial cells and liver stellate cells; c) allowing the gel precursor to gelate 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 the liver tissue, preferably wherein the liver tissue resides in the scaffold.

[0078]

[0075] Alternatively, in some embodiments, the method comprises the steps of A) providing a culturing device, wherein the culturing device at least comprises a culture chamber and wherein the culture chamber comprises a gel precursor comprising fibrinogen, liver endothelial cells and liver stellate cells; B) allowing the gel precursor to gelate to obtain a scaffold, wherein the scaffold comprises the cells of step (A);

[0079] C) contacting the scaffold of step (B) with a cell culture medium; and

[0080] D) culturing the cells in the scaffold to obtain the liver tissue, preferably wherein the liver tissue resides in the scaffold.

[0081]

[0076] Alternatively, a culturing device may be provided wherein the gel precursor has been allowed to gelate to obtain a scaffold and / or wherein fibrinogen has been converted to fibrin, e.g., by enzymatically converting fibrinogen, e.g., by thrombin- mediated proteolysis. In other words, in an alternate aspect there is provided for an in vitro method for obtaining a liver tissue, comprising the steps of:

[0082] AA) providing a culturing device, wherein the culturing device at least comprises a culture chamber and wherein the culture chamber comprises a scaffold, preferably a scaffold comprising fibrin and / or fibrinogen, liver endothelial cells and liver stellate cells;

[0083] BB) contacting the scaffold of step (A) with a cell culture medium; and

[0084] CC) culturing the cells in the scaffold to obtain the liver tissue, preferably wherein the liver tissue resides in the scaffold.

[0085]

[0077] Whereas in the description and claims reference will be made to first method described above (with e.g. steps (a) - (e)), the skilled person understands that that any method, use or composition described herein can likewise be implemented with respect to the methods presented with alternative wording (with e.g. steps (A) - (D) or (AA) - (CC)). The skilled person will also understand that were reference is made to cells comprised in in the gel precursor or comprised in the scaffold, these cells may in some embodiments have been provided via the gel precursor, and / or may in some embodiments have been provided to the gel. The skilled person also understands that in embodiments of the method according to the invention, initially particular cells are provided by being comprised in the gel precursor, whereas the same or distinct types of cells are provided after the gel precursor gelated, for example, during the culturing of the cells to obtain the liver tissue.

[0086]

[0078] In the method in accordance with the present invention, in step (a), a culturing device comprising a culturing chamber (may also be referred to as “graft chamber” or “culture chamber”) is provided. Culturing devices as used herein comprise any cell culture vessel known and described in the art. As provided herein the culturing device comprises at least a culturing chamber, in other words a container, channel or a plate where cell can be kept and cultivated, e.g., petri dish, culture flask or well. It is preferred that said culture chamber provides for an (at least partly) sealed chamber for cultivating cells. The culture chamber (also referred to as graft chamber) may have an opening, preferably a circular opening, that can be used for introducing of cells, gels, aqueous solutions, suspensions and / or dispersions and the like into the culture chamber. The opening in the culture chamber does not necessarily need to be circular but can comprise any shape suitable for providing an (additional) inlet to the graft chamber. This opening can also function as a capillary pressure barrier e.g., as described in WO2017216113 A2. The opening may be partially or fully closed by commonly used methods or tools to close an opening in a culturing chamber, e.g., film, lid and the like. Preferably, the opening is located on top of the culture chamber, but the opening can also be positioned on any other side, e.g., laterally (e.g., on the left or right side) of the culture chamber, as long as it allows for introduction of cells, gels, aqueous solutions, suspensions and / or dispersions and the like into the culture chamber. Multiple openings allowing for introducing cells, gels, aqueous solutions, suspensions and / or dispersions and the like into the culture chamber are also envisioned and encompassed herein. Alternatively, the culture chamber may comprise one or more channels connected to an opening suitable for allowing introduction of matter. In such embodiment, the culture chamber may itself be closed, except for the opening provided via the one or more channels. In other embodiment, the culture chamber is accessible, in addition to the one or more channels, via an closeable opening, for example, wherein the opening is positioned on the top side of the culture chamber. It may be that various ways on introducing matter into the culture chamber are combined in a culturing device. In the art, systems use standard culture plates and use various barrier inserts in an attempt to culture cells that more closely represent their in vivo characteristics (e.g., Transwell® permeable supports). It is herein contemplated that any culture plate may be used for the method of the invention. Alternatively, bioreactors or other devices, e.g., vessel-like devices, suitable for cultivating cells and at least comprising a culturing chamber may also be used in the method of the invention. However, in a preferred embodiment, the culture device is a microfluidic device at least comprising a culture chamber, and preferably comprising at least one channel fluidly connected to said culture chamber. It was found that the herein disclosed amounts of cells and volumes used in a microfluidic device allow for culturing cells in such a way that native liver tissue is closely resembled.

[0087]

[0079] Further, in step (b) a gel precursor is provided to said culturing chamber whereafter, in step (c), the gel precursor is allowed to gelate (i.e., solidify, e.g., polymerization) in the culturing device.

[0088]

[0080] 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. By way of example, the gel precursor, may be a hydrogel precursor, and is typically an extracellular matrix (ECM) gel precursor. ECM may for example comprise collagen, fibronectin, fibrinogen, and / or basement membrane extracts such as Matrigel or a synthetic gel. Provided herein, the gel precursor at least comprises fibrinogen. The gel precursor may, by way of example, be provided (e.g., introduced) into the culturing device with a pipette (typically a repeating pipette such as the Eppendorf Multipette® M4 (Eppendorf AG, Germany, catalogue number 4982 000.012) in combination with Eppendorf Combitips advanced® (Eppendorf AG, Germany, catalogue number 0030 089.405)). Preferably the gel precursor at least comprises fibrinogen, wherein preferably fibrinogen is present in the gel precursor in a concentration sufficient, e.g. between 1 mg / mL - 80 mg / mL, 1 mg / mL - 50 mg / mL, 1 mg / mL - 25 mg / mL, or 1 mg / mL - 10 mg / mL preferably at an end concentration of 3,4, 5, 6, 7, 8 mg / ml, more preferably 5 mg / mL for allowing the formation of a fibrin scaffold, and may further comprise a basement membrane extract, an extracellular matrix component, collagen, collagen I, collagen IV, fibronectin, laminin, vitronectin, D-lysine, entactin, heparan sulphide proteoglycans or combinations thereof.

[0089]

[0081] In a preferred embodiment, in the gel precursor, fibrinogen is the most abundant protein (by weight) that is present in the gel precursor. In an embodiment, in the gel, fibrin is the most abundant protein (by weight) that is present in the gel material (i.e. excluding any protein comprised in the cells that are included in the gel / gel precursor.

[0082] In another embodiment, in the gel precursor, fibrinogen constitutes, by weight, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the protein of the gel precursor. In an embodiment, in the gel, fibrin constitutes, by weight, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more (e.g. 100%) of the protein of the gel material. In some embodiments, as discussed herein, the gel precursor comprises a mixture of Matrigel and / or BME and fibrinogen. In some embodiments, as discussed herein, the gel (scaffold) comprises a mixture of Matrigel and / or BME and fibrin.

[0090]

[0083] In an embodiment the gel precursor comprises more fibrinogen (by weight) than collagen. In an embodiment the gel precursor comprises more fibrinogen (by weight) than elastin. In an embodiment the gel precursor comprises more fibrinogen (by weight) than fibronectin. In an embodiment the gel precursor comprises more fibrinogen (by weight) than laminin.

[0091]

[0084] It is contemplated that the gel precursor provided in the method of the invention should at least comprise fibrinogen so that gelating the gel precursor results in obtaining a scaffold at least comprising fibrinogen and / or fibrin. Preferably, the scaffold may comprise fibrin e.g., a fibrin gel or fibrin gel matrix (e.g., obtainable by enzymatically converting fibrinogen). It was found that a fibrin-based scaffold benefits the formation of vasculature by the cells in the method of the invention, therefore it is contemplated that a gel precursor comprising fibrinogen results in a liver tissue comprising an improved vasculature when compared to a method wherein no fibrinogen (and / or fibrin) is used in the gel precursor / gel. It is moreover preferred to use a fibrin scaffold because such a scaffold has a relatively low stiffness (e.g., elastic shear modulus below 150 Pa, preferably about or below 100 Pa) when compared to other scaffolds. It was found that low stiffness makes fibrin gel suitable for inducing and studying vasculogenesis in liver tissue in vitro. Likewise, the use of fibrinogen and / or fibrin in the gel precursor / gelated gel, is believed to promote the obtaining of the liver tissue according to the invention, and to provide for a liver tissue that closely resembles in vivo liver tissue, for example with respect to (spatial) organization and / or functionality.

[0092]

[0085] The gel precursor as provided in step (b) further comprises liver endothelial cells and liver stellate cells. It is preferred that the liver endothelial cells and / or liver stellate cells are primary cells, i.e., primary liver stellate cells and / or primary liver- derived endothelial cells, meaning that the cells are preferably liver-derived, e.g., isolated from an organ or a tissue for use in vitro. It is contemplated that primary cell lines are advantageous in the current method of the invention, e.g., beneficial for vasculogenesis, and therefore are preferred over (immortalized) cell lines or cells derived from pluripotent stem cells. In some embodiments, the liver stellate cells of step b) do not comprise LX-2 cells. It is possible that both the liver endothelial cells and liver stellate cells are passaged (e.g., cultivated) for one or more times prior to providing the cells in a gel precursor. In other words, the cells do not need to be directly, e.g., freshly, derived from a liver. Cells may have been frozen, e.g., in accordance with common cell processing protocols, and may need to be thawed prior to the method in accordance with the invention.

[0093]

[0086] It is understood that a gel precursor comprising fibrinogen, liver endothelial cells and / or liver stellate cells can be provided to the culture chamber as a mixture or suspension, for example in the form of an aqueous solution comprising at least the fibrinogen, liver endothelial cells and / or liver stellate cells. Alternatively, the gel precursor, fibrinogen, endothelial cells and / or stellate cells can be provided to the culture chamber separately and are mixed or suspended when in said culture chamber. However, it is highly preferred that gel precursor, comprising fibrinogen, and the cells are introduced together. After the gel precursor is allowed to gelate (forming a gel), preferably, at least some, 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 liver endothelial cells and / or liver stellate cells that are provided are embedded in the scaffold, thereby allowing culturing of the cells in the scaffold (and subsequent formation of, for example vasculature, bile canaliculi, and other liver tissue structures as disclosed herein in the gel (scaffold). It is contemplated that adding such a cell suspension and allowing the scaffold to form eventually results in a liver tissue that mimics the native liver closely. However, it is contemplated that some embodiments may require that part of the cells (for example part of the stellate cells, or part of the liver endothelial cells, or both, or part of any other cell that is to be included as part of the method of the invention), are added during and / or subsequent to the obtaining of the scaffold (i.e. during or after the gel precursor forms the gel) and / or are added on top of the gel(precursor) once the gel (precursor) has been introduced. However, without being bound by theory it may be that adding (a part of) the cells sequential to the gel precursor, albeit possible, may reduce the uniformity of distribution of the cells throughout the scaffold. This could negatively affect gelation, e.g., when in case of a gel precursor comprising fibrinogen thrombin needs to diffuse into the gel precursor and cells. Therefore, adding a suspension / dispersion of one or more gel precursors comprising fibrinogen and cells is preferred. Therefore, in some embodiments, the cells are more or less evenly distributed throughout the gel after gelation. In some embodiments, the cells are not evenly distributed throughout the gel after gelation.

[0094]

[0087] The mixing of gel precursor comprising fibrinogen and / or cells is typically done prior to that the gel precursor gelates at least partly, preferably completely, to for the (gel) scaffold in step I. It is preferred that a cell suspension, i.e., wherein the gel precursor at least comprises at least some liver endothelial cells or liver stellate cells, is introduced in the cell chamber thus allowing the embedding of cells in the scaffold. In some embodiment, the gel precursor (comprising the liver endothelial cells and liver stellate cells) is provided to the culture chamber as a whole. In some embodiment, first part of the gel precursor (for example, comprising the liver endothelial cells and / or liver stellate cells, or no cells) is provided to the culture chamber, and preferably allowed to gelate, before a subsequent part of the gel precursor (for example, comprising the liver endothelial cells and / or liver stellate cells, or no cells) is introduced in the cell culture chamber. In such embodiments, different spatial organization of the cells, including the liver endothelial cells and / or liver stellate cells throughout the scaffold may be obtained before culturing of the cells therein.

[0095]

[0088] In some embodiments, the cells are not provided in step b) of the method of the invention as viscous bodies of cells. In some embodiments, the method in accordance with the invention does not comprise the step of mixing the herein disclosed cells with collagen and heparin, prior to performing step a) of the herein disclosed method. In some preferred embodiments the invention does not comprise the step of centrifuging the herein disclosed cells, preferably a mixture comprising the cells, collagen and heparin, prior to and / or during performing the herein disclosed method, for example to obtain viscous bodies of cells.

[0096]

[0089] A skilled person is able to identify that liver endothelial cells and / or liver stellate cells are being used in the method of the invention by identification of cellular markers, such as (but not limited to) LYVE-1 and / or PLVAP for liver endothelial cells and / or DESMIN, aSMA and / or PDGFRp for liver stellate cells. Alternatively, a skilled person can identify the types of cells used by (single cell) sequencing techniques known in the art.

[0097]

[0090] The gel precursor may be introduced in the culturing device, e.g., in a microfluidic culturing device, by allowing the gel precursor to enter, and allowing to fill, at least (part of) the culturing chamber and optionally any further microfluidic channel network that may be present (see below). In embodiments wherein the culturing device is a microfluidic device, the gel precursor may be allowed to (partially) fill selected regions of the microfluidic device, such as at least one of the microfluidic channels that may be present , with help of commonly used patterning techniques such as for example by photolithographic patterning and patterning with capillary pressure techniques, potentially assisted by gravity. By way of example, it is possible to halt the gel precursor from filling certain parts of the culturing device, preferably the microfluidic device, with a capillary pressure barrier, e.g., a phaseguide (Vulto et al. (2011), Lab on a chip. 11. 1596-602) or similar suitable method of halting the transportation of the gel precursor. Capillary pressure barriers are not to be understood as a wall or a cavity which is filled with the gel precursor but consists of elements which make sure that the gel precursor due to the surface tension does not spread open. 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., an air-liquid meniscus, consisting of gel precursor will be achieved in the microfluidic device. By way of example the capillary pressure barrier can be a line of material, e.g., in the form of a rim, a groove, a hole, or combinations thereof, that prevents the gel precursor, e.g., by meniscus pinning, from flowing beyond the capillary pressure barrier. In another embodiment capillary pressure barriers can be created by pillars at selected intervals that are lining the area that is to be occupied by the gel. Particular advantageous capillary pressure barriers have been previously described by the applicant, e.g., in WO2017216113 A2 and WO2014038943 A1.

[0098]

[0091] Further, in step (c) the gel precursor as described herein is allowed to gelate into a scaffold. The gel precursor is allowed to gelate (solidify) in certain regions of the culturing device thereby occupying at least part of the device. It is understood that the gel precursor at least partly gelates in the culturing chamber, thereby occupying at least part of the culturing chamber in the form of a scaffold. It is preferred that subsequent to gelation at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% at least, 99% of the volume of the culturing chamber is occupied by the scaffold. With respect to the term “occupying” of at least part of the culturing device and at least the culture chamber, it will be understood by the skilled person that it is not required that scaffold is present throughout the culturing device, but preferably occupying certain areas, such that selected other regions remain accessible for introducing a further gel precursor, fluid or a growth medium, e.g. for a perfusion flow. It will also be understood that the scaffold should not block passage of growth medium in the device. A gel precursor may be solidified into a scaffold by for example using a photosensitive cross-linker, such that upon exposure to e.g., UV light, the gel precursor gelates. Alternatively, some gel precursors can self-assemble into scaffold by adding for example enzymes enabling conversion into a solid gel. Hence, in one preferred embodiment the gel precursor comprising fibrinogen is solidified into a scaffold by adding the enzyme thrombin, which cleaves fibrinogen into fibrin monomers allowing for self-assembly into a scaffold (Janmey et al. J. R. Soc. Interface (2009) 6, 1-10). It is well known in the art that the gelation of a gel precursor comprising fibrinogen can be tuned to alter gelation time and / or mechanical properties of the fibrin scaffold. For example, the culturing device comprising a gel precursor can be placed in a humidified incubator for a period of time. It is preferred that gelation (e.g., polymerization or solidification) of the gel precursor into a scaffold takes between 5 - 30 minutes, preferably 15 - 20 minutes after inducing gelation, e.g., by enzymatic conversion. Time to gelation may be depending on the type and / or concentration of the ECM, conditions, and / or concentration enzymes such as thrombin. It is preferred that gelation results in that the gel precursor is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% at least, 99% w / w gelated to a scaffold. In other words, it is preferred that at least part of the gel precursor gelates into a solid scaffold and it may be that still a part of the gel precursor has not (yet) solidified. After the gel precursor is provided, it is caused to gelate, preferably prior to introduction of a further fluid, aqueous solution, or culture medium. Several techniques for determining the mechanistic properties of a hydrogel have been described in the art (Handbook - Topics in Tissue Engineering, Vol. 4. (2008)). Whether a gel precursor has in part or fully gelated into a gel I scaffold can be measured by methods known to a skilled person, e.g., simple inversion of the culturing device.

[0099]

[0092] Further, in step (d) of the method in according to the invention the scaffold obtained in step (c) and comprising the cells of step (b) is contacted with a cell culture medium. The skilled person understands that multiple cell culture medium compositions are allowed to contact the scaffold comprising the cells, for example the scaffold is contacted with a first culture medium, e.g., suitable for supporting the growth of the cells, and subsequently is contacted with a second culture medium and even with a third or fourth culture medium, with similar of different compositions (as long as it allows the cells to grow and survive). In other words, cell culture medium can be replaced one or more times during cultivating of the cells. Typically, the cell culture medium is an aqueous medium. Preferably, the cell culture medium is a growth medium that is able to deliver sufficient nutrients and other compounds that are essential for the growth, differentiation and / or proliferation of the cells, but preferably does not contain compounds / agents that are potentially harmful to the growth, differentiation and / or proliferation of the cells.

[0100]

[0093] Further it is contemplated herein that depending on the cells used in the method in according to the invention the culture medium may be adjusted by a skilled person to enable a suitable growth medium for the cells of step (b). If so desired, the growth medium can be supplemented with additional (growth) factors. By way of example, liver endothelial cells and / or stellate cells benefit from the scaffold being contacted with EGM-2 I EGCM-2 medium, e.g., EGM-2 (Lonza™) and / or hepatocytes benefit from the scaffold being contacted with iHEP plating medium (Cellular Dynamins, Fujifilm). It may be that a suitable culture media promotes, i.e. , positively affects, the formation of vasculature in the method according to the invention. Other suitable media known to a skilled person are also possible and hence fully encompassed by the current invention. Therefore, it is preferred, but not necessary, to select a suitable, e.g., cell-specific, medium for cultivating, differentiating and / or proliferating cells. When performing step (d) the growth medium may be provided, in order to contact the scaffold, in a flow, e.g., by allowing a fluid flow to pass the scaffold through microfluidic channels and through any vasculature(-like) structures formed by cells in the scaffold. In the case of a flow, the growth medium can also be used to remove or dilute waste metabolites as produced by the cells. Alternatively, growth medium may be provided without using a flow to the scaffold, e.g., by directly providing a predefined volume of the medium to the scaffold, e.g., through a hole in the culture chamber.

[0101]

[0094] Further, in step e) of the method, the cells are cultured in the scaffold so as to obtain the liver tissue. In other words, the cells of step (b) that are at least partly embedded in the scaffold are allowed to be cultivated and / or proliferate and / or differentiate, at least until a part of a liver tissue is formed.

[0102]

[0095] It is contemplated that the method of the invention allows the liver stellate cells and endothelial cells to interact and proliferate such that is leads to the formation of a distinct morphological pattern of liver tissue that closely resembles native liver tissue with respect to spatial organisation and / or functioning, and that at least comprises a structure of a liver comprising liver stellate cells and liver endothelium.

[0103]

[0096] With respect to the formation of a liver tissue in the scaffold this encompasses the presence of a network of interacting, e.g., physically interacting and / or interacting through signal molecules, cells representing a liver tissue, which is preferably confluent or at least partially confluent. Such a confluent network comprises that, by way of example, about 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent of the cells that are present in the scaffold interact, e.g. by forming physically interacting cellular structures, namely (part of) one or more cells that are in direct contact with each other, that are spatially organized in the form of vasculature, tubes, bile canaliculi(-like) structures, fenestrations, bile ducts and / or clusters of cells, and thus form a liver tissue. 100 percent confluency, albeit preferred, is not necessary.

[0104]

[0097] It is preferred that the liver tissue is embedded, by way of example for at least by way of example, about 10, 20, 30, 40, 50, 60, 70, 80, or 90, or 100 percent of the cells forming the liver tissue , in the scaffold. Preferably, the embedding of the liver tissue in the scaffold resembles an arrangement of cells (e.g., spatial organization) that closely resembles in vivo tissue. By way of example, liver tissue comprises an arrangement of cells, such as (but not limited to) endothelial cells hepatocytes, Kupffer cells, stellate cells and the like, that is highly vascularized and comprises liver sinusoids, bile canaliculi and the like.

[0105]

[0098] It was found that by including hepatocytes, in addition to liver stellate cells and liver endothelial cells, in the gel precursor in step (b) an improved liver tissue is obtained by the method in accordance with the invention. In particular, it is surprisingly found that adding hepatocytes made the liver tissue even more closely resemble in vivo native liver tissue, for example by showing similar spatial arrangement as native liver tissue.

[0106]

[0099] It was particularly found that by including hepatocytes in the current method a liver tissue comprising functional (polarized) and spatially organized hepatocytes is obtained, preferably wherein at least a part of the hepatocytes is in direct contact and / or interacting through signal molecules with the liver stellate cells and / or liver endothelial cells. Therefore, in some embodiments, there is provided for a method in accordance to the invention, wherein the gel precursor of step (b) further comprises hepatocytes.

[0107]

[0100] As will be understood by a skilled person, when adding hepatocytes to the gel precursor in step (b) it is preferred that a cell culture medium suitable for cultivating (e.g., maintaining), differentiating and / or proliferating hepatocytes is (also) used in step (d). In an embodiment, the hepatocytes as provided herein are derived or differentiated from induced pluripotent stem cells and / or are (primary) human-derived hepatocytes. Albeit not preferred, it was found that liver endothelial cells and hepatocytes, without presence of liver stellate cells, also were able to form a structure that shared some resemblance with native liver tissue. Though the structure consisting only of liver endothelial cells and hepatocytes did not resemble the native tissue similarly robust and / or lacks benefits, that a combination of at least liver stellate cells and liver endothelial cells as set forth herein does.

[0108]

[0101] For example, it was found by the inventors that at least using liver stellate cells and liver endothelial cells enabled vasculogenesis in culture. In other words, it was found that liver endothelial cells, supported by stellate cells, are highly preferred to form liver tissue comprising a vasculature in a scaffold comprising at least fibrin, (e.g., a fibrin scaffold).

[0109]

[0102] In some embodiments, there is provided for a method in accordance with the invention, wherein the culturing in step (e) provides for obtaining a liver tissue comprising vasculature formed by at least the liver endothelial cells and the liver stellate cells. In some embodiments, at least the liver endothelial cells and the liver stellate cells are cultured and are allowed to proliferate and / or differentiate until at least a liver tissue comprising vasculature is formed. Optionally, during the method of the invention, a ((bio)chemical) gradient is provided, growth factors are provided and / or removed, culture media (components) are changed etc. Preferably, cells are provided in a number of total cells, comprised in the gel precursor provided in step (b), of at least 1.000 cells per microliter of gel precursor. Further, preferably liver endothelial cells and the liver stellate cells are provided in a ratio of liver endothelial cells : liver stellate cells of equal to or greater than 20 : 1. Also, preferably, cells are cultured for at least 3 days after the steps (a) - (c), preferably after commencing step (d) of the method in accordance with the invention.

[0103] In some embodiments, the herein formed vasculature may extend out of the obtained liver tissue. For example, (a part of) vasculature may extend out of a first group of cells forming a (part of a) liver tissue and may extend towards a second group of cells forming a (part of a) liver tissue or may extend towards (a part of) vasculature extending out of said second group of cells forming a (part of a) liver tissue. It may be that vasculature extends towards multiple (e.g., 3, 4, 5, 10, 100, 1.000, 10.000 etc.) (vasculatures of) such further groups of cells forming a (part of a) liver tissue. In other words, it may be that the cells (partly) forming the vasculature may form a vascular network (as part of a liver tissue) by directly (e.g., physically) and / or indirectly interacting with further cells in the scaffold. In some embodiments, the vasculature may extend out of the scaffold and / or obtained liver tissue, for example the vasculature may extend into other tissue structures, e.g., tubule(-like) structures formed by HLIVECs in one or more microfluidic channels. Alternatively, other tissue structures such as tubule(-like) structures formed by HLIVECs (and / or other endothelial cells) may extend into the scaffold and / or liver tissue thereby interacting with the cells comprised in the scaffold / liver tissue.

[0110]

[0104] Preferably vasculature also comprises the forming of at least one part of a tubule and / or a tubule-like structure. Preferably these tubules or tubule-like structures that allow for perfusion of a fluid through the tubule(-like) structure, e.g., through part of the vasculature.

[0111]

[0105] It is preferred that the vasculature comprises at least one tubule(-like) structure having an average diameter of between 1 - 100 pm, e.g., 1 , 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90, 100 pm, preferably 1 - 50 pm, preferably between 5 - 50 pm or even 5 - 30 pm. It was surprisingly found that the vasculature of the liver tissue as provided herein showed close resemblance to the average diameter of a vasculature in a native liver tissue.

[0112]

[0106] In some embodiments, there is provided for a method in accordance with the invention, wherein the culturing in step (e) provides for a liver tissue comprising one or more sinusoidal structures formed by at least the liver endothelial cells and the liver stellate cells. Preferably at least the liver endothelial cells and the liver stellate cells are cultured until a liver tissue, preferably comprising a vasculature wherein one or more sinusoidal structures, is formed.

[0107] In some embodiments, there is provided for the method according to the invention wherein, in addition to the stellate cells and the endothelial cells, hepatocytes are included in the method of the invention. The hepatocytes may be mixed together with the stellate cells and endothelial cells in the gel precursor and provided to the culture chamber In some embodiments, the hepatocytes are allowed to proliferate in the scaffold. It was found that the sinusoidal structures or sinusoid-like structures are at least partially surrounded by hepatocytes, as for example is exemplified in Figure 7 A - C. It is contemplated that the liver tissue comprising a vasculature and comprising one or more sinusoidal structures can comprise any other cell type that is commonly found in native liver tissue, such as other parenchymal cells and / or non-parenchymal cells, e.g., Kupffer cells, immune cells, biliary cells and the like. A skilled person is able to further include other suitable cells.

[0113]

[0108] Sinusoidal structures are capillaries that are typically found in native liver tissue. Sinusoidal structures have been described in the art, e.g., by Brunt et al. (Brunt, E M et al. (2014) Histopathology 64, 907-920). In in vivo hepatic sinusoids the endothelium is continuous, however some endothelial cells are loosely attached to one another allowing fluid and protein to leak through the intercellular junctions from one side of the endothelium to the other side of the endothelium. Further leaks can be enabled by formation of fenestrations in the liver endothelium. It was found that a liver tissue could be obtained with the method of the invention and that comprises sinusoidal structures and / or sinusoid-like structures by providing in step (b) of the method of the invention at least the liver stellate cells and liver endothelial cells. It is preferred that the liver tissue comprises a vasculature that comprises sinusoidal structures and / or sinusoid-like structures.

[0114]

[0109] Upon cultivating, differentiating and / or proliferating the cells in the method according to the invention, the liver endothelial cells form vasculature in the scaffold preferably comprising a vasculature structure that resembles liver sinusoidal(-like) structures, and preferably also comprising fenestrations. It was surprising that fenestrations were found as the presence of fenestrations indicates a healthy phenotype for liver endothelial cells, and particularly since such a healthy phenotype is rapidly lost when culturing liver endothelial cells, preferably liver-derived endothelial cells, by using standard cell culture methods. Thus, the method according to the invention suprisingly allows for the in vitro forming of a liver tissue, and wherein the liver tissue comprises fenestrations, resembling the presence of such fenestrations in healthy in vivo liver tissue.

[0115]

[0110] Fenestrations are known in the art and may be described as being transcellular holes, tiny openings or pores commonly found in an assembly of cells, particularly of endothelial cells. In an in vivo situation fenestrations as often found in the endothelium of organs where a higher rate of exchange between intra- and extravascular compartments is required, for example in tissues such as liver, pancreas, kidneys, small intestines, endocrine glands and the like. Generally, fenestrations enable movement of large molecules from and to the blood and / or extravascular compartment, e.g., fenestrations can be found within liver endothelial cells e.g. to enable the transfer of substrates (such as lipoproteins) between blood and hepatocytes. Usually, a single fenestration is about 50 - 300 nm or even 80 - 100 nm in diameter. In vivo, the majority of fenestrations are arranged in groups of 10-100 called “liver sieve plates".

[0116]

[0111] In one preferred aspect it was found that the average diameter of a sinusoid(- like) structure obtainable with the method of the invention, when using human cells, was between 1 - 50 pm, preferably between 5 - 30 pm, more preferably about 10 pm, thus resembling the average diameter of a human native liver sinusoid. The average diameter of a sinusoid(-like) structure provided by the method of the invention is about 2, 5, 10, 15, 20, 25, 27.5, 30, 35, 40, 45 or 50 pm. The skilled person understands that a sinusoid(-like) structure, formed by cells, is a naturally occurring structure and therefore does not comprise the same diameter over the whole of the sinusoid(-like) structure. In other words, the diameter of a sinusoid(-like) structure is not 10 pm in the whole sinusoid(-like) structure. On the contrary, it can be that in one part of the sinusoid(-like) structure the diameter is 60 pm, and in another part of the sinusoid(- like) structure the diameter is 1 pm, and in a further part of the sinusoid(-like) structure the diameter is 20 pm. The average diameter hence is herein considered as the average (estimated) diameter over the whole of the sinusoid(-like) structure, for example as determined by measuring the diameter at different positions in the sinusoid(-like) structure and, based thereupon determine an average diameter, using methods known to the skilled person.

[0117]

[0112] In one preferred embodiment, the cells comprised in the scaffold are cultured for at least 2 days, at least 4 days, 5 days, 6, days, preferably about 7 days to allow for the liver tissue comprising a vasculature with sinusoidal structures and / or sinusoidlike structures to form in the scaffold.

[0118]

[0113] In some embodiments, there is provided for a method in accordance with the invention, wherein the liver tissue further comprises polarized hepatocytes, wherein the polarized hepatocytes align such that a bile canaliculus or a bile-canaliculus-like structure is formed. As meant herein the term “aligning” means that the hepatocytes, when polarized, arrange in a line, e.g., a (mono)layer / sheet / group of hepatocytes, opposite to at least one other line, e.g., a further (mono)layer / sheet / group of hepatocytes, preferably with a bile canaliculus and / or bile canaliculus-like structure separating both, or more, lines of polarized hepatocytes. Hence, in some embodiments, there is provided for a method in accordance with the invention, wherein the polarized hepatocytes form a layer / sheet / group of cells, preferably wherein said layer / sheet / group is formed in the scaffold.

[0119]

[0114] It was found that, upon providing a gel precursor of step (b), further comprising hepatocytes, the hepatocytes in step (e) formed structures wherein the basal (basolateral) sides of hepatocytes are aligned such that they surround or are directed towards the vasculature and / or wherein the apical sides of the hepatocytes face other apical sides of hepatocytes. It was surprisingly found that the on the apical sides of the cultured hepatocytes in vitro formed structures that resemble bile canaliculi. As such, the hepatocytes form structures that resemble bile canaliculi, e.g., bile canaliculi or bile canaliculi-like structures. These bile canaliculi(-like) structures were identified by providing Calcein-AM (which is a substrate of the apically-expressed transporter MRP-2) into the culturing device and detecting the accumulation of Calcein-AM in the spaces between (apical sides) of polarized hepatocytes. Therefore, in some embodiments, there is provided for a method in accordance to the invention, wherein in the liver tissue, polarized hepatocytes are aligned such that the basal side of the polarized hepatocytes is adjacent to the vasculature (or directed towards the vasculature) and / or such that the apical side of the polarized hepatocytes is adjacent to, or is directed towards, a bile canaliculus or bile-canaliculus-like structure (i.e. together with the apical domains of other polarized hepatocytes form a bile canaliculus or bile-canaliculus-like structure). It is preferred that at least one bile canaliculus or bile canaliculus-like structure is obtained in the liver tissue obtained with the method according to the invention.

[0115] A skilled person is able to differentiate an apical side from a basal side by using methods known in the art, such as for example observing marker molecules that are indicative for the apical or basolateral side of a cell, e.g., of a hepatocyte.

[0120]

[0116] A suitable marker substance to establish the presence of polarized hepatocytes is for example is Calcein-AM, which is a substrate for MRP-2, an apically-expressed transported in (mature) hepatocytes. In the present invention it was found that, using detection of MRP-2 expression (or Calcein-AM accumulation), in the obtained liver tissue the apical sides of polarized hepatocytes aligned such that a bile canaliculus or a bile-canaliculus-like structure in the obtained liver tissue could be identified. A skilled person may also use other marker substances indicative for hepatocytes, e.g., for mature hepatocytes, to identify hepatocytes in the scaffold as provided herein. For example, albumin expression is used to identify mature hepatocytes.

[0121]

[0117] Also provided is that the hepatocytes, preferably polarized hepatocytes, are allowed to form a layer / group / sheet of cells, wherein the layer / group / sheet of cells comprises an apical and a basal side, the basal side being faced towards vasculature and / or the apical side facing towards bile canaliculi(-like) structures.

[0122]

[0118] In addition, it was found that the use of a perfusion flow, e.g., by applying a perfusion flow through the microfluidic device, benefited the formation of vasculature comprising polarized hepatocytes.

[0123]

[0119] In the present invention, the basolateral membrane of the hepatocyte is the membrane that faces a structure resembling vasculature, preferably a sinusoidal(-like) structure. It is herein contemplated that at least part of the polarized hepatocytes in the scaffold comprises a basal side adjacent to or directed towards the vasculature and / or comprise an apical side adjacent to or directed towards a bile canaliculi.

[0124]

[0120] Also provided is that the hepatocytes are positioned such in respect of a bile- canaliculus-like structure and / or bile canaliculus that the bile-canaliculus-like structure and / or bile canaliculus is at least partially positioned between apical sides of two or more layer / group / sheet of hepatocytes. It is contemplated that the bile-canaliculus-like structure and / or bile canaliculus preferably has a form or shape that is such that more than two layer / group / sheet of apical sides of polarized hepatocytes face the bile- canaliculus-like structure and / or bile canaliculus.

[0125]

[0121] In some embodiments, the method provides for the obtaining of a liver tissue comprising as a characteristic feature that a vascular network is formed, wherein the vascular network comprises a (clear) lumen and further comprises one or multiple liver sinusoids and further comprises (a layer of) (polarized) hepatocytes, the basolateral side thereof being adjacent to or directed towards a vascular structure, and the apical side of the hepatocytes forming a bile canaliculi .

[0126]

[0122] 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 resident liver immune cells, and tumor cells.

[0127]

[0123] More in particular, the immune cells are selected from neutrophils; eosinophils; basophils; lymphocytes, more in particular T cells, B cells and natural killer cells, monocytes, macrophages; a mixture comprising peripheral blood mononuclear cells (PBMCs); and combinations thereof.

[0128]

[0124] In a particular embodiment of resident liver immune cells, Kupffer cells are comprised in the gel precursor of step (b).

[0129]

[0125] In a particular embodiment the tumor cells are cells derived from liver tumors or from other types of tumors, preferably circulating tumor cells.

[0130]

[0126] The gel precursor comprising tumor cells allows to study a model of cancer in the obtained liver tissue, for example for the test of candidate therapeutic agents. It also provides a model to study the morphology and preferred tissue parts that are affected by a tumor. It also allows to characterize and monitor development of a tumor and its tumor (micro)environment over time and / or in response to (immune)therapy. Therefor in some embodiments there is provided for a liver tissue obtainable with the method of the invention, further comprising tumor cells and / or a tumor.

[0131]

[0127] In some embodiments of the method of obtaining a liver tissue, it further comprises, after the step (e) of culturing the cells in the scaffold to obtain the liver tissue, a step of adding one or more of undifferentiated cells, immune cells, infection agents, nucleic acid carriers, preferably gene-therapy vectors, organoids, tumoral cells and / or test compounds.

[0132]

[0128] In a more particular embodiment, the undifferentiated cells differentiate in the obtained liver tissue. Particular embodiments of undifferentiated cells comprise a source of monocytes, preferably added as one of the cell types in PBMCs. As illustrated in the figures (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 to differentiate to macrophages (cells expressing CD16 marker). In the same way, other undifferentiated cells may be added to differentiate in situ under (culture)condition known to the skilled person required to induce such differentiation.

[0133]

[0129] In another particular embodiment of the method other cell types may be added after step (e) in the method, i.e. , immune cells, preferably selected from neutrophils; eosinophils; basophils; lymphocytes, more in particular 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 to be added after step (e) in the method are T cells and macrophages.

[0134]

[0130] In another also particular embodiment of the method, after step (e) the method comprises the addition of one or more of an infectious agent (i.e., an agent or an organism unicellular or multicellular or that cause a disease), preferably selected from the group consisting of a bacteria, a virus, a fungus, a protozoa and a helminth, to the previously obtained liver tissue.

[0135]

[0131] In another also particular 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 virus vectors, preferably adeno-associated virus vectors. The addition of these nucleic acid carriers, preferably AAV, allow for the study of gene-therapy. In a more particular embodiment, the AAV are of a capsid serotype specific for liver cells, preferably AAV1 , AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10. In some embodiments, a nucleic acid is added to the obtained liver tissue, in some embodiments the nucleic acid is a DNA, in some embodiments the nucleic acid is a RNA. In some embodiments the RNA or DNA in encapsulated in a delivery vehicle, for example a liposome or nanoparticle. In some embodiments, the RNA or DNA is in the form of a vaccine.

[0136]

[0132] In another also particular embodiment of the method, after step (e) one or more organoids are added, preferably on top of the culturing device to obtain a grafted organoid. The organoid is selected from any origin and / or mimicking any organ at any stage of development. Preferably, the organoid is a liver organoid.

[0137]

[0133] In another also particular embodiment of the method, after step (e) one or more tumoral cells (cancer cells) are added. They may be, in particular, liver tumor cells or, in the alternative non-liver tumor cells. The addition of liver tumor cells allows for a model of a liver cancer. The addition of non-liver tumoral cells, allows for a model of metastases in the liver.

[0138]

[0134] In another also particular embodiment of the method, candidate test compounds are added, for example during or after step b,c,d or e, which compounds may be of any nature and may, for example, be used to screen for compounds that modulate liver development, or that provide a prophylactic or therapeutic effect in a liver model of disease. Therefore, and in some embodiments, there is provided for a screening method comprising testing compounds to be screened in a liver tissue obtained with the method of the invention or testing compounds in the method of obtaining a liver tissue as disclosed herein.

[0139]

[0135] For the addition of any of these cells, agents or compounds, for example after step (e), several approaches are possible. In one approach, these cells and / or agents and / or compounds can be added as ingredient of the culture media, for example on the top of the cell culturing chamber. In the alternative, they can be added to any lateral tube / channel of the culturing device, comprising or not a vasculature of endothelial cells (e.g., HLIVEC).

[0140]

[0136] Also provided is that, in some embodiments, the culturing device comprising the culture chamber is a microfluidic culturing device. Microfluidic cell culturing is an increasingly important technology. The technology finds its application in drug screening, tissue culturing, toxicity screening, and biologic research. A major advantage of microfluidic cell culturing is that it can add aspects such as perfusion flow, enhanced co-culturing and stable gradients to traditional cell culture, and may provide higher-quality data, reduced reagent consumption, and lower costs. It is preferred that the microfluidic culturing device comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 etc. microfluidic channels that are connected to the culture chamber. In the case of a microfluidic device the culture chamber can be in the form of a channel, preferably in the form of a channel that is the middle channel of two other channels. It may be that the microfluidic channel that serves as culture chamber is larger in size (e.g., comprising a larger diameter) than the adjacent channels. An exemplary microfluidic device is shown in Figure 2 of the current disclosure (not to scale). Provided herein the one or more microfluidic channels are preferably positioned lateral to the culture chamber. More preferably these channels are positioned substantially parallel to the culture chamber.

[0137] It is preferred that the culture chamber, when in the form of a channel, at least comprises an inlet.

[0141]

[0138] It is contemplated that the channels, preferably the channels not being the culture chamber, can be used to benefit the culturing chamber, for example by adding further cells, preferably endothelial cells such as HLIVECs, liver endothelial cells, and the like (or mixtures thereof), to the culturing device, thereby allowing the formation of further tubule(-like) structures in the channels, preferably in channels not being the culture chamber e.g. in the channels that are positioned lateral relative to the culturing chamber (in non-limiting examples, in Figure 2 two channels are positioned on both sides relative to a central culture chamber, and in Figure 3 the laterally positioned channels relative to the culturing chamber (in the example named “graft chamber”) are referred to as “perfusion channels”). Said tubule(-like) structures can for example be further used to supplement the culture in the channels and / or in the culture chamber with further aqueous culture medium, nutrients, fluid and the like and / or for removing and / or diluting waste metabolites as produced by the cells.

[0142]

[0139] Advantageously, within context of the invention, said microfluidic channels are used to allow for a perfusion or flow, for example to allow a flow (of, for example, a culture medium) through at least a part of the vasculature. In one preferred embodiment liver endothelial cells are added to the microfluidic device by using a channel, preferably at least two channels, to allow for the formation of a tubule(-like) structure in said two channel(s). This or these channel(s) are positioned laterally relative to the culture chamber. For example, in the case of two channels, one channel may be positioned on one lateral side of the culture chamber and another channel may be positioned on the opposing lateral side of the culture chamber. A benefit of allowing endothelial cells to form a tubule(-like) structure in the channel(s) is that this may be advantageous in the formation of vasculature in the scaffold. For example, endothelial cells in one or more of the lateral channels may interact, e.g., by directly and / or indirectly contacting, cells, e.g., endothelial cells, in the scaffold thereby connecting the endothelial cells in the channel to the cells in the scaffold, thereby allowing the forming of vasculature that can extend from the channel in and through the scaffold. In such embodiment, for example, a blood vessel in a microfluid channel is fluidly connected to the vascular bed in the culture chamber, allowing perfusion of both. Application of a flow or perfusion, for example with an aqueous solution or a culture medium may be further advantageous in the formation of vasculature in the scaffold.

[0143]

[0140] In another preferred embodiment one or more, preferably at least two, microfluidic channels are fluidically connected. It is further preferred that the microfluidic channels comprise an inlet and an outlet. The network formed by one or more microfluidic channels may be referred to as microfluidic channel network. In some embodiments, there is provided for a method in accordance with the invention, wherein the culturing device is a microfluidic culturing device comprising the culture chamber and further comprises at least one or at least two microfluidic channels that are fluidically connected to the culture chamber, and wherein in at least one or at least two microfluidic channels endothelial cells, preferably liver endothelial cells, are cultured. The endothelial cells may be provided in a gel precursor that, upon settling in the microfluidic channel, forms into a scaffold comprising the endothelial cells. It is preferred that the (further) endothelial cells are liver endothelial cells. Other (liver) cells can be provided together with the (liver) endothelial cells. Further it is 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 culturing device as provided in step (a) of the method. Also, it is contemplated that the further liver endothelial cells that are allowed to form the tubule(-like) structures in the microfluidic channels before, during (any step) or after performing the steps (a) - (e) in accordance to the method as provided herein. It is preferred that the tubule(-like) structures are formed during step (e) thereby forming an integral part of the obtained liver tissue.

[0144]

[0141] As described above, it is preferred that in the microfluidic culturing device comprising at least one or at least two microfluidic channels that are fluidically connected to the culture chamber, tubules and / or tubule-like structures are obtained, wherein the tubule(-like) structures are formed by cells, preferably endothelial cells.

[0145]

[0142] In one preferred embodiment the tubules and / or tubule-like structures have an average diameter that is similar to that of native liver tissue. The average diameter was found to be between 1 pm - 100 pm, e.g., 1 , 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90, 100 pm, preferably 1 - 50 pm, preferably between 5 - 30 pm or even 5 - 15 pm. It is preferred that the average diameter closely resembles that of blood vessels of native liver tissue.

[0143] Within the context of the current invention, a microfluidic channel network is a hollow volume defined by two side walls (surfaces), a bottom substrate, and a top substrate closing the channel network. Both side walls, top substrate and bottom substrate can be referred to as walls when being in contact with the microfluidic channel network. The channel network is furthermore connected to an inlet, typically a hole in the top substrate, that is used to fill the network from the outside world. The channel network may comprise one microfluidic channel or multiple microfluidic channels that are connected to one another. The microfluidic channel network can also be connected to further inlets or outlets.

[0146]

[0144] It may be the case that the microfluidic channel network, wherein cells may be further introduced, is characterized by the presence of a first part constructed to provide a fluid path to the cells and / or a second part constructed to provide a fluid path from said cells, preferably to and from the culture chamber comprising the liver stellate cells and liver endothelial cells. This allows for flow of growth medium through the channel and along the cells present in the channel, for example in the culture chamber. Therefore, in some embodiments, there is provided for a method in accordance to the invention, wherein the culturing device is a microfluidic culturing device comprising the culture chamber and at least two microfluidic channels, wherein the at least two microfluidic channels are, each independently, fluidically 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 a fluid to flow from one microfluidic channel to the other microfluidic channel through the scaffold comprising the cells, preferably through the obtained liver tissue provided by the method of the invention.

[0147]

[0145] It may even be the case that, adjacent to the scaffold a further microfluidic channel network is present that is in contact with the scaffold but wherein said channel is not in direct contact with a further microfluidic channel (not being the culture chamber) comprising cells, e.g., endothelial cells. For example, in case the scaffold is present in a channel that is adjacent to the channel wherein the cells will be introduced, the scaffold thus forms part of the wall of this channel. On the other side of the scaffold, a further channel may be present, and that may, for example, be used to provide the scaffold with nutrients or compounds, or that may be used to collect materials secreted by the cells. Alternatively, the scaffold may be present on two sides of a channel used for perfusion, e.g., a perfusion channel. This embodiment has the advantage that the maximum scaffold surface is directed and hence exposed towards a tubule(like)structure formed in a microfluidic channel.

[0148]

[0146] There is also provided for a method, wherein the culturing device is a microfluidic culturing device comprising the culture chamber and further comprising at least one or at least two microfluidic channels that are fluidically connected to the culture chamber, and wherein after step (e) of culturing the cells in the scaffold to obtain the liver tissue, one or more of undifferentiated cells, immune cells, infectious agents, nucleic acid carriers, tumoral cells and test compounds are added to the at least one microfluidic channel.

[0149]

[0147] There is also provided for a method, wherein the culture chamber is at least partly aligned and / or connected with a first reservoir and wherein the culture chamber is connected, preferably fluidically connected, to a second reservoir by at least one microfluidic channel. A channel may have at least one, two, more, reservoirs at each one or both ends of the channel. The reservoirs may serve different purposes but are preferred to serve the purpose of providing nutrients, fluid, agents, culture medium, cells etc. and / or collect waste, cells, fluid, agent metabolites, culture medium etc. to / of the cell culture in the culturing device.

[0150]

[0148] The reservoirs may have the form of a well (optionally of a microtiter plate), bottle, tube, flacon and the like. A reservoir may have any volume that is suitable for the setting in which the microfluidic device is used. The first and second reservoir may comprise different volumes. By way of example, a first reservoir may have a volume of about 15 pL and a second reservoir may have a volume of about 100 pL. Preferably the reservoirs are fixed and allow for the liquid, e.g., culture medium, in the reservoir can be replaced.

[0151]

[0149] There is also provided for a method, wherein the liver tissue extends, from the first reservoir to the second reservoir. Also provided for is a method, wherein the culture chamber is further connected, preferably fluidically connected, to a third reservoir by at least one microfluidic channel, and wherein a fluid flow is introduced between the second and third reservoir. For example, there can be provided for a fluid flow inlet anywhere between said second and third reservoir. It is preferred that the fluid flow is introduced by passive leveling, but alternative manners for providing a fluid flow are not excluded and may be appropriately applied by a skilled person. It is preferred that the liver tissue extends to the third reservoir. Further, it may be that the liver tissue extends to any further reservoir that forms a part of the microfluidic channel network and / or microfluidic device.

[0152]

[0150] Alternatively, cells can be added to any one or more of the reservoirs and be allowed to directly and / or indirectly interact with (cells forming) the liver tissue. For example, HLIVECs can be added in a reservoir and subsequently are allowed to grow on top of the liver tissue and / or allowed to migrate from the reservoir into the liver tissue, e.g., by migrating to the culture chamber.

[0153]

[0151] Also provided is that, in some embodiments of the method, the culturing device comprising the culture chamber, is an unidirectional flow microfluidic culturing device. A unidirectional flow microfluidic culturing device is defined as a microfluidic culturing device, in which the microfluidic networks are configured such that any of the liquids provided in reservoirs and / or in microfluidic channels, upon tilting the device, flow through the microfluidic channels and cell culture chamber only in one direction. Thus, the microfluidic networks are so configured that during reciprocal tilting of the device to two or more positions and angles, the perfusion flow through at least part of the microfluidic network and culture chamber, largely flows in a constant direction. This direction can sometimes be adjusted depending on the operating mode (i.e., positioning, titerplate angle) of the microfluidic culturing device. This is called unidirectional flow, as opposed to bidirectional flow, in which liquid flow changes directions when the tilting direction of the microfluidic device is changed. Any embodiment relating to the addition of further cells in the channels is also applicable to the method in which an unidirectional flow microfluidic culturing device is employed.

[0154]

[0152] In a preferred embodiment, the culturing device comprising the culture chamber, is an unidirectional flow microfluidic culturing device comprising at least one microfluidic network comprising:

[0155] (a) a microfluidic layer comprising a first flow channel and a second flow channel; and

[0156] (b) a reservoir layer disposed above the microfluidic layer and comprising first and second reservoirs, wherein the first reservoir and the second reservoir each have an access port to the first flow channel and an access port to the second flow channel, so that the first flow channel and the second flow channel form a flow circuit with the first reservoir and the second reservoir; wherein the access ports in the first reservoir and the second reservoir are spaced apart so that, in use, tilting the device (i.e. , titerplate) at a first angle induces a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir primarily via the first flow channel, and adjusting the tilt so that the device is titled at a second angle causes a volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir primarily via the second flow channel.

[0157]

[0153] In even a more particular embodiment of the method in which an unidirectional flow microfluidic culturing device is employed, the method comprises:

[0158] (a) tilting the device (i.e., titerplate) at a first angle by rotating around a first axis to induce a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir via the first flow channel; and

[0159] (b) adjusting the tilt so that the device (i.e., titerplate) is titled at a second angle by rotating around said 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 channel.

[0160]

[0154] Advantageously, the employment of an unidirectional flow microfluidic culturing device to carry out the method of the invention, i.e., the applications of unidirectional flow, provides for tissue grafts, in particular for liver tissue grafts, with an improved tissue structuration in the sense that the obtained tissue and any vasculature highly mimic in vivo tissue and vasculature. As illustrated in the Figures, when applying unidirectional flow, a tissue morphology highly resembling the in vivo tissue is obtained, even an improved tissue morphology may be obtained when compared to the culture obtained under bidirectional flow conditions. Application of unidirectional flow surprisingly also allows for the phenomenon of tissue zonation, in particular in the case of liver tissue a sinusoid zonation. As explained above, liver zonation is a well- known process in which liver cells have different, and sometimes opposing, functions depending on where they are located along the vascular connection between portal to central vein.

[0161]

[0155] Another particularly advantageous application when an unidirectional flow microfluidic culturing device is employed to carry out the method of the invention, is that metastasis in a liver tissue cancer model highly resembling the actual in vivo situation can be obtained. This is mainly because the liver tissue previously obtained is mimicking actual sinusoids and the liver vasculature at a high degree. Preferably, metastasis is induced by introducing tumor cells in the at least one microfluidic channel and allowing the tumor cells to perfuse into to the liver tissue, i.e., into the vasculature.

[0162]

[0156] In the same way, while applying unidirectional flow, the analysis or simulation of an actual infection process (e.g., bacteria, virus, etc.) can be provided. The particular infection with viral and other vector particles, such as adeno-associated virus particles provides, moreover, for the study of gene therapy in the liver and other tissues.

[0163]

[0157] The application of unidirectional flow allowed also for the observation that the cell alignment is tuned by the flow of the liquid involved in the process of obtaining the tissue model. The skilled person in the art will recognize that many tissues in the human body exhibit anisotropy, structural arrangement of the cells that imply directiondependent properties. All this is achieved by means of connectivity and migration at particular directions, usually accompanied by a particular spatial distribution of the constituents of the extracellular matrix (ECM).

[0164]

[0158] Several configurations can be provided to allow a microfluidic system to work as a unidirectional flow microfluidic device. An example of a preferred device for the obtention of living and other tissue models in a plate is schematically depicted in Figure 32, where the liquid stream flow during working is illustrated with arrows within the microfluidic channels. Herein , the device comprises one or more of a microfluidic network, said microfluidic network comprising: (a) a microfluidic layer comprising at least a first flow channel (1) and a second flow channel (2) that are fluidically connected to a cell culturing chamber (3); and (b) a reservoir layer, preferably disposed above the microfluidic layer, comprising at least a first (4) and second (5) reservoirs, wherein the at least first reservoir and the second reservoir each have an access port to the first flow channel and an access port to the second flow channel so that the first flow channel and the second flow channel form a flow circuit with the at least first reservoir and the second reservoir.

[0165]

[0159] Therefore, herewith is also disclosed an unidirectional flow microfluidic culturing device that comprises one or more of a microfluidic network, said microfluidic network comprising: (a) a microfluidic layer comprising at least a first flow channel (1) and a second flow channel (2) that are fluidically connected to a cell culturing chamber (3); and (b) a reservoir layer, preferably disposed above the microfluidic layer, comprising at least a first (4) and second (5) reservoirs adapted to receive a volume of fluid (i.e. , liquid), and each having an access port to the first flow channel and an access port to the second flow channel so that the first flow channel and the second flow channel form a flow circuit with the at least two reservoirs, (numbering in reference to Figure 32). The access ports in the first reservoir and the second reservoir are spaced apart so that, in use, tilting the device (i.e., titerplate) at a first angle induces a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir primarily via the first flow channel, and adjusting the tilt so that the device (i.e., titerplate) is titled at a second angle causes a volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir primarily via the second flow channel.

[0166]

[0160] In particular, this unidirectional flow microfluidic culturing device is used with a tilting system, preferably a rocker, in such a way that a first tilt angle is imposed to the device and this induces a volume of fluid in the at least a first reservoir to flow from the said first reservoir to the second reservoir via the first flow channel; and by adjusting the tilt to a second tilt angle, the volume of fluid in the at least second reservoir is caused to flow from the said second reservoir to the first reservoir via the second flow channel, and wherein at least tilting the device at the first angle induces the volume of fluid in the first reservoir to flow from the at least first reservoir, through the cell culture chamber to the second reservoir.

[0167]

[0161] In some embodiments, there is provided for a method in accordance to the invention, wherein an opening is created during step (c) and / or after step (e) in the scaffold. It is preferred that said opening allows for the introduction of a solution, a fluid, and / or an agent. The opening may comprise any suitable form for the intended purpose. For example, a hole may be created suitable for allowing the pipetting of an agent solubilized in a solution directly into the gel. For example, an agent may be added that has an effect on vasculogenesis by liver endothelial cells, for example VEGF. As such, a hole may provide access to, e.g., the luminal side of the vasculature for a fluid.

[0168]

[0162] In some embodiments, there is provided for a method in accordance to the invention, wherein the liver endothelial cells, liver stellate cells and / or hepatocytes are not separated from each other by an artificial membrane. For example, the liver stellate cells, liver endothelial cells and / or hepatocytes, optionally any further cells, can be in direct contact with each other, without the presence of any artificial membrane or support (i.e. forming a thin membrane or layer (e.g. 5 - 60 micrometer or less), for example based on polyester, polycarbonate, or polytetrafluoroethylene or collagen- coated polytetrafluoroethylene), for example a non-natural (plastic, glass etc.) and / or from the outside introduced membrane, such as the membranes used in Transwell systems. Moreover, it is contemplated that no artificial membrane or support is present between the scaffold as formed by gelation in the culture chamber and any adjacent (microfluidic) channel, thereby allowing for direct contact between tubule(-like) structures that may be formed by endothelial cells (as herein described) in a (microfluidic) channel and the liver tissue formed in the culture chamber. In preferred embodiments, no artificial (porous) membrane is used in the method according to the invention. Within the context of the current invention, the skilled person will understand that the gel precursor, gel and / or scaffold as used in the current invention is not considered an artificial membrane or support. Within the context of the current invention, the artificial membrane is not a membrane that is formed during performing the culturing method of the invention. The skilled person will understand that in embodiments, the artificial membrane is a non-biological membrane but man made. Therefore, within the context of the current invention, in a preferred embodiment, the cells used in the method according to the invention are not separated from each other by the presence of a membrane, such as an artificial membrane, that would disallow the cells to be in contact, in particular direct cell-cell contact, with each other. For example, in a preferred embodiment, no membrane, for example artificial membrane, separates the obtained polarized hepatocytes from the obtained vasculature. The invention is therefore in part based on the absence of any artificial membrane separating the cell in the obtained liver tissue, thereby allowing to obtain a perfusable network, e.g. perfusable vascular bed and / or blood vessels.

[0169]

[0163] In some embodiments, there is provided for a method in accordance to the invention, wherein the number of total cells comprised in the gel precursor provided in step (b) is at least 1 .000 cells per microliter. The number of cells preferably the number is at most 100.000 cells per microliter, more preferably 50.000 cells per microliter. It is contemplated that the total number of cells may likely be limited by the available nutrients and / or oxygen in the culture chamber. Also there is provided for method in accordance to the invention, wherein the ratio of the number of liver endothelial cells to liver stellate cells, preferably as provided in the gel precursor in step (b), is equal to or greater than 20: 1. Further there is provided for a method wherein the number of total cells comprised in the gel precursor is at least 1.000 cells per microliter and the ratio of the number of liver endothelial cells and liver stellate cells is equal to or greater than 20:1. It was found that the number of cells is advantageous for vasculogenesis. There may be provided at least 1.000 cells, 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 cells and / or at most 50.000 cells per microliter (and any amount of cells between 1.000 - 50.000 cells), preferably per microliter of gel precursor. In preferred embodiments, the number of total cells is at least 5.000 cells per microliter, more preferably at least 7.500 cells per microliter. It is preferred that the number of total cells is about between 8.000 - 15.000 cells per microliter, preferably at most 30.000 cells per microliter. Further, certain ratios of the amount liver stellate cells compared to liver endothelial cells seem to be highly advantageous for the forming of liver tissue according to the invention. It is preferred that stellate cells are present in a ratio of at least 1 stellate cell per 20 liver endothelial cells, meaning that, for example, in case 10.000 liver endothelial cells are provided at least 500 liver stellate cells are provided. Still, it was found that a ratio of equal to or greater than 20: 1 (liver endothelial cells : stellate cells), e.g., with increasing preference equal to or greater than 15:1 , equal to or greater dan 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 , resulted in beneficial vasculogenesis. As a non-limiting illustrative example several ratios (1 :1 , 2:1 , 4:1 and 20: 1) of liver endothelial cells to liver stellate cells have been tested and are shown in Figure 6 of the current disclosure. It may be that a ratio of liver endothelial cells and liver stellate cells is smaller than 20:1 , e.g., 25:1 , 30:1 etc. is used in the disclosed methods, although these smaller ratios of liver endothelial cells : liver stellate cells are not preferred for the current method. It was however found that a ratio of equal to or greater than 20: 1 (liver endothelial cells : stellate cells) allowed for robust vascular development. Most preferably, the ratio is about 4:1 , meaning that for every 4 liver endothelial cells there is 1 liver stellate cell in the gel precursor. It is particularly preferred that the herein referred numbers of cells and / or ratios are representative for the number of cells that are present in step (b) of the current method, i.e., before step c). The numbers of cells, for determining the ratio do not comprise proliferated and / or differentiated cells in accordance to step (e) of the current method, nor do the numbers of cells comprise the endothelial cells as further introduced in one or more microfluidic channels. The number of cells as provided herein comprises any further cells added in the gel precursor in accordance to step (b) i.e. , hepatocytes, Kupffer cells, immune cells, biliary cells and the like.

[0170]

[0164] In some embodiments, Kupffer cells can be added in the current method. It was found that adding of Kupffer cells results in obtaining a liver tissue comprising alive Kupffer cells. The addition of Kupffer cells benefits the inflammatory response in the obtained liver tissue. Hence, providing Kupffer cells allows for the herein obtained liver tissue to even more closely resemble native liver tissue. It was for example found that upon providing Kupffer cells in the method according to the invention, the cells were functional, e.g., when mimicking hepatic steatosis by providing free fatty acids.

[0171]

[0165] In some embodiments, Kupffer cells are added on top of the scaffold formed in step c) (e.g., during step c) and / or step d)) and / or on top of the obtained liver tissue in step e) and are allowed to migrate into the scaffold and / or liver tissue.

[0172]

[0166] In some embodiments, biliary cells can be added in the current method. It was found that adding of biliary cells results in a liver tissue comprising alive biliary cells. Hence, providing biliary cells allows for the herein obtained liver tissue to even more closely resemble native liver tissue.

[0173]

[0167] It is contemplated that the ratio does not need to comprise absolute numbers, e.g., round numbers such as 1 , as in “the ratio being 1 : 1”, but may for example comprise “1 ,00001 : 1” when referring to the ratio of the number of liver endothelial cells to the number of liver stellate cells.

[0174]

[0168] In some embodiments, there is provided for a method in accordance to the invention, wherein the ratio of the number of hepatocytes to the sum of the number of liver endothelial cells and liver stellate cells is greater or equal to than 1 :1. For example, when hepatocytes are provided in the method in accordance to the invention there are at least 2 hepatocytes for each 1 liver endothelial cells and 1 liver stellate cells (i.e., equal to a ratio of 1 : 1). As such, it is preferred that there are more hepatocytes present in the gel precursor than that there are liver endothelial cells and liver stellate cells in said gel precursor. Optionally, the numbers of cells, i.e., for determining the ratio, do not comprise proliferated and / or differentiated cells in accordance to step (e) of the current method, nor do the numbers of cells comprise the liver endothelial cells as further introduced in one or more microfluidic channels. For example, there may be about 8000 - 10.000 hepatocytes per microliter, between about 3.000 - 3.500 liver endothelial cells per microliter and between about 1.000 - 2.000 liver stellate cells per microliter.

[0175]

[0169] Further, it is preferred that the ratio of the number of hepatocytes to the sum of the number of liver endothelial cells and liver stellate cells is greater than 2:1. It is contemplated that an increased number of hepatocytes compared to liver endothelial cells and liver stellate cells is advantageous for the formation of liver specific structures, e.g., bile canaliculi(-like) structures. In a further embodiment the ratio of the number of hepatocytes to the sum of the number of liver endothelial cells and liver stellate cells is between (and including) 1 :1 and 30:1 , e.g. 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 liver endothelial cells and liver stellate cells between 1 :1 and 30:1 is encompassed by the invention. Preferably the ratio is between 1 :1 and 10: 1 , or even more preferably between 1 :1 and 5:1. It is contemplated that the ratio does not need to comprise absolute numbers, e.g., round numbers such as 1 : 1 , but may comprise 1 ,00001 : 1 when referring to the ratio of the number of hepatocytes to the sum of the number of liver endothelial cells and liver stellate cells.

[0176]

[0170] For example, in case the number of total cells I pl comprised in the gel precursor provided in step (b) is about 100.000 cells / pl, that the amount of hepatocytes to the sum of the amount liver endothelial cells and liver stellate cells may be, for example, about 50.000 hepatocytes : 50.000 liver endothelial cells and liver stellate cells, but can also be about 80.000 hepatocytes : 20.000 liver endothelial cells and liver stellate cells.

[0177]

[0171] In some preferred embodiments, there is provided for a method in accordance to the invention, wherein the gel precursor of step (b) comprises per microliter:

[0178] - between 1.000 and 10.000 liver endothelial cells; and

[0179] - between 100 and 10.000 liver stellate cells.

[0180] It is preferred that the gel precursor of step (b) comprises between 1.000 and 10.000 liver endothelial cells per microliter. Therefore, the gel precursor may comprise per microliter 1.000, 1.500, 2.500, 5.000, 7.500, 8.000, 9.000 or any other number of liver endothelial cells between 1.000 - 10.000 liver endothelial cells. It is contemplated that less liver endothelial cells may be used, e.g., 500 cells per microliter, and hence these lower numbers of liver endothelial cells are also encompassed by the current invention. An amount of between 1.000 and 10.000 liver endothelial cells per microliter is however preferred for the obtaining of a liver tissue as disclosed herein and preferably for vascular generation.

[0181]

[0172] It is preferred that the gel precursor of step (b) comprises between 100 and 10.000 liver stellate cells per microliter. Therefore, the gel precursor may per microliter comprise 500, 1.000, 2.500, 5.000, 7.500, 8.000, 9.000 or any other number of liver stellate cells between 100 - 10.000 liver stellate cells . It is contemplated that less liver stellate cells may be used, e.g., 50 cells per microliter, and hence is also encompassed by the current invention. An amount of between 100 and 10.000 liver stellate cells per microliter is however preferred for the obtaining of a liver tissue and preferably for vascular generation.

[0182]

[0173] It is further preferred that the gel precursor of step (b) comprises between 5.000 and 80.000, preferably between 5.000 - 50.000, hepatocytes per microliter. Preferably the gel precursor may, per microliter, comprise 5.500, 7.500, 8.000, 9.000, 10.000, 12.500, 25.000, 30.000, 40.000, 45.000 or any other number of hepatocytes between 5.000 - 50.000 hepatocytes. It is contemplated that less hepatocytes may be used, e.g., 500 cells per microliter, and hence these lower numbers of hepatocytes are also encompassed by the current invention. An amount of between 5.000 - 50.000 hepatocytes per microliter is however preferred for the obtaining of a liver tissue and preferably for vascular generation and / or formation of bile-canaliculi(-like) structures.

[0183]

[0174] In one particular preferred embodiment, there is provided for a method in accordance to the invention, wherein the gel precursor of step (b) comprises

[0184] - between 1.000 and 10.000 liver endothelial cells;

[0185] - between 100 and 10.000 liver stellate cells; and

[0186] - between 5.000 and 50.000 hepatocytes.

[0187]

[0175] In some embodiments, there is provided for a method in accordance to the invention, wherein the gel precursor of step (b) has a volume of between 0.1 pL and 50 pL. It is preferred that the gel precursor of step (b) has a volume preferably between 0.5 pL and 20 pL. The gel precursor of step (b) may have a volume of 0.5, 1 , 2, 5, 10, 15, 20, 25, 30, 40 microliter, or any other volume between 0.1 pL and 50 pL. A skilled person is able to adjust the amount of cells to match the volume of gel precursor. In one exemplary embodiment between 0.5 - 5 pL, preferably about 1.5 pL or about 1 .35 pL of gel precursor is provided. As disclosed herein, the volume of gel precursor that is provided to the culture chamber in step (b) may be provided at once (as a whole) or may be provided in separate sub volumes. In some embodiments it is preferred that the volume of gel precursor of step (b) is provided in a microfluidic culturing device.

[0188]

[0176] One advantage of using a relatively small volume of gel precursor is that it allows for a high cell density, namely a relatively large number of cells in a relatively small volume of gel precursor. For example, provided herein in step b) may be a gel precursor having a volume preferably of between 0.5 pL- 20 pL comprising a total amount of cells of at most 100.000 cells / pL. Thus, the volumes of gel precursor and amounts of cells provided herein allow for relatively high cell densities to be used in the method of the invention.

[0189]

[0177] In some embodiments, there is provided for a method in accordance to the invention, wherein the scaffold comprises a basement membrane matrix gel, an extracellular matrix gel, a collagen gel and / or a fibrin gel. In some embodiments, there is provided for a method in accordance to the invention, wherein the scaffold of step (c) further comprises a collagen scaffold.

[0190]

[0178] In some embodiments, there is provided for a method in accordance to the invention, wherein the gel precursor comprises a mixture of at least fibrinogen gel precursor and any one or more selected from the group consisting of basement membrane matrix gel precursors, extracellular matrix gel precursors and collagen gel precursors. For example, a further gel precursor may comprise a mixture of a collagen gel precursor and a fibrin gel precursor, or for example, may comprise a mixture of a basement membrane matrix gel precursor, collagen gel precursor and a fibrin gel precursor.

[0191]

[0179] In some embodiments, there is provided for a method in accordance to the invention, wherein the gel precursor of step (b) comprises fibrinogen in a concentration of at least 1 mg / mL. The gel precursor of step (b) preferably comprises fibrinogen in a concentration of at most 50 mg / mL. The gel precursor of step (b) hence can comprise between about 1 mg / mL - about 50 mg / mL. Thus, fibrinogen can be present in, 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 - about 50 mg / mL or between any two of the above-mentioned concentrations. It is even more preferred that the end concentration of fibrinogen in the gel precursor of step (b) is about 5 mg / mL.

[0180] In one non-limiting example, in order to achieve a suitable end concentration of fibrinogen the following mixture (of 60 pL) may be provided in step (b): 10 pL 8125 cells / pL stellate cells in DMEM (10% FBS, 1 % P / S) (end concentration 1.625 cells / pL), 10 pL 16.250 cells / pL liver endothelial cells in DMEM (10% FBS, 1 % P / S) (end concentration 3.250 cells / pL), 20 pL 25.000 cells / pL hepatocytes in DMEM (10% FBS, 1% P / S) (end concentration 10.000 cells / pL), 5 pL 50 mg / mL Fibrinogen (end concentration 5 mg / ml) and 5 pL 1 U / mL Thrombin (end concentration 0.1 U / mL).

[0192]

[0181] The concentration of fibrinogen in the gel precursor of step (b) may be achieved by adding a certain volume of a stock solution of fibrinogen to the gel precursor, preferably to the mixture / suspension / dispersion that will form the gel precursor. It is possible, though not preferred, to achieve the concentration of fibrinogen in the gel precursor as well by adding a certain volume of fibrinogen in step (b) and further add a volume of fibrinogen in step (c), at least insofar the gel precursor has not yet been allowed to completely gelate into a scaffold, i.e., when at least part of the gel precursor is not gelated. It is preferred that the gel precursor comprises sufficient fibrinogen for the gel precursor to gelate to obtain a scaffold, e.g., under influence of thrombin.

[0193]

[0182] In some embodiments, there is provided for a method in accordance to the invention, wherein the gel precursor of step (b) further comprises thrombin. Preferably the gel precursor of step (b) comprises thrombin in a concentration of at least 0.01 U / rnL or at most 1 U / rnL. The concentration of thrombin in the gel precursor of step b) may for example be 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 - 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 the enzymatical conversion, i.e., by thrombin-mediated proteolysis, of gel precursor comprising fibrinogen to a scaffold comprising fibrin. Moreover, a skilled person may want to adjust the concentration of thrombin depending on the solidity of the gel, e.g., it may be that for a certain use a less solid scaffold is desirable; in such a case the concentration of thrombin provided can be reduced, e.g., so that at least part of the concentration of fibrinogen in the gel precursor is not converted into fibrin.

[0194]

[0183] The concentration of thrombin in the gel precursor of step (b) may be achieved by adding a certain volume of a stock solution of thrombin to the gel precursor, preferably to the mixture / suspension / dispersion that will form the gel precursor. It is possible to achieve the concentration of thrombin in the gel precursor as well by adding a certain volume of thrombin in step (b) and further add a volume of thrombin in step (c), at least insofar the gel precursor has not yet been allowed to completely gelate into a scaffold, i.e. , when at least part of the gel precursor is not gelated. Alternatively, it is possible to achieve the concentration of thrombin in the gel precursor by adding a volume of thrombin in step (c), at least insofar the gel precursor has not yet been allowed to completely gelate into a scaffold.

[0195]

[0184] In some embodiments, there is provided for a method in accordance to the invention, wherein the culture medium of step (d) has a volume of between 20 and 500 pL, e.g., 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 490 pL, or any other volume between 20 - 500 pL. In some embodiments, there is provided for a method in accordance to the invention, wherein the culture chamber has a volume of between 0.1 pL and 50 pL, e.g., 0.5, 1 , 1.5, 2, 2.5, 5, 10, 15, 20, 25, 27.5, 30, 35, 40, 45, 49.5 pL It is even more preferred that the culture chamber has a volume of between 0.1 pL and 20 pL. In another embodiment, there is provided for a method in accordance to the invention, wherein the culture medium of step (d) has a volume of between 20 and 500 pL and wherein the culture chamber has a volume of between 0.1 pL and 50 pL.

[0196]

[0185] It may be that in step (d), and further in step (e), more than one culture medium, e.g., 2, 3, 4, 5, 6, 7, 8, 10 etc., is provided. It may be that one culture medium is provided multiple times in step (d). It may be that different culture media are provided (and replaced) multiple times. The invention is not particularly depending on a specific culture medium for contacting the scaffold comprising the cells and / or for culturing the cells in step (e) as long as it is suitable for growing cells within the context of the current invention. For example, cells may be provided in the gel precursor dispersed / suspended in a first culture medium, e.g., iHEP medium (Cellular Dynamics, Fujifilm), and the cells may subsequently, e.g., during step (b) and / or (c) and / or (d), be contacted with a second culture medium, and optionally with a third or fourth culture medium on day 1. In other words, the cells may be contacted with multiple culture media compositions during the method of the invention, and optionally may be contacted with one or more culture medium compositions on day 1. It is contemplated that culture media compositions can be mixed, although preferably different culture medium compositions are provided separately (e.g., at different time points) and optionally after removing of the previous culture medium composition to which cells have been exposed. Subsequently the scaffold comprising the cells may be contacted again with any one of the first or subsequent culture medium or with a different culture medium.

[0197]

[0186] A culture medium may for example comprise Dulbecco’s Modified Eagle Medium (DMEM, Gibco, cat. No. 11965-092) for example supplemented with further compounds such as 10% FBS (Gibco), penicillin-streptomycin (P / S, Sigma-Aldrich, cat. No. P4333-100ML) and sodium pyruvate (Thermo Fisher Scientific), or may comprise RPMI medium (Sigma-Aldrich, cat. No. R8758-500ML) for example supplemented with further compounds such as B27 supplement (Gibco, Cat. No. 17404-001 , Lot. No. 2209534), Oncostatin M, Dexamethasone (MP Biomed, cat. No. 194561), Gentamicin (Gibco, cat. No. 15750-060) and iCell Hepatocytes 2.0 Medium Supplement (Cellular Dynamics, Cat. No. M1024, Lot. No 106063) or may comprise Endothelial Cell Growth Basal Medium-2 (EBM-2, Lonza, cat. no. CC-3156) for example supplemented with further compounds such as FBS, hydrocortisone, hFGF- B, VEGF, R3-IGF-1 , ascorbic acid, hEGF, GA-1000 and Heparin. Other suitable media are commercially available, e.g., Hepatocyte Basal Medium (CC-3199) HCM™ SingleQuots™ Kit (CC-4182) (Lonza), William’s E medium etc.

[0198]

[0187] In some embodiments it is preferred that at least one of the culture media provided in the method according to the invention comprises one or more growth factors selected from FGF, VEGF and / or EGF, preferably in a concentration that benefits the formation of a vasculature.

[0199]

[0188] In some embodiments, there is provided for a method in accordance to the invention, wherein the culture medium of step (d) is (at least partially) replaced every 24 to 144 hours. In some embodiments, there is provided for a method in accordance to the invention, wherein the cells are cultured for at least, with increasing preference, 3, 4, 5, 6 or 7 days. Moreover, in some embodiments, there is provided for a method in accordance to the invention, wherein the culture medium of step (d) is replaced every 24 to 144 hours and there is provided for a method in accordance to the invention, wherein the cells are cultured for at least, with increasing preference, 3, 4, 5, 6 or 7 days.

[0200]

[0189] The culture medium provided in step (d) and / or the culture medium in which the cells are cultured in step (e) may be replaced at least every 24 hours and / or at most every 144 hours. The culture medium can for example be replaced 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 other timepoint between 24 to 144 hours is also encompassed by the current invention. The culture medium may be replaced by the same culture medium or by a different culture medium. It is preferred that medium is replaced once the nutrients in the culture medium have been (for the most part) taken up / used by the cells. Optionally, the culture medium is replaced to influence differentiation and / or proliferation of cells.

[0201]

[0190] It is preferred that cells are cultured for at least 3 days. The cells may be cultured for a longer period of time. It is preferred that after about 6 or 7 days of culturing the cells are suitable for being prepared for imaging (e.g., by adding an imaging agent such as Calcein AM to a culture medium). It was found that after about 3 days after commencing step (d) the cells start differentiating and / or proliferating and are forming a liver tissue, preferably the liver tissue comprising vasculature and / or comprising sinusoidal(-like) structures and / or bile canaliculi(-like) structures as disclosed herein. Hence, in some embodiments, it is contemplated that about 7 days, preferably after performing steps (a) - (c) of the current method, is sufficient a period of time to obtain a liver tissue as disclosed herein. Also, it is contemplated herein that any one or more of the steps (a) - (c) may be halted, e.g., by cryopreserving the culturing device comprising the gel precursor and / or scaffold comprising the cells. In such a case it is contemplated that, after thawing of said cryopreserved culturing device, a liver tissue and / or liver-like tissue may be obtained within 7 days of culturing. Also, it is contemplated that any one of steps (d) - (e) may be halted, e.g., by cryopreserving the culturing device comprising the cells and / or comprising the (in part) obtained liver tissue. Moreover, the method may comprise a step (f), wherein the obtained liver tissue is conserved, e.g., by cryopreservation, after step (e).

[0202]

[0191] In some embodiments, there is provided for a method in accordance to the invention, wherein the gel precursor of step (b) further comprises hepatocytes; and

[0203] - wherein the one or more, preferably all, cells are cultured for a period sufficient to allow the formation of a vasculature, preferably wherein the vasculature is accessible (e.g., fluidically and / or physically) and / or has a diameter of about 5 to 50 pm; and / or - wherein the one or more, preferably all, cells are cultured for a period sufficient to allow the formation of polarized hepatocytes, preferably a layer / sheet / group 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 a bile canaliculus and / or bile-canaliculus-like structure;

[0204] - wherein the one or more, preferably all, cells are cultured for a period sufficient to allow the formation of a bile canaliculus and / or bile-canaliculus-like structure; and / or

[0205] - wherein the one or more, preferably all, cells are cultured for a period sufficient to allow expression of LYVE-1 marker in the liver endothelial cells and / or expression of PLVAP marker in the liver endothelial cells and / or expression of MRP-2 in the hepatocytes; and / or

[0206] - wherein the one or more cells are cultured for a period sufficient to allow the formation of fenestrations in the liver tissue.

[0207]

[0192] In some embodiments, there is provided for a method in accordance to the invention, wherein the gel precursor of step (b) and / or the culture medium of step (d) comprises an antifibrinolytic agent. It is preferred that the antifibrinolytic agent is selected from the group consisting of a bovine pancreatic trypsin inhibitor, a lysine analogue, a coagulation factor (e.g., Factor XIII), or a serine protease inhibitor, e.g., aminocaproic acid, aprotinin, epsilon-aminocaproic acid, tranexamic acid. The agent may be a combination of any one of the herein referred antifibrinolytic agents. Alternative agents having an antifibrinolytic effect may be selected and used appropriately by a skilled person. It is preferred that the antifibrinolytic agent is aprotinin. It is preferred that an agent, and a concentration of said agent, is provided during step (b) and / or step (d) of the current method that at least partly prevents the degradation of the fibrinogen and / or fibrin in the scaffold. Moreover, it is preferred that the agent does not affect the growth, differentiation and / or proliferation of the cells and / or affects the obtaining of the liver tissue.

[0208]

[0193] As provided herein, the concentration of the agent depends on the various parameters, such as the volume of scaffold, concentration of fibrinogen and / or thrombin used etc. It is preferred that an amount of about 10 - 1000 kill (kallikrein inhibitor unit) of aprotinin is used and is provided in a suspension / solution of culture medium. For example, aprotinin may be provided in about 50 pL of culture medium. Thus, by way of example, aprotinin may be provided in a concentration of between about 0.2 klll / pL to about 20 klll / pL, e.g., a concentration of about 2 klll / pL of aprotinin is provided in either the culture medium, in the gel precursor, or both.

[0209]

[0194] There is also provided for a method wherein a culture medium, preferably the culture medium as used in step (d), comprises any one or more of the agents selected from: a glucose, a pyruvate, an amino acid, preferably a free amino acid, a vitamin, a salt, insulin, a protein, preferably a cytokine and / or a growth factor, such as, but not limited to Oncostatin M and / or VEGF. Preferably the growth factors at least comprise FGF, EGF and / or VEGF. The culture medium may be adjusted to benefit differentiation and / or proliferation of the cells. A skilled person is aware about how to adjust the culture medium accordingly comprising any one of the herein mentioned agents selected from the group consisting of glucose, a pyruvate, an amino acid, preferably a free amino acid, a vitamin, a salt, insulin, a protein, preferably a cytokine and / or a growth factor. Moreover, a skilled person is able to adjust, routinely, the concentration of any one of said agents depending on the amount of cells and or cell-type that is kept in culture in the culturing device.

[0210]

[0195] Moreover, in some aspects of the invention, the culture medium, preferably as used in step (d), does not comprise a TGF-p inhibitor, a hepatocyte growth factor, activin A, a R-spondin protein, a fibroblast growth factor, preferably fibroblast growth factor 10, epidermal growth factor, a GSK-3 inhibitor, preferably CHIR99021. It is contemplated that any one of these disclaimed agents may negatively affects the obtaining of a liver tissue by performing the method of the invention, for example, when used at certain concentrations in the culture medium.

[0211]

[0196] In some embodiments, there is provided for a method in accordance to the invention, wherein the liver endothelial cells, liver 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 cells derived from a human subject (for example 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.

[0212]

[0197] In some aspects the liver tissue as described herein is an artificial liver tissue. It is contemplated herein that an artificial liver tissue can refer to a liver tissue that mimics native tissue, but which has been engineered (e.g. obtained by using a method of tissue engineering, for example a method as provided herein), which is preferably cell-based, and may be partly stem cell derived, and wherein at least a part of the obtained liver tissue comprises a biopolymer matrix, such as a fibrin scaffold, to mimic part of a native liver tissue.

[0213]

[0198] In some embodiments, there is provided for a method in accordance to the invention, wherein the liver tissue that is obtained in step (e) allows for intracellular lipid droplet accumulation upon contacting the cells, preferably the obtained liver tissue, with free fatty acids for a period of between 12 and 48 hours. Preferably, a sufficient amount of free fatty acids is provided to allow the formation of lipid droplets. In one non-limiting example (Figure 12B) the obtained liver tissue was exposed to about 1000pM of free fatty acids (500pM oleic acid and 500pM palmitic acid). Free fatty acids may be provided together with or mixed in a culture medium. Any period between 12 - 48 hours for exposing the cells to free fatty acids can be considered and thus is herein encompassed, e.g., 12, 16, 20, 24, 26, 30, 32, 36, 40, 42, 48. Any free fatty acid and / or mixture of free fatty acids can be considered and thus is encompassed.

[0214]

[0199] In an alternative embodiment, specifically in case hepatocytes are provided in step (b) of the current method, the hepatocytes may have been exposed to free fatty acids prior to providing the cells in step (b). Thus, it may be that hepatocytes provided herein, e.g., in step (b), contain lipid droplets intracellularly. It is preferred that the lipid droplet accumulation in said hepatocytes provided in the current method still allows for at least some hepatocytes to proliferate and / or differentiate and optionally form into the at least one or more structures that are typically present in healthy native liver tissue. It is preferred in some uses of the current method that the concentration and / or period of exposing hepatocytes to free fatty acids is sufficient to induce hepatic steatosis, which for example is indicated by the accumulation of lipid droplets in the liver. The accumulation of such lipid droplets in the liver tissue as obtained and provided herein will be regarded by a skilled person as indicative for metabolically competent liver tissue.

[0215]

[0200] In some embodiments, there is provided for a method in accordance to the invention, wherein the method further comprises adding a compound to the cell culture and observing the effect thereof on the formation or functioning of the 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 hepatic 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. Further, a compound may be provided that is a parent compound, e.g., a drug, which is metabolized as a result of hepatic metabolism. Also, free fatty acids, e.g., supplemented to the medium, may be added for example suitable for studying / observing the accumulation of fat reserves. Further, fluorescent dyes (such as, CMFDA, Calcein-AM and the like) may be added for observing polarized transport, e.g., by adding them to the formed bile-canaliculi.

[0216]

[0201] The current invention further provides for a liver tissue that is obtained by the method as disclosed and embodied herein. It is preferred that the liver tissue comprises a vasculature. The liver tissue, and more specific a liver tissue comprising a vasculature, obtained in accordance with the method of the invention can comprise any one or more of the following characteristics: a) Viable population of at least endothelial cells and stellate cells, preferably of endothelial cells stellate cells and hepatocytes; b) Sinusoidal vascular network, preferably comprising a detectable lumen with a diameter between 5 and 30 pm and / or comprising an at least partly fenestrated endothelium; c) Compact parenchymal cell layers / sheets / groups at least partly allocated between the vascular structures, preferably comprising epithelial morphology; d) Polarized epithelium with transporters (e.g., the apical transporter MRP2) expressed either on the basolateral side facing blood vessel or on the apical side facing other parenchymal cells; e) Bile canaliculi(-like) structures between two layers / sheets of (polarized) hepatocytes; f) Fl uidical ly accessible liver tissue; g) Physically accessible liver tissue.

[0217]

[0202] Preferably, a viable population of cells is formed when at least about 30,%, 40% 50%, 60%, 70%, 75%, 80%, 90% of the total number of cells in the population is viable. Cell viability may be tested by using methods known and commonly used in the art. A cell population is considered viable when having at least a proportion of live, healthy cells within said population. A skilled person is able to determine and classify a cell population as being viable.

[0218]

[0203] It is further contemplated that the liver tissue obtained by the method in accordance to the invention can comprise a sinusoidal vascular network. It is preferred that this vascular network comprises a detectable lumen. Said lumen can be detected by common means used in the art, e.g., with a diameter of preferably between 5 and 30 pm, e.g., 10 pm, 15 pm, 20 pm etc. It is further preferred that the liver sinusoidal endothelial cells (i.e. , the endothelial cells herein comprised in forming liver sinusoid(- like) structures) comprises an at least partly fenestrated (i.e., comprising fenestrations and / or fenestration-like structures) endothelium.

[0219]

[0204] In addition, it is contemplated that, at least some of the herein provided parenchymal cells (i.e., mainly hepatocytes) form at least one, preferably more, layers / sheets / groups of hepatocytes. Preferably, the cells form similarly to native liver epithelial morphology, namely in compact or densely packed hepatocytes between at least two vessels and / or vessel-like structures.

[0220]

[0205] Moreover, preferably, the polarized epithelium that is formed as part of the liver tissue is at least partly, preferably for at least 50%, 60%, 70%, 75%, 80%, 90%, formed of hepatocytes. The polarized epithelium may comprise transporters that are also at least partly found in a native liver tissue. Hence, preferably the transport proteins comprise any one or more of Ntcp, Bsep, MRP-2, Mdr1 , Mdr2, AE2, AQP-8, ASBT, CFTR.

[0221]

[0206] Preferably the obtained liver tissue is fluidically and / or physically accessible. This may comprise for example that a perfusion flow can be realized through the obtained liver tissue, e.g., from a fluid-inlet to a fluid-outlet. A fluid flow may for example be realized by allowing a fluid to flow from one lateral microfluidic channel to another lateral microfluidic channel, e.g., thereby allowing fluid to flow in / through (vasculature of the) obtained liver tissue. Also, it may comprise that the obtained liver tissue allows for the adding and / or extracting of soluble factors or agents by e.g., mixing these with a fluid or culture medium. It may also be that the obtained liver tissue is accessible with by way of example certain agents and / or tools, such as, but not limited to, antibodies, nanobodies, cells, fluorescent staining reagents, nano capsules, nano sensors, and the like, to study the liver tissue and / or or to use a part of the liver tissue in a study, e.g., drug metabolism screening.

[0222]

[0207] The current invention further provides for a culturing device comprising a liver tissue obtained by the method as disclosed and embodied herein. It is preferred that the culturing device is a microfluidic culturing device. Preferably the microfluidic culturing device comprises any one of the features as disclosed and described herein. It is preferred that the microfluidic device comprises at least one culturing chamber and comprises at least one, preferably two, microfluidic channels adjacent to the culturing chamber. An exemplary schematic depiction (not in scale) of a suitable microfluidic device is found in Figure 2.

[0223]

[0208] Finally, there is also provided for uses of the method, the liver tissue and / or the culturing device as disclosed and embodied herein. The use can be a use an in vitro and / or ex vivo setting, for example, but not limited to any one of the following uses: studying liver toxicity, including drug-induced liver injury, nanoparticle toxicity, environmental exposure such as alcohol and diets, exposure to biologies; studying pharmacokinetics, including drug distribution, secretion and metabolism detoxification of xenobiotics, assessment of drug-drug interactions, study on gene and cell therapy, as well as enzyme replacement therapy for metabolic disorders, study on oxidative stress, studying uptake and biological effects of microparticles, studying liver functionality in each cell type, including lipid, amino acid and / or carbohydrate metabolism, vitamin and mineral storage and the production of macro- and micromolecules (e.g. albumin, vLDL, glucose, cholesterol), studying liver disease as a result of genetic, drug-induced or environmental factors such as steatosis, fibrosis, cirrhosis, hepatocellular carcinoma, cholestasis, hypertension, capillarization, cholangiocellular carcinoma, polycystic liver disease, studying liver disease as a result of infections such as hepatitis viruses, malaria parasites, bacteria, studying metastases where liver hosts the primary metastatic tumor or acts as a metastatic niece for circulating cancer cells, studying the interaction between liver cells and the immune component, studying liver infections by infectious agents (e.g., bacteria, viruses, fungi, protozoa), studying cell circulation of cells through the vasculature of liver tissue (e.g. immune cells), studying liver tissue cell transduction and / or transfection by nucleic acid carriers, preferably viral vectors (E.g. adeno-associated viruses), studying fibrosis in liver tissue, studying liver zonation, preferably sinusoid liver zonation, studying liver regeneration, studying liver development, studying polarized transport and bile production and secretion, studying molecules in the hepatic bile fraction, studying the storage of nutrients, study the effect of fluid flow and shear stress, study the effect of radiation, study host-graft interaction, study of the biological effect after exposure to patient-derived samples (e.g. serum, plasma, whole blood).

[0224]

[0209] It is preferred that the use is one selected from the group consisting of: studying liver toxicity, studying pharmacokinetics, studying the detoxification of xenobiotics, assessing drug-drug interactions, studying gene- and / or cell therapy, studying enzyme replacement therapy, studying oxidative stress, studying uptake and biological effects of (micro)particles, studying liver functionality, studying liver disease, studying cancer in a liver, studying metastases in the liver, studying cellular interaction, studying liver infections by infectious agents (e.g., bacteria, viruses, fungi, protozoa), studying cell circulation of cells through the vasculature of liver tissue (e.g. immune cells), studying liver tissue cell transduction and / or transfection by nucleic acid carriers, preferably viral vectors (E.g. adeno-associated viruses), studying fibrosis in liver tissue, studying liver zonation, preferably sinusoid liver zonation, studying liver regeneration, studying liver development, studying polarized transport and bile production and secretion, studying the hepatic bile fraction, studying the storage of nutrients, studying the effect of fluid flow and shear stress, studying the effect of radiation, studying host-graft interaction and studying effects after exposure to patient-derived samples (e.g. serum, plasma, whole blood).

[0225]

[0210] Also embodied herewith is a method of introducing cells to a liver tissue, or to a culturing device as disclosed in this description, comprising adding the cells to the obtained liver tissue, preferably by adding on top of the culturing chamber, under conditions to allow the cells to attach to the tissue, migrate and optionally differentiate or to specialize. Alternatively, the cells are introduced in the at least one microfluidic channel and allowed to perfuse to the vascularized liver tissue in the cell culturing chamber.

[0226]

[0211] The mode of adding these cells has been previously disclosed before in this description when referring to an embodiment of the method of obtaining liver tissue.

[0227]

[0212] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention 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 for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present 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.

[0228]

[0213] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[0229]

[0214] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0230]

[0215] It will be understood that all details, embodiments and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments and preferences for all aspect separately.

[0231]

[0216] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

[0232] EXAMPLES

[0233] Materials

[0234] Cells

[0235] Reagents Equipment

[0236] Medium preparation

[0237] Complete DMEM

[0238]

[0218] Dulbecco’s Modified Eagle Medium (DMEM, Gibco, cat. No. 11965-092) was supplemented with 10% FBS (Gibco), 1% penicillin-streptomycin (P / S, Sigma-Aldrich, cat. No. P4333-100ML) and 1 mM sodium pyruvate (Thermo Fisher Scientific). iHEP plating medium.

[0239]

[0219] IHep plating medium was prepared according to the manufacturer's (Cellular Dynamics, Fujifilm) instructions. Briefly, 96 mL of RPMI medium (Sigma-Aldrich, cat. No. R8758-500ML) were supplemented with 2 mL of B27 supplement 50X (Gibco, Cat. No. 17404-001 , Lot. No. 2209534) (final cone. 1X), 0.2 mL of Oncostatin M 10ug / ml (final concentration 20 ng / ml), 2 uL of Dexamethasone 5mM (MP Biomed, cat. No. 194561) (final concentration 0.1 uM), 50 ul of Gentamicin 50 mg / mL (Gibco, cat. No. 15750-060) (final concentration 25 ug / ml) and 2 mL of iCell Hepatocytes 2.0 Medium Supplement (Cellular Dynamics, Cat. No. M1024, Lot. No 106063) (final concentration 1X).

[0240] EGM-2 medium

[0241]

[0220] Endothelial Cell Growth Medium-2 BulletKit (EGM-2, Lonza, cat. no. CC-3162) was prepared as follows. Endothelial Cell Growth Basal Medium-2 (EBM-2, Lonza, cat. no. CC-3156) was supplemented with large-vessel endothelial cell growth medium SingleQuots and Growth Factors (Lonza, cat. co. CC-4146). The contents of this kit are 10 mL FBS, 0.20 mL Hydrocortisone, 2 mL hFGF-B, 0.5 mL VEGF, 0.5 mL R3- IGF-1 , 0.5 mL Ascorbic Acid, 0.5 mL hEGF, 0.5 mL GA-1000 and 0.5 mL Heparin. Procedure

[0242] Cell banking

[0243]

[0221] Cell banking for the LDECs (Liver endothelial cells, ScienCell) was performed as follows. Prior to thawing, a T75 orange cap flask was coated with fibronectin at 33.3ug / ml for 2 hours. To acquire the 33.3ug / ml solution of fibronectin, 150ul of 1 mg / ml fibronectin was diluted in 5ml PBS. Using 5ml of 33.3ug / ml fibronectin in a T75 flask results in a coverage of 1 ug / ml. Following 2H of incubation at 37°C, the fibronectin solution was removed. This fibronectin solution was routinely saved to be used once more for coating, whereafter it was tossed. Following the removal of the fibronectin solution, the flask was washed 1x with PBS. Thereafter, 14 ml of ECM (Sciencell) was added to the flask, after which the flask was placed back in the incubator to equilibrate the media. Next, a P1 vial of LDECs (Sciencell) was taken from -150°C storage and placed in a 37°C degree water bath momentarily. After the contents of the vial had thawed, they were transferred directly into the fibronectin coated T75 flask, which was subsequently placed back into an incubator. 18-24 Hours after the thawing procedure, the media was aspirated and replaced with fresh pre-warmed ECM (Sciencell). Subsequent media changes were performed every other day. LDECs were maintained in their flask until D6 (Thawing day = Day 0). On day 6, cell banking was commenced by aspirating the media and washing the cells with PBS. Hereafter, 0.013% trypsin / EDTA solution was prepared by diluting 0.5ml 0.25% trypsin / EDTA in 9.5ml PBS. The trypsin solution was added to the cells for 5 minutes at 37°C, whereafter 5ml Trypsin Neutralizing Solution (TNS) was added to the flask, and the total volume of 10 ML was transferred to a 50ml falcon containing 10ml FBS. Hereafter, the flask was washed with another 5ml TNS that was also transferred to the falcon. Cells were counted from the total volume of 25ml with the Luna Automated Cell counter. The cell solution was subsequently centrifuged for 5 minutes at 1000RPM, whereafter the supernatant was aspirated. The cells were then resuspended in a cold freezing solution consisting of 20% FBS, 10% DMSO and 70% ECM and transferred to cryo vials. The cryo vials were then placed in a Mr Frosty and placed in a -80°C freezer. 24 later, the cryo vials were moved to -150°C. The cell banking procedure for HHStECs was almost identical. Instead of a fibronectin coating, a poly-l-lysine coating was used at 1ug / cm2 and StECM was used instead of ECM. Additionally, the cells were trypsinized with 0.025% trypsin / EDTA instead of 0.013%. Cell subculturing

[0244]

[0222] HHSTeCs (HSC) (Liver stellate cells, ScienCell) (Passage 2>3 or 3>4) and LDECs (Passage 2>3) were thawed into 14mL warm StECM and ECM, respectively. The cells were then seeded in T75 orange flasks that were coated with poly-l-lysine (HHSTeCs or fibronectin

[0245]

[0223] HHSteCs and LDECs (Passage 2->3) were thawed in 14 mL warm SteCM and ECM, respectively. The cell suspension was transferred in T-75 flasks coated (for 2 hours) with 150uL Poly-L-Lysine in 10 mL MilliQ water (HHSteCs) or with 5 mL Fibronectin in PBS (HHSECs, Liver endothelial cells, ScienCell). Medium was refreshed 24 hours after plating and every 2-3 days for a total of 5 days after plating.

[0246] Cell suspension preparation

[0247]

[0224] iHep (iPSC-derived hepatocytes, Cellular Dynamics (Fujifilm)) were thawed in one 10 mL tube containing warm iHep plating medium. iHep vial was thawed in the waterbath for exactly 3 minutes, the content was then transferred to a tube containing 10 mL warm iHep plating medium. The tube was centrifuged 200 g for 3 minutes. Supernatant was discarded and the pellet resuspended in 2 mL plating medium and the cells were counted using the trypan blue exclusion method with LUNA cell counting device. IHeps were then resuspended in complete DMEM to a final concentration of 25’000 cells / ul.

[0248]

[0225] HHSteCs were dissociated using 9 mL PBS + 1 mL 2.5% Trypsin for 3 minutes. LDECs were dissociated with 8 mL PBS + 2 mL 2.5% Trypsin for 3-5 minutes. Trypsin was then neutralized with 5 mL TNS. Additional 5 mL TNS was used to rinse the flasks and collected in the same tube. The cells were then counted using the trypan blue exclusion method with LUNA cell counting device. The cell suspensions were centrifuged for 200 g for 5 minutes and resuspended to a final concentration of 8125 (HHSTeCs) and 16250 (LDECs) cells / ul in DMEM (10% FBS, 1 % P / S).

[0249] OrganoPlate seeding and maintenance.

[0250]

[0226] A mixture containing 10 ul HHSTeCs (end concentration 1625 cells / ul), 10 ul LDECs (end concentration 3250 cells / ul), 20 ul iHeps (end concentration 10000 cells / ul), 5 ul 50 mg / mL Fibrinogen (end concentration 5 mg / ml) and 5 ul 1 U / mL Thrombin (end concentration 0.1 U / mL) was prepared in a tube and vigorously mixed. 1.35 ul of this cell suspension was introduced through the graft chamber hole in a OrganoPlate Graft plate using a Sartorius P10 Pipet with minimum dispensing speed. The plate was then placed in a humidified incubator (37°C, 5% CO2) for 10 minutes. 50 ul of DMEM (10% FBS, 1% P / S) was added to the graft chamber. After 1-2 hours the medium in the graft chamber was replaced with iHep plating medium containing 100 kill Aprotinin. 50 ul of EGM-2 was added to each well of the perfusion channels. Plate was kept static in a humidified incubator and a full medium change was performed every 2-3 days for a total of 7 days.

[0251] Calcein-AM staining.

[0252]

[0227] On day 7 of culture, a mixture of 1 : 100 Calcein AM in EGM-2 was prepared and was added to each well of several chips. The plate was incubated 20 min in a humidified incubator and imaged with a confocal microscope (10x magnification).

[0253] Results

[0254]

[0228] The results obtained by the methods provided herein are shown in the accompanied Figures and Figure descriptions.

Claims

CLAIMS1. An in vitro method for obtaining a liver tissue, wherein the method comprises: a) providing a culturing device wherein the culturing device at least comprises a culture chamber; b) providing the culture chamber with a gel precursor, wherein the gel precursor at least comprises fibrinogen, and wherein the gel precursor further comprises liver endothelial cells and liver stellate cells; c) allowing the gel precursor to gelate 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 the liver tissue, preferably wherein the liver tissue resides in the scaffold.

2. The method of any of the previous claims, wherein the gel precursor of step (b) further comprises hepatocytes.

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

4. The method of any of the previous claims, wherein the culturing in step (e) provides for obtaining a liver tissue comprising a vasculature formed by at least the liver endothelial cells and the liver stellate cells.

5. The method of any of the previous claims, wherein the culturing in step (e) provides for a liver tissue, preferably comprising vasculature, comprising one or more sinusoidal structures formed by at least the liver endothelial cells and the liver stellate cells, preferably wherein a sinusoidal structure has an average diameter between 1 - 50 pm, more preferably between 5 - 30 pm, even more preferably about 10 pm.

6. The method of any of the previous claims, wherein the liver tissue, preferably comprising vasculature, further comprises polarized hepatocytes, wherein the polarized hepatocytes align such that a bile canaliculus or a bile-canaliculus-like structure is formed.

7. The method of any of the previous claims, 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 a bile canaliculus or bile-canaliculus- like structure.

8. The method of any of the previous claims, wherein the polarized hepatocytes form a layer / sheet / group of cells.

9. The method of any of the previous claims, wherein the bile canaliculus or bile-canaliculus-like structure is at least partially positioned between the apical sides of two or more layers / sheets / groups of polarized hepatocytes.

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

11. The method of any of the previous claims, wherein the culturing device comprising the culture chamber is a microfluidic culturing device, preferably wherein the microfluidic culturing device comprises at least one, preferably at least two, microfluidic channels that are fluidically connected to the culture chamber, preferably wherein the at least one, preferably at least two microfluidic channels comprise an inlet and an outlet.

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

13. The method of any of the previous claims, wherein the culturing device is a microfluidic culturing device comprising the culture chamber and further comprising at least one or at least two microfluidic channels that are fluidically connected to the culture chamber, and, wherein in at least one or at least two microfluidic channels, endothelial cells, preferably liver endothelial cells, are cultured, preferably to obtain a tubule or a tubule-like structure, more preferably a blood vessel and / or blood vessel-like structure.

14. The method of any of the previous claims, wherein the culturing device is a microfluidic culturing device comprising the culture chamber and at least two microfluidic channels, wherein the at least two microfluidic channels are, each independently, fluidically 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 of any of the previous claims, wherein the culturing device comprising the culture chamber, is an unidirectional flow microfluidic culturing device.

16. The method of any of the previous claims, wherein the culturing device comprising the culture chamber, is a unidirectional flow microfluidic culturing device comprising at least one microfluidic network comprising: (a) a microfluidic layer comprising a first flow channel and a second flow channel; and (b) a reservoir layer disposed above the microfluidic layer andcomprising first and second reservoirs, wherein the first reservoir and the second reservoir each have an access port to the first flow channel and an access port to the second flow channel, so that the first flow channel and the second flow channel form a flow circuit with the first reservoir and the second reservoir; and wherein the access ports in the first reservoir and the second reservoir are spaced apart so that, 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 primarily via the first flow channel, and adjusting the tilt so that the device is titled at a second angle causes a volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir primarily via the second flow channel.

17. The method of any of the previous claims, wherein the culturing device comprising the culture chamber, is a unidirectional flow microfluidic culturing device comprising at least one microfluidic network, and the method comprises(a) tilting the device at a first angle by rotating around a first axis to induce a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir via the first flow channel; and(b) adjusting the tilt so that the device is titled at a second angle by rotating around said 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 channel.

18. The method of any one of the previous claims, wherein an opening is created during step (c) and / or after step (e) in the scaffold, preferably wherein said opening allows for the introduction of a solution, a fluid, or an agent.

19. The method of any of the previous claims, wherein the liver endothelial cells, liver stellate cells and / or hepatocytes are not separated from each other by an artificial membrane.

20. The method of any of the previous claims, wherein the number of total cells comprised 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 8.000 - 15.000 cells per microliter and / or at most 100.000 cells per microliter, preferably at most 50.000 cells or 30.000 cells per microliter and / or wherein the ratio of the number of liver endothelial cells to liver 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. The method of any the previous claims, wherein the ratio of the number of hepatocytes to the sum of the number of liver endothelial cells and liver 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 method of any of the previous claims, wherein the gel precursor of step (b) comprises per microliter:• between 1.000 and 10.000 liver endothelial cells; and• between 100 and 10.000 liver stellate cells.

23. The method of any of the previous claims, further comprising:• Between 5.000 and 50.000 hepatocytes per microliter gel precursor.

24. The method of any of the previous claims, wherein the gel precursor of step (b) has a volume of between 0.1 pL and 50 pL, preferably between 0.5 pL and 20 pL.

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

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

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

28. The method of any of the previous claims, wherein the culture medium of step (d) has a volume of between 20 and 500 pL and / or wherein the culture chamber has a volume of between 0.1 pL and 50 pL, preferably between 0.1 pL and 20 pL.

29. The method of any of the previous claims, 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 a 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 of any of the previous claims, wherein the liver endothelial cells, liver stellate cells and / or hepatocytes are human cells, preferably human- derived cells.

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

32. The method of any of the previous claims, wherein the gel precursor of step (b) further comprises hepatocytes; and- wherein the one or more, preferably all, cells are cultured for a period sufficient to allow the formation of a vasculature, preferably wherein the vasculature is accessible and / or has a diameter of about 5 to 50 pm; and / or- wherein the one or more, preferably all, cells are cultured for a period sufficient to allow the formation of polarized hepatocytes, preferably a layer / sheet / group 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 a bile canaliculus and / or bile- canaliculus-like structure- wherein the one or more, preferably all, cells are cultured for a period sufficient to allow the formation of a bile canaliculus and / or bile-canaliculus- like structure; and / or- wherein the one or more, preferably all, cells are cultured for a period sufficient to allow expression of LYVE-1 marker in the liver endothelial cells and / or expression of PLVAP marker in the liver endothelial cells and / or expression of MRP-2 in the hepatocytes; and / or- wherein the one or more, preferably all, cells are cultured for a period sufficient to allow the formation of fenestrations in the liver tissue.

33. The method of any of the previous claims, wherein the liver tissue that is obtained in step (e) allows for intracellular lipid droplet accumulation upon contacting the cells with a free fatty acid for a period of between 12 and 48 hours.

34. The method of any of the previous claims, wherein the culturing device comprising the culture chamber, is an unidirectional flow preferably microfluidic culturing device, and the liver tissue that is obtained in step (e) allows for unidirectional flow of culture media through the liver tissue and results in absorption or metabolism of compounds in the medium mimicking the first pass effect observed in liver tissue in vivo.

35. The method of any of the previous claims, wherein the method further comprises adding a compound to the cell culture and observing the effect thereof on the formation or functioning of the liver tissue, preferably comprising a vasculature.

36. A liver tissue, preferably comprising a vasculature, obtained by the method of any of the previous claims.

37. The liver tissue according to claim 36, further comprising one or more sinusoidal structures, one or more bile canaliculi(-like) structures, one or more fenestrations and / or one or more bile duct(-like) structures.

38. A culturing device, preferably a microfluidic culturing device, comprising a liver tissue, preferably comprising a vasculature, obtained by the method of any of the previous claims.

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

40. Use of the method of any of the previous claims, the liver tissue of claim 36 or 37, and / or the culturing device of claim 38 or 39 in any one use selected from the group consisting of: studying liver toxicity, studying pharmacokinetics, studying the detoxification of xenobiotics, assessing drug-drug interactions, studying gene- and / or cell therapy, studying enzyme replacement therapy, studying oxidative stress, studying uptake and biological effects of (micro)particles, studying liver functionality, studying liver disease, studying cancer in a liver, studying cancer metastases, preferably cancer metastases in liver, studying cellular interaction, studying liver infections by infectious agents, studying cell circulation of cells through the vasculature of liver tissue, studying liver tissue cell transduction and / or transfection by nucleic acid carriers, preferably viral vectors, studying fibrosis in liver tissue, studying liver zonation, preferably sinusoid liver zonation, studying liver regeneration, studying liver development, studyingpolarized transport and bile production and secretion, studying the hepatic bile fraction, studying the storage of nutrients, studying the effect of fluid flow and shear stress, studying the effect of radiation, studying host-graft interaction and studying effects after exposure to patient-derived samples (e.g. serum, plasma, whole blood).

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

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

43. The method according to claim 41 or 42, wherein the introduced cells are allowed to fully or partially differentiate towards specific cell types, preferably Kupffer cells

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