System for in vitro modelling of biologically induced compression

Abstract

The invention provides a system (100) for in vitro modelling of biologically induced compression, wherein the system (100) comprises a channel (110), a first matrix material (121) and a second matrix material (122), wherein one or more of the first matrix material (121) and the second matrix material (122) comprises pancreatic stellate cells (126), wherein the channel (110) comprises cells (116), wherein the first matrix material (121) surrounds a first channel part (111) of the channel (110), and wherein the second matrix material (122) surrounds the first matrix material (121) and a second channel part (112) of the channel (110); wherein the first matrix material (121) and the second matrix material (122) both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl; wherein the first matrix material (121) comprises thrombin and n1 g / L fibrinogen, wherein n1 is selected from the range of 2 - 10, and wherein the second matrix material (122) comprises n2 g / L fibrinogen, wherein 0≤n2<n1.

Description

[0001] System for in vitro modelling of biologically induced compression

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a system for in vitro modelling of biologically induced compression and the use thereof. The invention further relates to a method for providing a system for in vitro modelling of biologically induced compression.

[0004] BACKGROUND OF THE INVENTION

[0005] Models of biological tissues are known in the art. US2004009589A1, for instance, describes a tissue engineering construct being formed by combining human embryonic endothelial cells with a cell support substrate. A polymer matrix may be infused with the cell support substrate. The polymer matrix may have any shape, for example, particles, tube, sponge, sphere, strand, coiled strand, capillary network, film, fiber, mesh, or sheet. The cell support substrate may be a gel and may be combined with a liquid carrier, for example, phosphate buffered saline. The gel may be Matrigel(TM) or a collagen-GAG gel. Alternatively, the gel may include one or more of collagen I, collagen IV, laminin, fibrin, fibronectin, proteoglycans, glycoproteins, glycoaminoglycans, proteinases, collagenases, chemotactic agents, or growth factors. An additional cell type may be combined with the human embryonic endothelial cells in the tissue engineering construct. For example, such cells may be muscle cells, nerve cells, connective tissue cells, or stem cells. The cell-support substrate may be a tube, for example, a decellularized blood vessel, a synthetic polymer tube, or a collagen tube, in which the cells are disposed on an inner surface.

[0006] SUMMARY OF THE INVENTION

[0007] The tumor microenvironment (TME) in fibrotic tumors such as pancreatic ductal adenocarcinoma (PDAC) is characterized by a high amount of desmoplasia. In PDAC, cancer- associated fibroblasts (CAFs) may form the most prevalent cell type accounting for around 80% of all cells in the TME and appear to be the main driving factor of this desmoplasia as they produce excessive amounts of extracellular matrix (ECM) proteins such as collagen, fibronectin or hyaluronic acid. Among several types of CAFs, myofibroblastic CAFs are a highly contractile and ECM regulating CAF type which mainly originate from pancreatic stellate cells (PSCs). Based on the crosstalk with tumor cells and environmental stimuli the PSCs obtain an activated CAF-like state. The abundance of CAFs and the resulting high amount of desmoplasia in PDAC creates a dense environment, which forms a nearly impenetrable barrier for therapeutics. Additionally, this desmoplasia, accompanied by an excess deposition of ECM, creates a high intratumoral stress causing the compression of blood vessels in and surrounding the tumor, which may prevent therapeutics from reaching the tumor site in the first place.

[0008] In recent years, several different biomaterials have been developed to mimic the ECM found in PDAC. In particular, the combination of such biomaterials with different biofabrication techniques has facilitated the generation of complex in vitro tumor models for the evaluation of treatment strategies. Currently, most biomaterial approaches aiming to mimic the ECM found in tumors through in vitro models may have difficulties with regards to controllability of the model. Furthermore, even though such in vitro models of tumors are becoming more complex to mimic the exact composition of the ECM, they may be unable to accurately represent biological and / or mechanical stimuli that occur in biological systems. In particular, prior art systems (or models) may be unsuitable for mimicking biologically (or physiologically) induced compression as occurs in tumors and other disease-affected tissues. Moreover, the complexity of prior art systems may make it difficult to interpret experimental observations, therewith limiting their suitability for drug development applications.

[0009] Furthermore, there appears to be a lack of standard protocols for the culture of in vitro pancreatic tumor models as well as a reported high heterogeneity within these models, which can further limit the suitability for drug development applications.

[0010] One alternative may be to research biologically induced compression through in vivo experiments rather than in vitro models. However, in vivo experiments suffer disadvantages such as difficulty in controlling variables and environment, relatively low reproducibility, relatively high cost, and ethical objections. Another alternative may be to research biologically induced compression through ex vivo experiments rather than in vitro models. However, ex vivo experiments may suffer from instability, therewith making such models unsuitable for researching progressive or chronic aspects of pathologies, and may also suffer from ethical objections.

[0011] Hence, it is an aspect of the invention to provide an alternative system for mimicking a biological tissue, such as for mimicking a tumor tissue, which system preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. In specific embodiments, the invention provides a system for in vitro modelling of biologically induced compression. In embodiments, the system may comprise a channel, a first matrix material and a second matrix material. Further, in embodiments, one or more of the first matrix material and the second matrix material may comprise fibroblasts and / or cells of mesenchymal origin, such as (at least) fibroblasts. Especially, the fibroblasts may comprise pancreatic stellate cells. In further embodiments, the channel may comprise (lining) cells. Moreover, in embodiments, a first matrix material surrounds a first channel part of the channel. Especially, a second matrix material surrounds the first matrix material and a second channel part of the channel. Further, in embodiments, the first matrix material and the second matrix material both may comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl. Moreover, in embodiments, the first matrix material may comprise thrombin and nl g / L fibrinogen. Furthermore, in embodiments, nl may be selected from the range of 2 - 10. In further embodiments, the second matrix material may comprise (thrombin and) n2 g / L fibrinogen. Moreover, in embodiments, 0 < n2 < nl, such as 0 < n2 < 0.5*nl.

[0012] Hence, in embodiments, the invention provides a system for in vitro modelling of biologically induced compression, wherein the system may comprise a channel, a first matrix material and a second matrix material, wherein one or more of the first matrix material and the second matrix material comprises pancreatic stellate cells, wherein the channel comprises cells, wherein a first matrix material surrounds a first channel part of the channel, and wherein a second matrix material surrounds the first matrix material and a second channel part of the channel; wherein the first matrix material and the second matrix material both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl; wherein the first matrix material comprises thrombin and nl g / L fibrinogen, wherein nl is selected from the range of 2 - 10, and wherein the second matrix material comprises n2 g / L fibrinogen, wherein 0 < n2 < nl.

[0013] The presence of thrombin and fibrinogen in matrix material may activate the fibroblasts, especially the pancreatic stellate cells (PSCs), resulting in induced compression (or “contraction”) of the matrix material (or “hydrogel”), which may lead to (about) a 10-fold contraction of the channel relative to a system without activated PSCs. Hence, the thrombin and fibrinogen may facilitate providing biologically induced compression in the system. Further, differences in the concentrations of fibrinogen and thrombin between the first matrix material and the second matrix material may facilitate simulating a local compression and / or spatially varying compressions. Such embodiments may be beneficial as the system consisting of PSCs embedded in a collagen and a collagen / fibrinogen (Col / Fib) matrix may provide a highly controllable environment to replicate the complex mechanical environment in PDAC. Especially, through the inclusion of a central endothelialized channel the system may facilitate studying cell-mediated contraction of vasculature in PDAC in relatively high detail. Hence, such a system may provide the benefit of serving as a tool for the in vitro evaluation of novel therapeutics that aim to modulate the mechanical environment in PDAC by comparing the efficacy of PSC-mediating therapeutics in vitro, in silico and in vivo. The system may especially comprise a simplified model of tumor tissues, therewith providing the benefit of improved reproducibility, improved controllability, and reduced complexity in interpretation of experimental observations. Although the system may be simplified, the system may nonetheless accurately represent biological and / or mechanical stimuli that occur in biological systems, therewith rendering the system relevant for fundamental and therapeutic analyses. Furthermore, the system may not only be limited to mimic PDAC, but may be translated to other fibrotic cancer types and other diseases related to vasculature compression. Hence, the system (and method, see further below) of the invention may provide a standard protocol for in vitro mimicking of biologically induced compression as a result of cancer and other pathologies. Yet further, the system may be produced in a relatively cheap and unintrusive manner.

[0014] In embodiments, the invention may provide a system for in vitro modelling of biologically induced compression. The term “in vitro” may herein indicate that the system may be applied for experimentation or measurement in or on cells from a biological organism, but especially in an external environment, i.e., outside of their normal biological environment. For modelling purposes as described herein, the external environment may preferably be designed such as to contain minimal alteration relative to the natural conditions of the tissue of origin of the cells. In embodiments, the system may essentially comprise an in vitro model of a biological tissue. In other words, in embodiments, the system may comprise a simplified synthetically engineered tissue that may respond to experiments or treatments in ways similar to those of a corresponding tissue of an organism. Therefore, the term “modelling” may herein refer to making and / or using a representation of something, such as making and / or using a representation of a process (e.g. compression of a blood vessel) in a biological organism.

[0015] The phrase “biologically induced compression” or simply “compression” may herein refer to compression of blood vessels in and surrounding tumors as a result of an abundance of cancer-associated fibroblasts (CAFs) and the resulting high amount of desmoplasia.

[0016] In embodiments, the system may comprise a channel (or “vessel”). Herein, the term “channel” may refer to a hollow structure or an enclosed passage. The channel may especially, in embodiments, have a (hollow) shape approximating a shape selected from the group comprising: a tubular shape, a semi-cylindrical shape, a spherical shape, an ellipsoidal shape, a (triangular, square, rectangular, or polygonal) prismatic shape, a frustum shape, a toric shape, and a dumbbell shape. In specific embodiments, the channel may especially approximate a (hollow) tubular shape, i.e., an elongated shape having an essentially circular cross-section perpendicular to a direction of elongation. It will be clear to the person skilled in the art that the channel being hollow does not preclude the presence of a liquid, e.g., a blood-like liquid, in the channel during use of the system (see below).

[0017] The term “approximate” and its conjugations herein, such as in “to approximate a shape”, may refer to being nearly identical to, especially identical to, the following term, for example nearly identical to a tubular shape or a (semi-)cylindrical shape. For example, a channel may define a tubular shape but for a defect. In particular, an object approximating a first shape may herein refer to: a first shape realization encompassing the object, wherein the first shape realization is defined as the smallest encompassing shape of the (2D or 3D, respectively) object wherein the first shape realization has the shape of the first shape, wherein a ratio of the area (volume) of the first shape realization to the area (volume) of the object may be < 1.2, especially < 1.1, such as <1.05, especially <1.02. For instance, a channel may approximate a (semi-)cylindrical shape, wherein the first shape realization may be defined as the smallest encompassing (semi-)cylindrical shape of the channel, wherein a ratio of the volume of the first shape realization to the volume of the channel is < 1.2, especially, especially < 1.1, such as <1.05, especially <1.02, including 1. Further, if the dimensions of the first shape are defined, the term approximate may refer to the object and the first shape being superimposable (in 2D or 3D, respectively) such that an intersection between the object and the first shape covers at least n% of the object and at least n% of the shape, wherein n is at least 90%, such as at least 95%, especially at least 98%, such as at least 99%, including 100%.

[0018] In embodiments, the channel may thus be an elongated channel. Therefore, the channel may, in embodiments, have a channel length (Lc) defined along the direction of elongation. Furthermore, in embodiments, the channel may have a channel diameter (De) defined in a first cross-sectional plane perpendicular to the direction of elongation (or an axis of elongation (A), see also further below). In some embodiments, the channel may be tubular (or cylindrical) and thus the cross-section may be circular. Therefore, the channel diameter (De) may, in embodiments, comprise an equivalent circular channel diameter (De).

[0019] The equivalent circular diameter (or ECD) (or “circular equivalent diameter”) of an (irregularly shaped) two-dimensional shape is the diameter of a circle of equivalent area. For instance, the equivalent circular diameter of a square with side a is 2*a*SQRT(l / 7t). For a circle, the diameter is the same as the equivalent circular diameter. Would a circle in an xy- plane with a diameter D be distorted to any other shape (in the xy-plane), without changing the area size, then the equivalent circular diameter of that shape would be D.

[0020] The channel may further, in embodiments, comprise an open end. Especially, in embodiments, the channel may comprise two open ends configured on opposite sides of the channel. In such embodiments, especially if the channel is hollow, the channel may provide a fluidic passage. Hence, in embodiments, fluid may flow into the channel from one open end and may pass through the channel towards the other open end. Hence, in embodiments, the channel may comprise two open ends configured in fluid contact with each other.

[0021] However, in other embodiments, the channel may be a (partially or even fully) closed channel.

[0022] Hence, in some embodiments, the channel may be fully enclosed by (first and / or second) matrix material. In such embodiments, there may be no (fluidic) interaction between the channel and the external of the matrix material. Moreover, in some embodiments, the channel may not be an elongated channel, but for example a spherical (or ovoid) structure, i.e., a closed structure (fully) surrounded by matrix material. In such embodiments, the channel length (Lc) may be defined as one or more of a diameter or a largest dimension of the channel. Hence, in some embodiments, the channel length (Lc) may be equal to or essentially the same as the channel diameter (De). However, in general, in embodiments, Lc>Dc, such as at least Lc>2Dc.

[0023] The channel length (Lc) may, in embodiments, be at least 0.2 mm, like at least 0.5 mm, such as at least 1 mm, especially at least 2 mm. Further, in embodiments, the channel length (Lc) may be at most 100 mm, especially at most 50 mm, such as at most 25 mm, like at most 15 mm. In embodiments, the channel length (Lc) may especially be selected from the range of 0.2 - 50 mm, such as from the range of 1 - 20 mm, like from the range of 4 - 16 mm, especially from the range of 2 - 8 mm.

[0024] The channel diameter (De) may, in embodiments, be at least 5 pm, like at least 10 pm, such as at least 0.1 mm, especially at least 0.5 mm. Further, in embodiments, the channel diameter (De) may be at most 15 mm, such as at most 10 mm, like at most 5 mm, especially at most 1 mm. Hence, in embodiments, the channel may have a(n equivalent circular) channel diameter (De) selected from the range of 10 pm - 20 mm, such as from the range of 50 gm -10 mm, like from the range of 0.1 mm - 5 mm, especially from the range of 10 pm - 1.5 mm. Such embodiments may be beneficial as such a channel diameter may closely resemble and therewith efficiently mimic biologically relevant systems such as blood vessels. Especially, blood vessels may have an average diameter of about 10 pm (for capillaries), 500 pm (for veins), or even 1 mm (for arteries).

[0025] In embodiments, the channel may be defined by a channel wall or a channel lining, especially, the channel may be defined by the (first and / or second) matrix material, see also further below, acting as a wall. Moreover, in embodiments, the channel may comprise cells. Especially, in embodiments, the channel may comprise eukaryotic cells, such as animal cells, especially human cells. Further, in embodiments, the cells may also be referred to as lining cells, i.e., the cells may be lined against (or “seeded onto”) at least part of the channel wall. In embodiments, the (lining) cells may comprise one or more of endothelial cells, pericytes, and other vascular cells. Hence, in specific embodiments, the channel (especially the matrix material defining the channel) may be lined with endothelial cells. Such embodiments may be beneficial as the presence of endothelial cells in the channel may further improve the resemblance of the system to biologically relevant systems such as blood vessels. Therefore, the presence of endothelial cells in the channel may improve the efficiency of the system as a tool in therapeutic development and / or testing efforts. More especially, in embodiments, the channel may (essentially) comprise a blood vessel. For example, in embodiments, the channel may comprise one of a capillary, a vein, and an artery.

[0026] The channel may be defined by the matrix material and may, in embodiments, comprise a first channel part and a second channel part. In embodiments, the first channel part may have a first channel part length (Lei). The first channel part may, in embodiments, be a singular part. However, in other embodiments, the first channel part may be comprised of multiple separated parts. Therefore, the first channel part length (Lei) may comprise a total (cumulative) length of all first channel parts combined. In embodiments, the first channel part length (Lei) may be selected from the range of 0.2 - 100 mm, especially from the range of 0.2 - 50 mm, such as from the range of 0.5 - 20 mm, especially from the range of 1 - 6 mm. Similarly, in embodiments, the second channel part may have a second channel part length (LC2). The second channel part may, in embodiments, be a singular part. However, in other embodiments, the second channel part may be comprised of multiple separated parts, e.g. multiple second channel parts separated by one or more first channel parts. Therefore, the second channel part length (Lc2) may comprise a total (cumulative) length of all second channel parts combined. In embodiments, the second channel part length (Lc2) may be selected from the range of 0.2 - 100 mm, especially from the range of 0.2 - 50 mm, such as from the range of 0.5 - 20 mm, especially from the range of 1 - 6 mm. In embodiments, the first channel part(s) and the second channel part(s) may thus form the channel. Therefore, in embodiments, LC=LCI+LC2. For example, in embodiments, the channel may consist of a first channel part and a second channel part configured adjacent to each other. In other embodiments, the channel may, e.g., consist of two second channel parts separated by a singular first channel part.

[0027] In some embodiments, it may be desired to have a first channel part smaller than the (one or more) second channel part(s). For example, in embodiments, the system may comprise a first channel part configured between two second channel parts, wherein Lci< 0.5 * LC2. Hence, in embodiments, Lci< 0.75 * Lc2, such as Lci< 0.5 * Lc2, like Lci< 0.25 * Lc2, especially Lci< 0.2 * Lc2. Such embodiments may be beneficial as the system comprising a first channel part (where compression may be induced) smaller than the second channel part may more accurately mimic blockages of biological tissues such as blood vessel. However, in other embodiments, Lci> 0.5 * Lc2, such as Lci> Lc2, like Lci> 1.5 * Lc2.

[0028] The system may especially, in embodiments, comprise a first matrix material and a second matrix material. The first matrix material may, in embodiments, (at least partially) surround the first channel part. In specific embodiments, the first matrix material may (essentially fully) enclose the first channel part, i.e., the first channel part may be fully defined by the first matrix material. Further, in embodiments, the second matrix material may (at least partially) surround the first matrix material and the (one or more) second channel part(s) of the channel. In specific embodiments, the second matrix material may (essentially fully) enclose (both) the first matrix material and the second channel part(s), i.e., the second channel part(s) may be fully defined by the second matrix material.

[0029] The term “surround”, such as in “the first matrix material may at least partially surround the first channel part” may herein refer to the surrounding element (e.g., the first matrix material) effectively covering a substantial proportion, such as at least 60%, of a surface of the surrounded element (e.g., the first channel part), either directly or by (directly) covering one or more intermediate layers. For instance, with respect to embodiments wherein the channel is elongated, in a cross-sectional plane perpendicular to the axis of elongation the first matrix material may cover at least 60% of a surface outline of the first channel part, such as at least 80, especially at least 90%, including 100%. In particular, in further embodiments, for at least 60% of a first channel part length (see below), the first matrix material may cover at least 60% of a surface outline of the first channel part (in a plane perpendicular to the axis of elongation), such as at least 80%, especially at least 90%, including 100%. In general, the matrix materials may fully surround the channel, i.e., the first matrix material may fully surround the first channel part and the second matrix material may fully surround the second channel part.

[0030] The term “fully surround” or “enclose” may herein refer to the surrounding element essentially fully covering the surrounded element.

[0031] In embodiments, one or more of the first matrix material and the second matrix material may comprise fibroblasts. Additionally or alternatively, in embodiments, one or more of the first matrix material and the second matrix material may comprise cells of mesenchymal origin, such as cardiac myocytes, smooth muscle cells, myoepithelial cells, mesenchymal stem cells, myofibroblasts etc. Especially, in embodiments, at least the first matrix material may comprise fibroblasts (and / or cells of mesenchymal origin). The fibroblasts (and / or cells of mesenchymal origin) may, in embodiments, comprise pancreatic stellate cells. In other embodiments, the fibroblasts (and / or cells of mesenchymal origin) may (also) comprise one or more of the group comprising: hepatic stellate cells, lung stellate cells, and renal stellate cells. Optionally, in embodiments, one or more of the first matrix material and the second matrix material may comprise other cells, such as e.g. epithelial cells, which combination may better mimic normal tissue. For example, in embodiments, the first matrix material may comprise (fibroblasts, especially) pancreatic stellate cells and the second matrix material may comprise epithelial cells. In other embodiments, for example, the first matrix material may comprise (fibroblasts, especially) pancreatic stellate cells and the second matrix material may comprise both fibroblasts (e.g. pancreatic stellate cells) and epithelial cells.

[0032] Further, in embodiments, the first matrix material and the second matrix material may both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl. Hence, in embodiments, the first matrix material may comprise one or more of collagen, gelatin and gelatin methacryloyl. The first matrix material may, in embodiments, especially comprise a hydrogel, such as e.g. a collagen hydrogel. Similarly, in embodiments, the second matrix material may comprise one or more of collagen, gelatin and gelatin methacryloyl. The second matrix material may, in embodiments, especially comprise a hydrogel, such as e.g. a collagen hydrogel. In further embodiments, the first matrix material and the second matrix material may comprise the same material selected from the group comprising collagen, gelatin and gelatin methacryloyl, e.g., the first matrix material and the second matrix material may both comprise collagen or may both comprise gelatin. The first matrix material may, in embodiments, comprise nl g / L fibrinogen and thrombin, and optionally further also Ca2+. The presence of thrombin and fibrinogen may activate the (fibroblasts, especially the) pancreatic stellate cells, resulting in (biologically) induced compression (or “contraction”) of the hydrogel. The (biologically) induced compression may especially be progressive compression (rather than acute compression), which may facilitate simulating chronic pathological processes. In embodiments, nl may be at least 1, such as at least 2, like at least 3, especially at least 4. Further, in embodiments, nl may be at most 15, such as at most 12, like at most 10, especially at most 9. Especially, in embodiments, the first matrix material may comprise (an amount of) fibrinogen selected from the range of 1 - 15 g / L, such as from the range of 2 - 10 g / L, like from the range of 2 - 8 g / L, especially from the range of 4 - 6 g / L. Further, in embodiments, the first matrix material may comprise (an amount of) thrombin selected from the range of 0.5 - 15 units / mL, such as from the range of 1 - 10 units / mL, like from the range of 2 - 9 units / mL, especially from the range of 2 - 8 units / mL, more especially from the range of 3 - 7 units / mL. Optionally, in embodiments, the first matrix material may further comprise (an amount of) Ca2+selected from the range of 0.5 - 15 mM, such as from the range of 1 - 10 mM, like from the range of 2 - 8 mM. Addition of calcium ions to the matrix material may especially aid in inducing contraction of the channel, similar to how calcium ions control induction and relaxation of blood vessels in vivo. In embodiments, however, instead of adding the calcium ions to the matrix material, calcium ions may also be provided to the system through one or more of perfusion and (in case of a double open-ended channel) providing a flow of calcium ions through the channel.

[0033] In embodiments, the first matrix material may especially comprise a first weight ratio of fibrinogen to thrombin (Fib:Thr). Especially, in embodiments, the first weight ratio of fibrinogen to thrombin may be selected from the range of 12:1 to 8: 1, such as e.g. a weight ratio of 10: 1. Furthermore, in embodiments, the first matrix material may comprise a second weight ratio of collagen to fibrinogen (Col Lib). In embodiments, the second weight ratio of collagen to fibrinogen may be selected from the range of 1 :5 to 1 :2, such as from the range of 1 :4 to 1 :2, such as e.g. 7:20. Yet further, in embodiments, the first matrix material may comprise a combined weight ratio of collagen and fibrinogen combined to thrombin (ColFib:Thr). In embodiments, the combined weight ratio of collagen and fibrinogen combined to thrombin may be selected from the range of 250: 1 to 25: 1, such as from the range of 200: 1 to 50: 1, like from the range of 150: 1 to 75: 1. For example, in embodiments, the first matrix material may comprise a second weight ratio of collagen to fibrinogen (CokFib) of 1 :3, i.e., three times as much fibrinogen as compared to collagen. In such embodiments, the thrombin may then be added to the mixture of collagen and fibrinogen in a combined weight ratio of collagen and fibrinogen combined to thrombin (ColFib:Thr) of 100: 1, i.e., there may be a hundred times as much collagen-fibrinogen mixture as compared to thrombin.

[0034] Hence, in specific embodiments, the first matrix material may comprise a weight ratio of collagen to fibrinogen selected from the range of 1 :4 to 1 :2, and the first matrix material may comprise a weight ratio of collagen and fibrinogen combined to thrombin selected from the range of 200: 1 to 50:1. Such embodiments may be beneficial as the combination of fibrinogen and thrombin in the matrix material may induce the activation of PSCs, contraction of the channel, and extracellular matrix (ECM) production of PSCs. Especially, the indicated weight ratios of collagen, fibrinogen and thrombin may increase mechanical stiffness and may provide enhanced activation of PSCs in the system, therewith providing improved mimicking of vasculature compression (in pancreatic tumors). In particular, such embodiments may induce activation of PSCs leading to a 10-fold contraction of the channel relative to a system without activated PSCs.

[0035] Optionally, in embodiments, the second matrix material may (also) comprise one or more of thrombin and fibrinogen. Especially, in embodiments, the second matrix material may comprise n2 g / L fibrinogen. In embodiments, n2 may be at least 1, such as at least 2, like at least 3, especially at least 4. Further, in embodiments, n2 may be at most 14, such as at most 12, like at most 10, especially at most 9. Yet further, in embodiments, n2 may (also) be essentially zero, i.e., there may not necessarily be fibrinogen present in the second matrix material. Especially, in embodiments, there may be less fibrinogen present in the second matrix material relative to the first matrix material, i.e., n2<nl, such as < 0.5*nl, especially < 0.25*nl, such as < 0.1*nl. More especially, in embodiments, 0<n2<nl. In embodiments, the second matrix material may comprise (an amount of) fibrinogen selected from the range of 1 - 14 g / L, such as from the range of 1 - 10 g / L, like from the range of 2 - 8 g / L, especially from the range of 2 - 6 g / L. Further, in embodiments, the second matrix material may comprise (an amount of) thrombin selected from the range of 0.5 - 14 units / mL, such as from the range of 1 - 10 units / mL, like from the range of 2 - 9 units / mL, especially from the range of 2 - 8 units / mL. Especially, in embodiments, there may be less thrombin present in the second matrix material relative to the first matrix material. Optionally, in embodiments, the second matrix material may comprise essentially no thrombin.

[0036] Note that, in embodiments, other (bio)materials may also (alternatively to the combination of fibrinogen with thrombin) be applied in the system to (help) mimic biologically induced compression. For example, in embodiments, one or more of the first matrix material and the second matrix material may comprise a compression-inducing compound, which may induce (physical, rather than physiological and / or pathological) contraction in the system. However, such compression-inducing compounds may in particular be convenient for mimicking acute compression, such as seen in e.g. stroke, rather than progressive compression. In embodiments, the compression-inducing compounds may be selected from the group comprising hyaluronic acids and / or glycosaminoglycans (GAGs). Such compounds may especially induce compression through the absorption of water in the matrix material. Additionally or alternatively, in embodiments, the compression-inducing compounds may comprise materials such as e.g.: elastin-like polypeptide, electroactive polymers such as polypyrrole and polythiophene, pH-dependent polymer such as polyacrylic acid, thermoresponsive polymer such as poly(N-isopropylacrylamide), and light-responsive polymers such as azobenzene-based polymers. Such compounds may especially induce compression through (reversible) changes in conformation or structure in response to temperature-, pH-, or light-stimuli. The (reversible) changes in conformation or structure of the compound may cause the compounds to reduce (or increase) in size (or “occupied space”), therewith manipulating a compression pressure in the system. As such, the invention may provide a system for modelling biologically induced compression as seen in one or more pathologies selected from the group comprising: pancreatic tumors, lung tumors (using lung stellate cells), liver tumors (using hepatic stellate cells), systemic sclerosis, cirrhosis and fibrosis. Such embodiments may be beneficial as the system may thus help provide valuable insights into such pathologies and improve understanding of their underlying mechanisms.

[0037] Hence, in embodiments, the system may comprise a channel, a first matrix material and a second matrix material. In embodiments, one or more of the first matrix material and the second matrix material may comprise cells, such as fibroblasts and / or cells of mesenchymal origin. Especially, in embodiments, the fibroblasts and / or cells of mesenchymal origin may comprise one or more of the group comprising: cardiac myocytes, smooth muscle cells, myoepithelial cells, mesenchymal stem cells, myofibroblasts, pericytes, pancreatic stellate cells, hepatic stellate cells, and lung stellate cells. Further, in embodiments, the first matrix material may surround a first channel part of the channel and the second matrix material may surround the first matrix material and a second channel part of the channel. The first matrix material and the second matrix material may both, in embodiments, comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl. Furthermore, in embodiments, the first matrix material may comprise thrombin and nl g / L fibrinogen. Furthermore, in embodiments, nl may be selected from the range of 2 - 10. In further embodiments, the second matrix material may comprise (thrombin and) n2 g / L fibrinogen. Moreover, in embodiments, 0 < n2 < nl, such as 0 < n2 < 0.5*nl.

[0038] Hence, in embodiments, the system for in vitro modelling of biologically induced compression comprises a channel, a first matrix material, and a second matrix material, wherein one or more of the first matrix material and the second matrix material comprises cells, wherein the first matrix material surrounds a first part of the channel, and wherein the second matrix material surrounds the first matrix material and a second channel part of the channel, wherein the first matrix material and the second matrix material both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl, wherein the first matrix material comprises thrombin and nl g / L fibrinogen, wherein nl is selected from the range of 2 - 10, and wherein the second matrix material comprises n2 g / L fibrinogen, and wherein 0 < n2 < nl.

[0039] In embodiments, the system (as a whole) may have a diameter (Ds) and a length (Ls). The (system) diameter (Ds) may, in embodiments, be defined in the first cross-sectional plane. The (system) length (Ls) may, in embodiments, be defined perpendicular to the first cross-sectional plane. The (system) length (Ls) may especially, in embodiments, be defined in the same direction as the channel length (Lc), i.e., in the direction of elongation of the channel. In some embodiments, the (system) length (Ls) may essentially be the same as the channel length (Lc), i.e., Ls=Lc. However, in other embodiments, the (system) length (Ls) may be larger than the channel length (Lc), i.e., Ls>Lc. Especially, in embodiments, the (system) length (Ls) may be at least 0.2 mm, like at least 0.5 mm, such as at least 1 mm, especially at least 2 mm. Further, in embodiments, the (system) length (Ls) may be at most 100 mm, such as at most 50 mm, like at most 25 mm, especially at most 15 mm. In embodiments, the (system) length (Ls) may especially be selected from the range of 0.2 - 100 mm, especially from the range of 0.2- 50 mm, such as from the range of 1 - 20 mm, like from the range of 4 - 16 mm, especially from the range of 2 - 8 mm. The (system) diameter (Ds) may, in embodiments, be at least 0.1 mm, like at least 0.5 mm, such as at least 1 mm, especially at least 2 mm. Further, in embodiments, the system diameter (Ds) may be at most 25 mm, such as at most 15 mm, like at most 5 mm. In embodiments, the (system) diameter (Ds) may especially be selected from the range of 0.1 - 50 mm, such as from the range of 0.5 - 20 mm, like from the range of 1 - 16 mm, especially from the range of 2 - 8 mm. Hence, in embodiments, the system may have a diameter (Ds) selected from the range of 2 - 8 mm, and the system may have a length (Ls) selected from the range of 4 - 16 mm. Such embodiments may be beneficial as a too large system may result in hypoxia and a lack of nutrition (e.g. through perfusion) for the cells. In contrast a too small system may result in reproducibility issues between different systems and between experiments using the system. It may especially be desirable to make the system, when modelling a blood vessel, as close to the native environment and its respective measurements as possible.

[0040] In a further aspect the invention provides a use of the above described system for modelling vasculature compression. Especially, in embodiments, the channel may comprise a blood vessel (model), and the (first) matrix material, especially the fibrinogen and thrombin in the (first) matrix material may stimulate the fibroblasts (or cells of mesenchymal origin) in the system causing the (channel, especially) blood vessel to be compressed. Different types of experiments may be performed to gain understanding of the compression and underlying mechanisms as well as to develop potential therapies and / or therapeutics. In embodiments, the fibroblasts (or cells of mesenchymal origin) in the system may comprise specific cell types, such as pancreatic stellate cells. Hence, in such embodiments, the system may be used to model biologically induced vasculature compression as seen in pancreatic tumors. Furthermore, in embodiments, the system may be used for simulating one or more other pathologies, such as e.g. pathologies selected from the group comprising: pancreatic tumors, lung tumors (using lung stellate cells), liver tumors (using hepatic stellate cells), systemic sclerosis, cirrhosis, and fibrosis. In embodiments, the invention may thus comprise the use of the system as described herein for modelling vasculature compression. Especially, in embodiments, the invention may comprise the use of the system as described herein for simulating a pancreatic tumor. Such embodiments may be beneficial as using the system for modelling pathologies or diseases as indicated above may provide valuable insights into such pathologies and improve understanding of the underlying mechanism. Further, using the system for modelling such pathologies or diseases and experimenting with potential therapeutics may be less invasive than existing (in vivo) experiments.

[0041] In a further aspect, the invention provides a method for providing a system for in vitro modelling of biologically induced compression. In embodiments, the method may comprise a preparation stage comprising providing a sacrificial structure, a first matrix precursor and a second matrix precursor. Especially, in embodiments, the first matrix precursor and the second matrix precursor may (both) comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl. Further, in embodiments, the first matrix precursor may comprise thrombin and nl g / L fibrinogen. Similarly, in embodiments, the second matrix precursor may comprise (thrombin and) n2 g / L fibrinogen. Moreover, in embodiments, 0<n2<nl. Furthermore, in embodiments, one or more of the first matrix precursor and the second matrix precursor may comprise pancreatic stellate cells. Additionally, in embodiments, the method may comprise a first layer deposition stage comprising (i) providing the first matrix precursor to a first sacrificial part of the sacrificial structure, and (ii) (at least partially) solidifying the first matrix precursor to provide a first layer of first matrix material. Additionally, in embodiments, the method may comprise a second layer deposition stage comprising (i) providing the second matrix precursor to the first layer and to a second sacrificial part of the sacrificial structure, and (ii) (at least partially) solidifying the second matrix precursor to provide a second layer of second matrix material. Additionally, in embodiments, the method may comprise a separation stage comprising separating the sacrificial structure from the first layer and the second layer to provide a channel.

[0042] Hence, in embodiments, the invention may provide a method for providing a system for in vitro modelling of biologically induced compression, wherein the method may comprise: (A) a preparation stage comprising providing a sacrificial structure, a first matrix precursor and a second matrix precursor, wherein the first matrix precursor and the second matrix precursor may both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl, and wherein the first matrix precursor may comprise thrombin and nl g / L fibrinogen, and wherein the second matrix precursor may comprise n2 g / L fibrinogen, wherein 0<n2<nl, and wherein one or more of the first matrix precursor and the second matrix precursor may comprise pancreatic stellate cells; (B) a first layer deposition stage comprising (i) providing the first matrix precursor to a first sacrificial part of the sacrificial structure, and (ii) solidifying the first matrix precursor to provide a first layer of first matrix material; (C) a second layer deposition stage comprising (i) providing the second matrix precursor to the first layer and to a second sacrificial part of the sacrificial structure, and (ii) solidifying the second matrix precursor to provide a second layer of second matrix material; (D) a separation stage comprising separating the sacrificial structure from the first layer and the second layer to provide a channel.

[0043] Such embodiments may be beneficial as the method may provide a more streamlined and convenient standard protocol for the fabrication of in vitro tumor models. Especially, the method may provide a system (especially an in vitro tumor model) that may achieve improved levels of complexity, more consistent results, and controllability relative to existing models. Further, the method may provide a system (especially an in vitro tumor model) with relatively low internal heterogeneity compared to existing systems (or models), therewith improving their suitability for drug development applications.

[0044] In embodiments, the method may comprise a preparation stage, a first layer deposition stage, a second layer deposition stage, and a separation stage. The term “stage” and similar terms used herein may refer to a (time) period (also “phase” or “step”) of a method and / or an operational mode. The different stages may (partially) overlap (in time). For example, the preparation stage may, in embodiments, partially overlap in time with the first layer deposition stage. However, for example, the first layer deposition stage may typically be completed prior to the second layer deposition stage. It will be clear to the person skilled in the art how the stages may be beneficially arranged in time. Here below, the different stages are described in further detail.

[0045] The preparation stage may, in embodiments, especially comprise providing a sacrificial structure, a first matrix precursor, and a second matrix precursor.

[0046] Herein, the term “sacrificial structure” may refer to a (semi-)solid structure or a rigid hollow structure. In embodiments, the sacrificial structure may have an axis of elongation (A). Further, in embodiments, the sacrificial structure may have an equivalent circular diameter (Dss). The (sacrificial structure) equivalent circular diameter may, in embodiments, especially be defined in a first cross-sectional plane defined perpendicular to the axis of elongation (A) (or the direction of elongation, see also further above). Especially, in embodiments, the (sacrificial structure) equivalent circular diameter (Dss) may be at least 5 pm, like at least 10 pm, such as at least 0.1 mm, especially at least 0.5 mm. Further, in embodiments, the (sacrificial structure) equivalent circular diameter (Dss) may be at most 15 mm, such as at most 10 mm, like at most 5 mm, especially at most 1 mm. Yet further, in embodiments, along at least 60%, such as at least 70%, especially at least 80%, like at least 90%, including 100%, of a length (Lss, see also further below) of the sacrificial structure along the axis of elongation (A) the sacrificial structure may have an equivalent circular diameter (Dss) selected from the range of 10 pm - 20 mm, such as from the range of 50 pm -10 mm, like from the range of 0.1 mm - 5 mm, especially from the range of 10 pm - 1.5 mm. Hence, in embodiments, the sacrificial structure may have an axis of elongation (A), wherein along at least 60% of a length (Lss) of the sacrificial structure along the axis of elongation (A), the sacrificial structure has an equivalent circular diameter (Dss) selected from the range of 10 pm - 1.5 mm.

[0047] The sacrificial structure may further, in embodiments, have a shape approximating a shape selected from the group comprising: a tubular shape, a semi-cylindrical shape, a spherical shape, an ellipsoid shape, a (triangular, square, rectangular, or polygonal) prismatic shape, a frustum shape, a toric shape, and a dumbbell shape. In specific embodiments, the sacrificial structure may especially have a shape selected from the group comprising a rod and a sphere. Yet further, in embodiments, the sacrificial structure may comprise a first sacrificial part and a second sacrificial part. In embodiments, the first sacrificial part may have a first sacrificial part length (Lsi). The first sacrificial part may, in embodiments, be a singular part. However, in other embodiments, the first sacrificial part may be comprised of multiple separated parts. Therefore, the first sacrificial part length (Lsi) may comprise a total (cumulative) length of all first sacrificial parts combined. In embodiments, the first sacrificial part length (Lsi) may be selected from the range of 0.2 - 100 mm, especially from the range of 0.2 - 50 mm, such as from the range of 0.5 - 20 mm, especially from the range of 1 - 6 mm. Similarly, in embodiments, the second sacrificial part may have a second sacrificial part length (Lsi). The second sacrificial part may, in embodiments, be a singular part. However, in other embodiments, the second sacrificial part may be comprised of multiple separated parts, e.g. multiple second sacrificial parts separated by one or more first sacrificial parts. Therefore, the second sacrificial part length (Lsi) may comprise a total (cumulative) length of all second sacrificial parts combined. In embodiments, the second sacrificial part length (Lsi) may be selected from the range of 0.2 - 50 mm, such as from the range of 0.5 - 20 mm, especially from the range of 1 - 6 mm. In embodiments, the first sacrificial part(s) and the second sacrificial part(s) may thus form the sacrificial structure. Therefore, in embodiments, the sacrificial structure may have a sacrificial structure length (Lss) defined in an elongation direction of the sacrificial structure, especially Lss=Lsi+Lsi. For example, in embodiments, the sacrificial structure may consist of rod comprising a first sacrificial part and a second sacrificial part configured adjacent to each other. In other embodiments, the sacrificial structure may, e.g., consist of a rod comprising two second sacrificial parts separated by a singular first sacrificial part.

[0048] In embodiments, the preparation stage may especially comprise arranging the sacrificial structure in a first mold. Especially, in embodiments, the sacrificial structure may be arranged in the first mold such that at most 40% of a total surface of the sacrificial structure, like at most 25%, such as at most 10%, especially at most 5%, more especially at most 2% is in physical contact with the first mold. Especially, in embodiments, the sacrificial structure may be arranged in the first mold such that at least 60%, such as at least 75%, like at least 80%, especially at least 90% of the total surface of the sacrificial structure is exposed to the environment (e.g. to the air).

[0049] The first mold may, in embodiments, have a first mold diameter (Dmi) and a first mold length (Lmi). In embodiments, the first mold diameter (Dmi) may be defined in the first cross-sectional plane. The first mold diameter (Dmi) may especially be selected from the range of 0.5 - 20 mm, such as from the range of 1 - 15 mm, like from the range of 1 - 8 mm, especially from the range of 1.5 - 5 mm. In embodiments, the first mold length (Lmi) may be defined perpendicular to the first cross-sectional plane. The first mold length (Lmi) may especially be selected from the range of 0.5 - 50 mm, such as from the range of 1 - 20 mm, like from the range of 2 - 10 mm, especially from the range of 2 - 8 mm.

[0050] Further, in embodiments, the preparation stage may comprise providing a first matrix precursor and a second matrix precursor. In embodiments, the first matrix precursor and the second matrix precursor may both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl. Hence, in embodiments, the first matrix precursor may comprise one or more of collagen, gelatin and gelatin methacryloyl. The first matrix precursor may, in embodiments, especially comprise a hydrogel, such as e.g. a collagen hydrogel. Similarly, in embodiments, the second matrix precursor may comprise one or more of collagen, gelatin and gelatin methacryloyl. The second precursor material may, in embodiments, especially comprise a hydrogel, such as e.g. a collagen hydrogel.

[0051] The first matrix precursor may, in embodiments, comprise nl g / L fibrinogen and thrombin, and optionally further also Ca2+. In embodiments, nl may be at least 1, such as at least 2, like at least 3, especially at least 4. Further, in embodiments, nl may be at most 15, such as at most 12, like at most 10, especially at most 9. Especially, in embodiments, the first matrix precursor may comprise fibrinogen selected from the range of 1 - 15 g / L, such as from the range of 2 - 10 g / L, like from the range of 2 - 8 g / L, especially from the range of 4 - 6 g / L. Further, in embodiments, the first matrix precursor may comprise thrombin selected from the range of 0.5 - 15 units / mL, such as from the range of 1 - 10 units / mL, like from the range of 2

[0052] - 9 units / mL, especially from the range of 2 - 8 units / mL, more especially from the range of 3

[0053] - 7 units / mL. Optionally, in embodiments, the first matrix precursor may further comprise Ca2+selected from the range of 0.5 - 15 mM, such as from the range of 1 - 10 mM, like from the range of 2 - 8 mM.

[0054] In embodiments, the first matrix precursor may especially comprise a first weight ratio of fibrinogen to thrombin (Fib:Thr). Especially, in embodiments, the first weight ratio of fibrinogen to thrombin may be selected from the range of 12:1 to 8: 1, such as e.g. a weight ratio of 10: 1. Furthermore, in embodiments, the first matrix precursor may comprise a second weight ratio of collagen to fibrinogen (CokFib). In embodiments, the second weight ratio of collagen to fibrinogen may be selected from the range of 1 :5 to 1 :2, such as from the range of 1 :4 to 1 :2, such as e.g. 7:20. Yet further, in embodiments, the first matrix precursor may comprise a combined weight ratio of collagen and fibrinogen combined to thrombin (ColFib:Thr). In embodiments, the combined weight ratio of collagen and fibrinogen combined to thrombin selected from the range of 250: 1 to 25: 1, such as from the range of 200: 1 to 50: 1, like from the range of 150: 1 to 75:1.

[0055] Optionally, in embodiments, the second matrix precursor may (also) comprise one or more of thrombin and fibrinogen. Especially, in embodiments, the second matrix precursor may comprise n2 g / L fibrinogen. In embodiments, n2 may be at least 1, such as at least 2, like at least 3, especially at least 4. Further, in embodiments, n2 may be at most 14, such as at most 12, like at most 10, especially at most 9. Yet further, in embodiments, n2 may (also) be essentially zero, i.e., there may not necessarily be fibrinogen present in the second matrix precursor. Especially, in embodiments, there may be less fibrinogen present in the second matrix precursor relative to the first matrix precursor, i.e., n2<nl. More especially, in embodiments, 0<n2<nl. In embodiments, the second matrix precursor may comprise fibrinogen selected from the range of 1 - 14 g / L, such as from the range of 1 - 10 g / L, like from the range of 2 - 8 g / L, especially from the range of 2 - 6 g / L. Further, in embodiments, the second matrix precursor may comprise thrombin selected from the range of 0.5 - 14 units / mL, such as from the range of 1 - 10 units / mL, like from the range of 2 - 9 units / mL, especially from the range of 2 - 8 units / mL. Especially, in embodiments, there may be less thrombin present in the second matrix precursor relative to the first matrix precursor. Optionally, in embodiments, the second matrix precursor may comprise essentially no thrombin.

[0056] Note that, in embodiments, other (bio)materials may also (alternatively to the combination of fibrinogen with thrombin) be applied in the system to (help) mimic biologically induced compression, see also above.

[0057] Further, in embodiments, one or more of the first matrix precursor and the second matrix precursor may comprise fibroblasts. Additionally or alternatively, in embodiments, one or more of the first matrix precursor and the second matrix precursor may comprise cells of mesenchymal origin, such as cardiac myocytes, smooth muscle cells, myoepithelial cells, mesenchymal stem cells, myofibroblasts etc.. Especially, in embodiments, at least the first matrix precursor may comprise fibroblasts (and / or cells of mesenchymal origin). The fibroblasts (and / or cells of mesenchymal origin) may, in embodiments, comprise pancreatic stellate cells. In other embodiments, the fibroblasts (and / or cells of mesenchymal origin) may (also) comprise one or more of the group comprising: hepatic stellate cells, lung stellate cells, and renal stellate cells. Optionally, in embodiments, one or more of the first matrix precursor and the second matrix precursor may comprise other cells, such as e.g. epithelial cells, which mimic normal tissue. For example, in embodiments, the first matrix precursor may comprise (fibroblasts, especially) pancreatic stellate cells and the second matrix precursor may comprise epithelial cells. In other embodiments, for example, the first matrix precursor may comprise (fibroblasts, especially) pancreatic stellate cells and the second matrix precursor may comprise both fibroblasts (e.g. pancreatic stellate cells) and epithelial cells.

[0058] In embodiments, the first layer deposition stage may comprise providing the first matrix precursor to the first sacrificial part of the sacrificial structure. Especially, in embodiments, the first layer deposition stage may comprise providing the first matrix precursor to the first mold to at least partially surround the sacrificial structure. More especially, the first layer deposition stage may comprise providing the first matrix precursor to the first mold to at least partially surround, especially fully surround, the first sacrificial part of the sacrificial structure. In embodiments, the first matrix precursor may along at least 60% of a first sacrificial part length, such as along at least 80% of the first sacrificial surface part length, cover at least 30% of a surface outline of the first sacrificial surface part in a plane perpendicular to the axis of elongation, such as at least 50%, like at least 70%, especially at least 90%. Especially, in embodiments, the first matrix precursor may be provided (to the first mold, especially to the (one or more) first sacrificial part(s)) such that the (one or more) first sacrificial part(s) may be surrounded by the first matrix precursor. Especially, in some embodiments, the first matrix precursor may be provided (to the first mold, especially to the (one or more) first sacrificial part(s)) such that the (one or more) first sacrificial part(s) may essentially be enclosed (i.e., 100% surrounded) by the first matrix precursor.

[0059] Subsequently, in embodiments, the first layer deposition stage may comprise (at least partially) solidifying the first matrix precursor to provide a first layer of first matrix material. Especially, in embodiments, the first layer deposition stage may comprise solidifying at least 50% of the first matrix precursor, such as at least 60%, like at least 70%, especially at least 80%, more especially at least 90%, including 100% of the first matrix precursor to provide the first layer of first matrix material. The first layer deposition stage may especially, in embodiments, comprise solidifying the first matrix precursor to provide a single layer construct comprising the first layer of first matrix material. In such embodiments, the first layer of first matrix material may especially surround at least part of the sacrificial structure. Especially, in embodiments, the first layer of first matrix material may surround (or even fully enclose) at least the (one or more) first sacrificial part(s). In embodiments, the solidifying may comprise exposing the (first / second) matrix precursor to suitable solidification conditions, e.g., a suitable temperature, a suitable pH, and / or a suitable humidity. Especially, in embodiments, solidifying the first matrix precursor may comprise exposing the first matrix precursor to a first temperature. More especially, in embodiments, the first temperature may be selected from the range of 10 - 80°, such as from the range of 20 - 70°, like from the range of 30 - 60°, especially from the range of 35 - 45°, like from the range of 35 - 40°. For example, in embodiments, the first layer deposition stage may comprise solidifying the first matrix precursor by exposing the first matrix precursor to a first temperature of 37°. Additionally or alternatively, in embodiments, solidifying the first matrix precursor may comprise exposing the first matrix precursor to a first pH. Especially, in embodiments, the first pH may be selected from the range of 7.10 - 7.70, such as from the range of 7.20 - 7.60, like from the range of 7.30 - 7.50, especially from the range of 7.35 - 7.45, like from the range of 7.38 - 7.42. For example, in embodiments, the first layer deposition stage may comprise solidifying the first matrix precursor by exposing the first matrix precursor to a first pH selected from the range of 7.35 - 7.45. Additionally or alternatively, in embodiments, solidifying the first matrix precursor may comprise exposing the first matrix precursor to a first (relative) humidity. Especially, in embodiments, the first (relative) humidity may be selected from the range of 25 - 75%, such as from the range of 30 - 70%, like from the range of 35 - 65%, especially from the range of 40 - 60%, like from the range of 45 - 55%. For example, in embodiments, the first layer deposition stage may comprise solidifying the first matrix precursor by exposing the first matrix precursor to a first (relative) humidity selected from the range of 40 - 60%. Hence, in embodiments, the first layer deposition stage may comprise solidifying the first matrix precursor by exposing the first matrix precursor to one or more of a temperature selected from the range of 30° - 60°, a pH selected from the range of 7.35 - 7.45, and a humidity selected from the range of 40 - 60%.

[0060] In embodiments, the first layer deposition stage may comprise only partial solidification (i.e. semi-solidification) of the first matrix precursor. The second layer deposition stage may, in such embodiments, be executed while the first matrix precursor may still be in a semi-solidified state, especially the fibrinogen may have crosslinked, therewith keeping the fibroblasts and / or cells of mesenchymal origin in the first matrix precursor in place, while the collagen may still be in a semi-solidified state. Such embodiments may be beneficial as keeping the first matrix precursor in a semi-solidified state may improve proper attachment of the second matrix precursor to the first matrix precursor. Furthermore, such embodiments may be beneficial as time required for the fabrication process may be kept to a minimum, which may help increase cell survival in the system. Semi-solidification of the first matrix material may be achieved, in embodiments, by exposing the first matrix precursor to above indicated suitable conditions for an initial solidification duration (Ti). In embodiments, the initial solidification duration (Ti) may be selected from the range of 15 - 60 minutes, especially from the range of 25 - 50 min, such as from the range of 30 - 45 minutes. In contrast, (full) solidification of the first (and optionally the second) matrix precursor may be achieved, in embodiments, by exposing the first matrix precursor to above indicated suitable conditions for a solidification duration (Ts). In embodiments, the solidification duration (Ts) may be selected from the range of 45 - 105 minutes, especially from the range of 60 - 90 min, such as from the range of 70 - 90 minutes. In embodiments, the first layer deposition stage may comprise initial solidification of the first matrix precursor, while (full) solidification of the first matrix precursor may occur simultaneously with solidification of the second matrix precursor as described below for the second layer deposition stage. However, in embodiments, (full) solidification of the first matrix precursor may also occur prior to the second layer deposition stage, such as during the first layer deposition stage. In embodiments, the second layer deposition stage may comprise providing the second matrix precursor to the first layer and to the second sacrificial part of the sacrificial structure. Especially, in embodiments, the second layer deposition stage may comprise (rapidly) transferring the single layer construct to a second mold. In embodiments, the single layer construct may be arranged in the second mold such that at most 40%, like at most 25%, such as at most 10%, especially at most 5%, more especially at most 2%, of a total surface of the single layer construct is in physical contact with the second mold. Especially, in embodiments, the single layer construct may be arranged in the second mold such that at least 60%, such as at least 75%, like at least 80%, especially at least 90%, of the total surface of the single layer construct is exposed to the environment (e.g. to the air).

[0061] The second mold may, in embodiments, have a second mold diameter (Dm2) and a second mold length (Lnu). Especially, in embodiments, the second mold may be larger than the first mold in at least one dimension. Therefore, in embodiments, one or more of the following may apply: (i) the second mold length (Lnu) may be larger than the first mold length (Lmi), i.e., Lm2>Lmi, and (ii) the second mold diameter (Dm2) may be larger than the first mold diameter (Dmi), i.e., Dm2>Dmi. Especially, in embodiments, Lm2>Lmiand Dm2>Dmi. In embodiments, the second mold diameter (Dm2) may be defined in the first cross-sectional plane. The second mold diameter (Dm2) may especially be selected from the range of 0.5 - 20 mm, such as from the range of 1 - 15 mm, like from the range of 2 - 10 mm, especially from the range of 4 - 12 mm. In embodiments, the second mold length (Lnu) may be defined perpendicular to the first cross-sectional plane (and parallel to the first mold length (Lmi)). The second mold length (Lnu) may especially be selected from the range of 1 - 50 mm, such as from the range of 2 - 20 mm, like from the range of 4 - 16 mm, especially from the range of 4 - 12 mm.

[0062] Further, in embodiments, the first mold and the second mold may comprise a mold material. Especially, in embodiments, the first mold and the second mold may comprise one or more mold materials (individually) selected from the group comprising polydimethylsiloxane (PDMS), Teflon, polyurethane, poly(lactic-co-glycolic acid) (PLGA) and elastomeric materials such as Sylgard 184, polyethylene (PE), polypropylene (PP) and polycaprolactone (PCL). In embodiments, the first mold and the second mold may comprise essentially the same (combination of) mold material(s). In other embodiments, the first mold and the second mold may comprise a different (combination of) mold material(s).

[0063] The second layer deposition stage may thus, in embodiments, comprise providing the second matrix precursor to the second mold to at least partially surround the single layer construct. More especially, the second layer deposition stage may comprise providing the second matrix precursor to the second mold to at least partially surround the first layer and the second sacrificial part of the sacrificial structure. In embodiments, the second matrix precursor may along at least 60% of a first layer length, such as along at least 80% of the first layer length, cover at least 30% of a surface outline of the first layer in a plane perpendicular to the axis of elongation, such as at least 50%, like at least 70%, especially at least 90%. Similarly, in embodiments, the second matrix precursor may along at least 60% of a second sacrificial part length, such as along at least 80% of the second sacrificial part length, cover at least 30% of a surface outline of the second sacrificial part in a plane perpendicular to the axis of elongation, such as at least 50%, like at least 70%, especially at least 90%. Especially, in embodiments, the second matrix precursor may be provided (to the second mold, especially to the first layer and the (one or more) second sacrificial part(s)) such that the first layer and the (one or more) second sacrificial part(s) may be surrounded by the second matrix precursor. Especially, in some embodiments, the second matrix precursor may be provided (to the second mold, especially to the first layer and the (one or more) second sacrificial part(s)) such that the first layer and the (one or more) second sacrificial part(s) may essentially be enclosed by the second matrix precursor.

[0064] Subsequently, in embodiments, the second layer deposition stage may comprise (at least partially) solidifying the second matrix precursor to provide a second layer of second matrix material. Especially, in embodiments, the second layer deposition stage may comprise solidifying at least 50% of the second matrix precursor, such as at least 60%, like at least 70%, especially at least 80%, more especially at least 90%, including 100% of the second matrix precursor to provide the second layer of second matrix material. The second layer deposition stage may especially, in embodiments, comprise solidifying the second matrix precursor to provide a bilayer construct comprising the first layer and the second layer surrounding at least part of the first layer and at least part of the sacrificial structure. Hence, in such embodiments, the second layer of second matrix material may especially surround at least part of the sacrificial structure. Especially, in embodiments, the second layer of second matrix material may surround (or even fully enclose) at least the (one or more) second sacrificial part(s). In embodiments, solidifying the second matrix precursor may comprise exposing the second matrix precursor to a second temperature. Especially, in embodiments, the second temperature may be selected from the range of 10 - 80°, such as from the range of 20 - 70°, like from the range of 30 - 60°, especially from the range of 35 - 45°, like from the range of 35 - 40°. For example, in embodiments, the second layer deposition stage may comprise solidifying the second matrix precursor by exposing second first matrix precursor to a second temperature of 37°. Additionally or alternatively, in embodiments, solidifying the second matrix precursor may comprise exposing the second matrix precursor to a second pH. Especially, in embodiments, the second pH may be selected from the range of 7.10 - 7.70, such as from the range of 7.20 - 7.60, like from the range of 7.30 - 7.50, especially from the range of 7.35 - 7.45, like from the range of 7.38 - 7.42. For example, in embodiments, the second layer deposition stage may comprise solidifying the second matrix precursor by exposing the second matrix precursor to a second pH selected from the range of 7.35 - 7.45. Additionally or alternatively, in embodiments, solidifying the second matrix precursor may comprise exposing the second matrix precursor to a second (relative) humidity. Especially, in embodiments, the second (relative) humidity may be selected from the range of 25 - 75%, such as from the range of 30 - 70%, like from the range of 35 - 65%, especially from the range of 40 - 60%, like from the range of 45 - 55%. For example, in embodiments, the second layer deposition stage may comprise solidifying the second matrix precursor by exposing the second matrix precursor to a second (relative) humidity selected from the range of 40 - 60%. Hence, in embodiments, the second layer deposition stage may comprise solidifying the second matrix precursor by exposing the second matrix precursor to one or more of a temperature selected from the range of 30° - 60°, a pH selected from the range of 7.35 - 7.45, and a humidity selected from the range of 40 - 60%.

[0065] The separation stage may, in embodiments, comprise separating the sacrificial structure from the first layer and the second layer to provide a channel. Especially, in embodiments, the separation stage may comprise separating the sacrificial structure from the bilayer construct to provide a channel in the bilayer construct. Therefore, in embodiments, the separation stage may comprise removing the sacrificial structure from the bilayer construct, e.g., through pushing and / or pulling the sacrificial structure out of the bilayer construct. However, in embodiments, the sacrificial structure may also be separated from the (bilayer construct, especially the) first layer and the second layer through (self-)dissolving the sacrificial structure. Therefore, in embodiments, the sacrificial structure may comprise a (self-)dissolvable material. Potential (self-)dissolvable materials are known in the art. For example, in embodiments, the sacrificial structure may comprise a (self-)dissolvable material selected from the group comprising pluronic F127, polycaprolactone (PCL), polylactic acid (PLA), polyglactin, and polydioxane. Hence, in some embodiments, the sacrificial structure may comprise a self-dissolvable material. In other embodiments, especially wherein the channel may comprise two open ends, the channel may be washed (or flushed) with a solvent suitable for dissolving the dissolvable material.

[0066] In embodiments, the channel may be defined by the (first and / or second) matrix material, see also further above, acting as a wall. The method may, in embodiments, further comprise a seeding stage. In embodiments, the seeding stage may comprise providing cells into the channel to provide (at least part of) a channel lining. Hence, in embodiments, the cells may also be referred to as lining cells, i.e., the cells may be lined against (or “seeded onto”) at least part of the channel wall. The cells may especially, in embodiments, comprise eukaryotic cells, such as animal or human cells. In embodiments, the cells may comprise one or more of endothelial cells, pericytes, and other vascular cells, such as at least endothelial cells. Hence, in embodiments, the method may further comprise a seeding stage comprising providing cells into the channel to provide a channel lining, wherein the cells comprise endothelial cells.

[0067] As indicated above, in embodiments, the first matrix precursor may comprise a first weight ratio of fibrinogen to thrombin (Fib:Thr), a second weight ratio of collagen to fibrinogen (CoFFib), and a combined weight ratio of collagen and fibrinogen combined to thrombin (ColFib:Thr). In embodiments of the method, the weight ratios of the first matrix precursor may especially be selected such, that the first matrix material may especially comprise the first weight ratio of fibrinogen to thrombin (Fib:Thr), as indicated above. Especially, in embodiments, the first weight ratio of fibrinogen to thrombin may be selected from the range of 12: 1 to 8: 1, such as e.g. a weight ratio of 10: 1. Furthermore, in embodiments of the method, the weight ratios of the first matrix precursor may especially be selected such, that the first matrix material may comprise the second weight ratio of collagen to fibrinogen (CoFFib), as indicated above. Especially, in embodiments, the second weight ratio of collagen to fibrinogen may be selected from the range of 1 :5 to 1 :2, such as from the range of 1 :4 to 1 :2, such as e.g. 7:20. Yet further, in embodiments of the method, the weight ratios of the first matrix precursor may especially be selected such, that the first matrix material may comprise a combined weight ratio of collagen and fibrinogen combined to thrombin (ColFib:Thr), as indicated above. Especially, in embodiments, the combined weight ratio of collagen and fibrinogen combined to thrombin may be selected from the range of 250: 1 to 25: 1, such as from the range of 200: 1 to 50: 1, like from the range of 150: 1 to 75: 1. Hence, in embodiments, the first matrix material may comprise a weight ratio of collagen to fibrinogen selected from the range of 1 :4 to 1 :2, and the first matrix material may comprise a weight ratio of collagen and fibrinogen combined to thrombin selected from the range of 200:1 to 50: 1. Such embodiments may be beneficial as the combination of fibrinogen and thrombin in the matrix material may induce the activation of PSCs, contraction of the channel, and extracellular matrix (ECM) production of PSCs. Especially, the indicated weight ratios of collagen, fibrinogen and thrombin may increase mechanical stiffness and may provide enhanced activation of PSCs in the system, therewith providing improved mimicking of vasculature compression (in pancreatic tumors). In particular, such embodiments may induce activation of PSCs leading to a 10-fold contraction of the channel relative to a system without activated PSCs.

[0068] In embodiments, the method may thus provide a system comprising the channel, the first layer of first matrix material and the second layer of second matrix material. The channel may, in embodiments, have essentially the same dimensions as the sacrificial structure. Further, in embodiments, the first layer may have a first average thickness (dl) defined in the first cross-sectional plane. Especially, in embodiments, the first average thickness (dl) may be at most 50 mm, especially at most 30 mm, such as at most 20 mm, especially at most 15 mm, like at most 10 mm, such as at most 8 mm. Further, in embodiments, the first average thickness (dl) may be at least 0.1 mm, such as at least 0.5 mm, especially at least 1 mm, like at least 2 mm, such as at least 3 mm. Especially, in embodiments, the first average thickness (dl) may be selected from the range of 1 - 10 mm.

[0069] Similarly, in embodiments, the second layer may have a second average thickness (d2) defined in the first cross-sectional plane. Especially, in embodiments, the second average thickness (d2) may be at most 50 mm, especially at most 30 mm, such as at most 20 mm, especially at most 15 mm, like at most 10 mm, such as at most 8 mm. Further, in embodiments, the second average thickness (d2) may be at least 0.1 mm, such as at least 0.5 mm, especially at least 1 mm, like at least 2 mm, such as at least 3 mm. Especially, in embodiments, the second average thickness (d2) may be selected from the range of 1 - 10 mm. Hence, in embodiments, the first layer may have a first average thickness (dl) selected from the range of 1 - 10 mm. In further embodiments, the second layer may have a second average thickness (d2) selected from the range of 1 - 10 mm.

[0070] Therefore, in embodiments, the system (as a whole) may have a diameter (Ds) as defined above. Especially, in embodiments, the (system) diameter (Ds) may be at least 0.1 mm, like at least 0.5 mm, such as at least 1 mm, especially at least 2 mm. Further, in embodiments, the (system) diameter (Ds) may be at most 25 mm, such as at most 15 mm, like at most 5 mm, especially at most 1 mm.

[0071] The embodiments described herein are not limited to a single aspect of the invention. For example, an embodiment describing the system may further relate to the use of the system. In particular, an embodiment of the use describing an operation (of the system) may indicate that the system may, in embodiments, be configured for and / or be suitable for the operation. Similarly, an embodiment of the method of providing the system may further relate to embodiments of the system, and vice versa.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig.l schematically depicts some embodiments of the system and the use thereof. Figs. 2A-2B schematically depict embodiments and aspects of the method of the invention. The schematic drawings are not necessarily on scale.

[0074] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0075] Fig. 1 schematically depicts the system 100 for in vitro modelling of biologically induced compression. In the depicted embodiment, the system 100 comprises a channel 110 and a matrix material 120. Especially, in embodiments, the system 100 (especially the matrix material 120) may comprise a first matrix material 121 and a second matrix material 122. Furthermore, in embodiments, one or more of the first matrix material 121 and the second matrix material 122 may comprise fibroblasts or cells of mesenchymal origin 125. Especially, in embodiments, one or more of the first matrix material 121 and the second matrix material 122 may comprise (fibroblasts or cells of mesenchymal origin 125 especially comprising) pancreatic stellate cells 126. In specific embodiments, at least the first matrix material 121 may comprise (fibroblasts or cells of mesenchymal origin 125 especially comprising) pancreatic stellate cells 126.

[0076] In embodiments, the channel 110 may have a shape approximating a shape selected from the group comprising: a tubular shape, a cylindrical shape, a spherical shape, and other shapes as indicated above. The channel 110 may, in embodiments, be an elongated channel. Especially, in embodiments as depicted here, the channel 110 may have a tubular or cylindrical shape. Therefore, in embodiments, the channel 110 may have a(n equivalent circular) channel diameter De defined in a first cross-sectional plane 50 defined perpendicular to the direction of elongation (or the axis of elongation A, see also further below). The (equivalent circular) channel diameter De may, in embodiments, especially be selected from the range of 10 pm - 1.5 mm. The (tubular) channel 110 may further have a channel length Lc defined along the direction of elongation. The channel length Lc may, in embodiments, especially be selected from the range of 4 - 16 mm. Note that in some embodiments, not depicted here, the channel may not be an elongated channel, but for example a spherical structure. In such embodiments, the channel length Lc may be defined as one or more of a diameter or a largest dimension of the channel. Hence, in some embodiments, the channel length Lc may be equal to or essentially the same as the channel diameter De.

[0077] The channel 110 may, in embodiments, comprise a first channel part 111, having a first channel part length Lei, and a second channel part 112, having a second channel part length LC2. Especially, in embodiments, Lc = Lei + Lc2. In embodiments, the second channel part 121 may be configured as two (or more) separate parts, especially two (or more) separate second channel parts 121 separated by the (one or more) first channel part(s) 111 (such as depicted here). Therefore, in embodiments, the first channel part length Lei may comprise the cumulative length of all first channel parts 111. Likewise, in embodiments, the second channel part length Lc2 may comprise the cumulative length of all second channel parts 112. However, in other embodiments, the first channel part 111 and the second channel part may 112 also be configured as singular parts adjacent to each other.

[0078] In embodiments, the first matrix material 121 may especially surround the first channel part 111 of the channel 110. As depicted here, the first matrix material 121 may surround the centrally located first channel part 111 of the tubular channel 110. Hence, in embodiments, the channel 110 may be defined by the matrix material 120. Further, in embodiments, the second matrix material 122 may surround the first matrix material 121 and the (one or more) second channel part(s) 112 of the channel 110. As depicted here, the second matrix material 121 may surround the outer second channel parts 112 of the tubular channel 110 as well as the first matrix material 121.

[0079] Further, in embodiments, the channel 110 may comprise cells 116, especially (biological) cells 116 lining the (matrix material 120 defining the) channel 110. In specific embodiments, the channel 110(, especially the matrix material 120 defining the channel 110) may be lined with one or more of endothelial cells 117, pericytes, and other vascular cells, especially at least endothelial cells 117. Yet further, in embodiments, the channel 110 may comprise a blood vessel 118.

[0080] The first matrix material 121 and the second matrix material 122 may, in embodiments, both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl. Furthermore, in embodiments, the first matrix material 121 may comprise thrombin and nl g / L fibrinogen. Especially, nl may be selected from the range of 2 - 10. Further, in embodiments, the second matrix material 122 may (also) comprise (thrombin and) n2 g / L fibrinogen. Moreover, in embodiments, 0<n2<nl.

[0081] Further, in embodiments, the first matrix material 121 may comprise a (first) weight ratio of collagen to fibrinogen (Col:Fib) selected from the range of 1 :4 to 1 :2, especially of about 7:20. In further embodiments, the first matrix material 121 may comprise a (third) weight ratio of collagen and fibrinogen combined to thrombin (ColFib:Thr) selected from the range of 200: 1 to 50: 1.

[0082] The system 100 may, in embodiments, have a (system) diameter Ds defined in the first cross-sectional plane 50. Especially, in embodiments, the (system) diameter Ds may be selected from the range of 2 - 8 mm. Further, in embodiments, the system 100 may have a (system) length Ls defined perpendicular to the first cross-sectional plane 50. Especially, in embodiments, the (system) length Ls may be selected from the range of 4 - 16 mm.

[0083] The invention may further provide a use of the system 100 as described above for modelling vasculature compression. Fig. 1 especially schematically depicts such vasculature compression. In the left part of the figure, the system is depicted having pancreatic stellate cells 126 in a non-activated state. In the right part of the figure, the system is depicted having pancreatic stellate cells 126 in an activated state, especially having activated pancreatic stellate cells 126 in the first matrix material 121. Therefore, in such embodiments, the first channel part 111 (being surrounded by the first matrix material 121) may be compressed, as can be seen in the top right box depicting the first cross-sectional plane 50a. The presence of thrombin and fibrinogen in the first matrix material 121 may activate the fibroblasts or cells of mesenchymal origin 125, especially the pancreatic stellate cells 236, resulting in induced compression (or “contraction”) of the channel 111. In specific embodiments as depicted here, the use may thus especially be for simulating a pancreatic tumor.

[0084] Further, in embodiments, the invention provides a method for providing a system for in vitro modelling of biologically induced compression. In embodiments, the method may comprise a preparation stage comprising providing a sacrificial structure 10, a first matrix precursor 21 and a second matrix precursor 22, as depicted in Figs. 2A-B. Especially, in embodiments, the first matrix precursor 21 and the second matrix precursor 22 both may comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl. Further, in embodiments, the first matrix precursor 21 may comprise thrombin and nl g / L fibrinogen. Yet further, the second matrix precursor 22 may comprise n2 g / L fibrinogen. Moreover, in embodiments, 0<n2<nl. In further embodiments, one or more of the first matrix precursor 21 and the second matrix precursor 22 may comprise pancreatic stellate cells 126, such as especially at least the first matrix precursor 21. The method may especially, in embodiments, make use of a mold 40. In specific embodiments, such as depicted in Fig. 2A, the preparation stage may comprise arranging the sacrificial structure 10 in a first mold 41.

[0085] Furthermore, in embodiments, the method may comprise a first layer deposition stage comprising providing the first matrix precursor 21 to a first sacrificial part 11 of the sacrificial structure 10. Especially, in embodiments, the first layer deposition stage may comprise providing the first matrix precursor 21 to the first mold 41 to at least partially surround the sacrificial structure. Additionally, in embodiments, the first layer deposition stage may comprise (at least partially) solidifying the first matrix precursor 11 to provide a first layer 31 of first matrix material 121. Especially, in embodiments, the first layer deposition stage may comprise solidifying the first matrix precursor 21 to provide a single layer construct 35 comprising a first layer 31 of first matrix material 121 surrounding at least part of the sacrificial structure 10. In specific embodiments, the first layer deposition stage may comprise solidifying the first matrix precursor 21 by exposing the first matrix precursor 21 to one or more of a temperature selected from the range of 30° - 60°, a pH selected from the range of 7.35 - 7.45, and a humidity selected from the range of 40-60%.

[0086] Yet further, in embodiments, the method may comprise a second layer deposition stage comprising providing the second matrix precursor 22 to the first layer 31 and to a second sacrificial part 12 of the sacrificial structure 10. Especially, in embodiments, the second layer deposition stage may comprise transferring the single layer construct 35 to a second mold 42. Additionally, in embodiments, the second layer deposition stage may comprise (at least partially) solidifying the second matrix precursor 22 to provide a second layer 32 of second matrix material 122. Especially, in embodiments, the second layer deposition stage may comprise providing the second matrix precursor 22 to the second mold 42 to at least partially surround the single layer construct 35. Subsequently, in embodiments, the second layer deposition stage may comprise solidifying the second matrix precursor 22 to provide a bilayer construct 30 comprising the first layer 31 and a second layer 32 surrounding at least part of the first layer 31 and at least part of the sacrificial structure 10. In specific embodiments, the second layer deposition stage may comprise solidifying the second matrix precursor 22 by exposing the second matrix precursor 22 to one or more of a temperature selected from the range of 30° - 60°, a pH selected from the range of 7.35 - 7.45, and a humidity selected from the range of 40-60%.

[0087] In further embodiments, the method may comprise a separation stage comprising separating the sacrificial structure 10 from the first layer 31 and the second layer 32 to provide a channel 110. Especially, in embodiments, the separation stage may comprise removing the sacrificial structure 10 from the bilayer construct 30 to provide a channel 110 in the bilayer construct 30.

[0088] In embodiments, the first mold 41 may have a first mold diameter Dmidefined in the first cross-sectional plane 50. Especially, in embodiments, the first mold diameter Dmimay be selected from the range of 1 - 8 mm. Further, in embodiments, the first mold 41 may define a first mold length Lmidefined perpendicular to the first cross-sectional plane 50. Especially, in embodiments, the first mold length Lmimay be selected from the range of 2 - 10 mm.

[0089] Similarly, in embodiments, the second mold 42 may have a second mold diameter Dm2 defined in the first cross-sectional plane 50. Especially, in embodiments, the second mold diameter Dm2 may be selected from the range of 1 - 8 mm. Further, in embodiments, the second mold 42 may define a second mold length Lm2 defined perpendicular to the first cross-sectional plane 50. Especially, in embodiments, the second mold length Lm2 may be selected from the range of 2 - 10 mm.

[0090] In specific embodiments, the second mold 42 may be larger than the first mold 41 in at least one dimension.

[0091] Further, in embodiments, the method may comprise a seeding stage comprising providing (human) cells 116 into the channel 110 to provide (at least part of) a channel lining 115. Further, in embodiments, the (human) cells 116 may comprise endothelial cells 117. Further, in embodiments, such as depicted here, the sacrificial structure 10 may have a shape selected from the group comprising a rod and a sphere, here especially a rod. Therefore, in embodiments, the sacrificial structure 10 may have an axis of elongation A. Moreover, in embodiments, along at least 60% of a length of the sacrificial structure along the axis of elongation A, the sacrificial structure may have an equivalent circular diameter Dss. Especially, in embodiments, the (sacrificial structure) equivalent circular diameter Dss may be selected from the range of 10 pm - 1.5 mm.

[0092] In specific embodiments, the first layer 31 may have a first average thickness dl. Especially, in embodiments, the first average thickness dl may be selected from the range of 1 - 10 mm. Similarly, in embodiments, the second layer 32 may have a second average thickness d2. Especially, in embodiments, the second average thickness d2 may be selected from the range of 1 - 10.

[0093] Experiments

[0094] Unless specified otherwise, the experiments described herein are performed using the materials and methods described hereinafter.

[0095] Generation of collagen, fibrinogen and collagen / fibrinogen (Col / Fib) gels - Collagen gels were prepared according to the manufacturer’s instructions. In brief, to prepare 1 mL of gel solution, 45 pl of 1 M NaOH (Sigma-Aldrich), 50 pl of 1 M 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES, Sigma-Aldrich) buffer, 5 pl of sterile water, 100 pl of lOx Medium (Sigma- Aldrich) were mixed with 800 pl of collagen solution (0.5%, type 1 from bovine calfskin, Matrix Biosciences, Morlenbach, Germany) to obtain a final collagen concentration of 4 mg / mL. The hydrogel solution was further diluted with sterile Dulbecco’s phosphate buffered saline (DPBS, Sigma-Aldrich) to achieve a final concentration of 3.5 mg / mL. The gels were transferred to a custom-made PDMS mold (7 mm x 7 mm x 5 mm (length x width x height)) and allowed to fully solidify at 37 °C in a humidified atmosphere. Collagen / fibrinogen (Col / Fib) hybrid gels were prepared by first mixing the collagen stock solution (4 mg / mL, prepared as aforementioned) with fibrinogen stock solution (80 mg / mL) in a ratio of 7: 1 to achieve a final concentration of 3.5 mg / mL collagen and 10 mg / mL fibrinogen. Next, thrombin was added in a ratio of 1 : 100 to achieve a final concentration of lU / mL, the solution was homogenously mixed and transferred to custom made PDMS molds (7 mm x 7 mm x 5 mm) and allowed to solidify at 37 °C in a humidified atmosphere.

[0096] Channel contraction in PSC-laden collagen or Col / Fib disks - PSC-laden gels were prepared as aforementioned using a custom-made PDMS mold of a 06mm and 2 mm height. Each disk was punctured using an Accupunch (Electron Microscopy Sciences) of a size of 01mm to achieve a channel in the hydrogel of the same size. The disks were cultured for a duration of 3 days while being imaged on daily basis. The channel size was determined using ImageJ and the contraction was determined by standardizing the observed size for each day to the initial size on day 0. The presence of thrombin and fibrinogen in the PSC-laden collagen / fibrinogen (Col / Fib) hybrid gels led to activation of the (fibroblasts, especially the) pancreatic stellate cells (PSCs), resulting in induced compression (or “contraction”) of the hydrogel. In particular, a 10-fold contraction of the channel relative to the PSC-laden collagen gels (without thrombin and fibrinogen) was observed.

[0097] Generation of vascularized collagen + Col / Fib multi-layered constructs - Multilayered constructs were prepared using a sandwich method. First, a PSC-laden collagen / fibrinogen (single layer) construct was prepared using a custom-made PDMS first mold (Lmi = 8mm, Dmi= 4 mm) including a blunt needle (Dss = 1 mm) in the middle serving as sacrificial structure for the central channel. After initial solidification where the fibrinogen component is already crosslinked while the collagen is still in a semi-solidified state, the PSC- laden Col / Fib gels (i.e., the first layer) including the sacrificial structure were rapidly transferred to a custom-made PDMS second mold (Lm2 = 8mm, Dm2 = 10 mm) before being surrounded by a PSC-laden collagen solution (i.e., second matrix precursor). The multi-layered construct was allowed to fully solidify at 37 °C in a humidified atmosphere. Next, the sacrificial structure was carefully removed from the construct to obtain a final channel of about De = 1 mm. In the meantime, Human umbilical vein endothelial cells (HUVECs) were labelled with CellTracker Orange CMRA (Thermofisher Scientific, 20 pM in FBS-free culture medium for 30 min). Afterwards, labelled HUVECs (5 x 106 cells / mL) were seeded into the channel and allowed to attach for 6 h before not-attached HUVECs were washed out and the multi-layered construct (i.e., the system) was cultured.

[0098] The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

[0099] The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0100] The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

[0101] The term “and / or” especially relates to one or more of the items mentioned before and after “and / or”. For instance, a phrase “item 1 and / or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

[0102] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0103] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0104] The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0105] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

[0106] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0107] Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0108] The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0109] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0110] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

[0111] The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:

1. A system (100) for in vitro modelling of biologically induced compression, wherein the system (100) comprises a channel (110), a first matrix material (121) and a second matrix material (122), wherein one or more of the first matrix material (121) and the second matrix material (122) comprises pancreatic stellate cells (126), wherein the channel (110) comprises cells (116), wherein the first matrix material (121) surrounds a first channel part (111) of the channel (110), and wherein the second matrix material (122) surrounds the first matrix material (121) and a second channel part (112) of the channel (110); wherein the first matrix material (121) and the second matrix material (122) both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl; wherein the first matrix material (121) comprises thrombin and nl g / L fibrinogen, wherein nl is selected from the range of 2 - 10, and wherein the second matrix material (122) comprises n2 g / L fibrinogen, wherein 0<n2<nl.

2. The system (100) according to claim 1, wherein the channel (110) is lined with endothelial cells (117).

3. The system (100) according to any one of the preceding claims, wherein the channel (110) comprises a blood vessel (118).

4. The system (100) according to any one of the preceding claims, wherein the first matrix material (121) comprises a first weight ratio of collagen to fibrinogen selected from the range of 1 :4 to 1 :2, wherein the first matrix material (121) comprises a combined weight ratio of collagen and fibrinogen combined to thrombin selected from the range of 200: 1 to 50: 1, and wherein 0 < n2 < 0.5*nl.

5. The system (100) according to any one of the preceding claims, wherein the system (100) has a diameter (Ds) selected from the range of 2 - 8 mm, and wherein the system (100) has a length (Ls) selected from the range of 4 - 16 mm.

6. The system (100) according to any one of the preceding claims, wherein the channel (110) has a channel diameter (De) selected from the range of 10 pm - 1.5 mm.

7. Use of the system (100) according to any one of the preceding claims for modelling vasculature compression.

8. The use according to claim 7 for simulating a pancreatic tumor.

9. A method for providing a system for in vitro modelling of biologically induced compression, wherein the method comprises: a preparation stage comprising providing a sacrificial structure (10), a first matrix precursor (21) and a second matrix precursor (22), wherein the first matrix precursor (21) and the second matrix precursor (22) both comprise a material independently selected from the group comprising collagen, gelatin and gelatin methacryloyl, and wherein the first matrix precursor (21) comprises thrombin and nl g / L fibrinogen, and wherein the second matrix precursor (22) comprises n2 g / L fibrinogen, wherein 0<n2<nl, and wherein one or more of the first matrix precursor (21) and the second matrix precursor (22) comprises pancreatic stellate cells (126); a first layer deposition stage comprising (i) providing the first matrix precursor (21) to a first sacrificial part (11) of the sacrificial structure (10), and (ii) solidifying the first matrix precursor (11) to provide a first layer (31) of first matrix material (121); a second layer deposition stage comprising (i) providing the second matrix precursor (22) to the first layer (31) and to a second sacrificial part (12) of the sacrificial structure (10), and (ii) solidifying the second matrix precursor (22) to provide a second layer (32) of second matrix material (122); a separation stage comprising separating the sacrificial structure (10) from the first layer (31) and the second layer (32) to provide a channel (110).

10. The method according to claim 9, wherein the method further comprises: a seeding stage comprising providing cells (116) into the channel (110) to provide a channel lining (115), wherein the cells (116) comprise endothelial cells (117).

11. The method according to any one of the preceding claims 9-10, wherein the first matrix material (121) comprises a first weight ratio of collagen to fibrinogen selected from the range of 1 :4 to 1 :2, wherein the first matrix material (121) comprises a combined weight ratioof collagen and fibrinogen combined to thrombin selected from the range of 200: 1 to 50: 1, and wherein 0 < n2 < 0.5*nl.

12. The method according to any one of the preceding claims 9-11, wherein the sacrificial structure (10) has an axis of elongation (A), wherein along at least 60% of a length of the sacrificial structure along the axis of elongation (A), the sacrificial structure has an equivalent circular diameter (Dss) selected from the range of 10 pm - 1.5 mm.

13. The method according to any one of the preceding claims 9-12, wherein the first layer (31) has a first average thickness (dl) selected from the range of 1 - 10 mm, and wherein the second layer (32) has a second average thickness (d2) selected from the range of 1 - 10 mm.

14. The method according to any one of the preceding claims 9-13, wherein the first layer deposition stage comprises solidifying the first matrix precursor (21) by exposing the first matrix precursor (21) to one or more of a temperature selected from the range of 30° - 60°, a pH selected from the range of 7.35 - 7.45, and a humidity selected from the range of 40-60%.

15. The method according to any one of the preceding claims 9-14, wherein the second layer deposition stage comprises solidifying the second matrix precursor (22) by exposing the second matrix precursor (22) to one or more of: a temperature selected from the range of 30° - 60°, a pH selected from the range of 7.35 - 7.45, and a humidity selected from the range of 40-60%.

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