Milli-scale vessel-like structure, method and device

Abstract

It forms an object of the present invention a hollowed scaffold and a method to obtain the same, said hollowed scaffold being a milli-scale vessel-like structure comprising a microenvironment, a lumen, and a vessel wall, said microenvironment and said vessel wall being made, independently the one from the other, of a matrix, wherein in said matrix constituting said microenvironment stromal cells are embedded, and wherein in said matrix constituting said vessel walls endothelial cells are embedded. Additional objects of the present invention are a bioreactor comprising the hollowed scaffold and method to study cells in tissue-like vascularised constructs.

Description

[0001] "MILLI-SCALE VESSEL-LIKE STRUCTURE, METHOD AND DEVICE"

[0002] BACKGROUND

[0003] Among the challenges of tissue engineering, vascularization is one of the most critical, being fundamental to establish complex and long-term reliable three- dimensional models. However, current manufacturing technologies for vessels structure reproduction still lacks standardization to enable a wider adoption of such systems and a deeper characterization of different pathophysiological conditions.

[0004] Extrusion-based bioprinting (EBB) has been evaluated, as an example in Ryma M et al. Adv. Mater. 2022, 34, 2200653, but failed in generating a mechanically stable 3D structure, causing lumen collapse during or soon after printing, even when using sacrificial materials as Pluronics F-127 or alternative printing orientation strategies.

[0005] Casting has been tested, too. As an example, Li X et al. Pios One 2015, 10(5): e0126797 and, for a summary, Miri AK et al. Biomaterials 2019, 198:204-216. However, the generated channels have a diameter of about 100 jim or less. Difficulties arise when facing with larger size.

[0006] Gensler et al. 2020 https: / / doi.org / 10.1371 / journal.pone.0242615 describe a bioreactor in which the lumen geometry and shape of the hosted are heavily compromised upon perfusion.

[0007] WO2018237132A1 describes a hollow scaffold to investigate cells extravasation. However, the method is very strict in term of geometry and dimension of the obtained channels, therefore does not allow to study turbulent flow. Moreover, the physiological vascular system is not reproduced, wherein endothelial cells are seeded within the channel, creating a monolayer which is then exposed to flux.

[0008] US2023357709A1 discloses a scaffold suitable for culture of vascular tissue, comprising an internal lumen.

[0009] US2023348830 discloses a device suitable to receive and to allow culture on a cylindrical scaffold.

[0010] There is the need for hollowed scaffolds capable to reproduce the physiological microenvironment with vessel wall and lumen (tuneable in thickness, diameter, and length) which is stable over time and methods for the study of circulating cells extravasation / trafficking in the microenvironment compartment of said scaffolds, suitable for studying tumor cells, too.

[0011] DESCRIPTION

[0012] In a first embodiment, it is here claimed a hollowed scaffold which is a milli-scale vessel-like structure. In a second embodiment, it is here claimed a method to obtain in vitro a vascularised tissue model.

[0013] In additional embodiments, a device to house said hollowed scaffold and a bioreactor comprising said device and said hollowed scaffold are claimed. The device is designed to easily interchange its components, allowing the housing of vessels adjustable in length and diameter. Said device hosts hollowed scaffolds with variable hollow cavities, as casted or bioprinted ones. In an embodiment, the bioreactor comprises a double optical access, to allow live-imaging analysis of the cells hosted into the same.

[0014] In a further embodiment, it is here claimed a method to study cells in tissue-like vascularized environment.

[0015] DRAWINGS

[0016] Figure 1: An embodiment of the device. (A) perspective view; (B) exploded view; (C) front sectional view.

[0017] Figure 2: An embodiment of the adapter, perspective view.

[0018] Figure 3: An embodiment of the pin, perspective view.

[0019] Figure 4: An embodiment of the scaffold, schematic (A) top view and (B) perspective view.

[0020] Figure 5: An embodiment of the bioreactor. (A) perspective cross sectional schematic view. (B) representative picture of the bioreactor in operating conditions.

[0021] Figure 6: Generation of a hollowed scaffold according to the method of the present invention: (A) a plastic rod (pin) is inserted in the hydrogel which is then photo crosslinked; (B) the plastic rod is removed, thus generating the hollow channel.

[0022] Figure 7: 3D bio printed Primary Lymphatic Fibroblasts, Normal, Human cell line (HLF) shows sustained viability up to 28 days. Live&Dead assay on HLF 28 days after printing in VitroINK RGD (A) and GelXA LAMININK411 (B). Images were acquired with AXIO zl Observer (A) and Olympus FluoVIEW 3000 RS confocal microscope (B).

[0023] Figure 8: Exemplificative pictures of vessel lumen (A) and vessel wall (B), top view, with embedded HUVEC and HLF stained for phalloidin and Hoechst 33342 displaying homogeneous distribution 7 days post-manufacturing. Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope.

[0024] Figure 9: Medium delivery within the hollowed scaffold housed in the device. Gradually increasing color intensity after 0 (A), 60 minutes (B) and O / N perfusion (C).

[0025] Figure 10: Embedded endothelial cells and fibroblasts show sustained viability up to 14 days post-casting. Live&Dead assay on HUVEC and HLF after 7 (A) and 14 (B) days of static culture in a multiwell plate. At both time points, cells display a homogeneous distribution and sustained viability throughout the entire hollowed scaffold. Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope.

[0026] Figure 11: Vessel maturation assessment after 14 days of static culture. Hybrid hollowed scaffolds maintain a defined vessel lumen up to 14 days of static culture, together with a high cell density. Selective endothelial marker CD31 (short arrow) and collagen IV production (long arrow) is illustrated in the top view at 4X and 10X magnification (A, B). VE-cadherin (long arrow) and vWF (short arrow) expression is visualized from the bottom view of the vessel lumen at 4X and 20X magnification (C, D). Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope.

[0027] Figure 12: Cell distribution and vascular morphology in mature vessels after 3 and 7 days of dynamic perfusion. Immunofluorescence staining of hybrid hollowed scaffolds perfused for 3 and 7 days at lOOpL / min after 14 days of maturation in static culture. Bottom view of the vessel wall shows a tight layer of CD31+ cells (A), together with a high cell density in the surrounding microenvironment (B) 3 days post-perfusion. Phalloidin staining (C) of the vascularized scaffold displays homogeneous cell distribution in the whole construct, together with CD31 localization at lumen wall, as well as in the microenvironment (D) 3 days post-perfusion. Top view of the scaffold 7 days post-perfusion shows undamaged lumen cavity and dense cell network both in the lumen wall (short arrow) and in the microenvironment (long arrow) (E). Magnification of microenvironmental cells confirms proper cell morphology and distribution over -time (F). Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope.

[0028] Figure 13: (A) An embodiment of the lid, front sectional view. (B) An embodiment of the adapter, front sectional view.

[0029] Figure 14: MEC1-GFP CLL cells extravasate from the circulating medium to the microenvironment compartment (A) and show modulation of CD38 (B) and CD49d (C).

[0030] Figure 15: CLL primary cells that circulate within the system are allowed to extravasate and show modulation of CXCR4 (A), involved in cell trafficking, and CD23 (B).

[0031] Figure 16: MEC-GFP CLL cells are able to exit the scaffold and enter the circulation when exposed to a chemo attracting agent, which in this case is represented by CCL19.

[0032] DETAILED DESCRIPTION

[0033] In a first embodiment, and with reference to Figure 4, it is here claimed a hollowed scaffold which is a milli-scale vessel-like structure. In the context of the present application, milli-scale means having cross-sectional dimensions ranging from about 1 mm to 5 mm, preferably 1.5 mm - 3 mm, or about 2 mm, or about 2.5 mm. Said hollowed scaffold comprises a microenvironment 4, a lumen 1, and a vessel wall 3, said microenvironment 4 and said vessel wall 3 being made, independently the one from the other, of a matrix, wherein in said matrix constituting said microenvironment stromal cells are embedded, and wherein in said matrix constituting said vessel walls endothelial cells are embedded.

[0034] In an embodiment, said matrix constituting said microenvironment is made by a material selected into the group comprising: gelatine, alginate or other natural polymers, synthetic polymers, or combinations of them.

[0035] Said cells embedded in said microenvironment are at least one cell population selected in the group comprising Lymphatic Fibroblasts (LF), Bone Marrow Stromal Cells (BMSC), Mesenchymal Stem Cells (MSC), neurons and glial cells, Pancreatic Stellate Cells (PSC), Hematopoietic Stem Cells (HSC), hepatocytes, preferably said cells are human cells, preferably are humanLF.

[0036] In an embodiment, said matrix constituting said vessel walls is made of a material selected into the group comprising: chemically, physically or thermally cross linkable materials, which may contain matrix proteins (e.g., collagen, laminins) or be supplemented with pro-angiogenic factors such as epidermal growth factor EGF, platelet-derived growth factor PDGF, nerve growth factor NGF, basic fibroblast growth factor FGF-2, transforming growth factor-beta TGF-beta and insulin-like growth factor IGF.

[0037] Said cells embedded in said vessel walls are at least one cell population selected in the group comprising Human Umbilical Vein Endothelial Cells (HUVEC), Smooth Muscle Cells (SMC), pericytes, Microvascular Endothelial Cells, Brain Microvascular Endothelial Cells (BMEC), Lymphatic Endothelial Cells (LEC), LF, preferably said cells are human cells, preferably are HUVEC and HLF. In an embodiment, said vessel walls 3 comprises HUVEC and HLF cells in a ratio comprised between 2:1 and 4:1, preferably 3:1. In an embodiment, said vessel walls 3 comprises HUVEC and SMC cells in a ratio comprised between 2:1 and 4:1, preferably 3:1.

[0038] In an embodiment, said cells are embedded in said matrices in an amount comprised between 10xl05- 10xl07, preferably about 10xl06cells / ml.

[0039] In an embodiment, said microenvironment further comprises tumoral cells, preferably selected into the group comprising: Chronic lymphocytic leukaemia (CLL) cells, neuroendocrine tumor cells.

[0040] In an embodiment, said lumen has a diameter comprised in the range 1 - 5 mm, preferably about 2 mm, 2.5 mm and it has a smooth surface or a threaded surface.

[0041] In an embodiment, said vessel walls have a thickness comprised in the range 500 jim - 3 mm, preferably about 500 m or about 1 mm.

[0042] In an embodiment, said vessel walls are a monolayer. Alternatively, said vessel walls are multilayer. In an embodiment, said vessel walls consist of three different layers, each one comprising a different cell population, wherein each one of said three layers has a thickness of about 333 pm.

[0043] In an embodiment, said vessel walls have a stiffness which is different from the stiffness of said microenvironment. As an example, said different stiffness is obtained by using different materials with peculiar physical composition (intrinsic stiffness) or exposing photosensitive biomaterials (i.e., GelMA Fibrin) to UV light for variable time.

[0044] It forms a further object of the present invention a method to obtain a vascularised tissue model in vitro. Said method is a hybrid method, wherein hybrid method in the present application means a two steps method, comprising extrusion based bioprinting and casting.

[0045] The method to obtain a milli-scale vessel-like structure, which is a hollowed scaffold 5 comprising a microenvironment 4 a lumen 1 and vessel walls 3, comprises:

[0046] - Making available at least one stromal cell population and at least one matrix;

[0047] - Via extrusion-based bioprinting (EBB), embedding said at least one stromal cell population in said matrix, obtaining a solid figure comprising a cylindrical hole, said solid figure being the microenvironment 4;

[0048] - Casting in said cylindrical hole a liquid cross linkable biomaterial pre-mixed with at least one endothelial cell population;

[0049] - Inserting a pin in said cylindrical hole;

[0050] Crosslinking said biomaterial, thus obtaining the lumen walls 3;

[0051] - Removing said pin, thus generating the lumen 1.

[0052] In an embodiment, wherein said vessel walls are a multilayer, said steps iii) -vi) are repeated as many times as there are desired layers, each time using a pin having a central body with a smaller diameter.

[0053] In step ii), cells are directly embedded in the matrix in a spatially controlled geometry (parallelepiped with a central cylindrical hole), by way of EBB.

[0054] In step iii), a liquid chemically, physically, or thermally cross linkable material premixed with the cells of interest, as an example a photocrosslinkable material, is casted in the hole of the above-described scaffold. Immediately, the pin 15 is placed in the centre of the structure, generating a hole which is the vessel lumen, after biomaterial photo crosslinking and pin removal.

[0055] As an example, Figure 6A is a representative picture of said second step, wherein the pin is inserted into the hole of the scaffold, where the liquid chemically, physically, or thermally cross linkable material pre-mixed with the cells was added. Figure 6B is a representative picture of the final hollowed scaffold, having a central cylindrical hole which is the vessel lumen.

[0056] With reference to Figure 3, said pin 15 comprises a central body 9 and peripheral legs 8. Said central body 9 is cylindrical, to allow the formation of a hollowed scaffolds. Said legs, three in the representative embodiment of Figure 3, allow the pin to stand. In an embodiment (not shown) said body comprises a thread.

[0057] The hollowed scaffold obtained according to the here described method is claimed, too.

[0058] In a further embodiment, and with reference to Figure 1, it is here described a device 10 to house a hollowed scaffold and to study stromal and immune cells homing, migration, and transmigration processes in a tissue-like vascularized construct.

[0059] Said device 10, according to a preferred embodiment, depicted in Figure 1, comprises: a base 12, a lid 11, at least two adapters 13.

[0060] Said base 12 is bottom closed and top open, wherein said lid 11, when positioned on the top of said base, closes the same. Said lid seals said base, in one embodiment it is screwed onto said base. In one embodiment, it is interlocking.

[0061] Said lid 11 comprises a central hole 16, wherein said central hole 16 is closed by a glass coverslip.

[0062] In an embodiment, said base is cylindrical.

[0063] Said homing module 14, when in function, houses a scaffold.

[0064] On the lateral wall of said base 12 are comprised two openings 17, diametrically opposite each other. Said openings house the two adapters 13, wherein said adapters are passing tubes, in an embodiment, reference is made to Figure 2, they are silicone tubes, comprising an external portion 20 and an internal portion 21. Said two adapters allows for a sealed connection. As an example, they can be screwed, or connected by way of gaskets, or O-rings.

[0065] Said adapters 13, once inserted through said openings 17 into said base 12, put in fluidic connection the external environment and the homing module 14, wherein said internal portion 21 reaches said homing module 14. The two adapters conveniently and alternatively work as loading or unloading region, to enter fluid into the scaffold once housed in said homing module or to recover the outgoing fluid.

[0066] The length and the diameter of said adapters can be varied, thus regulating the system fluid dynamics. In an embodiment, silicone gaskets are put on the lid and the adapters' seat to ensure sealing and pressure maintenance inside the bioreactor, as well as selective medium convey in luminal cavities. This configuration allows to selectively perfuse vascularized scaffolds.

[0067] Said homing module 14 comprises two cavities 18, diametrically opposite each other. Each one of said two cavities 18 conveniently houses the internal portion 21 of one of said adapters 13. In the centre of said homing module, there is a casing 19 in which the hollowed scaffold is housed, when in operating conditions.

[0068] In an embodiment, with reference to Figure 13A, said lid 110 comprises a curved cavity 111. When screwing down the lid, said curved cavity allow pressure release. In this embodiment, the lid does not have a central hole and it is useful when completely filling the bioreactor chamber, with no selective perfusion. This avoids contaminations, and the use of external reservoirs.

[0069] In an embodiment, with reference to Figure 13B, said adapter 130 comprises two flat plastic lugs 131 at the base, useful to tighten the connectors. Moreover, an integrated luer-lock male connector 132 improves silicone tubes fitting and a slight chamfer 133 on the tip of the connector improves the fit of the lumen-like structure in the vascularized scaffold.

[0070] Said hollowed scaffold to be housed in said device 10 is a prefabricated (e.g., electrospinning, gas foaming), casted (mold), 3D bioprinted or a hybrid-method obtained hollowed scaffold. Said hollowed scaffold comprises at least one support matrix (hydrogels or rigid scaffolds) and the cells of interest.

[0071] In a further embodiment, with reference to Figure 5A for a representative cross- sectional view, it is here claimed a bioreactor comprising:

[0072] - a hollowed scaffold which is a milli-scale vessel-like structure, comprising a microenvironment, a lumen and vessel walls, wherein said microenvironment and said vessel walls are made, independently the one from the other, of at least one matrix comprising at least one cell population;

[0073] - a device comprising: o a base which is bottom closed and top open, comprising, on the lateral wall, two openings, diametrically opposite each other, wherein each one of said openings house one adapter, wherein said adapters are passing tubes; o a homing module housed inside said base; o a lid closing said base, comprising a central hole opened on said homing module; said hollowed scaffold being housed in said homing module comprised in said device.

[0074] In a further embodiment, it is here claimed a method to study cells in tissue-like vascularized constructs, wherein said method comprises:

[0075] - making available a bioreactor according to the present description;

[0076] - perfusing said hollowed scaffold, through tubes connected to said adapters;

[0077] - monitoring cells homing, migration, transmigration processes and response to stimuli or drugs.

[0078] Said perfusion fluid is a cell culture media. In this media, cells could be suspended. As an example, among the cells that are conveniently suspended in the perfusion fluid are tumoral cells, wild-type or engineered immune cells, such as CAR-T cells, and / or stem / progenitor cells, such as mesangioblasts.

[0079] The bioreactor is capable to promote a functional perfusion, as demonstrated by assessing media perfusion within the hollowed scaffold over-time, thus demonstrating that the entire construct and the embedded cells could be gradually reached by the medium. Moreover, the bioreactor is stable, wherein cells remain alive and express CD31, Ve-Cadherin and vWF endothelial-specific markers over time, also producing extracellular matrix, as collagen IV.

[0080] The man skilled in the art know how to monitor cells over-time in this system, wherein the glass coverslip opened on said device allows to evaluate cells via optical microscopy.

[0081] The system allows for a complex co-culture, comprising more than one cellular component, in very defined compartments. As an example, the interactions among a cell population in the environment, a cell population in the vessel walls and a cell population suspended in the perfusion fluid could be tested thank to the instant method. As an example, said bioreactor, when said cells are CLL, is suitable for engineering vasculature in vitro with surrounding leukemic microenvironment.

[0082] In an embodiment, said method it to study the capability of therapeutics to cross the vessel walls. For example, the perfusion is made with a perfusion fluid comprising engineered lymphocytes, such as CAR-T cells, and the method allows to monitor the migration of said therapeutic cells.

[0083] In an embodiment, the method is to study the capability of both tumoral and healthy immune cells to infiltrate. In this case, a perfusion fluid comprising cancer cells, as an example CLL cells, or mesangioblasts is used.

[0084] In an embodiment, wherein the microenvironment of the hollowed scaffold comprises tumor cells, such as CLL, the method is to study the capability of said cells to pass into vessels.

[0085] As an example, primary cells obtained from a subject tumor are used, in the microenvironment and / or in the perfusion media, thus allowing for a personalised medicine.

[0086] In an embodiment, said method comprises varying the T of said bioreactor during time, wherein said variation is obtained via an external heat source, i.e., placing said bioreactor in an incubator, or on a hotplate.

[0087] In an embodiment, useful to mimic a circadian rhythm, said bioreactor allows to adjust the illumination of said scaffold over time, for example by means of LEDs conveniently housed on said lid.

[0088] In an embodiment, said method is performed placing said bioreactor on a plane surface. Alternatively, said bioreactor is inclined at an angle between 0 and 90°C with respect to the plane, to account for the impact of gravity on the perfusion steps.

[0089] Advantages

[0090] Hollowed scaffolds according to the present invention allow to co-culture cells in a milli-scale vessel-like environment. The system has proven to be stable over time, allowing to monitor a complex system in a static, as an example, by placing said hollowed scaffolds in a culture plate comprising culture media, or in a dynamic situation, by housing said hollowed scaffolds in a device according to the present description.

[0091] The combination, in the hybrid method, of the two-step, i.e., EBB and casting, surprisingly allows to precisely control cell distribution in the surrounding microenvironment by 3D bioprinting and generate a defined vessel lumen by casting of pre-mixed endothelial cells with different vessel walls thickness and geometry.

[0092] Advantageously, Gelatine methacryoyl (GelMA) Fibrin casting promoted an extremely precise and stable structure, while VitroINK RGD, a bioink system modified with RGD cell adhesive peptide, and GelXA LAMININK411, a bioink comprising several laminins, showed higher compatibility with HLF cells, which represent the surrounding lymphoid microenvironment in a here presented embodiment.

[0093] Advantageously, in an embodiment, a hollowed scaffold with variable stiffness around the vessel allows to reconstruct the tumor microenvironment more specifically, depending on the pathology to be studied.

[0094] Advantageously, the bioreactor according to the present invention allows to monitor in a system resembling the physiological one, the cells in the microenvironment and in the vessel walls, upon perfusion with a media, optionally comprising actives of interest.

[0095] The man skilled in the art envisages that the above invention will give a more accurate study of in vivo stromal, immune and tumoral cells effects by using the in vitro model for experiments to see the effect of various external factors on same. For example, studying the effect of therapeutics, particularly in the field of tumour therapy, will be greatly assisted by this invention. By way of not limiting example, the assay can be used to determine tumours and tissue penetration.

[0096] A significant advantage of the method according to the present invention is the ability to apply shear forces through perfusion which more accurately represent the in vivo state, whereby physiologically relevant blood pressure can be applied thanks to the fact that said device is capable to work under controlled pressure.

[0097] Moreover, a turbulent flow can be generated, when using the described hollowed scaffolds with coiled luminal cavities. The Authors of the present invention have surprisingly demonstrated that luminal cavities of the hollowed scaffold generated by using pins, when housed in the device according to the present invention, are easily perfused without compromising their geometry and / or integrity.

[0098] EXPERIMENTAL SECTION

[0099] The present invention is described in connection with certain specific embodiments. However, it is understood that the following examples are for exemplificative purpose only, not limiting the scope of protection, wherein the scope of protection of the present invention is defined by the appended claims.

[0100] MATERIALS AND METHODS

[0101] Device fabrication

[0102] The device 10, named VesselBox, was designed with Fusion 360 (Autodesk) and 3D printed (FDM) with a polylactic acid-based biocompatible material. The device has been conceived with a modular configuration, making it possible to easily tune dimensions, diameters, and shapes of the hosted hollowed scaffolds. The VesselBox 10 is made up by a base 12, a screw-down lid 11 with optical access 16, a homing module 14 for scaffolds.

[0103] Cell culture

[0104] MEC1 cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) and was genotyped as following: 10 ng of DNA from MEGI cells was purified with QiAmp DNA Mini Kit (Qiagen, Dusseldorf, Germany) and amplified through PCR with GenePrint ® 10 System (Qiagen, Dusseldorf, Germany) and sold Eurofins Genomics Standard FLA Service to perform genotyping. Data were analysed with DSMZ Online STR Analysis. The identity of the analysed cell line was confirmed. MEC1 cells and GFP-Tagged MEC1 cells (MEC1-GFP) were cultured in RPMI 1640 medium (EuroClone, Pero, Italy) supplemented with 10% (v / v) Foetal Bovine Serum (FBS) and 15 mg / ml Gentamicin (complete RPMI) at 37°C and 5% CO2.

[0105] Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in EGM-2 medium (EuroClone, Pero, Italy) and used between passages 1 and 5. Before plating, standard culture flasks were coated with 1.8% bovine type B serum from bovine skin (Sigma-Aldrich, Missouri, USA) and washed 1 time with PBS.

[0106] Human Lymphatic Fibroblasts (HLF) (ScienceCell, California, USA) cells were cultured in FM medium (ScienceCell, California, USA) and used between passages 3 and 15. Before plating, standard culture flasks were coated with poly-L-lysine (Sigma- Aldrich, Missouri, USA) for Ih, then washed once with PBS.

[0107] Scaffolds preparation and perfusion

[0108] Hosted scaffolds have been generated with a hybrid technique, combining 3D bioprinting and casting methods. Briefly, VitroINK RGD (TheWell Bioscience, New Jersey, USA) or GelXA LAMININK411 (CELLINK AB, Gothenburg, Sweden) were mixed with HLF (10xl06cells / mL) and bioprinted with the BioX bioprinter (CELLINK AB, Gothenburg, Sweden) to generate a 7x7x6 mm3 scaffold, with a central channel having a diameter of 5.5 mm, said diameter being the external diameter of the vessel to be created. GelMA Fibrin (CELLINK AB, Gothenburg, Sweden) was heated up at 37°C in a laboratory bath until complete dissolution, mixed with HUVEC and HLF cells (3:1 ratio, final cell concentration 10xl06cells / mL) and manually casted to fill the hollow cavity mentioned above.

[0109] Immediately, a 3D printed tool (pin diameter 2 or 2.5mm) was placed inside the casted material and photo-crosslinked with 405nM UV light for 2 minutes, tilting the scaffold 180° along the z-axis after 60 seconds (total crosslinking time: 120 seconds).

[0110] By gently removing the tool we generated a 2- or 2.5-mm lumen 1, wherein the cross-linked material creates the vessel walls 3. The scaffolds were placed in EGM-2 medium added of lOU / mL thrombin (Merck, New Jersey, USA) O / N at 37°C, 5% CO2. The following day, the thrombin solution was replaced with fresh medium, and scaffolds kept in static culture (multiwell plate) for 14 days before perfusion with EMG2 + FM medium (3:1 ratio), with medium changes every 3 days. After 14 days of static culture in multiwell plates, the scaffolds were placed in the VesselBox device according to the following steps: the cell-laden scaffold was gently placed in the homing module and moved in the bioreactor base. One adapter was screwed in the base to fit the lumen in the scaffold. Before tightening the second adapter, the lid was screwed down and the circuit filled with EGM-2 + FM medium (3:1 ratio) medium. The flow perfusion (lOOpL / min) was ensured by the R100-1J peristaltic pump (React4Life, Vimodrone, Italy). Simultaneously, fresh medium circulation throughout the culturing period was guaranteed by the presence of an external reservoir filled with lOmL EGM-2 + FM medium (3:1 ratio). Half medium was changed every 3 days. A scaffold in a multiwell plate (static condition) was used as a control for each time-point (day 14, day 17 compared to day 3 dynamic and day 21 compared to day 21 dynamic).

[0111] Perfusion test

[0112] A perfusion test was performed with Trypan blue. Briefly, after O / N culture in a multiwell plate in DMEM (Dulbecco's Modified Eagle Medium, EuroClone, Pero, Italy), we perfused the empty scaffold (no embedded cells) with DMEM (7mL) added of ImL of Trypan blue at 100uL / min with the R100-1J peristaltic pump (React4Life, Vimodrone, Italy). Medium diffusion was assessed after 0 / 30 / 60 minutes and O / N perfusion and demonstrated by the gradually intensified purple colour of the construct.

[0113] Live / Dead assay

[0114] To assess HUVEC and HLF viability after manufacturing and before to perfuse the scaffolds, we used the LIVE / DEAD® Cell Imaging Kit (Thermo Fisher Scientific, Massachusetts, USA), which allows for the visualization of live (green) and dead (red) cells. Briefly, the scaffolds were washed one time (30 minutes) with DMEM without serum (Thermo Fisher Scientific, Massachusetts, USA) and Live / Dead reagent was added in a 1:3 ratio (reagent: DMEM without serum). After Ih of incubation at 37°C, 5% CO2 the constructs were washed one time with DMEM without serum and observed with the Olympus FluoVIEW 3000 RS confocal microscope and then processed using FIJI (ImageJ) software.

[0115] Immunofluorescence (IFs) images

[0116] At defined time-points, cell-laden scaffolds either from static (multiwell) or dynamic culture were fixed with 4% PFA in PBS 2h at room temperature. After fixation, scaffolds were washed twice with HBSS (Euroclone, Pero, Italy) for 5 minutes RT, manually cut in half (or more parts) lengthwise and eventually stained for the markers of interest. Briefly, the two halves were permeabilized (Img / mL BSA, 10% FBS and 0.3% Triton X in PBS) for 30 minutes RT and then stained overnight 4°C with primary antibodies for CD31 (Abeam, Cambridge, UK), VE-Cadherin (Cell Signalling, Massachusetts, USA) diluted 1:100 in blocking solution (Img / mL BSA, 10% FBS in PBS) and vWF (Santa Cruz Biotechnology, Texas, USA) diluted 1:50 in blocking solution. The following day, the scaffolds were washed twice with HBSS (Euroclone, Pero, Italy) for 5 minutes RT and incubated RT for 2h with 488 or 674 AlexaFluor secondary antibodies (Thermo Fisher Scientific, Massachusetts, USA) diluted 1:500 and phalloidin Atto 565 (Sigma-Aldrich, Missouri, USA) diluted 1:100 in blocking solution. After washing 2 times with HBSS (Euroclone, Pero, Italy), Hoechst 33342 in PBS (1:2000) was added for Ih RT. Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope and then processed using FIJI (ImageJ) and Arivis software.

[0117] Live imaging analysis

[0118] To perform live imaging analysis, the VesselBox bioreactor has been specifically modified to obtain a double optical access. After 14 days of static culture in a multiwell plate, 500pL of 70kDa Dextran-FITC (12.5ug / mL in EGM-2 + FM, 3:1 ratio) were manually injected in the vascularized scaffold to assess endothelial barrier integrity. Fluorescent images were acquired with Olympus FluoVIEW 3000 RS confocal microscope after defined time-points post-injection and then processed using FIJI (ImageJ) software. A scaffold without HUVEC and HLF in the GelMA Fibrin compartment was used as control. To prove the whole system is useful to investigate leukemic cells behaviour and migration, MEC-GFP cells (0,6x106 cells / mL) were putin the reservoir, making them able to recirculate for 3 days. After this period, the scaffold was fixed as mentioned above and imaged with Olympus FluoVIEW 3000 RS confocal microscope to define the position of extravasated fluorescent leukemic cells.

[0119] Example 1: 3D bio printed HLF viability assessment and generation of the vessel lumen

[0120] 3D bio printed HLF viability was evaluated up to 28 days after printing in VitroINK RGD and GelXA LAMININK411. Representative images are reported in Figure 7A and B, respectively, where living cells are shown in grey. In both matrices, cells display a homogeneous distribution and sustained viability throughout the entire hollowed scaffold. Embedded cells in GelXA LAMININK411 display a more stretched and natural morphology (B), when compared to VitroINK RGD (A).

[0121] Figure 6 shows 3D bio printed hollow scaffolds with HLF in VitroINK RGD filled by manual casting technique with HUVEC+HLF in GelMA Fibrin. The pin according to the present description is inserted and the scaffold is then photo-crosslinked (A). After gently removing the pin, the hollow channel is generated (B) and can be perfused in the VesselBox device without leakage after vascular maturation. Upon scaffolds generation, cell density and distribution have been monitored over-time by immunofluorescence. Figure 8A, 8B clearly shows embedded cells homogeneously populating the matrix seven days post-manufacturing.

[0122] Example 2: scaffold perfusion in the device according to the present invention

[0123] The system is capable to promote a functional perfusion. This has been tested experimentally by assessing the change in color intensity of the hollowed scaffold over- time, during perfusion with Trypan Blue diluted in PBS to assess medium delivery to more distant regions of the constructs, thus demonstrating that the entire construct and, consequently, the embedded cells, are gradually reached by the medium after overnight perfusion. The bioreactor geometries allow to convey the culture medium to encapsulated cells, even to those lying far from the central vascular flow. Representative images reported in Figure 9 show color diffusion at 0 (A), 60 minutes (B) and after O / N perfusion (C).

[0124] In order to gain more insights in this perspective and reproduce leukemic cells dissemination in the vascular compartment, MEC-GFP cells were manually injected in a mature vascular-laden scaffold (at day 14 post-manufacturing), being then imaged with a tailored support for the VesselBox device live-imaging version. In Figure 8, fluorescent leukemic cells 10 / 15 / 30 minutes post-injection are shown mainly confined in the lumen, while some of them seem to migrate soon after injection in the microenvironment compartment, which may indicate tumor-microenvironment crosstalk and consequent malignant cells recruitment.

[0125] To date, many studies brought to light the central role of flow-induced forces, as wall shear stress, on vascular morphogenesis and remodeling, thus supporting the idea that dynamic perfusion of hybrid vascularized constructs is necessary to reproduce physiologically relevant conditions in vitro, also enabling for a deeper understanding of several pathophysiological processes in more complex models.

[0126] Hence, after vessel maturation in static conditions was confirmed, the constructs were subjected to dynamic flow perfusion in the VesselBox device for 3 and 7 days with flow speed set at 100 jiL / min to test selective medium conveying in the channel without leakage and investigate flow-related effects on cell distribution and vascular morphology. Perfusion parameters and device architecture supported cell viability at both time-points, avoiding critical culture conditions for the cells.

[0127] The extravasation of the tumor cells from the circulating medium to the microenvironment, depicted in Figure 14A, is capable to elicit a modulation of CD38 (Figure 14B) and of CD49d (Figure 14B), two critical markers for cell migration and homing to tissues.

[0128] CLL primary cells circulating within the system are allowed to extravasate and show modulation of CXCR4 (Figure 15A), involved in cell trafficking, and CD23 (Figure 15B), an indicator of cell activation and proliferation.

[0129] The system is capable to show cells intravasation, i.e., the movement of cells from the surrounding scaffold to the circulating medium. MEC-GFP CLL cell line are able to exit the scaffold and enter the circulation when exposed to a chemoattracting agent, which in this case is represented by CCL19 (Figure 16). Moreover, dynamic cultured constructs display a different organization of embedded cells, if compared to static cultured ones.

[0130] In particular, after 3 days of selective perfusion, a tight layer of CD31+ cells can be observed, as shown in Figure 12A. Interestingly, at day 7 post-perfusion, cells laying in close proximity to the lumen are increased in number (Figure 12), if compared to static cultured scaffolds (Figure 11A, B), also showing a dense network infiltration in the microenvironment compartment (Figure 9C), where HLF are present in a consistent number.

[0131] For comparative purpose, HUVEC cells were seeded in casted GelMA Fibrin luminal structure, according to the casting method described by Li X et al. Pios One 2015 (cit). Poor cell adhesion and cell-cell contact were observed, even at high cell density, probably due to the large channel diameter (2 / 2.5mm).

[0132] Example 3: compatibility tests

[0133] Selection of specific cell types for the generation of the vascular barrier and microenvironment compartment was performed carrying out compatibility tests.

[0134] The following materials have been tested with the indicated cell types to engineer the vascular compartment: a. HUVEC cells:

[0135] GelXA Laminink 411, Cellink RGD, Cellink Bioink, Cellink Fibrin, Vitroink RGD, PureCol, GelXA Fibrin, GelMA Fibrin: any one of the tested materials revealed suitable to work with HUVEC cells. The results were advantageous when using GelMA Fibrin. b. HUVEC + hMSC (human Mesenchymal Stem Cells): cells were co-cultured at the ratio 2: 1. For this co-culture, the suitable materials were GelXA Laminink 411 and GelMA Fibrin. c. HUVEC + SMCs (Smooth Muscle Cells): cells were co-cultured at the ratio 3: 1. For this co-culture, the most suitable material was

[0136] GelMA Fibrin.

[0137] Reference numbers:

[0138] 1. vessel lumen or hole

[0139] 3. vessel wall

[0140] 4. microenvironment

[0141] 5. hollowed scaffold

[0142] 6. bioreactor 8. legs

[0143] 9. central body

[0144] 10. device

[0145] 11. lid 12. base

[0146] 13. adapters

[0147] 14. homing module

[0148] 15. pin

[0149] 16. central hole 17. openings

[0150] 18. cavities

[0151] 19. casing

[0152] 20. external portion

[0153] 21. internal portion

Claims

CLAIMS1. A hollowed scaffold (5) which is a milli-scale vessel-like structure comprising a microenvironment (4), a lumen (I ), and a vessel wall (3), said microenvironment (4) and said vessel wall (3) being made, independently the one from the other, of a matrix, wherein in said matrix constituting said microenvironment stromal cells are embedded, and wherein in said matrix constituting said vessel walls (3) endothelial cells are embedded.

2. The hollowed scaffold according to claim 1, wherein said matrix constituting said microenvironment is made by a material selected into the group comprising: gelatine, alginate or other natural polymers, synthetic polymers or combinations of them and said matrix constituting said vessel walls is made of a material selected into the group comprising: chemically, physically or thermally cross linkable materials, which may contain matrix proteins (e.g., collagen, laminins) or be supplemented with pro-angiogenic factors such as epidermal growth factor EGF, platelet-derived growth factor PDGF, nerve growth factor NGF, basic fibroblast growth factor FGF-2, transforming growth factor-beta TGF-beta and insulin-like growth factor IGF.

3. The hollowed scaffold according to claim 1 or 2, wherein said vessel wall (3) has a stiffness which is different from the stiffness of said microenvironment (4).

4. The hollowed scaffold according to any of the claims 1-3, wherein said cells embedded in said microenvironment are at least one cell population selected in the group comprising Lymphatic Fibroblasts (LF), Bone Marrow Stromal Cells (BMSC), Mesenchymal Stem Cells (MSC), neurons and glial cells, Pancreatic Stellate Cells (PSC), Hematopoietic Stem Cells (HSC), hepatocytes, preferably said cells are human cells, preferably are humanLF (hLF).

5. The hollowed scaffold according to any of the claims 1-4, wherein said cells embedded in said vessel walls are at least one cell population selected in the group comprising Human Umbilical Vein Endothelial Cells (HUVEC), Smooth Muscle Cells (SMC), pericytes, Microvascular Endothelial Cells, Brain Microvascular Endothelial Cells (BMEC), Lymphatic Endothelial Cells (LEC), preferably said cells are human cells, preferably are HUVEC and hLF, preferably HUVEC and hLF cells in a ratio comprised between 2:1 and 4:1, preferably 3:1, more preferable areHUVEC and SMC cells in a ratio comprised between 2:1 and 4:1, preferably 3:1.

6. The hollowed scaffold according to one of claims 1-5, said cells being embedded in said matrices in an amount comprised between 10xl05- 10xl07, preferably about 10xl06cells / ml.

7. The hollowed scaffold according to one of claims 1-6, wherein said microenvironment further comprises tumoral cells, preferably selected into the group comprising: CLL cells, neuroendocrine tumor cells.

8. The hollowed scaffold according to any one of the claims 1-7, wherein said lumen has a diameter comprised in the range 1 - 5 mm, preferably about 2 mm, 2.5 mm and said vessel walls have a thickness comprised in the range 500 gm - 3 mm, preferably about 500 gm or about 1 mm.

9. A method to obtain a hollowed scaffold (5) which is a milli-scale vessel-tike structure comprising a microenvironment (4), a lumen (1) and vessel walls (3), the method comprising:Making available at least one stromal cell population and at least a matrix;- Via extrusion-based bioprinting (EBB), embedding said at least one stromal cell population in said matrix, obtaining a solid figure comprising a cylindrical hole, said solid figure being the microenvironment (4);- Casting in said cylindrical hole a liquid cross linkable biomaterial pre-mixed with at least one endothelial cell population;Inserting a pin (15) in said cylindrical hole;- Crosslinking said biomaterial, thus obtaining the lumen walls (3);Removing said pin, thus generating the lumen (1).

10. A hollowed scaffold obtained according to the method of claim 9.

11. The hollowed scaffold according to any one of the claims 1-8, 10 housed in a device (10) comprising: a. a base (12) which is bottom closed and top open, comprising, on the lateral wall, two openings (17), diametrically opposite each other, wherein each one of said openings (17) house one adapter (13), wherein said adapters are passing tubes; b. a homing module (14) housed inside said base (12); c. a lid (11) closing said base, comprising a central hole (16) opened on saidhoming module (14).

12. A device (10) to house a hollowed scaffold and to study cells in tissue-like vascularized constructs, wherein said device comprises: a. a base (12) which is bottom closed and top open, comprising, on the lateral wall, two openings (17), diametrically opposite each other, wherein each one of said openings (17) house one adapter (13), wherein said adapters are passing tubes; b. a homing module (14) housed inside said base (12); c. a lid (11) closing said base, comprising a central hole (16) opened on said homing module (14).

13. The device according to claim 12, wherein said homing module (14) comprises two cavities (18), diametrically opposite each other, and each one of said two cavities (18) conveniently houses the internal portion (21) of one of said adapters (13).

14. A bioreactor comprising: a hollowed scaffold (5) according to one of the claims 1-8, 10; a device (10) according to one of the claims 12, 13, said hollowed scaffold being housed in said homing module (14) comprised in said device (10).

15. Use of the bioreactor according to claim 14, to study cells in tissue-like vascularized constructs.

16. A method to study cells in tissue-like vascularized constructs, wherein said method comprises:- making available a bioreactor which is a hollowed scaffold housed in a device, according to claim 11;- perfusing said hollowed scaffold;- monitoring cells homing, migration, transmigration processes and response to stimuli or drugs.

17. The method according to claim 16, wherein said perfusion is made with a perfusion fluid comprising tumoral cells, wild-type or engineered immune cells, such as CAR-T cells, and / or stem / progenitor cells, such as mesangio blasts.

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