Controlled perfusion of a vascularized microenvironment within a 3D printed bioreactor to study leukemia cells trafficking ex-vivo

Controlled perfusion of a vascularized microenvironment within a 3D printed bioreactor to study leukemia cells trafficking ex-vivo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Controlled perfusion of a vascularized microenvironment within a 3D printed bioreactor to study leukemia cells trafficking ex-vivo Riccardo Pinos, Margherita Pauri, Federica Barbaglio, Giulia Maria Di Gravina, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6503832/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in Western countries, marked by the accumulation of CD5 + B cells in blood and lymphoid tissues. The vascular system plays a critical role, as malignant cells interact with endothelial cells through poorly understood mechanisms. To study CLL dissemination, we developed VesselBox, a modular bioreactor for selective perfusion of a milli-scale vessel-like structure within a lymphoid microenvironment scaffold, created using 3D bioprinting and casting. Numerical simulations optimized perfusion parameters for cell homeostasis. Vessel maturation, confirmed by endothelial markers (CD31, Ve-cadherin, Von Willebrand Factor, collagen IV), showed VesselBox sustains perfusion for up to 7 days. By recirculating CLL cells, we validated its use for studying extravasation and immunophenotype characterization. This pioneering device enables ex vivo analysis of CLL dissemination, offering potential to uncover new therapeutic targets by examining circulating and extravasated cells with or without drugs. Biological sciences/Cancer/Cancer microenvironment Biological sciences/Biological techniques/Biological models/Cancer models Biological sciences/Immunology/Lymphocytes/B cells Biological sciences/Cancer/Haematological cancer/Leukaemia/Chronic lymphocytic leukaemia Chronic Lymphocytic Leukemia bioprinting trafficking vascularization bioreactor microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 INTRODUCTION Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. The trafficking of malignant B cells (CD19 + /CD5 + ) between peripheral blood (PB), bone marrow, and secondary lymphoid tissues is pivotal for their survival, proliferation, 1 and response/resistance to therapies. 2 , 3 Although the introduction of novel targeted therapies (e.g., BTK and BCL2 inhibitors), 4 , 5 , 6 has revolutionized patient management and significantly improved outcomes, patients experience treatment resistance and the disease remains incurable. CLL is a dynamic and heterogeneous disease that progresses thanks to an ill-defined cross-talk with a supportive microenvironment, that involves a variety of interactions including those with endothelial cells (ECs) present in the blood vessels and into the lymphoid tissues. 1 , 7 Accordingly, some studies suggest that the interaction between endothelial cells and CLL cells through the CD31/CD38 engagement supports the proliferation and survival of leukemia cells. This interaction likely occurs in peripheral tissues (e.g., lymph nodes), where malignant B cells can maintain cellular contacts for prolonged periods. 8 , 9 In addition to the direct interaction between CD31 and CD38, soluble factors such as Chromogranin A have been shown to regulate the trafficking of CLL cells. Chromogranin A has been associated with increased vascular barrier function and integrity while also playing a central role in migration of leukemic cells. 10 In recent years, there has been a shift from traditional 2D culture to more reliable 3D models to gain insights into the onset, progression and resistance to treatment in CLL. To recapitulate the impact of the microenvironment in CLL, a scaffold-based approach 11 has been successfully used to study patient-specific responses to a BTK-inhibitor in a bioreactor under microgravity conditions, in the presence of bone-marrow stromal cells. 12 Building on this study and given the limited viability of primary CLL cells in vitro , we exploited 3D bioprinting to establish a 3D long-term model for CLL cells. Our results demonstrate that CLL cells can survive up to 28 days in 3D conditions compared to those cultured in 2D. This increased viability can be partially explained by changes in gene expression in pro- and anti-apoptotic genes such as BAX and BCL2, among others. 13 Remarkably, these models have great potential to help dissect, to some extent, driver mechanisms underlying CLL. However, it is clear that the endothelial compartment must be present during the trafficking process to accurately mimic CLL dynamics in vitro . To date, only one study has recapitulated the trafficking process of malignant B cells in vitro , showing modulation of surface markers upon extravasation. However, this model lacked the tissue microenvironment that provides several biochemical cues to tumor cells. 14 In the present work, we designed, developed, and manufactured a milli-fluidic system capable of perfusing vascularized constructs embedded with the leukemia microenvironment. In this setting, we can monitor CLL cells extravasation and characterize their immunophenotype over-time, paving the way for advanced dynamic in vitro models for CLL, which can also be translated to other pathophysiological conditions. RESULTS Bioreactor fabrication The first step towards the reconstruction of a complex and advanced in vitro dynamic system to study leukemic cells trafficking was the design and manufacturing of a brand-new bioreactor: the VesselBox. Specifically, the device has been conceived with a modular configuration, making it possible to easily tune dimensions, diameters, shapes of the hosted scaffolds and embedded vascular channels. The VesselBox is made up by a base, a screw-down lid with optical access, a homing module for prefabricated, casted, 3D bioprinted or hybrid scaffolds and two adapters equipped with silicone tubes acting as medium inlet and outlet ( Fig. 1 A, B ) . Silicon gaskets have been put on the screw-down lid and the adapters’ seats to ensure sealing and pressure maintenance inside the bioreactor, as well as selective medium convey in luminal cavities. All the components were 3D printed with a FDM 3D printer, upon accurate selection of a material that allowed us to sterilize all the components before to culture our scaffolds and to reuse them for at least three times after autoclave sterilization. Since 3D printed parts are known to be subjected to different drawbacks, 15 especially due to its layer-by-layer fabrication process, we carefully tuned all the parameters in the slicing step, such as infill density and line directions, number of printed perimeters, top and bottom layers, in order to avoid medium leakage during perfusion and components delamination after sterilization. In the next sections, we will deepen the vascularized scaffolds fabrication process to be hosted within the VesselBox device, as well as the computational support throughout the design and development of the bioreactor. Establishment of a hybrid method to engineer vessels surrounded by a lymphoid microenvironment in vitro Among the strategies to obtain the milli-scale vessel-like structure, we set up and selected a hybrid technique combining extrusion-based bioprinting (EBB) and casting methods. In particular, EBB alone failed in generating a mechanically stable 3D structure, causing lumen collapse during or soon after printing, even when using sacrificial materials such as Pluronics F-127 16 or different printing orientation strategies. Moreover, bioinks that we previously tested to engineer the whole structure by EBB were not supporting cell viability and morphology properly, also giving significant background signal in immunofluorescence imaging (see Supplementary Materials Fig. 1). Conversely, GelMA Fibrin casting promoted an extremely precise and stable structure for HUVEC cells, while VitroINK RGD and GelXA LAMININK 411 – that has been selected as most suitable material - showed higher compatibility with HLF cells, which represent the surrounding lymphoid microenvironment in our 3D model ( Fig. 2 A, B ) . HUVECs seeding in casted GelMA Fibrin luminal structure resulted in poor cell adhesion and cell-cell contact even at high cell density and different incubation/rotation time, probably due to the large channel diameter (2/2.5mm) (see Supplementary Materials Fig. 2) . For this reason, the combination of a two-step hybrid method has been chosen, relying on the possibility to precisely control cell distribution in the surrounding microenvironment by 3D bioprinting, concurrently generating a defined central lumen by casting of pre-mixed HUVEC and HLF cells in GelMA Fibrin. Briefly, we first 3D bioprint HLFs in the microenvironment compartment leaving the hollow cavity in vertical position ( Fig. 3 A ) , which is then filled with the endothelial cells and fibroblasts mix ( Fig. 3 B ) . Immediately, a self-standing plastic rod is inserted to create space for the vascular lumen ( Fig. 3 C ) , then gently removed after photocrosslinking ( Fig. 3 D ) . The scaffold is placed within the VesselBox ( Fig. 3 E ) ., which is then connected to a peristaltic pump to selectively perfuse the biofabricated scaffold ( Fig. 3 F, G ) . Increasing cell concentration and different HUVEC:HLF ratio have been tested to optimize the vessel wall formation, concluding that 3:1 ratio with a final cell concentration of 10x10 6 cells/mL were the most appropriate settings for the lumen and 10x10 6 cells/mL (HLFs only) for the surrounding tumor environment (data not shown). Upon scaffold generation, cell density and distribution have been monitored over-time by immunofluorescence, which clearly showed embedded cells homogeneously populating the matrix already 1 day post-manufacturing ( Fig. 4 A ) , as well as after 14 days ( Fig. 4 B ) of static culture. Moreover, resident cells displayed a more stretched morphology and dense network formation at later time-points (14 days, Fig. 4 D), if compared to day 1 ( Fig. 4 C ) , suggesting an increasing adaptation and distribution in a time-related manner. Numerical simulations and perfusion tests allowed to demonstrate selective perfusion of vascularized hybrid scaffolds As support in the VesselBox design, a priori evaluations were performed to assess its suitability for 3D cell culture before carrying out experimental tests requiring time and costs. Particularly, shear stress and oxygen concentration were the two main parameters of interest, experimentally difficult to measure, but essential in the view of the correct functioning of the bioreactor. 17 As a first step, we carried out a parametric study in which different geometries and inflows were considered (see Supplementary Materials Section 1) . Then, computational analyses were performed by focusing on the prototype that was selected and printed for testing the performance of the device from a biological point of view, characterized by a diameter and length channel of 2 mm and 6 mm, respectively, a scaffold equal to 7x7x6mm, and perfused by a flow rate equal to 100 or 500 \(\:uL/min\) . Particularly, simulations consisting in coupling fluid-dynamics equations with mass transport equations were performed to estimate the two target parameters, namely shear stress and oxygen concentration. The line plot of shear stress, reported in Fig. 5 A, shows a constant trend (with a value equal to 0.0165 and 0.08 dyn/cm 2 for 100 and 500 \(\:\mu\:\text{L}\) /min, respectively) at the level of the wall channel, providing in this way a homogenous stimulation to endothelial cells here located. Although the wall shear stress is lower than one experienced by endothelial cells in vivo , it is coherent with the values found in literature. 18 Moreover, shear stress rapidly reduced to values close to zero on the interior part of the scaffold as the fluid has difficulty to flow inside due to the porous nature of the hydrogel, thus indicating a shielding effect of the hydrogel matrix for the embedded cells. Then, the numerical evaluation allowed assessing the effect of the design of the milli-fluidic device for oxygen consumption. In the simulation, two different cell types were considered: endothelial cells forming a monolayer around the bioreactor channel and fibroblasts inside the construct. We assumed 0.04mM as a critical value under which cells are considered to be no more functional in human tissues/organs. 19 As illustrated in Fig. 5 B, oxygen concentration showed a decreasing trend along the perpendicular direction to the central axis of the channel, varying from a maximum of 0.2 along the wall channel to a minimum of 0.1mol/m 3 at the chamber edge (i.e., h = 3.5mm), creating in this way an oxygen gradient along the perfusion system. However, no oxygen depletion was revealed from the computational results with both the flow rate used, demonstrating in this way the optimal performance of the bioreactor. Finally, it was possible to observe that the flow pattern, represented by velocity streamlines, is able to reach, even if with difficulty, the outermost regions of the scaffold with the value of hydrogel's permeability and porosity considered ( Fig. 5 C ) . Moreover, no flow stagnation was detected. Figure 5 D shows the distribution of the pressure within the perfusion system and low pressure values in the range between 0.25 and 0.038Pa were calculated inside the chamber. Following numerical simulations, we proved experimentally with several perfusion tests without the hosted scaffold at different flow rates, namely 0.5, 1, 5, and 10mL/min, that our system could easily withstand low to high medium speed, and consequently proportional pressure variations inside the chamber, without culture medium leakage, which in our prototypes has been the main cause of contamination. Conversely, the selective perfusion was assessed at lower flow speed (up to 1mL/min), since higher flow rates caused complete disruption of the scaffold. Total volume of medium within the system has been measured over-time to ensure that no leakage was present. Specifically, the ability of the system to promote a functional and selective perfusion was assessed by the change in color intensity of the scaffold after 0 ( Fig. 6 A ) , 60 minutes ( Fig. 6 B ) and overnight perfusion ( Fig. 6 C ) , thus demonstrating that the entire construct (and consequently the embedded cells) could be gradually reached by the medium after overnight perfusion. Indeed, bioreactor geometries allow the conveying of culture medium to encapsulated cells, even to those lying far from the central vascular flow. Embedded cells express functional markers in the VesselBox bioreactor over-time In order to generate a functional and thick 3D model in vitro , it is fundamental to check if, besides scaffold architectural preservation, viability and selective cell markers are maintained and expressed during time. To demonstrate that the entire manufacturing process was fully compatible with embedded cells, further integrating the results shown by Fig. 4 , we assessed cell viability at different time-points with the Live&Dead assay. As illustrated in Fig. 7 , static cultured cells show sustained viability 7 days after manufacturing in the top view, as well as a homogeneous distribution throughout the whole matrix, although they need more time to assume a proper morphology, as illustrated above. Through the two-step hybrid process described in the second section, we managed to reproduce a tightly organized and confluent layer of ECs on the lumen walls after 14 days of static culture, as demonstrated by the expression of CD31 in the lumen wall, together with collagen IV, which is dispersed within the vascular compartment ( Fig. 8 A, B ) . Interestingly, at this time-point, ECs form a continuous and organized network within the same compartment, as shown by Ve-Cadherin and vWF markers expression ( Fig. 8 C, D and Supplementary movie 1) . Importantly, the above-mentioned markers have been associated with fine regulation of vascular maturation and permeability. 20 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. 21 Moreover, dynamic perfusion is pivotal to investigate leukemia cells trafficking ex vivo , which is our goal. Hence, after vessel maturation in static conditions was confirmed, the constructs were exposed to dynamic flow perfusion in the VesselBox device up to 7 days with flow speed set at 100 \(\:uL/min\) to further prove medium transport in the channel without leakage and investigate flow-related effects on cell distribution and vascular morphology before making leukemic cells recirculating within the system. As predicted by numerical simulations, perfusion parameters and device architecture supported cell viability at both time-points, avoiding critical culture conditions for the cells (data not shown). Moreover, dynamic cultured constructs display a different organization of embedded cells, as compared to static cultured ones. In particular, after 3 days of selective perfusion, CD31 + cells are branching towards the microenvironment side and can be observed also in the periphery of the scaffold, as shown in Fig. 8 E. Interestingly, at day 7 post-perfusion, ECs laying in close proximity to the lumen are more elongated and dispersed in the whole matrix ( Fig. 8 F ) , if compared to the previous time-point. Immunophenotype characterization of circulating CLL cells in the engineered leukemia microenvironment As previously discussed, malignant B cells trafficking between blood, bone marrow and secondary lymphoid tissues is pivotal for the progression of the disease and resistance to therapy. In order to gain more insights in this perspective and mirror the leukemic cells dissemination across the vascular compartment, the CLL cell line MEC1-GFP or primary CLL cells were allowed to recirculate in mature vascular-laden scaffold (at day 14 post-manufacturing), being then retrieved for immunophenotype characterization after 1, 3 and 7 days of dynamic culture. These cells were compared with the same cells circulating in the empty bioreactor, i.e. without the vascularized construct and the microenvironment, in order to discriminate between flow-induced and microenvironment-induced changes on surface markers. Interestingly, MEC1-GFP cells can be visualized after circulation and extravasation by confocal imaging ( Fig. 9 A ) and show modulation of critical surface markers for activation and homing processes. Importantly, CD38, which may contribute to CLL cells extravasation and homing, 9 is increased from day 1 to day 7 of circulation in the presence of the microenvironment ( Fig. 9 B ) . Regarding CD49d, 22 the subunit of VLA-4 complex involved in CLL cells homing in the lymph nodes, we could determine a significantly increased expression at day 7 post-circulation in the presence of the microenvironment ( Fig. 9 C ) . Imaging analyses on selectively perfused scaffolds with primary cells show their extravasation in the vascular compartment already after 3 days of circulation at ( Fig. 10 D ) . In order to assess the potential mechanisms underlying the process of extravasation of primary cells (n = 3), we evaluated by flow cytometry the expression of CXCR4 and CD5 on primary CLL cells circulating in the bioreactor with the scaffold, whose levels are known to be associated with extravasation and homing of CLL cells. 23 Furthermore, we compared those levels to the circulating cells in the empty bioreactor, from now on defined as circulation without the scaffold, and to the basal level measured on the cells freshly isolated from the peripheral blood or just thawed. After 1, 3 and 7 days of circulation in the bioreactor, we detected the presence of a CXCR4 high /CD5 high cell population, which has been described as more prone to tissue homing. Interestingly this population is absent at basal level and in the dynamic setting without the scaffold/microenvironment ( Fig. 10 A ) . This result is indicative that a cross-talk is occurring between the microenvironment and CLL cells while trafficking between the different compartments as demonstrated in vivo. 24 , 25 Moreover, we investigated the expression of CD62L, since this marker is involved in homing and migration of lymphocytes to lymphoid organs and is known for its essential role in controlling the extravasation of CLL cells through the High Endothelial Venules (HEVs) in the lymph nodes. We observed upregulation in both surface and gene expression in presence of the scaffold, indicating a potential interaction between leukemic and endothelial cells ( Fig. 10 B ). Lastly, patient cells also show an increased expression of the CD23 high /CD5 high population 26 at day 1 and day 3 post-circulation in the VesselBox in the presence of the scaffold, as compared to the same dynamic settings without the scaffold ( Fig. 10 C ) . CD23 is amongst the surface markers used for CLL diagnosis 27 and it is involved in normal and CLL B cells activation and growth. 28 All the markers have also been evaluated by RT-PCR. Interestingly, CXCR4 and CD62L gene levels mirror flow cytometry analyses, while CD23 gene expression seems not to follow protein levels which may indicate a different regulatory mechanism that is time-dependent (see Supplementary Materials Fig. 3) DISCUSSION In the present work, we developed a brand-new device, together with a novel method to engineer complex 3D vascularized scaffolds, enabling the investigation of leukemic cells trafficking in a relevant ex vivo environment. Dynamic culture systems in biomedical research often refer to microfluidic chips, which are typically used to ensure fluid perfusion for seeded cells inside hollow cavities. Due to their affordability and versatility, microfluidic chips have been adopted over the years for various purposes, such as studying shear stress effects on endothelial monolayers, 29 reconstructing the blood-brain-barrier (BBB) to gain more insights into neurovascular disorders, 30 and investigating cancer biology and responses to drugs. 31 However, these promising tools are usually manufactured with inert materials, such as PDMS, which makes it impossible to study cell extravasation and cross-talk with stromal compartments. 32 These processes are critical in several diseases, especially in hematological cancers like CLL. In this regard, we previously demonstrated that both CLL cell lines and primary CLL cells can survive for prolonged period in 3D bioprinted constructs, also showing molecular fingerprints of adaptation to the tissue-like structure. 13 On top of that, we increased the complexity of the system by introducing the vascular component into the scaffold through a hybrid manufacturing method. This allows for a deeper investigation of leukemic cells trafficking in peripheral blood-like settings (circulating medium) and secondary lymphoid tissues-like settings (3D bioprinted microenvironment). To facilitate this, we designed and produced a novel bioreactor, the VesselBox, which enables the selective perfusion of these scaffolds using a peristaltic pump. With CFD analysis supporting the entire development stage of the device, we demonstrated that the VesselBox ensures nutrients delivery throughout the entire matrix while simultaneously supporting cell survival and proper morphology within the hosted scaffold. Consequently, embedded endothelial cells maintain the expression of selective markers such as CD31, Ve-cadherin, VWF and collagen IV, with CD31 being highly represented at the periphery of dynamically cultured scaffolds after 7 days. This model was then used to assess MEC1-GFP and primary patients cells’ extravasation over-time while also allowing for their immunophenotype characterization. According to available studies, 24 , 25 in vivo monitoring of CLL cells trafficking shows different surface patterns on their homing to tissues. CXCR4 has been described as fundamental for homing leukemic cells within lymphoid tissues. 23 Our results indicate that circulating primary cells in the bioreactor with the scaffold - and thus with the microenvironment - lead to the emergence of a double positive cell population expressing CXCR4 high /CD5 high . Remarkably, CXCR4 high /CD5 high cells have recently been identified as actively proliferating cells based on experiments conducted on primary cells and in CLL mouse model. 33 Consistent with these findings, our data show that circulating cells in presence of the scaffold upregulate CD23, a marker involved in the activation and proliferation of CLL cells. 34 As further evidence of the interaction occurring between circulating CLL cells and microenvironmental cells represented by the scaffold, we observed an increase of CD62L on cells circulating with the scaffold. CD62L has been described as the mediator for binding CLL cells to high endothelial venules cells in lymph nodes, which is essential for CLL cells dissemination to these sites. 35 Thus, the presence of a microenvironment under dynamic conditions is pivotal for changes of CLL cells immunophenotype during circulation. These changes are not observed when cells are only exposed to dynamic flow without the scaffold microenvironment. It is important to highlight that the effects of modulation by the microenvironment on both surface markers and gene expression are more pronounced when using fresh cells directly isolated from patients compared to frozen and thawed cells, which show lower modulation of markers. Moreover, given the high versatility of the bioreactor, we will be able to adjust scaffold and vessel dimensions as well as types of microenvironments, ultimately applying multimodal stimulations to better mimic real-life complexity. MATERIALS AND METHODS Human Ethics Statement Patients with CLL were diagnosed according to the updated National Cancer Institute Working Group (NCIWG) guidelines. 36 Peripheral blood (PB) samples were obtained after informed consent from patients who were untreated or off treatment for at least 6 months. The study was approved by the Ospedale San Raffaele (OSR) ethics committee under the protocol CLL-BIO. All the experiments were performed in accordance with relevant guidelines and regulations. Cell culture MEC1 cell line 37 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) and was recently genotyped as follows: 10 ng of DNA from MEC1 cells was purified with QiAmp DNA Mini Kit (Qiagen, Düsseldorf, Germany) and amplified through PCR with GenePrint ® 10 System (Qiagen, Düsseldorf, Germany) and sold Eurofins Genomics Standard FLA Service to perform genotyping. Data was analyzed with DSMZ Online STR Analysis. We confirmed the identity of the cell line analyzed. MEC1 cells and GFP-Tagged MEC1 (MEC1-GFP) cells 38 were cultured in RPMI 1640 medium (EuroClone, Pero, Italy) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 15 mg/ml Gentamicin (complete RPMI) at 37°C and 5% CO 2 . Human Umbilical Vein Endothelial Cells (HUVEC) (Lonza, Basel, Switzerland) were cultured in EGM-2 medium (EuroClone, Pero, Italy) and used between passage 1 and 5. Before plating the cells, standard culture flasks were coated with 1.8% type B gelatine from bovine skin (Sigma-Aldrich, Missouri, USA). Human Lymphatic Fibroblasts (HLF) (ScienceCell, California, USA) cells were cultured in FM medium (ScienceCell, California, USA) and used between passage 3 and 15. Before plating the cells, standard culture flasks were coated with poly-L-lisine (Sigma-Aldrich, Missouri, USA) for 1h, then washed one time with sterile water. Bioreactor design and fabrication The bioreactor (VesselBox) was designed with Fusion 360 (Autodesk, California, USA), and all the components were exported in . stl format. These files have been sliced to . gcode extension with the software Ultimaker Cura (Ultimaker, Utrecht, Netherlands) before being 3D printed (FDM) with a biocompatible material (Extrudr, Lauterach, Austria). Scaffold preparation and perfusion Before engineering the whole structure, we performed compatibility tests for HUVEC cells and HLFs in several biomaterials. In particular, the first cell type has been tested in GelXA LAMININK 411, CELLINK RGD, CELLINK Bioink, CELLINK Fibrin, Vitroink RGD, PureCol, GelXA Fibrin, GelMA Fibrin; selecting the latter as best material in terms of cell morphology and viability. For HLFs, we tested CELLINK Bioink, VitroInk RGD, GelXA Fibrin GelXA LAMININK 411, selecting the latter as best material according to the criteria listed above. Then, hosted scaffolds have been generated with a hybrid technique, combining 3D bioprinting and casting methods. Briefly, GelXA LAMININK 411 (CELLINK AB, Gothenburg, Sweden) was mixed with HLF (10x10 6 cells/mL) and bioprinted with the BIO X bioprinter (CELLINK AB, Gothenburg, Sweden) to generate a 7x7x6mm 3 scaffold, with a 5.5mm in diameter central channel. 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 10x10 6 cells/mL) and manually cast to fill the hollow cavity. Immediately, a 3D printed tool (diameter 2 or 2.5mm) was placed inside the casted material and photo-crosslinked with 405nM UV light for 120 seconds, tilting the scaffold 180° along the z -axis after 60 seconds. We generated a 2 or 2.5mm lumen in diameter by gently removing the tool. The scaffolds were placed in EGM-2 medium supplemented with 10U/mL thrombin (Merck, New Jersey, USA) overnight (O/N) at 37°C, 5% CO 2 . The following day, the thrombin solution was replaced with fresh medium (EGM-2 + FM medium, 3:1 is the best ratio defined to support high viability of both cell types) and the scaffolds were kept in static culture (multiwell plate) before perfusion, 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 into the bioreactor base. The adapters were screwed in the base to fit the lumen in the scaffold. The system was then filled with EGM-2 + FM medium (3:1 ratio) and finally, the lid tightened. The flow perfusion (100 \(\:\mu\:\text{L}\) /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 10mL EGM-2 + FM medium (3:1 ratio). In dynamic settings, when no patients’ cells are present, 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 17 and 21 in static condition compared to day 3 and 7 in dynamic conditions, respectively). In order to validate computational fluid-dynamics simulations, we performed a perfusion test in a hollow scaffold with Trypan blue. Briefly, after O/N culture in a multiwell plate in DMEM (EuroClone, Pero, Italy), we perfused the scaffold with DMEM (7mL) supplemented with 1mL of Trypan blue at 100 \(\:\mu\:\text{L}\) /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 color of the construct. Computational simulations To support the bioreactor design, computational simulations were performed to assess both the fluid-dynamics of the medium flowing inside the bioreactor and the oxygen (O 2 ) diffusion throughout the scaffold by using the commercial software COMSOL MultiPhysics® v5.6. For the numerical fluid-dynamic assessment by Computational Fluid Dynamics (CFD) simulations, two domains were identified: The fluid volume within the lumen, where the culture medium is free to flow and the fluid motion was modeled with the Navier-Stokes equations (Eq. 1). \(\:\rho\:\bullet\:\left[\frac{\delta\:v}{\delta\:t}+\left(v\bullet\:\:\nabla\:\right)\:v\right]=-\nabla\:p+\mu\:\:{\nabla\:}^{2}\:v=F\) The hydrogel around the hollow channel, which was modeled as a homogeneous and isotropic porous medium defined by the properties of permeability and porosity; in this domain, Stokes-Brinkman equation was adopted to describe the fluid perfusion (Eq. 2). \(\:\rho\:\:\left(\varvec{v}\bullet\:\:\nabla\:\right)\varvec{v}=\nabla\:\cdot\:[p\varvec{I}+K]+\varvec{F}\) where \(\:v\) the velocity field, 𝑝 the pressure (Pa), and F the mass forces vector field such as gravity (N m −3 ), I is the identity matrix, K is the permeability tensor of the porous material ( \(\:\epsilon\:\) is the porosity), defined as follows (Eq. 3): $$\:\varvec{K}=\mu\:\:\left(\frac{1}{\epsilon\:}\right)(\nabla\:v+\nabla\:{v}^{T})-\frac{2}{3}\mu\:\left(\frac{1}{\epsilon\:}\right)(\nabla\:\cdot\:v)\varvec{I}$$ In both domains, the culture media was considered as an incompressible, Newtonian fluid defined by density and dynamic viscosity. Different flow rates in the range of the values chosen for the experimental tests (i.e., 100 and 500 \(\:\mu\:\text{L}\) /min) were applied at the inlet, taking as reference values that successfully exert a change on endothelial cells in vascularized scaffolds. 18 To estimate the O 2 concentration within the medium and hydrogel, the mass transport equation was used (Eq. 4): \(\:\nabla\:\bullet\:(-\:D\:\nabla\:c)+\:\varvec{v}\cdot\:\nabla\:c=R\) where c is the concentration, R the O 2 volumetric consumption rate, D the diffusion coefficient of oxygen in the fluid and in the hydrogel, which is supposed to be mainly formed by GelMA and alginate. 39 The O 2 volumetric consumption rate of the cellular component was modelled with Michaelis–Menten kinetics as following (Eq. 5). $$\:R={V}_{max}\bullet\:\left(\frac{c}{{K}_{m}+c}\right)$$ where \(\:{V}_{max}\) is the maximum molar consumption rate, c is the local oxygen concentration function and \(\:{K}_{m}\) is the Michaelis–Menten constant, corresponding to the oxygen concentration at which the consumption is half of \(\:{V}_{max}\) . \(\:{V}_{max}\) depends on the cellular density ( \(\:{\rho\:}_{c}\) ) and single-cell maximum oxygen consumption rate ( \(\:sOCR\) ) (Eq. 6): $$\:{V}_{max}={\rho\:}_{c}\bullet\:sOCR\:\:\:$$ Both \(\:sOCR\) and \(\:{K}_{m}\) are specific for the considered cell types. In our simulation, we replicated the same cell types and densities present in the experimental set-up, i.e., HUVEC and fibroblasts around the channel and only fibroblasts within the hydrogel-based construct. An O 2 concentration value equal to 0.22mol/m 3 was set at the bioreactor inlet. 40 All numerical values of the parameters and boundary conditions used for the simulations are reported in Tables 1 and 2, respectively. An automatic extra-fine triangular mesh was used. Velocity, pressure fields, shear stress, and O 2 concentration distribution throughout the perfusion system were computed and analyzed. Dimensionless parameters, such as Reynolds (Re) and Graez (Gz) numbers, were calculated. Reynolds number (Eq. 7) describes the state of the fluid motion: laminar when < 2300 and turbulent if 2300. It is calculated as: $$\:Re=(\rho\:\bullet\:\stackrel{-}{w}\bullet\:{D}_{h})/\mu\:$$ where \(\:\rho\:\) is the fluid density, \(\:w\) the velocity vector, and \(\:{D}_{h}\) is the hydraulic perimeter. The Graetz number (Eq. 8) represents the relationship between the characteristic time of diffusion, \(\:\:{t}_{diff}\) and the characteristic time of convection, \(\:{t}_{conv}\) , $$\:Gr=\:{t}_{diff}/{t}_{conv}=({{D}_{h}}^{2}\bullet\:\stackrel{-}{w})/D\bullet\:L$$ where \(\:\rho\:\) is the fluid density, \(\:w\) the average fluid velocity, \(\:{D}_{h}\) the hydraulic perimeter, \(\:\mu\:\) the fluid viscosity, L the length channel, and D the oxygen diffusion coefficient. We performed the same simulation performed for replicating the experimental experiments (only considering a flow rate equal to 100 \(\:\mu\:\text{L}\) /min), but by setting a higher and smaller value of diffusion coefficient for oxygen (See Supplementary Table 1) . If we assumed 0.04mM as a critical value under which cells are considered to be no more functional in human tissues/organs, also with the lower value of diffusion coefficient this threshold is satisfied. However, far from the perfused channel, the discrepancy in terms of oxygen concentration is more evident when a lower value of this parameter is used. For example, compared to the “normal condition” (with D = 2.30 x 10 9 m 2 s − 1 ), at a distance of 2mm from the perfused channel, the decrease of O 2 concentration is equal to 15%, while at the edge of the construct (h = 3.5mm from the perfused channel) the oxygen decrease is equal to 26%. Live/Dead assay To assess embedded cells viability after manufacturing and before scaffold perfusion (days 14 post-printing), 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 and phenol red (Thermo Fisher Scientific, Massachusetts, USA) and Live/Dead reagent was added in a 1:3 ratio (reagent:medium). After 30 minutes of incubation at 37°C, 5% CO 2 the constructs were washed one time with DMEM without serum and phenol red, observed with the Olympus FluoVIEW 3000 RS confocal microscope and then processed using FIJI (ImageJ) software. Immunofluorescence (IF) At defined time-points, cell-laden scaffolds either from static (multiwell) or dynamic (VesselBox) culture were fixed with 4% PFA in Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, Texas, USA) 2h at room temperature (RT). After fixation, the scaffolds were washed twice with Hank’s Balanced Salt Solution (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 slices were permeabilized (1mg/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 (Abcam, Cambridge, UK), VE-Cadherin (Cell Signaling, Massachusetts, USA), collagen IV (Invitrogen, Massachusetts, USA) diluted 1:100 in blocking solution (1mg/mL BSA, 10% FBS in PBS) and vWF (Santa Cruz Biotechnology, Texas, USA) diluted 1:50 in blocking solution. Scaffolds perfused with leukemic primary cells were also stained for CD45 (BD Biosciences, New Jersey, USA) diluted 1:100 in blocking solution. The following day, the scaffolds were washed twice with HBSS (Euroclone, Pero, Italy) for 5 minutes RT and incubated always at 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 1:250 in blocking solution. After washing 2 times with HBSS (Euroclone, Pero, Italy), Hoechst 33342 (Invitrogen, Massachusetts, USA) in PBS (1:2000) was added for 10 minutes RT. Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope and then processed using FIJI (ImageJ) and Arivis software. Extravasation studies To prove the whole system is useful for investigating leukemic cells’ behavior and migration, MEC1-GFP cells (1x10 6 cells/mL) or CLL primary cells (5x10 6 cells/mL) were resuspended in the medium reported above, put in the reservoir and made them recirculate within the system for 1, 3 and 7 days after 14 days of static maturation of the scaffolds. At defined time-points, the circulating cells were recovered and analyzed by flow cytometry, while the perfused scaffolds were fixed and eventually stained to localize extravasated leukemic cells, together with the architecture of the microenvironment. Flow cytometry After 1, 3 and 7 days of selective perfusion within the vascularized scaffold hosted in the VesselBox device, leukemic cell line MEC1-GFP and primary cells from patients were recovered and stained 25 minutes RT for the following markers: CD19, IgD, IgM, CD62L, CD29, CD49d, CXCR4, CD45, CCR7, CD40, CD69 (Miltenyi, Bergisch Gladbach, Germany), CD80, CD38, CD23 (BD Biosciences, New Jersey, USA), CD5 (Beckman Coulter, California, USA). After washing with PBS for 5 minutes, 1500 RPM, cells were analyzed with CytoFlex LX flow cytometer (Beckman Coulter, California, USA). Data were processed with FCS Express (De Novo Software, California, USA). RNA extraction and real-time PCR (RT-qPCR) RNA extraction was performed on primary cells at basal level (for freshly used cells the basal level corresponds to the isolated cells from the peripheral blood, for frozen cells the basal level corresponds to thawed cells after 1 hour of adaptation in static culture) and at different time-points (1, 3 and 7 days of selective perfusion) using ReliaPrep RNA Cell Miniprep System® (Promega, Madison, USA) following the manufacturer’s protocol. cDNA was synthesized according to the manufacturer’s protocol using the RevertAid® H Minus First Strand DNA Synthesis kit (Thermo Fisher Scientific, Massachusetts, USA). RT-qPCR analysis was performed using TaqMan™ Fast Advanced Master Mix (Applied Biosystems, Massachusetts, USA) and TaqMan™ gene expression probes (Applied Biosystems, Massachusetts, USA) with CFX96 Real-Time PCR Detection System (BioRad, California, USA). The analysis was performed in duplicate. Quantification of CXCR4, CD62L, and CD23 transcripts (Applied Biosystem probes) was performed according to the Ct method, 41 using YWHAZ as the housekeeping gene. Declarations ACKNOWLEDGEMENTS C.S. acknowledges financial support from EHA Advanced Research Grant 2020. Associazione Italiana per la Ricerca sul Cancro AIRC under IG 2018 - ID. 21332 and Special Program on Metastatic Disease – 5 per mille #2119. We acknowledge Alembic and Fractal facilities. Schemes in figure 3 have been created with BioRender.com. AUTHOR CONTRIBUTION RP, MP, FB performed the experiments; RP, MC, CS analyzed the data; CS and MC supervised the activity; RP, GMDG, CS wrote the manuscript; LS, PG, provided patients' and clinical information; MC, CS, PG revised the manuscript. COMPETING INTERESTS RP and CS are inventors of the patent pending bioreactor technology. DATA AVAILABILITY The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 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A miniaturized hydrogel-based in vitro model for dynamic culturing of human cells overexpressing beta-amyloid precursor protein. J Tissue Eng 11 , (2020). Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 , 402–408 (2001). Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementarymaterialFigureandTable.docx Movie1.avi Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6503832","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":450170407,"identity":"08f2310c-7dda-4604-8297-f011ec89fd80","order_by":0,"name":"Riccardo Pinos","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Pinos","suffix":""},{"id":450170408,"identity":"a63ce8d9-edc3-41c7-bd8a-79ae0f9f79e4","order_by":1,"name":"Margherita Pauri","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Margherita","middleName":"","lastName":"Pauri","suffix":""},{"id":450170409,"identity":"362a0794-fb16-4652-ac35-e49c9d64424a","order_by":2,"name":"Federica Barbaglio","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Federica","middleName":"","lastName":"Barbaglio","suffix":""},{"id":450170410,"identity":"92ac95b3-690d-4b28-a331-c9a2634ef1c8","order_by":3,"name":"Giulia Maria Di Gravina","email":"","orcid":"","institution":"University of Pavia","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"Maria Di","lastName":"Gravina","suffix":""},{"id":450170411,"identity":"4679a7dd-0bc4-4168-922e-358b246cd189","order_by":4,"name":"Lydia Scarfò","email":"","orcid":"","institution":"Vita-Salute San Raffaele University","correspondingAuthor":false,"prefix":"","firstName":"Lydia","middleName":"","lastName":"Scarfò","suffix":""},{"id":450170412,"identity":"17cbfbfa-94dd-424f-8c79-721d53281642","order_by":5,"name":"Paolo Ghia","email":"","orcid":"","institution":"Vita-Salute San Raffaele University","correspondingAuthor":false,"prefix":"","firstName":"Paolo","middleName":"","lastName":"Ghia","suffix":""},{"id":450170414,"identity":"d8fd67cf-c41d-4e20-905f-902e74331a94","order_by":6,"name":"Michele Conti","email":"","orcid":"","institution":"University of Pavia","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Conti","suffix":""},{"id":450170415,"identity":"1bb2939b-8086-4954-967d-5230ee939fe5","order_by":7,"name":"Cristina Scielzo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIie2PsYrCQBCGZ7GwCaQdG/MKI9sqvsoEwWssLK9UAkkj2C74EgcH1oEtbMLVgbPwEKwXrrHYwk3QwiJra7FfNQPz7f4/QCDwlogVnB4DEECMzUIvFH4MpbscqFbxOnBXHGVzXIP/m6TQ6xPbMcQ7nf2b5TGRv8XFwNJ2KlSlGXE0B/xJcyzpMtofK6l8wQhFjowaKBK566LFvl70vF0SJYorU6tkxinTb/Vx9tev3ePMrbJywXT6hSy9StMFuZxHWLmEFemZqhcSmGR3sOLwZ4wdD+NN/2w+rZ5sm2DGDruD3YmeV34pBAKBQMDHDeXDUuU9+WIEAAAAAElFTkSuQmCC","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":true,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Scielzo","suffix":""}],"badges":[],"createdAt":"2025-04-22 11:38:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6503832/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6503832/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81802857,"identity":"a0638a83-4979-4f04-90a4-ae9bb0f32696","added_by":"auto","created_at":"2025-05-02 06:24:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the VesselBox device and its components.\u003cbr\u003e\n \u003c/strong\u003eRendering of the exploded view of the VesselBox device \u003cstrong\u003e(A)\u003c/strong\u003eshowing threads on lid (I) and adapters (V) to guarantee tight sealing of the device, together with the optical window (II) on the lid to directly monitor the dynamic cultured scaffold over-time. The homing module (III) is made to host custom scaffolds within the base (IV), in which the adapters enter a little in order to maintain selective perfusion over-time, as shown in the assembled bioreactor \u003cstrong\u003e(B)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/5b7b6025138d8e9166b4f719.png"},{"id":81803403,"identity":"051c1be6-4c72-49ff-b50b-e4bd5db4a4a0","added_by":"auto","created_at":"2025-05-02 06:32:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1913535,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D bioprinted HLFs show sustained viability up to 28 days.\u003cbr\u003e\n \u003c/strong\u003eLive\u0026amp;Dead assay on HLFs 28 days after printing in VitroINK RGD \u003cstrong\u003e(A)\u003c/strong\u003eand GelXA LAMININK 411 \u003cstrong\u003e(B)\u003c/strong\u003e. In both matrices, cells display an homogeneous distribution and sustained viability throughout the entire scaffold. Embedded cells in GelXA LAMININK 411 display a more stretched and physiological morphology \u003cstrong\u003e(B)\u003c/strong\u003e, when compared to VitroINK RGD \u003cstrong\u003e(A)\u003c/strong\u003e. Images were acquired with AXIO z1 Observer \u003cstrong\u003e(A)\u003c/strong\u003e and Olympus FluoVIEW 3000 RS confocal microscope \u003cstrong\u003e(B)\u003c/strong\u003e.\u003cbr\u003e\nScale bar: 500μm \u003cem\u003eLiving cells (green), dead cells (red).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/28bc32987e96990843bc10de.png"},{"id":81802856,"identity":"984c6155-5e25-474f-85f8-4d705d17047d","added_by":"auto","created_at":"2025-05-02 06:24:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequential steps required for the generation and perfusion of multi-material and multi-cellular vascularized constructs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3D bioprinted hollow scaffold with HLFs in GelXA LAMININK 411 \u003cstrong\u003e(A)\u003c/strong\u003e is filled by manual casting technique with HUVEC+HLF cells in GelMA Fibrin \u003cstrong\u003e(B)\u003c/strong\u003e, which is then photocrosslinked after inserting a self-designed plastic rod \u003cstrong\u003e(C)\u003c/strong\u003e. The hollow channel is generated after gently removing the plastic rod\u003cstrong\u003e (D)\u003c/strong\u003eand the scaffold is placed within the VesselBox \u003cstrong\u003e(E)\u003c/strong\u003e. The bioreactor is then connected to a peristaltic pump to selectively perfuse the biofabricated scaffold \u003cstrong\u003e(F, G)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/6a017623d69f7c4e6c4ae894.png"},{"id":81804045,"identity":"ab867799-5a09-4a00-9ed6-c9c89d600190","added_by":"auto","created_at":"2025-05-02 06:40:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":770578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHybrid scaffolds in static culture show homogeneous cell distribution after manufacturing.\u003cbr\u003e\n \u003c/strong\u003eTop view of vessel cavity and lumen wall with embedded HUVECs and HLFs stained for phalloidin displaying homogeneous distribution 1 \u003cstrong\u003e(A)\u003c/strong\u003e and 14 days \u003cstrong\u003e(B)\u003c/strong\u003e post-manufacturing. Zoomed images clearly show increasing adaptation and maturation of the construct starting from day 1 \u003cstrong\u003e(C)\u003c/strong\u003e up to day 14 \u003cstrong\u003e(D)\u003c/strong\u003e. Images were acquired with Olympus FluoVIEW 3000μm RS confocal microscope. Scale bar: 1000 \u003cem\u003ePhalloidin (red).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/960311033cf86cdcb0fe1fb8.png"},{"id":81803397,"identity":"8b63523c-0734-40e0-9c62-47d1592575dc","added_by":"auto","created_at":"2025-05-02 06:32:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":346375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTop view of the milli-fluidic device and graphical representations of computed parameters.\u003cbr\u003e\n (A) \u003c/strong\u003eShear stress: side view of the color map (left) and line plot at the wall channel (right) at the different heights considered from the channel axis (flow rate = 100 and 500μL/min). \u003cstrong\u003e(B)\u003c/strong\u003e Side view of the color map (left) and line plot at a height equal to 1, 1.5, 2, 2.5, 3 3.5mm from the central axis of the perfused channel (right) showing oxygen concentration (flow rate = 100 and 500μL/min). \u003cstrong\u003e(C)\u003c/strong\u003e Side view of the color map showing the velocity field (left) and velocity streamlines (right) (flow rate = 100μL/min). \u003cstrong\u003e(D)\u003c/strong\u003e Side view of the color map showing the pressure drop along the perfusion system (left) and its line plot at a height equal to 1,3mm, 2mm, 3.5mm from the central axis of the perfused channel (right) (flow rate = 100μL/min).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/3e583ab86db31e9817cdff53.png"},{"id":81802850,"identity":"622c0301-c7d8-4333-b65a-cfa2f57c77f6","added_by":"auto","created_at":"2025-05-02 06:24:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":296444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePerfusion test for channel permeability and selective convey assessment.\u003cbr\u003e\n \u003c/strong\u003eHybrid scaffolds are perfused within the VesselBox device with Trypan blue diluted in PBS to assess medium delivery to more distant regions of the constructs. Gradually increasing color intensity after 0 \u003cstrong\u003e(A)\u003c/strong\u003e, 60 minutes \u003cstrong\u003e(B)\u003c/strong\u003e and O/N perfusion \u003cstrong\u003e(C)\u003c/strong\u003e demonstrates effective perfusion and diffusion within the hydrogel matrix.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/e089c3fefac6102f93cbe9df.png"},{"id":81802873,"identity":"ec779e0b-caee-47cf-9bc9-48244f357fd5","added_by":"auto","created_at":"2025-05-02 06:24:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":433784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmbedded endothelial cells and fibroblasts show sustained viability up to 7 days post-casting.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLive\u0026amp;Dead assay on 3D bioprinted HUVECs and HLFs after 7 days of static culture in a multiwell plate. Cells display homogeneous distribution and sustained viability throughout the entire scaffold. Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope. Living cells \u003cstrong\u003e(A)\u003c/strong\u003e, dead cells \u003cstrong\u003e(B)\u003c/strong\u003e, merge \u003cstrong\u003e(C)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLiving cells (green), dead cells (red). \u003c/em\u003eScale bar: 500μm\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/8d0614a440bbd3a50fe07078.png"},{"id":81802849,"identity":"ad87059e-189b-4c75-83c4-01729384e831","added_by":"auto","created_at":"2025-05-02 06:24:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1257195,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVessel maturation assessment after 14 days of static culture and effects of the dynamic culture.\u003cbr\u003e\n \u003c/strong\u003eHybrid scaffolds maintain a defined luminal cavity up to 14 days of static culture, together with a high cell density.Selective endothelial marker CD31 (white) and collagen IV production (green) is illustrated in the top view at 4X and 10X magnification \u003cstrong\u003e(A, B)\u003c/strong\u003e. VE-cadherin (white) 19and vWF (green) expression is visualized from the bottom view of the lumen at 4X and 20X magnification \u003cstrong\u003e(C, D)\u003c/strong\u003e. Embedded cells show stretched morphology throughout the entire scaffold, with increased peripheral CD31 expression 7 days after perfusion \u003cstrong\u003e(F)\u003c/strong\u003e, if compared to 3 days in the same settings \u003cstrong\u003e(E)\u003c/strong\u003eas indicated by yellow arrows. Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope. \u003cem\u003eCD31/Ve-cadherin (white), Collagen IV/vWF (green), phalloidin (red), Hoechst 33342 (blue). \u0026nbsp;\u003c/em\u003eScale bar A-C: 500μm; Scale bar D: 100μm; Scale bar E, F: 500μm\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/4b5cbd81ce554d51a1a6be54.png"},{"id":81802884,"identity":"c6c755ca-531d-4772-b961-e9c6ce032009","added_by":"auto","created_at":"2025-05-02 06:24:02","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":247591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirculating MEC1-GFP cells show modulation of surface markers involved in cell adhesion and migrate towards the vascular compartment.\u003cbr\u003e\n \u003c/strong\u003eMEC1-GFP cells (green) extravasated from the lumen to the vascular and microenvironment compartments \u003cstrong\u003e(A)\u003c/strong\u003e. CD38 \u003cstrong\u003e(B)\u003c/strong\u003e and CD49d \u003cstrong\u003e(C)\u003c/strong\u003eshow an increasing trend from day 1 to day 7 in circulating cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGFP(green) \u0026nbsp;\u003c/em\u003eScale bar: 500μm\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/8ee943d33bf60af94e555c44.png"},{"id":81802890,"identity":"8123a916-7a57-4896-aa21-ecfb67562856","added_by":"auto","created_at":"2025-05-02 06:24:02","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1678883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunophenotype modulation of surface markers involved in activation and homing processes in circulating CLL primary cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCXCR4\u003csup\u003ehigh\u003c/sup\u003e/CD5\u003csup\u003ehigh\u003c/sup\u003e population representation is increased on circulating primary cells in the presence of the scaffold, if compared to the same cells at basal level or circulating without the scaffold; plots gated on CD19\u003csup\u003e+\u003c/sup\u003e cells. CXCR4\u003csup\u003ehigh\u003c/sup\u003e/CD5\u003csup\u003ehigh\u003c/sup\u003e CLL primary cells circulating in the presence (pink line) or absence (black line) of the scaffold (A). CD62L\u003csup\u003e+\u003c/sup\u003e cells are increased in the presence of the scaffold, if compared to the same cells at basal level or circulating without the scaffold; plots gated on CD19\u003csup\u003e+\u003c/sup\u003e cells. CD62L\u003csup\u003e+\u003c/sup\u003e CLL primary cells circulating in the presence (pink line) or absence (black line) of the scaffold (B). CD23\u003csup\u003ehigh\u003c/sup\u003e cell population is higher in CLL cells circulating in the VesselBox with the scaffold, if compared to the same cells circulating in the bioreactor without scaffold; plots gated on CD19\u003csup\u003e+\u003c/sup\u003e cells. CD23\u003csup\u003e+\u003c/sup\u003e CLL primary cells circulating in the presence (pink line) or absence (black line) of the scaffold (C) (n=3). CLL primary cells (green) extravasated from the lumen to the vascular compartment after 3 days of circulation (D).\u003cbr\u003e\nData are represented as mean ± SEM (n=3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCD31 (white), CD45 (green), phalloidin (red), Hoechst 33342 (blue). \u003c/em\u003eScale bar: 100μm\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/df478b66f763f5f39848f6b5.png"},{"id":89487881,"identity":"a2e06d02-52c4-4c7c-bd1b-83e8590f1b20","added_by":"auto","created_at":"2025-08-20 13:09:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9246856,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/8fcdc309-975c-4121-9c95-d374535023f0.pdf"},{"id":81802869,"identity":"605b3019-59ee-45ea-bde2-b6e39453d9aa","added_by":"auto","created_at":"2025-05-02 06:24:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3530882,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialFigureandTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/3ba15655d1c7b3b8c1e1f9c1.docx"},{"id":81802846,"identity":"8656a8e7-4292-48ed-b439-c2557943263c","added_by":"auto","created_at":"2025-05-02 06:24:00","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4792474,"visible":true,"origin":"","legend":"","description":"","filename":"Movie1.avi","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/11ee431a2bba62f05c9da1cd.avi"},{"id":81802894,"identity":"1e9708a0-6a7f-4bc9-991d-732f392e2f98","added_by":"auto","created_at":"2025-05-02 06:24:03","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2864919,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6503832/v1/8f5d7a445c039ebc18ffa988.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Controlled perfusion of a vascularized microenvironment within a 3D printed bioreactor to study leukemia cells trafficking ex-vivo","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eChronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. The trafficking of malignant B cells (CD19\u003csup\u003e+\u003c/sup\u003e/CD5\u003csup\u003e+\u003c/sup\u003e) between peripheral blood (PB), bone marrow, and secondary lymphoid tissues is pivotal for their survival, proliferation,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and response/resistance to therapies.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Although the introduction of novel targeted therapies (e.g., BTK and BCL2 inhibitors),\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e has revolutionized patient management and significantly improved outcomes, patients experience treatment resistance and the disease remains incurable.\u003c/p\u003e \u003cp\u003eCLL is a dynamic and heterogeneous disease that progresses thanks to an ill-defined cross-talk with a supportive microenvironment, that involves a variety of interactions including those with endothelial cells (ECs) present in the blood vessels and into the lymphoid tissues.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAccordingly, some studies suggest that the interaction between endothelial cells and CLL cells through the CD31/CD38 engagement supports the proliferation and survival of leukemia cells. This interaction likely occurs in peripheral tissues (e.g., lymph nodes), where malignant B cells can maintain cellular contacts for prolonged periods.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e In addition to the direct interaction between CD31 and CD38, soluble factors such as Chromogranin A have been shown to regulate the trafficking of CLL cells. Chromogranin A has been associated with increased vascular barrier function and integrity while also playing a central role in migration of leukemic cells.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn recent years, there has been a shift from traditional 2D culture to more reliable 3D models to gain insights into the onset, progression and resistance to treatment in CLL.\u003c/p\u003e \u003cp\u003eTo recapitulate the impact of the microenvironment in CLL, a scaffold-based approach\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e has been successfully used to study patient-specific responses to a BTK-inhibitor in a bioreactor under microgravity conditions, in the presence of bone-marrow stromal cells.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBuilding on this study and given the limited viability of primary CLL cells \u003cem\u003ein vitro\u003c/em\u003e, we exploited 3D bioprinting to establish a 3D long-term model for CLL cells. Our results demonstrate that CLL cells can survive up to 28 days in 3D conditions compared to those cultured in 2D. This increased viability can be partially explained by changes in gene expression in pro- and anti-apoptotic genes such as BAX and BCL2, among others.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRemarkably, these models have great potential to help dissect, to some extent, driver mechanisms underlying CLL. However, it is clear that the endothelial compartment must be present during the trafficking process to accurately mimic CLL dynamics \u003cem\u003ein vitro\u003c/em\u003e. To date, only one study has recapitulated the trafficking process of malignant B cells \u003cem\u003ein vitro\u003c/em\u003e, showing modulation of surface markers upon extravasation. However, this model lacked the tissue microenvironment that provides several biochemical cues to tumor cells.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the present work, we designed, developed, and manufactured a milli-fluidic system capable of perfusing vascularized constructs embedded with the leukemia microenvironment. In this setting, we can monitor CLL cells extravasation and characterize their immunophenotype over-time, paving the way for advanced dynamic \u003cem\u003ein vitro\u003c/em\u003e models for CLL, which can also be translated to other pathophysiological conditions.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBioreactor fabrication\u003c/h2\u003e \u003cp\u003eThe first step towards the reconstruction of a complex and advanced \u003cem\u003ein vitro\u003c/em\u003e dynamic system to study leukemic cells trafficking was the design and manufacturing of a brand-new bioreactor: the VesselBox.\u003c/p\u003e \u003cp\u003eSpecifically, the device has been conceived with a modular configuration, making it possible to easily tune dimensions, diameters, shapes of the hosted scaffolds and embedded vascular channels. The VesselBox is made up by a base, a screw-down lid with optical access, a homing module for prefabricated, casted, 3D bioprinted or hybrid scaffolds and two adapters equipped with silicone tubes acting as medium inlet and outlet \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. Silicon gaskets have been put on the screw-down lid and the adapters\u0026rsquo; seats to ensure sealing and pressure maintenance inside the bioreactor, as well as selective medium convey in luminal cavities. All the components were 3D printed with a FDM 3D printer, upon accurate selection of a material that allowed us to sterilize all the components before to culture our scaffolds and to reuse them for at least three times after autoclave sterilization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince 3D printed parts are known to be subjected to different drawbacks,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e especially due to its layer-by-layer fabrication process, we carefully tuned all the parameters in the slicing step, such as infill density and line directions, number of printed perimeters, top and bottom layers, in order to avoid medium leakage during perfusion and components delamination after sterilization. In the next sections, we will deepen the vascularized scaffolds fabrication process to be hosted within the VesselBox device, as well as the computational support throughout the design and development of the bioreactor.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstablishment of a hybrid method to engineer vessels surrounded by a lymphoid microenvironment\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong the strategies to obtain the milli-scale vessel-like structure, we set up and selected a hybrid technique combining extrusion-based bioprinting (EBB) and casting methods. In particular, EBB alone failed in generating a mechanically stable 3D structure, causing lumen collapse during or soon after printing, even when using sacrificial materials such as Pluronics F-127\u003csup\u003e16\u003c/sup\u003e or different printing orientation strategies. Moreover, bioinks that we previously tested to engineer the whole structure by EBB were not supporting cell viability and morphology properly, also giving significant background signal in immunofluorescence imaging \u003cb\u003e(see Supplementary Materials Fig.\u0026nbsp;1).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eConversely, GelMA Fibrin casting promoted an extremely precise and stable structure for HUVEC cells, while VitroINK RGD and GelXA LAMININK 411 \u0026ndash; that has been selected as most suitable material - showed higher compatibility with HLF cells, which represent the surrounding lymphoid microenvironment in our 3D model \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHUVECs seeding in casted GelMA Fibrin luminal structure resulted in poor cell adhesion and cell-cell contact even at high cell density and different incubation/rotation time, probably due to the large channel diameter (2/2.5mm) \u003cb\u003e(see Supplementary Materials Fig.\u0026nbsp;2)\u003c/b\u003e. For this reason, the combination of a two-step hybrid method has been chosen, relying on the possibility to precisely control cell distribution in the surrounding microenvironment by 3D bioprinting, concurrently generating a defined central lumen by casting of pre-mixed HUVEC and HLF cells in GelMA Fibrin. Briefly, we first 3D bioprint HLFs in the microenvironment compartment leaving the hollow cavity in vertical position \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, which is then filled with the endothelial cells and fibroblasts mix \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Immediately, a self-standing plastic rod is inserted to create space for the vascular lumen \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, then gently removed after photocrosslinking \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. The scaffold is placed within the VesselBox \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e., which is then connected to a peristaltic pump to selectively perfuse the biofabricated scaffold \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIncreasing cell concentration and different HUVEC:HLF ratio have been tested to optimize the vessel wall formation, concluding that 3:1 ratio with a final cell concentration of 10x10\u003csup\u003e6\u003c/sup\u003e cells/mL were the most appropriate settings for the lumen and 10x10\u003csup\u003e6\u003c/sup\u003e cells/mL (HLFs only) for the surrounding tumor environment (data not shown).\u003c/p\u003e \u003cp\u003eUpon scaffold generation, cell density and distribution have been monitored over-time by immunofluorescence, which clearly showed embedded cells homogeneously populating the matrix already 1 day post-manufacturing \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, as well as after 14 days \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e of static culture. Moreover, resident cells displayed a more stretched morphology and dense network formation at later time-points (14 days, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), if compared to day 1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, suggesting an increasing adaptation and distribution in a time-related manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNumerical simulations and perfusion tests allowed to demonstrate selective perfusion of vascularized hybrid scaffolds\u003c/h3\u003e\n\u003cp\u003eAs support in the VesselBox design, \u003cem\u003ea priori\u003c/em\u003e evaluations were performed to assess its suitability for 3D cell culture before carrying out experimental tests requiring time and costs. Particularly, shear stress and oxygen concentration were the two main parameters of interest, experimentally difficult to measure, but essential in the view of the correct functioning of the bioreactor.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAs a first step, we carried out a parametric study in which different geometries and inflows were considered \u003cb\u003e(see Supplementary Materials Section 1)\u003c/b\u003e. Then, computational analyses were performed by focusing on the prototype that was selected and printed for testing the performance of the device from a biological point of view, characterized by a diameter and length channel of 2 mm and 6 mm, respectively, a scaffold equal to 7x7x6mm, and perfused by a flow rate equal to 100 or 500\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:uL/min\\)\u003c/span\u003e\u003c/span\u003e. Particularly, simulations consisting in coupling fluid-dynamics equations with mass transport equations were performed to estimate the two target parameters, namely shear stress and oxygen concentration.\u003c/p\u003e \u003cp\u003eThe line plot of shear stress, reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, shows a constant trend (with a value equal to 0.0165 and 0.08 dyn/cm\u003csup\u003e2\u003c/sup\u003e for 100 and 500\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\text{L}\\)\u003c/span\u003e\u003c/span\u003e/min, respectively) at the level of the wall channel, providing in this way a homogenous stimulation to endothelial cells here located. Although the wall shear stress is lower than one experienced by endothelial cells \u003cem\u003ein vivo\u003c/em\u003e, it is coherent with the values found in literature.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Moreover, shear stress rapidly reduced to values close to zero on the interior part of the scaffold as the fluid has difficulty to flow inside due to the porous nature of the hydrogel, thus indicating a shielding effect of the hydrogel matrix for the embedded cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, the numerical evaluation allowed assessing the effect of the design of the milli-fluidic device for oxygen consumption. In the simulation, two different cell types were considered: endothelial cells forming a monolayer around the bioreactor channel and fibroblasts inside the construct. We assumed 0.04mM as a critical value under which cells are considered to be no more functional in human tissues/organs.\u003csup\u003e19\u003c/sup\u003e As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, oxygen concentration showed a decreasing trend along the perpendicular direction to the central axis of the channel, varying from a maximum of 0.2 along the wall channel to a minimum of 0.1mol/m\u003csup\u003e3\u003c/sup\u003e at the chamber edge (i.e., h\u0026thinsp;=\u0026thinsp;3.5mm), creating in this way an oxygen gradient along the perfusion system. However, no oxygen depletion was revealed from the computational results with both the flow rate used, demonstrating in this way the optimal performance of the bioreactor.\u003c/p\u003e \u003cp\u003eFinally, it was possible to observe that the flow pattern, represented by velocity streamlines, is able to reach, even if with difficulty, the outermost regions of the scaffold with the value of hydrogel's permeability and porosity considered \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Moreover, no flow stagnation was detected. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD shows the distribution of the pressure within the perfusion system and low pressure values in the range between 0.25 and 0.038Pa were calculated inside the chamber.\u003c/p\u003e \u003cp\u003eFollowing numerical simulations, we proved experimentally with several perfusion tests without the hosted scaffold at different flow rates, namely 0.5, 1, 5, and 10mL/min, that our system could easily withstand low to high medium speed, and consequently proportional pressure variations inside the chamber, without culture medium leakage, which in our prototypes has been the main cause of contamination. Conversely, the selective perfusion was assessed at lower flow speed (up to 1mL/min), since higher flow rates caused complete disruption of the scaffold. Total volume of medium within the system has been measured over-time to ensure that no leakage was present. Specifically, the ability of the system to promote a functional and selective perfusion was assessed by the change in color intensity of the scaffold after 0 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, 60 minutes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e and overnight perfusion \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, thus demonstrating that the entire construct (and consequently the embedded cells) could be gradually reached by the medium after overnight perfusion. Indeed, bioreactor geometries allow the conveying of culture medium to encapsulated cells, even to those lying far from the central vascular flow.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEmbedded cells express functional markers in the VesselBox bioreactor over-time\u003c/h3\u003e\n\u003cp\u003eIn order to generate a functional and thick 3D model \u003cem\u003ein vitro\u003c/em\u003e, it is fundamental to check if, besides scaffold architectural preservation, viability and selective cell markers are maintained and expressed during time.\u003c/p\u003e \u003cp\u003eTo demonstrate that the entire manufacturing process was fully compatible with embedded cells, further integrating the results shown by Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, we assessed cell viability at different time-points with the Live\u0026amp;Dead assay. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, static cultured cells show sustained viability 7 days after manufacturing in the top view, as well as a homogeneous distribution throughout the whole matrix, although they need more time to assume a proper morphology, as illustrated above.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThrough the two-step hybrid process described in the second section, we managed to reproduce a tightly organized and confluent layer of ECs on the lumen walls after 14 days of static culture, as demonstrated by the expression of CD31 in the lumen wall, together with collagen IV, which is dispersed within the vascular compartment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. Interestingly, at this time-point, ECs form a continuous and organized network within the same compartment, as shown by Ve-Cadherin and vWF markers expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, D \u003cb\u003eand Supplementary movie 1)\u003c/b\u003e. Importantly, the above-mentioned markers have been associated with fine regulation of vascular maturation and permeability.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo 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 \u003cem\u003ein vitro\u003c/em\u003e, also enabling for a deeper understanding of several pathophysiological processes in more complex models.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Moreover, dynamic perfusion is pivotal to investigate leukemia cells trafficking \u003cem\u003eex vivo\u003c/em\u003e, which is our goal. Hence, after vessel maturation in static conditions was confirmed, the constructs were exposed to dynamic flow perfusion in the VesselBox device up to 7 days with flow speed set at 100\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:uL/min\\)\u003c/span\u003e\u003c/span\u003e to further prove medium transport in the channel without leakage and investigate flow-related effects on cell distribution and vascular morphology before making leukemic cells recirculating within the system. As predicted by numerical simulations, perfusion parameters and device architecture supported cell viability at both time-points, avoiding critical culture conditions for the cells (data not shown).\u003c/p\u003e \u003cp\u003eMoreover, dynamic cultured constructs display a different organization of embedded cells, as compared to static cultured ones. In particular, after 3 days of selective perfusion, CD31\u003csup\u003e+\u003c/sup\u003e cells are branching towards the microenvironment side and can be observed also in the periphery of the scaffold, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE. Interestingly, at day 7 post-perfusion, ECs laying in close proximity to the lumen are more elongated and dispersed in the whole matrix \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e, if compared to the previous time-point.\u003c/p\u003e\n\u003ch3\u003eImmunophenotype characterization of circulating CLL cells in the engineered leukemia microenvironment\u003c/h3\u003e\n\u003cp\u003eAs previously discussed, malignant B cells trafficking between blood, bone marrow and secondary lymphoid tissues is pivotal for the progression of the disease and resistance to therapy. In order to gain more insights in this perspective and mirror the leukemic cells dissemination across the vascular compartment, the CLL cell line MEC1-GFP or primary CLL cells were allowed to recirculate in mature vascular-laden scaffold (at day 14 post-manufacturing), being then retrieved for immunophenotype characterization after 1, 3 and 7 days of dynamic culture. These cells were compared with the same cells circulating in the empty bioreactor, i.e. without the vascularized construct and the microenvironment, in order to discriminate between flow-induced and microenvironment-induced changes on surface markers. Interestingly, MEC1-GFP cells can be visualized after circulation and extravasation by confocal imaging \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and show modulation of critical surface markers for activation and homing processes. Importantly, CD38, which may contribute to CLL cells extravasation and homing,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e is increased from day 1 to day 7 of circulation in the presence of the microenvironment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Regarding CD49d,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e the subunit of VLA-4 complex involved in CLL cells homing in the lymph nodes, we could determine a significantly increased expression at day 7 post-circulation in the presence of the microenvironment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Imaging analyses on selectively perfused scaffolds with primary cells show their extravasation in the vascular compartment already after 3 days of circulation at \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to assess the potential mechanisms underlying the process of extravasation of primary cells (n\u0026thinsp;=\u0026thinsp;3), we evaluated by flow cytometry the expression of CXCR4 and CD5 on primary CLL cells circulating in the bioreactor with the scaffold, whose levels are known to be associated with extravasation and homing of CLL cells.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Furthermore, we compared those levels to the circulating cells in the empty bioreactor, from now on defined as circulation without the scaffold, and to the basal level measured on the cells freshly isolated from the peripheral blood or just thawed. After 1, 3 and 7 days of circulation in the bioreactor, we detected the presence of a CXCR4\u003csup\u003ehigh\u003c/sup\u003e/CD5\u003csup\u003ehigh\u003c/sup\u003e cell population, which has been described as more prone to tissue homing. Interestingly this population is absent at basal level and in the dynamic setting without the scaffold/microenvironment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. This result is indicative that a cross-talk is occurring between the microenvironment and CLL cells while trafficking between the different compartments as demonstrated in vivo.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Moreover, we investigated the expression of CD62L, since this marker is involved in homing and migration of lymphocytes to lymphoid organs and is known for its essential role in controlling the extravasation of CLL cells through the High Endothelial Venules (HEVs) in the lymph nodes. We observed upregulation in both surface and gene expression in presence of the scaffold, indicating a potential interaction between leukemic and endothelial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLastly, patient cells also show an increased expression of the CD23\u003csup\u003ehigh\u003c/sup\u003e/CD5\u003csup\u003ehigh\u003c/sup\u003e population\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e at day 1 and day 3 post-circulation in the VesselBox in the presence of the scaffold, as compared to the same dynamic settings without the scaffold \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. CD23 is amongst the surface markers used for CLL diagnosis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and it is involved in normal and CLL B cells activation and growth.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAll the markers have also been evaluated by RT-PCR. Interestingly, CXCR4 and CD62L gene levels mirror flow cytometry analyses, while CD23 gene expression seems not to follow protein levels which may indicate a different regulatory mechanism that is time-dependent \u003cb\u003e(see Supplementary Materials Fig.\u0026nbsp;3)\u003c/b\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn the present work, we developed a brand-new device, together with a novel method to engineer complex 3D vascularized scaffolds, enabling the investigation of leukemic cells trafficking in a relevant \u003cem\u003eex vivo\u003c/em\u003e environment.\u003c/p\u003e \u003cp\u003eDynamic culture systems in biomedical research often refer to microfluidic chips, which are typically used to ensure fluid perfusion for seeded cells inside hollow cavities. Due to their affordability and versatility, microfluidic chips have been adopted over the years for various purposes, such as studying shear stress effects on endothelial monolayers,\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e reconstructing the blood-brain-barrier (BBB) to gain more insights into neurovascular disorders,\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and investigating cancer biology and responses to drugs.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e However, these promising tools are usually manufactured with inert materials, such as PDMS, which makes it impossible to study cell extravasation and cross-talk with stromal compartments.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e These processes are critical in several diseases, especially in hematological cancers like CLL.\u003c/p\u003e \u003cp\u003eIn this regard, we previously demonstrated that both CLL cell lines and primary CLL cells can survive for prolonged period in 3D bioprinted constructs, also showing molecular fingerprints of adaptation to the tissue-like structure.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOn top of that, we increased the complexity of the system by introducing the vascular component into the scaffold through a hybrid manufacturing method. This allows for a deeper investigation of leukemic cells trafficking in peripheral blood-like settings (circulating medium) and secondary lymphoid tissues-like settings (3D bioprinted microenvironment). To facilitate this, we designed and produced a novel bioreactor, the VesselBox, which enables the selective perfusion of these scaffolds using a peristaltic pump. With CFD analysis supporting the entire development stage of the device, we demonstrated that the VesselBox ensures nutrients delivery throughout the entire matrix while simultaneously supporting cell survival and proper morphology within the hosted scaffold. Consequently, embedded endothelial cells maintain the expression of selective markers such as CD31, Ve-cadherin, VWF and collagen IV, with CD31 being highly represented at the periphery of dynamically cultured scaffolds after 7 days. This model was then used to assess MEC1-GFP and primary patients cells\u0026rsquo; extravasation over-time while also allowing for their immunophenotype characterization.\u003c/p\u003e \u003cp\u003eAccording to available studies,\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e \u003cem\u003ein vivo\u003c/em\u003e monitoring of CLL cells trafficking shows different surface patterns on their homing to tissues. CXCR4 has been described as fundamental for homing leukemic cells within lymphoid tissues.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Our results indicate that circulating primary cells in the bioreactor with the scaffold - and thus with the microenvironment - lead to the emergence of a double positive cell population expressing CXCR4\u003csup\u003ehigh\u003c/sup\u003e/CD5\u003csup\u003ehigh\u003c/sup\u003e. Remarkably, CXCR4\u003csup\u003ehigh\u003c/sup\u003e/CD5\u003csup\u003ehigh\u003c/sup\u003e cells have recently been identified as actively proliferating cells based on experiments conducted on primary cells and in CLL mouse model.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Consistent with these findings, our data show that circulating cells in presence of the scaffold upregulate CD23, a marker involved in the activation and proliferation of CLL cells.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAs further evidence of the interaction occurring between circulating CLL cells and microenvironmental cells represented by the scaffold, we observed an increase of CD62L on cells circulating with the scaffold. CD62L has been described as the mediator for binding CLL cells to high endothelial venules cells in lymph nodes, which is essential for CLL cells dissemination to these sites.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThus, the presence of a microenvironment under dynamic conditions is pivotal for changes of CLL cells immunophenotype during circulation. These changes are not observed when cells are only exposed to dynamic flow without the scaffold microenvironment.\u003c/p\u003e \u003cp\u003eIt is important to highlight that the effects of modulation by the microenvironment on both surface markers and gene expression are more pronounced when using fresh cells directly isolated from patients compared to frozen and thawed cells, which show lower modulation of markers.\u003c/p\u003e \u003cp\u003eMoreover, given the high versatility of the bioreactor, we will be able to adjust scaffold and vessel dimensions as well as types of microenvironments, ultimately applying multimodal stimulations to better mimic real-life complexity.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHuman Ethics Statement\u003c/h2\u003e \u003cp\u003ePatients with CLL were diagnosed according to the updated National Cancer Institute Working Group (NCIWG) guidelines.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Peripheral blood (PB) samples were obtained after informed consent from patients who were untreated or off treatment for at least 6 months. The study was approved by the Ospedale San Raffaele (OSR) ethics committee under the protocol CLL-BIO. All the experiments were performed in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eMEC1 cell line\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) and was recently genotyped as follows: 10 ng of DNA from MEC1 cells was purified with QiAmp DNA Mini Kit (Qiagen, D\u0026uuml;sseldorf, Germany) and amplified through PCR with GenePrint \u0026reg; 10 System (Qiagen, D\u0026uuml;sseldorf, Germany) and sold Eurofins Genomics Standard FLA Service to perform genotyping. Data was analyzed with DSMZ Online STR Analysis. We confirmed the identity of the cell line analyzed. MEC1 cells and GFP-Tagged MEC1 (MEC1-GFP) cells\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e were cultured in RPMI 1640 medium (EuroClone, Pero, Italy) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 15 mg/ml Gentamicin (complete RPMI) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eHuman Umbilical Vein Endothelial Cells (HUVEC) (Lonza, Basel, Switzerland) were cultured in EGM-2 medium (EuroClone, Pero, Italy) and used between passage 1 and 5. Before plating the cells, standard culture flasks were coated with 1.8% type B gelatine from bovine skin (Sigma-Aldrich, Missouri, USA).\u003c/p\u003e \u003cp\u003eHuman Lymphatic Fibroblasts (HLF) (ScienceCell, California, USA) cells were cultured in FM medium (ScienceCell, California, USA) and used between passage 3 and 15. Before plating the cells, standard culture flasks were coated with poly-L-lisine (Sigma-Aldrich, Missouri, USA) for 1h, then washed one time with sterile water.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBioreactor design and fabrication\u003c/h2\u003e \u003cp\u003eThe bioreactor (VesselBox) was designed with Fusion 360 (Autodesk, California, USA), and all the components were exported in .\u003cem\u003estl\u003c/em\u003e format. These files have been sliced to .\u003cem\u003egcode\u003c/em\u003e extension with the software Ultimaker Cura (Ultimaker, Utrecht, Netherlands) before being 3D printed (FDM) with a biocompatible material (Extrudr, Lauterach, Austria).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eScaffold preparation and perfusion\u003c/h2\u003e \u003cp\u003eBefore engineering the whole structure, we performed compatibility tests for HUVEC cells and HLFs in several biomaterials. In particular, the first cell type has been tested in GelXA LAMININK 411, CELLINK RGD, CELLINK Bioink, CELLINK Fibrin, Vitroink RGD, PureCol, GelXA Fibrin, GelMA Fibrin; selecting the latter as best material in terms of cell morphology and viability. For HLFs, we tested CELLINK Bioink, VitroInk RGD, GelXA Fibrin GelXA LAMININK 411, selecting the latter as best material according to the criteria listed above. Then, hosted scaffolds have been generated with a hybrid technique, combining 3D bioprinting and casting methods. Briefly, GelXA LAMININK 411 (CELLINK AB, Gothenburg, Sweden) was mixed with HLF (10x10\u003csup\u003e6\u003c/sup\u003e cells/mL) and bioprinted with the BIO X bioprinter (CELLINK AB, Gothenburg, Sweden) to generate a 7x7x6mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e scaffold, with a 5.5mm in diameter central channel. GelMA Fibrin (CELLINK AB, Gothenburg, Sweden) was heated up at 37\u0026deg;C in a laboratory bath until complete dissolution, mixed with HUVEC and HLF cells (3:1 ratio, final cell concentration 10x10\u003csup\u003e6\u003c/sup\u003e cells/mL) and manually cast to fill the hollow cavity.\u003c/p\u003e \u003cp\u003eImmediately, a 3D printed tool (diameter 2 or 2.5mm) was placed inside the casted material and photo-crosslinked with 405nM UV light for 120 seconds, tilting the scaffold 180\u0026deg; along the \u003cem\u003ez\u003c/em\u003e-axis after 60 seconds.\u003c/p\u003e \u003cp\u003eWe generated a 2 or 2.5mm lumen in diameter by gently removing the tool. The scaffolds were placed in EGM-2 medium supplemented with 10U/mL thrombin (Merck, New Jersey, USA) overnight (O/N) at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. The following day, the thrombin solution was replaced with fresh medium (EGM-2\u0026thinsp;+\u0026thinsp;FM medium, 3:1 is the best ratio defined to support high viability of both cell types) and the scaffolds were kept in static culture (multiwell plate) before perfusion, 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 into the bioreactor base. The adapters were screwed in the base to fit the lumen in the scaffold. The system was then filled with EGM-2\u0026thinsp;+\u0026thinsp;FM medium (3:1 ratio) and finally, the lid tightened. The flow perfusion (100\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\text{L}\\)\u003c/span\u003e\u003c/span\u003e/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 10mL EGM-2\u0026thinsp;+\u0026thinsp;FM medium (3:1 ratio). In dynamic settings, when no patients\u0026rsquo; cells are present, 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 17 and 21 in static condition compared to day 3 and 7 in dynamic conditions, respectively).\u003c/p\u003e \u003cp\u003eIn order to validate computational fluid-dynamics simulations, we performed a perfusion test in a hollow scaffold with Trypan blue. Briefly, after O/N culture in a multiwell plate in DMEM (EuroClone, Pero, Italy), we perfused the scaffold with DMEM (7mL) supplemented with 1mL of Trypan blue at 100\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\text{L}\\)\u003c/span\u003e\u003c/span\u003e/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 color of the construct.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComputational simulations\u003c/h2\u003e \u003cp\u003eTo support the bioreactor design, computational simulations were performed to assess both the fluid-dynamics of the medium flowing inside the bioreactor and the oxygen (O\u003csub\u003e2\u003c/sub\u003e) diffusion throughout the scaffold by using the commercial software COMSOL MultiPhysics\u0026reg; v5.6.\u003c/p\u003e \u003cp\u003eFor the numerical fluid-dynamic assessment by Computational Fluid Dynamics (CFD) simulations, two domains were identified:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe fluid volume within the lumen, where the culture medium is free to flow and the fluid motion was modeled with the Navier-Stokes equations (Eq.\u0026nbsp;1).\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\rho\\:\\bullet\\:\\left[\\frac{\\delta\\:v}{\\delta\\:t}+\\left(v\\bullet\\:\\:\\nabla\\:\\right)\\:v\\right]=-\\nabla\\:p+\\mu\\:\\:{\\nabla\\:}^{2}\\:v=F\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe hydrogel around the hollow channel, which was modeled as a homogeneous and isotropic porous medium defined by the properties of permeability and porosity; in this domain, Stokes-Brinkman equation was adopted to describe the fluid perfusion (Eq.\u0026nbsp;2).\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\rho\\:\\:\\left(\\varvec{v}\\bullet\\:\\:\\nabla\\:\\right)\\varvec{v}=\\nabla\\:\\cdot\\:[p\\varvec{I}+K]+\\varvec{F}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:v\\)\u003c/span\u003e\u003c/span\u003e the velocity field, \u0026#119901; the pressure (Pa), and F the mass forces vector field such as gravity (N m\u003csup\u003e\u0026minus;3\u003c/sup\u003e), \u003cb\u003eI\u003c/b\u003e is the identity matrix, \u003cb\u003eK\u003c/b\u003e is the permeability tensor of the porous material (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\epsilon\\:\\)\u003c/span\u003e\u003c/span\u003e is the porosity), defined as follows (Eq.\u0026nbsp;3):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{K}=\\mu\\:\\:\\left(\\frac{1}{\\epsilon\\:}\\right)(\\nabla\\:v+\\nabla\\:{v}^{T})-\\frac{2}{3}\\mu\\:\\left(\\frac{1}{\\epsilon\\:}\\right)(\\nabla\\:\\cdot\\:v)\\varvec{I}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn both domains, the culture media was considered as an incompressible, Newtonian fluid defined by density and dynamic viscosity. Different flow rates in the range of the values chosen for the experimental tests (i.e., 100 and 500\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\text{L}\\)\u003c/span\u003e\u003c/span\u003e/min) were applied at the inlet, taking as reference values that successfully exert a change on endothelial cells in vascularized scaffolds.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo estimate the O\u003csub\u003e2\u003c/sub\u003e concentration within the medium and hydrogel, the mass transport equation was used (Eq.\u0026nbsp;4):\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\nabla\\:\\bullet\\:(-\\:D\\:\\nabla\\:c)+\\:\\varvec{v}\\cdot\\:\\nabla\\:c=R\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere c is the concentration, R the O\u003csub\u003e2\u003c/sub\u003e volumetric consumption rate, D the diffusion coefficient of oxygen in the fluid and in the hydrogel, which is supposed to be mainly formed by GelMA and alginate.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe O\u003csub\u003e2\u003c/sub\u003e volumetric consumption rate of the cellular component was modelled with Michaelis\u0026ndash;Menten kinetics as following (Eq.\u0026nbsp;5).\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:R={V}_{max}\\bullet\\:\\left(\\frac{c}{{K}_{m}+c}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{V}_{max}\\)\u003c/span\u003e\u003c/span\u003e is the maximum molar consumption rate, \u003cem\u003ec\u003c/em\u003e is the local oxygen concentration function and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{m}\\)\u003c/span\u003e\u003c/span\u003e is the Michaelis\u0026ndash;Menten constant, corresponding to the oxygen concentration at which the consumption is half of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{V}_{max}\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:{V}_{max}\\)\u003c/span\u003e \u003c/span\u003edepends on the cellular density (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\rho\\:}_{c}\\)\u003c/span\u003e\u003c/span\u003e) and single-cell maximum oxygen consumption rate (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:sOCR\\)\u003c/span\u003e\u003c/span\u003e) (Eq.\u0026nbsp;6):\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:{V}_{max}={\\rho\\:}_{c}\\bullet\\:sOCR\\:\\:\\:$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eBoth \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:sOCR\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{m}\\)\u003c/span\u003e\u003c/span\u003e are specific for the considered cell types. In our simulation, we replicated the same cell types and densities present in the experimental set-up, i.e., HUVEC and fibroblasts around the channel and only fibroblasts within the hydrogel-based construct. An O\u003csub\u003e2\u003c/sub\u003e concentration value equal to 0.22mol/m\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e was set at the bioreactor inlet.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAll numerical values of the parameters and boundary conditions used for the simulations are reported in Tables\u0026nbsp;1 and 2, respectively. An automatic extra-fine triangular mesh was used. Velocity, pressure fields, shear stress, and O\u003csub\u003e2\u003c/sub\u003e concentration distribution throughout the perfusion system were computed and analyzed.\u003c/p\u003e \u003cp\u003eDimensionless parameters, such as Reynolds (Re) and Graez (Gz) numbers, were calculated.\u003c/p\u003e \u003cp\u003eReynolds number (Eq.\u0026nbsp;7) describes the state of the fluid motion: laminar when \u0026lt;\u0026thinsp;2300 and turbulent if 2300. It is calculated as:\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:Re=(\\rho\\:\\bullet\\:\\stackrel{-}{w}\\bullet\\:{D}_{h})/\\mu\\:$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\rho\\:\\)\u003c/span\u003e\u003c/span\u003e is the fluid density, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:w\\)\u003c/span\u003e\u003c/span\u003e the velocity vector, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{D}_{h}\\)\u003c/span\u003e\u003c/span\u003e is the hydraulic perimeter.\u003c/p\u003e \u003cp\u003eThe Graetz number (Eq.\u0026nbsp;8) represents the relationship between the characteristic time of diffusion,\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{t}_{diff}\\)\u003c/span\u003e\u003c/span\u003e and the characteristic time of convection, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{t}_{conv}\\)\u003c/span\u003e\u003c/span\u003e,\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:Gr=\\:{t}_{diff}/{t}_{conv}=({{D}_{h}}^{2}\\bullet\\:\\stackrel{-}{w})/D\\bullet\\:L$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\rho\\:\\)\u003c/span\u003e\u003c/span\u003e is the fluid density, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:w\\)\u003c/span\u003e\u003c/span\u003e the average fluid velocity, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{D}_{h}\\)\u003c/span\u003e\u003c/span\u003e the hydraulic perimeter, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003e the fluid viscosity, L the length channel, and D the oxygen diffusion coefficient.\u003c/p\u003e \u003cp\u003eWe performed the same simulation performed for replicating the experimental experiments (only considering a flow rate equal to 100\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\text{L}\\)\u003c/span\u003e\u003c/span\u003e/min), but by setting a higher and smaller value of diffusion coefficient for oxygen \u003cb\u003e(See Supplementary Table\u0026nbsp;1)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIf we assumed 0.04mM as a critical value under which cells are considered to be no more functional in human tissues/organs, also with the lower value of diffusion coefficient this threshold is satisfied. However, far from the perfused channel, the discrepancy in terms of oxygen concentration is more evident when a lower value of this parameter is used. For example, compared to the \u0026ldquo;normal condition\u0026rdquo; (with D\u0026thinsp;=\u0026thinsp;2.30 x 10\u003csup\u003e9\u003c/sup\u003e m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), at a distance of 2mm from the perfused channel, the decrease of O\u003csub\u003e2\u003c/sub\u003e concentration is equal to 15%, while at the edge of the construct (h\u0026thinsp;=\u0026thinsp;3.5mm from the perfused channel) the oxygen decrease is equal to 26%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLive/Dead assay\u003c/h2\u003e \u003cp\u003eTo assess embedded cells viability after manufacturing and before scaffold perfusion (days 14 post-printing), we used the LIVE/DEAD\u0026reg; 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 and phenol red (Thermo Fisher Scientific, Massachusetts, USA) and Live/Dead reagent was added in a 1:3 ratio (reagent:medium). After 30 minutes of incubation at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e the constructs were washed one time with DMEM without serum and phenol red, observed with the Olympus FluoVIEW 3000 RS confocal microscope and then processed using FIJI (ImageJ) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF)\u003c/h2\u003e \u003cp\u003eAt defined time-points, cell-laden scaffolds either from static (multiwell) or dynamic (VesselBox) culture were fixed with 4% PFA in Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, Texas, USA) 2h at room temperature (RT). After fixation, the scaffolds were washed twice with Hank\u0026rsquo;s Balanced Salt Solution (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 slices were permeabilized (1mg/mL BSA, 10% FBS and 0.3% Triton X in PBS) for 30 minutes RT and then stained overnight 4\u0026deg;C with primary antibodies for CD31 (Abcam, Cambridge, UK), VE-Cadherin (Cell Signaling, Massachusetts, USA), collagen IV (Invitrogen, Massachusetts, USA) diluted 1:100 in blocking solution (1mg/mL BSA, 10% FBS in PBS) and vWF (Santa Cruz Biotechnology, Texas, USA) diluted 1:50 in blocking solution. Scaffolds perfused with leukemic primary cells were also stained for CD45 (BD Biosciences, New Jersey, USA) diluted 1:100 in blocking solution. The following day, the scaffolds were washed twice with HBSS (Euroclone, Pero, Italy) for 5 minutes RT and incubated always at 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 1:250 in blocking solution. After washing 2 times with HBSS (Euroclone, Pero, Italy), Hoechst 33342 (Invitrogen, Massachusetts, USA) in PBS (1:2000) was added for 10 minutes RT. Images were acquired with Olympus FluoVIEW 3000 RS confocal microscope and then processed using FIJI (ImageJ) and Arivis software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExtravasation studies\u003c/h2\u003e \u003cp\u003eTo prove the whole system is useful for investigating leukemic cells\u0026rsquo; behavior and migration, MEC1-GFP cells (1x10\u003csup\u003e6\u003c/sup\u003e cells/mL) or CLL primary cells (5x10\u003csup\u003e6\u003c/sup\u003e cells/mL) were resuspended in the medium reported above, put in the reservoir and made them recirculate within the system for 1, 3 and 7 days after 14 days of static maturation of the scaffolds. At defined time-points, the circulating cells were recovered and analyzed by flow cytometry, while the perfused scaffolds were fixed and eventually stained to localize extravasated leukemic cells, together with the architecture of the microenvironment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eAfter 1, 3 and 7 days of selective perfusion within the vascularized scaffold hosted in the VesselBox device, leukemic cell line MEC1-GFP and primary cells from patients were recovered and stained 25 minutes RT for the following markers: CD19, IgD, IgM, CD62L, CD29, CD49d, CXCR4, CD45, CCR7, CD40, CD69 (Miltenyi, Bergisch Gladbach, Germany), CD80, CD38, CD23 (BD Biosciences, New Jersey, USA), CD5 (Beckman Coulter, California, USA). After washing with PBS for 5 minutes, 1500 RPM, cells were analyzed with CytoFlex LX flow cytometer (Beckman Coulter, California, USA). Data were processed with FCS Express (De Novo Software, California, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and real-time PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eRNA extraction was performed on primary cells at basal level (for freshly used cells the basal level corresponds to the isolated cells from the peripheral blood, for frozen cells the basal level corresponds to thawed cells after 1 hour of adaptation in static culture) and at different time-points (1, 3 and 7 days of selective perfusion) using ReliaPrep RNA Cell Miniprep System\u0026reg; (Promega, Madison, USA) following the manufacturer\u0026rsquo;s protocol. cDNA was synthesized according to the manufacturer\u0026rsquo;s protocol using the RevertAid\u0026reg; H Minus First Strand DNA Synthesis kit (Thermo Fisher Scientific, Massachusetts, USA). RT-qPCR analysis was performed using TaqMan\u0026trade; Fast Advanced Master Mix (Applied Biosystems, Massachusetts, USA) and TaqMan\u0026trade; gene expression probes (Applied Biosystems, Massachusetts, USA) with CFX96 Real-Time PCR Detection System (BioRad, California, USA). The analysis was performed in duplicate. Quantification of CXCR4, CD62L, and CD23 transcripts (Applied Biosystem probes) was performed according to the Ct method,\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e using YWHAZ as the housekeeping gene.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC.S. acknowledges financial support from EHA Advanced Research Grant 2020. Associazione Italiana per la Ricerca sul Cancro AIRC under IG 2018 - ID. 21332 and Special Program on Metastatic Disease \u0026ndash; 5 per mille #2119. We acknowledge Alembic and Fractal facilities. Schemes in figure 3 have been created with BioRender.com.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAUTHOR CONTRIBUTION\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRP, MP, FB performed the experiments; RP, MC, CS analyzed the data; CS and MC supervised the activity; RP, GMDG, CS wrote the manuscript; LS, PG, provided patients\u0026apos; and clinical information; MC, CS, PG revised the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRP and CS are inventors of the patent pending bioreactor technology.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003cbr\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCrassini, K., Shen, Y., Mulligan, S. \u0026amp; Giles Best, O. 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Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. \u003cem\u003eMethods\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 402\u0026ndash;408 (2001).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chronic Lymphocytic Leukemia, bioprinting, trafficking, vascularization, bioreactor, microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-6503832/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6503832/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in Western countries, marked by the accumulation of CD5\u003csup\u003e+\u003c/sup\u003e B cells in blood and lymphoid tissues. The vascular system plays a critical role, as malignant cells interact with endothelial cells through poorly understood mechanisms. To study CLL dissemination, we developed VesselBox, a modular bioreactor for selective perfusion of a milli-scale vessel-like structure within a lymphoid microenvironment scaffold, created using 3D bioprinting and casting. Numerical simulations optimized perfusion parameters for cell homeostasis. Vessel maturation, confirmed by endothelial markers (CD31, Ve-cadherin, Von Willebrand Factor, collagen IV), showed VesselBox sustains perfusion for up to 7 days. By recirculating CLL cells, we validated its use for studying extravasation and immunophenotype characterization. This pioneering device enables \u003cem\u003eex vivo\u003c/em\u003e analysis of CLL dissemination, offering potential to uncover new therapeutic targets by examining circulating and extravasated cells with or without drugs.\u003c/p\u003e","manuscriptTitle":"Controlled perfusion of a vascularized microenvironment within a 3D printed bioreactor to study leukemia cells trafficking ex-vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 06:23:53","doi":"10.21203/rs.3.rs-6503832/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab0343e0-dbc9-40b5-b6cd-66d9f8b9d5fb","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47904093,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":47904094,"name":"Biological sciences/Biological techniques/Biological models/Cancer models"},{"id":47904095,"name":"Biological sciences/Immunology/Lymphocytes/B cells"},{"id":47904096,"name":"Biological sciences/Cancer/Haematological cancer/Leukaemia/Chronic lymphocytic leukaemia"}],"tags":[],"updatedAt":"2025-08-20T13:08:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-02 06:23:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6503832","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6503832","identity":"rs-6503832","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}