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The human vasculature is essential in organs and tissues for the transport of nutrients, metabolic waste products, and the maintenance of homeostasis. The integration of vessels in in vitro organs-on-chip may, therefore, improve the similarity to the native organ microenvironment, ensuring proper physiological functions and reducing the gap between experimental research and clinical outcomes. This gap is particularly evident in drug testing and the use of vascularized models may provide more realistic insights into human responses to drugs in the pre-clinical phases of the drug development pipeline. In this context, different vascularized liver models have been developed to recapitulate the architecture of the hepatic sinusoid, exploiting either porous membranes or bioprinting techniques. In this work, we developed a method to generate perfusable vascular channels with a circular cross section within organs-on-chip without any interposing material between the parenchyma and the surrounding environment. Through this technique, vascularized liver sinusoid-on-chip systems with and without the inclusion of the space of Disse were designed and developed. The recapitulation of the Disse layer, therefore, a gap between hepatocytes and endothelial cells physiologically present in the native liver milieu, seems to enhance hepatic functionality (e.g., albumin production) compared to when hepatocytes are in close contact with endothelial cells. These findings pave the way to numerous further uses of microfluidic technologies coupled with vascularized tissue models (e.g., immune system perfusion) as well as the integration within multiorgan-on-chip settings. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is a deadly cancer with limited treatment options. The development of new therapies is urgently needed and Organ-On-Chip has emerged as a promising tool for studying diseases in a more reliable 3D environment. In this study, we evaluated the reliability of CCA-on-chip as a patient-specific platform by integrating the immune cells and assessing their migration based on patient characteristics. Primary CCA cells were isolated from surgically resected patients at Humanitas Research Hospital. The CCA microenvironment was recapitulated in the device by co-culturing CCA cells and cancer-associated fibroblasts (CAFs) in the central channel, flanked by endothelial cells in one lateral channel. T-cells exhibited a high ability to migrate within the tumor niche, spreading throughout the central channel. Two culture conditions were compared to investigate the influence of crosstalk between CCA cells and CAFs on T-cell migration. In the monoculture, T-cells showed greater trafficking compared to the co-culture. Furthermore, T-cells formed aggregates surrounding tumor spheroids in the monoculture, while they appeared dispersed in the co-culture. Interestingly, the co-culture exhibited higher levels of immunosuppressive molecules, suggesting that CAFs could contribute to an immunosuppressive microenvironment. Furthermore, in immunohistochemistry, CCA patient-derived cells were divided into two groups according to CD3+ cells: high-infiltrating (HOT) patients showed increased T-cell migration compared to low-infiltrating (COLD) patients. T-cells exhibited higher migration in the monoculture in HOT and COLD patients compared to their respective co-culture, further corroborating the role of CAFs in influencing immune cell recruitment and immunosuppression. Indeed, the expression of chemoattractant or immunosuppressive molecules, such as CXCL9, CXCL10, IL6 and IL10, varied between HOT and COLD patients and between culture conditions, suggesting their roles in immune cell recruitment and immunosuppression. Our CCA-on-chip platform recapitulates the heterogeneity of the tumor microenvironment, demonstrating differences in T-cell trafficking and the expression of immunomodulatory molecules between high-infiltrating and low-infiltrating patients. [ABSTRACT FROM AUTHOR]

Intrahepatic cholangiocarcinoma (iCCA) is a deadly cancer of biliary epithelium with generally unsuccessful therapeutic options. This highlights the importance of deeper decipher iCCA mechanisms to develop new effective therapeutic strategies. Nevertheless, in-vivo cell interaction complexity has hindered an effective recapitulation of the in-vitro human milieu through 2D culture systems. In the last years, strong efforts were focused on the Organ-On-Chip (OoC) as promising models to faithfully recapitulate the in-vivo tumor niche. In this study, we aimed to develop an in-vitro 3D microfluidic device by co-culturing three cell types involved in iCCA. Primary iCCA cells were isolated from patients surgically resected at Humanitas Research Hospital. The microfluidic device was fabricated at Polytechnic of Milan, composed of three microfluidically interconnected channels. iCCA microenvironment was recapitulated by co-culturing iCCA cells and cancer-associated fibroblasts (CAFs) in the central channel with an ad hoc medium and embedded in an optimized hydrogel, flanked by an endothelial tubule in the lateral channel. The 3D cellular organization was visualized using confocal microscopy and the significant increase in the expression of key phenotypic cell markers was assessed by q RT-PCR, compared to 2D culture system. Diffusion assays at small and large molecules showed the high biocompatibility of this platform and the functional integrity of the endothelial tubule. Subsequently, the mechanical and biological proprieties of the platform were evaluated overtime in culture, showing that the cross-talk established between iCCA cells and CAFs within the chip led to a deep extracellular matrix remodeling. Indeed, scanning electron microscopy (SEM) allowed to measure the dimension of the matrix pores and immunofluorescence assay revealed a significant increase in the collagen IV deposition within the hydrogel. Our results showed that iCCA-on-chip provides a reliable 3D platform able to mimic the in-vivo iCCA microenvironment and may represent useful tool to investigate patient-specific therapeutic strategies. [ABSTRACT FROM AUTHOR]

Thrombosis of ventricular assist devices (VADs) compromises their performance, with associated risks of systemic embolization, stroke, pump stop and possible death. Anti-thrombotic (AT) drugs, utilized to limit thrombosis, are largely dosed empirically, with limited testing of their efficacy. Further, such testing, if performed, typically examines efficacy under static conditions, which is not reflective of actual shear-mediated flow. Here we adopted our previously developed Device Thrombogenicity Emulation methodology to design microfluidic platforms able to emulate representative shear stress profiles of mechanical circulatory support (MCS) devices. Our long-term goal is to utilize these systems for point-of-care (POC) personalized testing of AT efficacy under specific, individual shear profiles. First, we designed different types of microfluidic channels able to replicate sample shear stress patterns observed in MCS devices. Second, we explored the flexibility of microfluidic technology in generating dynamic shear stress profiles by modulating the geometrical features of the channels. Finally, we designed microfluidic channel systems able to emulate the shear stress profiles of two commercial VADs. From CFD analyses, the VAD-emulating microfluidic systems were able to replicate the main characteristics of the shear stress waveforms of the macroscale VADs (i.e. shear stress peaks and duration). Our results establish the basis for development of a lab-on-chip POC system able to perform device-specific and patient-specific platelet activation state assays. [ABSTRACT FROM AUTHOR]