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With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human hepatic tissues in vitro. Here, we introduce a platform for routine grafting of liver and other tissues on an in vitro grown microvascular bed. The platform consists of 64 microfluidic chips patterned underneath a 384-well microtiter plate. Each chip allows the formation of a microvascular bed between two main lateral vessels by inducing angiogenesis. Chips consist of an open-top microfluidic chamber, which enables addition of a target tissue by manual or robotic pipetting. Upon grafting a liver microtissue, the microvascular bed undergoes anastomosis, resulting in a stable, perfusable vascular network. Interactions with vasculature were found in spheroids and organoids upon 7 days of co-culture with space of Disse-like architecture in between hepatocytes and endothelium. Veno-occlusive disease was induced by azathioprine exposure, leading to impeded perfusion of the vascularized spheroid. The platform holds the potential to replace animals with an in vitro alternative for routine grafting of spheroids, organoids, or (patient-derived) explants.

The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here we present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. We generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel-tumor cell coculture assay.

Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1-3 weeks), as well as for generating and characterizing tubuloid cell-derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Its incidence is increasing, and is closely related to advanced liver disease. Interactions in the HCC microenvironment between tumor cells and the associated stroma actively regulate tumor initiation, progression, metastasis, and therapy response. In the present study, we used the OrganoPlate graft to establish a co-culture system consisting of dissociated HCC tumors (HCC 1-8) and cell lines, HCC derived fibroblasts and vasculature. Cultures were prepared and validated by assessing their response to Sorafenib and Lenvatinib (72 hours). Cultures had their viability (alamar blue assay), and chemokine/cytokine levels in the supernatant (Luminex) determined. In addition, the organization of the vasculature in the tumor compartment was studied through immunostainings, confocal imaging, and subsequent morphological analyses. HCC models were characterized by a range of specific markers, tumor (albumin), endothelial (CD31 and VE-Cadherin) and stromal (aSMA) cells. CD31 immunostained cultures were imaged, and morphology changes quantified. Sorafenib and Lenvatinib induced changes in the tumor vasculature area and organization. Hereby, we present vascularized patient-derived HCC models that include relevant cellular players of the HCC microenvironment. These co-cultures are highly suitable for studying specific cell types as well as patient-specific responses. We envision that this patient derived model will evolve to become a platform for understanding the interplay between angiogenesis, stroma and immune infiltrate in HCC. Citation Format: Orsola Mocellin, Abbie Robinson, Aleksandra Olczyk, Stephane Treillard, Thomas Olivier, Chee Ng, Jeroen Heijmans, Désiréé Goubert, Arthur Stok, Gilles van Tienderen, Monique Verstegen, Sebastian Trietsch, Henriëtte Lanz, Paul Vulto, Jos Joore, Karla S. Queiroz. Sorafenib and Lenvatinib induce vascular responses in patient derived HCC on-Chip models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5876.

Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease affecting upper and lower motor neurons with no cure available. Clinical and animal studies reveal that the neuromuscular junction (NMJ), a synaptic connection between motor neurons and skeletal muscle fibers, is highly vulnerable in ALS and suggest that NMJ defects may occur at the early stages of the disease. However, mechanistic insight into how NMJ dysfunction relates to the onset and progression of ALS is incomplete, which hampers therapy development. This is, in part, caused by a lack of robust in vitro models. The ability to combine microfluidic and induced pluripotent stem cell (iPSC) technologies has opened up new avenues for studying molecular and cellular ALS phenotypes in vitro. Microfluidic devices offer several advantages over traditional culture approaches when modeling the NMJ, such as the spatial separation of different cell types and increased control over the cellular microenvironment. Moreover, they are compatible with 3D cell culture, which enhances NMJ functionality and maturity. Here, we review how microfluidic technology is currently being employed to develop more reliable in vitro NMJ models. To validate and phenotype such models, various morphological and functional read-outs have been developed. We describe and discuss the relevance of these read-outs and specifically illustrate how these read-outs have enhanced our understanding of NMJ pathology in ALS. Finally, we share our view on potential future directions and challenges.

Development of efficient drugs and therapies for the treatment of inflammatory conditions in the intestine is often hampered by the lack of reliable, robust, and high-throughput in vitro and in vivo models. Current models generally fail to recapitulate key aspects of the intestine, resulting in low translatability to the human situation. Here, an immunocompetent 3D perfused intestine-on-a-chip platform was developed and characterized for studying intestinal inflammation. Forty independent polarized 3D perfused epithelial tubular structures were grown from cells of mixed epithelial origin, including enterocytes (Caco-2) and goblet cells (HT29-MTX-E12). Immune cells THP-1 and MUTZ-3, which can be activated, were added to the system and assessed for cytokine release. Intestinal inflammation was mimicked through exposure to tumor necrosis factor-α (TNFα) and interleukin (IL)-1β. The effects were quantified by measuring transepithelial electrical resistance (TEER) and proinflammatory cytokine secretion on the apical and basal sides. Cytokines induced an inflammatory state in the culture, as demonstrated by the impaired barrier function and increased IL-8 secretion. Exposure to the known anti-inflammatory drug TPCA-1 prevented the inflammatory state. The model provides biological modularity for key aspects of intestinal inflammation, making use of well-established cell lines. This allows robust assays that can be tailored in complexity to serve all preclinical stages in the drug discovery and development process.

Background: Renal ischemia/reperfusion injury (rIRI) is one of the major causes of acute kidney injury (AKI). While animal models are suitable for investigating systemic symptoms of AKI they are limited in translatability. Human in vitro models are crucial in giving mechanistic insights into rIRI, however, they miss out on crucial aspects as reperfusion injury and the multi tissue aspect of AKI. Methods: We advanced the current renal proximal tubule-on-a-chip model to a coculture model with a perfused endothelial vessel separated by an extracellular matrix (ECM). The coculture was characterized for its three-dimensional structure, protein expression, and response to nephrotoxins. Then, rIRI was captured through control of oxygen levels, nutrient availability, and perfusion flow settings. Injury was quantified through morphological assessment, caspase 3/7 activation, and cell viability. Results: The combination of low oxygen, reduced glucose, and interrupted flow was potent to disturb the proximal tubules. This effect was strongly amplified upon reperfusion. Endothelial vessels were less sensitive to the ischemia-reperfusion parameters. Adenosine treatment showed a protective effect on the disruption of the epithelium and on the caspase 3/7 activation. Conclusions: A human in vitro rIRI model was developed using a coculture of a proximal tubule and blood vessel on-a-chip, which was used to characterize the renoprotective effect of adenosine. The robustness of the model and assays in combination with the throughput of the platform make it ideal to advance pathophysiological research and enable the development of novel therapeutic modalities.

The study of epithelial barrier properties in the human body is of paramount interest to a range of disciplines, including disease modeling, drug transport studies, toxicology, developmental biology, and regenerative biology. Current day in vitro studies largely rely on growing epithelial cells in a static environment on membrane cell culture inserts. With the advancement of microfluidic and organ-on-a-chip techniques it became possible to culture 3D intestinal tubules directly against an extracellular matrix (ECM) under flow and without the need for artificial membranes. Here we describe detailed protocols for culturing epithelial tubules in a high-throughput format, assessing their permeability and marker expression. The platform harbors 40 independent microfluidic chips in a microtiter plate format. The resulting 40 epithelial tubules are analyzed in parallel using a high-content microscopy. Protocols described here allow for adoption and routine application of microfluidic techniques by nonspecialized end-users.

The study of epithelial barrier properties in the human body is of paramount interest to a range of disciplines, including disease modeling, drug transport studies, toxicology, developmental biology, and regenerative biology. Current day in vitro studies largely rely on growing epithelial cells in a static environment on membrane cell culture inserts. With the advancement of microfluidic and organ-on-a-chip techniques it became possible to culture 3D intestinal tubules directly against an extracellular matrix (ECM) under flow and without the need for artificial membranes. Here we describe detailed protocols for culturing epithelial tubules in a high-throughput format, assessing their permeability and marker expression. The platform harbors 40 independent microfluidic chips in a microtiter plate format. The resulting 40 epithelial tubules are analyzed in parallel using a high-content microscopy. Protocols described here allow for adoption and routine application of microfluidic techniques by nonspecialized end-users.

Assessment of epithelial barrier function is critically important for studying healthy and diseased biological models. Here we introduce an instrument that measures transepithelial electrical resistance (TEER) of perfused epithelial tubes in the microfluidic OrganoPlate platform. The tubules are grown in microfluidic channels directly against an extracellular matrix, obviating the need for artificial filter membranes. We present TEER measurements on Caco-2 intestinal and renal proximal tubule epithelium. Forty tubules on one single plate were interrogated in less than a minute. We show that TEER measurement is significantly more sensitive than a fluorescent reporter leakage assay in response to staurosporine. We demonstrate a 40-channel time-lapse data acquisition over a 25 hour time period under flow conditions. We furthermore observed a 50% reduction in Caco-2 TEER values following exposure to a cocktail of inflammatory cytokines. To our best knowledge, this is the first instrument of its kind that allows routine TEER studies in perfused organ-on-a-chip systems without interference by artificial filter membranes. We believe the apparatus will contribute to accelerating routine adoption of perfused organ-on-a-chip systems in academic research and in industrial drug development.

We report a method to generate a 3D motor neuron model with segregated and directed axonal outgrowth. iPSC-derived motor neurons are cultured in extracellular matrix gel in a microfluidic platform. Neurons extend their axons into an adjacent layer of gel, whereas dendrites and soma remain predominantly in the somal compartment, as verified by immunofluorescent staining. Axonal outgrowth could be precisely quantified and was shown to respond to the chemotherapeutic drug vincristine in a highly reproducible dose-dependent manner. The model was shown susceptible to excitotoxicity upon exposure with excess glutamate and showed formation of stress granules upon excess glutamate or sodium arsenite exposure, mimicking processes common in motor neuron diseases. Importantly, outgrowing axons could be attracted and repelled through a gradient of axonal guidance cues, such as semaphorins. The platform comprises 40 chips arranged underneath a microtiter plate providing both throughput and compatibility to standard laboratory equipment. The model will thus prove ideal for studying axonal biology and disease, drug discovery and regenerative medicine.

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1-3 weeks), as well as for generating and characterizing tubuloid cell-derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

Recent clinical success of immune checkpoint inhibitors and chimeric antigen receptor T cells has highly increased the attention for the field of immunotherapy. However, identifying responders to these therapies is challenging underscoring the necessity for translational models that increase understanding of tumor-immune responses. In the present study, a co-culture system containing immune cells and vasculature was established. Both are essential components of the tumor microenvironment and very often lacking in in vitro tumor models, highlighting the added value of our co-culture platform. We focused on optimizing endothelial and CD8+ T cell co-cultures and subsequently assessing T cell migration from the endothelial tubes via endothelial sprouts towards various chemoattractants. In order to generate stratified 3D co-cultures, the Mimetas OrganoPlate Graft containing 64 microfluidic culture units was used. The microfluidic units in this platform are composed of two parallel microfluidics channels and a central chamber. The two parallel microfluidic channels were used for generating parallel endothelial tubules, whilst angiogenic factors (S1P, VEGF, bFGF and PMA) were added to the central chamber of the culture unit resulting in a generation of a gradient and sprouting of the endothelial tubes towards the central chamber. Generated sprouts were stable and perfusable. The central chamber is designed for culturing complex microtissues such as spheroids, organoids and explants. Angiogenic endothelial tubules formed vascular beds in presence of added factors within 3-5 days. Once vascular beds were formed, activated and fluorescently labeled CD8+ T cells were loaded in the endothelial tubules and followed in culture for 48 hours. CD8+ T cell migration was observed both via the sprouts as well as by crossing the endothelial barrier, and increased in presence of gradients of CCL2, CCXl12 and CCL9. Highest CD8+ T cell numbers were observed in presence of a gradient generated with a mix of these three chemokines. Therefore, we present a high throughput co-culture system containing angiogenic endothelial tubules and CD8+ T cells. These co-cultures are highly suitable for studying T cell migration, event which precedes the detection and recognition of antigens at the surface of antigen-presenting cells and for interactions with other cells involved in the immune response. In addition, these co-cultures serve as a platform for understanding the interplay between T cell migration and angiogenesis in the tumor microenvironment. Furthermore, we envision that this model will evolve into an immunocompetent patient-derived tumor model that can be used to study immune responses to tumors. Citation Format: Luuk de Haan, Sacha Spelier, Johnny Suijker, Erik Wallinga, Lenie van den Broek, Henriette Lanz, Jos Joore, Paul Vulto, Karla Queiroz. Modelling T Cell-Vasculature co-culture in a high-throughput microfluidic platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2777.

The field of Organ-on-a-Chip is rapidly shifting from academic proof-of-concept studies to real-world solutions. The challenge is now to enhance end-user adoption by improving user friendliness, compatibility, assay ability and product readiness of these solutions. This review evaluates Organ-on-a-Chip efforts published over the last two years in light of such end-user adoption aspects. Elegant platforms have been reported including a microtiter plate-based 3D cell culture platform and a platform of cantilevers with integrated gauge sensors for contractility measurement. Also functional assays for angiogenesis, calcium imaging of neurons and neuro-muscular contractility were reported. Compatibility with standard analysis techniques such as sequencing, fluorescent activated cell sorting and mass spectrometry were reported only in rare cases. It is concluded that the elements that enable the leap towards end-user adoption are in place, but only few systems have managed to incorporate all aspects, and are able to answer biological questions.

Background One of the major functions of the human intestine is to provide a protective epithelial barrier between the body and digestive environment. Additionally, the interplay of commensal microbes of the gut microbiome with the gut tissue and host immune system significantly contributes to intestinal homeostasis. Crohn’s disease and ulcerative colitis, collectively referred to as inflammatory bowel diseases, are both associated with increased permeability of the epithelial barrier and dysregulated immune response. Great efforts have been made to develop both in vitro and in vivo models of the human intestine in order to understand the development and underlying pathogenesis of IBD. These efforts have provided valuable insights into multiple aspects of the disease. However, none of these models has been able to capture the complexity and multifactorial nature of IBD. Animal models generally fail to accurately predict the efficacy and toxicity of novel compounds in human tissues, while in vitro human intestinal models developed on porous membranes within Transwell inserts fail to accurately recapitulate and mimic key aspects of the in vivo situation. Methods Here, we present the development and characterisation of a 3D multicellular perfused intestine-on-a-chip model in a microfluidic platform, the OrganoPlate®, and its application for investigating intestinal inflammation. The model described here comprises a coculture of Caco-2 and mucus-secreting HT29-MTX cells in the top compartment of the chip and a coculture of immune-competent cells THP-1 and MUTZ-3 in the bottom compartment, lining a collagen-I ECM in the middle. Results We show that the Caco-2 and HT29-MTX coculture form confluent and polarised tubular structures against the collagen-I ECM in the OrganoPlate®, with a stable barrier function over time as well as the capability to secrete mucus. By exposing the cultures to TNFα and/or IL-1β, we were able to induce an inflammatory state, characterised by cytokine release (IL-8) and a decrease in trans-epithelial electrical resistance. Finally, we proved the applicability of the model in screening anti-inflammatory compounds by its reversibility. Using a well-known anti-inflammatory drug, TPCA-1, we were able to prevent cytokine-induced inflammation. This result was evident from the decreased secretion of IL-8 and retention of barrier function in treated cultures, similar to that observed in untreated cultures. Conclusion Overall, this complex 3D multicellular perfused intestine-on-a-chip model provides the versatile modularity of mimicking key features of intestinal inflammation and can, therefore, further support drug screening efforts and provide a platform for personalised medicine.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most lethal cancers due to a high chemoresistance and poor vascularization, which results in an ineffective systemic therapy. PDAC is characterized by a high intratumoral pressure, which is not captured by current 2D and 3D in vitro models. Here, we demonstrated a 3D microfluidic interstitial flow model to mimic the intratumoral pressure in PDAC. We found that subjecting the S2-028 PDAC cell line to interstitial flow inhibits the proliferation, while maintaining a high viability. We observed increased gemcitabine chemoresistance, with an almost nine-fold higher EC50 as compared to a monolayer culture (31 nM versus 277 nM), and an alleviated expression and function of the multidrug resistance protein (MRP) family. In conclusion, we developed a 3D cell culture modality for studying intratissue pressure and flow that exhibits more predictive capabilities than conventional 2D cell culture and is less time-consuming, and more scalable and accessible than animal models. This increase in microphysiological relevance might support improved efficiency in the drug development pipeline.

Contains fulltext : 170854.pdf (Publisher’s version ) (Closed access) Improved model systems to predict drug efficacy, interactions, and drug-induced kidney injury (DIKI) are crucially needed in drug development. Organ-on-a-chip technology is a suitable in vitro system because it reproduces the 3D microenvironment. A kidney-on-a-chip can mimic the structural, mechanical, transport, absorptive, and physiological properties of the human kidney. In this review we address the application of state-of-the-art microfluidic culturing techniques, with a focus on culturing kidney proximal tubules, that are promising for the detection of biomarkers that predict drug interactions and DIKI. We also discuss high-throughput screening and the challenges for in vitro to in vivo extrapolation (IVIVE) that will need to be overcome for successful implementation.

Head and neck squamous cell carcinoma (HNSCC) cancer is a broad category of tumor types arising from the inner lining of various anatomic structures including the oral cavity, pharynx, larynx and salivary glands. Treatment often involves an intensive combination of surgery, radiotherapy and chemotherapy. Despite this, tumor recurrence rates remain high and survival rates are relatively poor. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate®, a microfluidic based 3D-culture system, and HN cancer-derived organoids. MIMETAS develops Organ-on-a-Chip-based models for evaluation of new medicines. Our unique microfluidic technology enables testing of compounds on miniaturized 3D organ models in high-throughput. Hereby we show the establishment of HNSCC cancer-derived organoids in 2-lane OrganoPlate®, and its usefulness for phenotypic drug screenings. The aim of the study is to capture differential sensitivity of HN organoid lines to either a common platinum-based chemotherapy or to a targeted drug still not used in the clinic. Organoid lines (T1, T3 and T5) were embedded in the 2-lane OrganoPlate® as single cells in an extracellular matrix gel. At day 2, cultures were treated with cisplatin or PARP-inhibitor niraparib for 120 hours. Additionally, cultures exposed to high doses of cisplatin were evaluated after one week of recovery in order to determine treatment failure and recapitulate potential patient relapse. Drug response was evaluated by assessment of morphology (phase contrast), cell viability (total nuclei count, alamar blue assay), proliferation (EdU incorporation) and DNA damage (phosphorylated histone H2AX). Organoid cultures grow well under medium perfusion in the 2-lane OrganoPlate® and selective sensitivity to cisplatin and niraparib is revealed by the used readouts. The high-throughput, microfluidic 2-lane OrganoPlate® platform offers an attractive method for growing HNSCC cancer-derived organoids, supporting development of individualized tumour models for phenotypic drug screenings. Citation Format: Silvia Bonilla, Else Driehuis, Henriëtte Lanz, Hans Clevers, Jos Joore, Karla Queiroz, Paul Vulto. A novel HNSCC organoid-on-a-chip platform for preclinical investigation and drug screening [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2794.

Predicting clinical response to anticancer drugs remains a major challenge in cancer therapy research. The current treatment of patients with ovarian cancer involves surgery and platinum-based chemotherapy, which still result in recurrence on advanced stages of the disease. During tumor progression, cancerous cells continuously accumulate mutations giving rise to a heterogeneous population of cells. Additionally, spatial distribution of stromal, endothelial and immune cells in the microenvironment affects tumor progression, cell morphology, and ultimately drug response. In order to predict clinical outcome of chemotherapy in vitro, tumor heterogeneity and microenvironment constituents must be conserved. Current models of tumor biology and microenvironment consist in xenografts of human tumors implanted in immunodeficient mice. These models allow studying systemic treatment response, but its application is limited to small developmental studies. Here, we present a high throughput in vitro 'grafting' platform where we co-culture blood vessels with tumor explants. Each unit in this platform is composed of two parallel microfluidic channels and a central chamber. Two endothelial tubules are generated in the microfluidic channels and cultured in presence of a gradient of angiogenic factors (S1P, VEGF, bFGF and PMA) added to the central chamber of the culture unit. Angiogenic tubules form vascular beds within 3-5 days, after which tumor explants are loaded to the central chamber on top of the vascular beds. The model shown in this study consists of ovarian serous papillary adenocarcinoma collected after xenograft growth in immunodeficient mice. This ovarian cancer explant was previously characterized as resistant to Paclitaxel. In this study, we observe how the vascular bed remodels in the presence of the explant and closely interacts with ovarian tumor tissue. Vessel perfusion and stabilization of vascular bed was monitored by real time imaging of 150 kDa FITC-Dextran. Cultures were evaluated by assessment of morphology and presence of endothelial and tumor cell biomarkers. Moreover, co-culture response to Sorafenib (anti-angiogenic), Palbociclib and Paclitaxel was detected by distinctive proliferation rates as compared to control conditions. The established ovary cancer-on-a-chip platform enables the study of fundamental aspects of tumor disease and progression. In addition, these co-cultures serve as a platform for understanding tumor-endothelial cell crosstalk and its consequences for tumor aggressiveness. Moreover, these models constitute a suitable platform for drug screenings of anti-cancer and anti-angiogenic compounds, making them a powerful translational tool for drug selection in personalized medicine applications. Citation Format: Silvia Bonilla, Jean-François Mirjolet, Pauline Berger, Elodie Rajon, Erik Walinga, Remko van Vught, Henriëtte Lanz, Jos Joore, Paul Vulto, Fabrice Viviani, Karla Queiroz. Ex vivo modelling of vascularized pdx-derived ovarian tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2795.

Intestinal organoids have emerged as the new paradigm for modelling the healthy and diseased intestine with patient-relevant properties. In this study, we show directed differentiation of induced pluripotent stem cells towards intestinal-like phenotype within a microfluidic device. iPSCs are cultured against a gel in microfluidic chips of the OrganoPlate, in which they undergo stepwise differentiation. Cells form a tubular structure, lose their stem cell markers and start expressing mature intestinal markers, including markers for Paneth cells, enterocytes and neuroendocrine cells. Tubes develop barrier properties as confirmed by transepithelial electrical resistance (TEER). Lastly, we show that tubules respond to pro-inflammatory cytokine triggers. The whole procedure for differentiation lasts 14 days, making it an efficient process to make patient-specific organoid tubules. We anticipate the usage of the platform for disease modelling and drug candidate screening.

Angiogenic sprouting, the growth of new blood vessels from pre-existing vessels, is orchestrated by cues from within the cellular microenvironment, such as biochemical gradients and perfusion. However, many of these cues are missing in current in vitro models of angiogenic sprouting. We here describe an in vitro platform that integrates both perfusion and the generation of stable biomolecular gradients and demonstrate its potential to study more physiologically relevant angiogenic sprouting and microvascular stabilization. The platform consists of an array of 40 individually addressable microfluidic units that enable the culture of perfused microvessels against a three-dimensional collagen-1 matrix. Upon the introduction of a gradient of pro-angiogenic factors, the endothelial cells differentiated into tip cells that invaded the matrix. Continuous exposure resulted in continuous migration and the formation of lumen by stalk cells. A combination of vascular endothelial growth factor-165 (VEGF-165), phorbol 12-myristate 13-acetate (PMA), and sphingosine-1-phosphate (S1P) was the most optimal cocktail to trigger robust, directional angiogenesis with S1P being crucial for guidance and repetitive sprout formation. Prolonged exposure forces the angiogenic sprouts to anastomose through the collagen to the other channel. This resulted in remodeling of the angiogenic sprouts within the collagen: angiogenic sprouts that anastomosed with the other perfusion channel remained stable, while those who did not retracted and degraded. Furthermore, perfusion with 150 kDa FITC-Dextran revealed that while the angiogenic sprouts were initially leaky, once they fully crossed the collagen lane they became leak tight. This demonstrates that once anastomosis occurred, the sprouts matured and suggests that perfusion can act as an important survival and stabilization factor for the angiogenic microvessels. The robustness of this platform in combination with the possibility to include a more physiological relevant three-dimensional microenvironment makes our platform uniquely suited to study angiogenesis in vitro. Electronic supplementary material The online version of this article (10.1007/s10456-018-9647-0) contains supplementary material, which is available to authorized users.

Background Receptor-mediated transcytosis is one of the major routes for drug delivery of large molecules into the brain. The aim of this study was to develop a novel model of the human blood–brain barrier (BBB) in a high-throughput microfluidic device. This model can be used to assess passage of large biopharmaceuticals, such as therapeutic antibodies, across the BBB. Methods The model comprises human cell lines of brain endothelial cells, astrocytes, and pericytes in a two-lane or three-lane microfluidic platform that harbors 96 or 40 chips, respectively, in a 384-well plate format. In each chip, a perfused vessel of brain endothelial cells was grown against an extracellular matrix gel, which was patterned by means of surface tension techniques. Astrocytes and pericytes were added on the other side of the gel to complete the BBB on-a-chip model. Barrier function of the model was studied using fluorescent barrier integrity assays. To test antibody transcytosis, the lumen of the model’s endothelial vessel was perfused with an anti-transferrin receptor antibody or with a control antibody. The levels of antibody that penetrated to the basal compartment were quantified using a mesoscale discovery assay. Results The perfused BBB on-a-chip model shows presence of adherens and tight junctions and severely limits the passage of a 20 kDa FITC-dextran dye. Penetration of the antibody targeting the human transferrin receptor (MEM-189) was markedly higher than penetration of the control antibody (apparent permeability of 2.9 × 10−5 versus 1.6 × 10−5 cm/min, respectively). Conclusions We demonstrate successful integration of a human BBB microfluidic model in a high-throughput plate-based format that can be used for drug screening purposes. This in vitro model shows sufficient barrier function to study the passage of large molecules and is sensitive to differences in antibody penetration, which could support discovery and engineering of BBB-shuttle technologies. Electronic supplementary material The online version of this article (10.1186/s12987-018-0108-3) contains supplementary material, which is available to authorized users.

Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20 kDa dextran but not to 150 kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNFα and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature.

Background Breast cancer is the most common invasive cancer among women. Currently, there are only a few models used for therapy selection, and they are often poor predictors of therapeutic response or take months to set up and assay. In this report, we introduce a microfluidic OrganoPlate® platform for extracellular matrix (ECM) embedded tumor culture under perfusion as an initial study designed to investigate the feasibility of adapting this technology for therapy selection. Methods The triple negative breast cancer cell lines MDA-MB-453, MDA-MB-231 and HCC1937 were selected based on their different BRCA1 and P53 status, and were seeded in the platform. We evaluate seeding densities, ECM composition (Matrigel®, BME2rgf, collagen I) and biomechanical (perfusion vs static) conditions. We then exposed the cells to a series of anti-cancer drugs (paclitaxel, olaparib, cisplatin) and compared their responses to those in 2D cultures. Finally, we generated cisplatin dose responses in 3D cultures of breast cancer cells derived from 2 PDX models. Results The microfluidic platform allows the simultaneous culture of 96 perfused micro tissues, using limited amounts of material, enabling drug screening of patient-derived material. 3D cell culture viability is improved by constant perfusion of the medium. Furthermore, the drug response of these triple negative breast cancer cells was attenuated by culture in 3D and differed from that observed in 2D substrates. Conclusions We have investigated the use of a high-throughput organ-on-a-chip platform to select therapies. Our results have raised the possibility to use this technology in personalized medicine to support selection of appropriate drugs and to predict response to therapy in a real time fashion. Electronic supplementary material The online version of this article (10.1186/s12885-017-3709-3) contains supplementary material, which is available to authorized users.

The Lab-on-a-Chip concept aims at miniaturizing laboratory processes to enable automation and/or parallelization via microfluidic chips that are capable of handling minute sample volumes. Mass spectrometry is nowadays the detection method of choice, because of its selectivity, sensitivity and wide application range. We review the most interesting examples over the last two-and-a-half years where the two techniques were used for bioanalytical applications. Furthermore, we discuss the merits and limitations of such hyphenated systems. We inventorize the reported applications and approaches. We see an ongoing trend towards chip-based liquid chromatography-mass spectrometry usage and small volume analysis applications, particularly in the field of proteomics where bottom-up approaches profit from chip-based technologies and hyphenation with complex cell cultures.

Parkinson’s disease is a slowly progressive neurodegenerative disease characterised by dysfunction and death of selectively vulnerable midbrain dopaminergic neurons leading mainly to motor dysfunction, but also other non-motor symptoms. The development of human in vitro cellular models with similar phenotypic characteristics to selectively vulnerable neurons is a major challenge in Parkinson’s disease research. We constructed a fully automated cell culture platform optimised for long-term maintenance and monitoring of induced pluripotent stem cell derived neurons in three dimensional microfluidic cell culture devices. The system can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess neuronal activity. Using this system, we continuously monitored the differentiation of Parkinson’s disease patient derived human neuroepithelial stem cells into midbrain specific dopaminergic neurons. Calcium imaging confirmed the electrophysiological activity of differentiated neurons and immunostaining confirmed the efficiency of the differentiation protocol. This system is the first example of a fully automated Organ-on-a-Chip culture and enables a versatile array of in vitro experiments for patient-specific disease modelling.

In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories., Efforts to determine the effects of drugs on epithelial barriers could benefit from better in vitro models. Here the authors develop a microfluidic device supporting the growth and function of extracellular matrix-supported intestinal tubules, and evaluate the effect of staurosporine and acetylsalicylic acid on barrier integrity.

Isotachophoresis (ITP) and electric field gradient focusing (EFGF) are two powerful approaches for simultaneous focusing and separation of charged compounds. Remarkably, in many EFGF methods, isotachophoretic hallmarks have been found, including observations of plateau concentrations and contiguous analyte bands. We discuss the similarities between ITP and EFGF and describe promising possibilities to transfer the functionality and applications developed on one platform to other platforms. Of particular importance is the observation that single-electrolyte isotachophoretic separations with tunable ionic mobility window can be performed, as is illustrated with the example of depletion zone isotachophoresis (dzITP). By exploiting the rapid developments in micro- and nanofluidics, many interesting combinations of ITP and EFGF features can be achieved, yielding powerful analytical platforms for sample preparation, biomarker discovery, molecular interaction assays, drug screening, and clinical diagnostics.

With great advances in the field of in vitro brain modelling, the challenge is now to implement these technologies for development and evaluation of new drug candidates. Here we demonstrate a method for culturing three-dimensional networks of spontaneously active neurons and supporting glial cells in a microfluidic platform. The high-throughput nature of the platform in combination with its compatibility with all standard laboratory equipment allows for parallel evaluation of compound effects.