Microfluidic 3D Cytotoxic Assay.

Microfluidic-based cytotoxic assays provide high physiological relevance with the potential to replace conventional animal experiments and two-dimensional (2D) assays. Here, a 3D method utilizing a microfluidic platform for analysis of lymphocyte cytotoxicity is introduced in detail, including platform design, cell culture method, real-time cytotoxic assay setup, and image-based analysis. A 2D experimental method is used for comparison, which effectively demonstrates the advantages of 3D microfluidic platforms in closely recapitulating immune responses within the tumor microenvironment. Moreover, a wide range of experimental possibilities and applications using microfluidic 3D cytotoxic assays is introduced in this chapter, along with their capabilities, limitations, and future outlook.

Vascularized tissue on mesh-assisted platform (VT-MAP): a novel approach for diverse organoid size culture and tailored cancer drug response analysis.

This study presents the vascularized tissue on mesh-assisted platform (VT-MAP), a novel microfluidic in vitro model that uses an open microfluidic principle for cultivating vascularized organoids. Addressing the gap in 3D high-throughput platforms for drug response analysis, the VT-MAP can host tumor clusters of various sizes, allowing for precise, size-dependent drug interaction assessments. Key features include capability for forming versatile co-culture conditions (EC, fibroblasts and colon cancer organoids) that enhance tumor organoid viability and a perfusable vessel network that ensures efficient drug delivery and maintenance of organoid health. The VT-MAP enables the culture and analysis of organoids across a diverse size spectrum, from tens of microns to several millimeters. The VT-MAP addresses the inconsistencies in traditional organoid testing related to organoid size, which significantly impacts drug response and viability. Its ability to handle various organoid sizes leads to results that more accurately reflect patient-derived xenograft (PDX) models and differ markedly from traditional in vitro well plate-based methods. We introduce a novel image analysis algorithm that allows for quantitative analysis of organoid size-dependent drug responses, marking a significant step forward in replicating PDX models. The PDX sample from a positive responder exhibited a significant reduction in cell viability across all organoid sizes when exposed to chemotherapeutic agents (5-FU, oxaliplatin, and irinotecan), as expected for cytotoxic drugs. In sharp contrast, PDX samples of a negative responder showed little to no change in viability in smaller clusters and only a slight reduction in larger clusters. This differential response, accurately replicated in the VT-MAP, underscores its ability to generate data that align with PDX models and in vivo findings. Its capacity to handle various organoid sizes leads to results that more accurately reflect PDX models and differ markedly from traditional in vitro methods. The platform's distinct advantage lies in demonstrating how organoid size can critically influence drug response, revealing insights into cancer biology previously unattainable with conventional techniques.

Patient-derived tumor spheroid-induced angiogenesis preclinical platform for exploring therapeutic vulnerabilities in cancer.

This study addresses the demand for research models that can support patient-treatment decisions and clarify the complexities of a tumor microenvironment by developing an advanced non-animal preclinical cancer model. Based on patient-derived tumor spheroids (PDTS), the proposed model reconstructs the tumor microenvironment with emphasis on tumor spheroid-driven angiogenesis. The resulting microfluidic chip system mirrors angiogenic responses elicited by PDTS, recapitulating patient-specific tumor conditions and providing robust, easily quantifiable outcomes. Vascularized PDTS exhibited marked angiogenesis and tumor proliferation on the microfluidic chip. Furthermore, a drug that targets the vascular endothelial growth factor receptor 2 (VEGFR2, ramucirumab) was deployed, which effectively inhibited angiogenesis and impeded tumor invasion. This innovative preclinical model was used for investigating distinct responses for various drug combinations, encompassing HER2 inhibitors and angiogenesis inhibitors, within the context of PDTS. This integrated platform could potentially advance precision medicine by harmonizing diverse data points within the tumor microenvironment with a focus on the interplay between cancer and the vascular system.

Angio-Net: deep learning-based label-free detection and morphometric analysis of in vitro angiogenesis.

Despite significant advancements in three-dimensional (3D) cell culture technology and the acquisition of extensive data, there is an ongoing need for more effective and dependable data analysis methods. These concerns arise from the continued reliance on manual quantification techniques. In this study, we introduce a microphysiological system (MPS) that seamlessly integrates 3D cell culture to acquire large-scale imaging data and employs deep learning-based virtual staining for quantitative angiogenesis analysis. We utilize a standardized microfluidic device to obtain comprehensive angiogenesis data. Introducing Angio-Net, a novel solution that replaces conventional immunocytochemistry, we convert brightfield images into label-free virtual fluorescence images through the fusion of SegNet and cGAN. Moreover, we develop a tool capable of extracting morphological blood vessel features and automating their measurement, facilitating precise quantitative analysis. This integrated system proves to be invaluable for evaluating drug efficacy, including the assessment of anticancer drugs on targets such as the tumor microenvironment. Additionally, its unique ability to enable live cell imaging without the need for cell fixation promises to broaden the horizons of pharmaceutical and biological research. Our study pioneers a powerful approach to high-throughput angiogenesis analysis, marking a significant advancement in MPS.

Angiogenesis-on-a-chip coupled with single-cell RNA sequencing reveals spatially differential activations of autophagy along angiogenic sprouts.

Several functions of autophagy associated with proliferation, differentiation, and migration of endothelial cells have been reported. Due to lack of models recapitulating angiogenic sprouting, functional heterogeneity of autophagy in endothelial cells along angiogenic sprouts remains elusive. Here, we apply an angiogenesis-on-a-chip to reconstruct 3D sprouts with clear endpoints. We perform single-cell RNA sequencing of sprouting endothelial cells from our chip to reveal high activation of autophagy in two endothelial cell populations- proliferating endothelial cells in sprout basements and stalk-like endothelial cells near sprout endpoints- and further the reciprocal expression pattern of autophagy-related genes between stalk- and tip-like endothelial cells near sprout endpoints, implying an association of autophagy with tip-stalk cell specification. Our results suggest a model describing spatially differential roles of autophagy: quality control of proliferating endothelial cells in sprout basements for sprout elongation and tip-stalk cell specification near sprout endpoints, which may change strategies for developing autophagy-based anti-angiogenic therapeutics.

Engineering choroid plexus-on-a-chip with oscillatory flow for modeling brain metastasis.

The human brain choroid plexus (ChP) is a highly organized secretory tissue with a complex vascular system and epithelial layers in the ventricles of the brain. The ChP is the body's principal source of cerebrospinal fluid (CSF); it also functions as a barrier to separate the blood from CSF, because the movement of CSF through the body is pulsatile in nature. Thus far, it has been challenging to recreate the specialized features and dynamics of the ChP in a physiologically relevant microenvironment. In this study, we recapitulated the ChP structure by developing a microfluidic chip in accordance with established design rules. Furthermore, we used image processing and analysis to mimic CSF flow dynamics within a rlcking system; we also used a hydrogel containing laminin to mimic brain extracellular matrix (ECM). Human ChP cells were cultured in the ChP-on-a-chip with in vivo-like CSF dynamic flow and an engineered ECM. The key ChP characteristics of capillaries, the epithelial layer, and secreted components were recreated in the adjusted microenvironment of our human ChP-on-a-chip. The drug screening capabilities of the device were observed through physiologically relevant drug responses from breast cancer cells that had spread in the ChP. ChP immune responses were also recapitulated in this device, as demonstrated by the motility and cytotoxic effects of macrophages, which are the most prevalent immune cells in the ChP. Our human ChP-on-a-chip will facilitate the elucidation of ChP pathophysiology and support the development of therapeutics to treat cancers that have metastasized into the ChP.

Characterizing On-Chip Angiogenesis Induction in a Microphysiological System as a Functional Measure of Mesenchymal Stromal Cell Bioactivity.

Mesenchymal stromal cells (MSCs) continue to be proposed for clinical investigation to treat myriad diseases given their purported potential to stimulate endogenous regenerative processes, such as angiogenesis. However, MSC functional heterogeneity has hindered clinical success and still poses a substantial manufacturing challenge from a product quality control perspective. Here, a quantitative bioassay based on an enhanced-throughput is described, microphysiological system (MPS) to measure the specific bioactivity of MSCs to stimulate angiogenesis as a potential measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages are co-cultured with human umbilical vein endothelial cells and exhibit significant heterogeneity in angiogenic potency between donors and cell passage. Depending on donor source and cellular passage number, MSCs varied in their ability to stimulate tip cell dominant or stalk cell dominant phenotypes in angiogenic sprout morphology which correlated with expression levels of hepatocyte growth factor (HGF). These findings suggest that MSC angiogenic bioactivity may be considered as a possible potency attribute in MSC quality control strategies. Development of a reliable and functionally relevant potency assay for measuring clinically relevant potency attributes of MSCs will help to improve consistency in quality and thereby, accelerate clinical development of these cell-based products.

3D microengineered vascularized tumor spheroids for drug delivery and efficacy testing.

Tumor angiogenesis is regarded as a promising target for limiting cancer progression because tumor-associated vasculature supplies blood and provides a path for metastasis. Thus, in vitro recapitulation of vascularized tumors is critical to understand the pathology of cancer and identify the mechanisms by which tumor cells proliferate, metastasize, and respond to drugs. In this study, we microengineered a vascularized tumor spheroid (VTS) model to reproduce the pathological features of solid tumors. We first generated tumor-EC hybrid spheroids with self-assembled intratumoral vessels, which enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed only with cancer cells. Notably, the hybrid spheroids also exhibited expression profiles associated with aggressive behavior. The blood vessels sprouting around the hybrid spheroids on the VTS chip displayed the distinctive characteristics of leaky tumor vessels. With the VTS chip showing a progressive tumor phenotype, we validated the suppressive effects of axitinib on tumor growth and angiogenesis, which depended on exposure dose and time, highlighting the significance of tumor vascularization to predict the efficacy of anticancer drugs. Ultimately, we effectively induced both lymphangiogenesis and angiogenesis around the tumor spheroid by promoting interstitial flow. Thus, our VTS model is a valuable platform with which to investigate the interactions between tumor microenvironments and explore therapeutic strategies in cancer. STATEMENT OF SIGNIFICANCE: We conducted an integrative study within a vascularized tumor spheroid (VTS) model. We first generated tumor-EC hybrid spheroids with self-assembled intratumoral vessels, which enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed only with cancer cells. Through RNA sequencing, we elucidated that the tumor-EC hybrid spheroids exhibited expression profiles associated with aggressive behavior such as cancer progression, invasion and metastasis. The blood vessels sprouting around the hybrid spheroids on the VTS chip displayed the distinctive characteristics of leaky tumor vessels. We further validated the suppressive effects of axitinib on tumor growth and angiogenesis, depending on exposure dose and time. Ultimately, we effectively induced both lymphangiogenesis and angiogenesis around the tumor spheroid by promoting interstitial flow.

U-IMPACT: a universal 3D microfluidic cell culture platform.

The development of organs-on-a-chip has resulted in advances in the reconstruction of 3D cellular microenvironments. However, there remain limitations regarding applicability and manufacturability. Here, we present an injection-molded plastic array 3D universal culture platform (U-IMPACT) for various biological applications in a single platform, such as cocultures of various cell types, and spheroids (e.g., tumor spheroids, neurospheres) and tissues (e.g., microvessels). The U-IMPACT consists of three channels and a spheroid zone with a 96-well plate form factor. Specifically, organoids or spheroids (~500 µm) can be located in designated areas, while cell suspensions or cell-laden hydrogels can be selectively placed in three channels. For stable multichannel patterning, we developed a new patterning method based on capillary action, utilizing capillary channels and the native contact angle of the materials without any modification. We derived the optimal material hydrophilicity (contact angle of the body, 45-90°; substrate, <30°) for robust patterning through experiments and theoretical calculations. We demonstrated that the U-IMPACT can implement 3D tumor microenvironments for angiogenesis, vascularization, and tumor cell migration. Furthermore, we cultured neurospheres from induced neural stem cells. The U-IMPACT can serve as a multifunctional organ-on-a-chip platform for high-content and high-throughput screening.

A microphysiological system-based potency bioassay for the functional quality assessment of mesenchymal stromal cells targeting vasculogenesis.

Mesenchymal stromal cells (MSCs) continue to be proposed for use in clinical trials to treat various diseases due to their therapeutic potential to pleiotropically influence endogenous regenerative processes, such as vasculogenesis. However, the functional heterogeneity of MSCs has hampered their clinical success and poses a significant manufacturing challenge with respect to MSC quality control. Here, we evaluated and qualified a quantitative bioassay based on an enhanced-throughput, microphysiological system to measure the specific paracrine bioactivity of MSCs to stimulate vasculogenesis as a measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages were co-cultured with human umbilical vein endothelial cells (HUVECs) and exhibited significant heterogeneity in vasculogenic potency between donors and cell passage. Using our microphysiological system (MPS)-based platform, we demonstrated that variations in MSC vasculogenic bioactivity were maintained when assayed across laboratories and operators. The differences in MSC vasculogenic bioactivity were also correlated with the baseline expression of several genes involved in vasculogenesis (hepatocyte growth factor (HGF), angiopoietin-1 (ANGPT)) or the production of matricellular proteins (fibronectin (FN), insulin-like growth factor-binding protein 7 (IGFBP7)). These findings emphasize the significant functional heterogeneity of MSCs in vasculogenic bioactivity and suggest that changes in baseline gene expression of vasculogenic or matricellular protein genes during manufacturing may affect this bioactivity. The development of a reliable and functionally relevant potency assay for measuring the specific vasculogenic bioactivity of manufactured MSCs will help to reliably assure their quality when used in appropriate clinical trials.

Dynamics of axonal ß-actin mRNA in live hippocampal neurons.

Localization of mRNA facilitates spatiotemporally controlled protein expression in neurons. In axons, mRNA transport followed by local protein synthesis plays a critical role in axonal growth and guidance. However, it is not yet clearly understood how mRNA is transported to axonal subcellular sites and what regulates axonal mRNA localization. Using a transgenic mouse model in which endogenous ß-actin mRNA is fluorescently labeled, we investigated ß-actin mRNA movement in axons of hippocampal neurons. We cultured neurons in microfluidic devices to separate axons from dendrites and performed single-particle tracking of axonal ß-actin mRNA. Compared with dendritic ß-actin mRNA, axonal ß-actin mRNA showed less directed motion and exhibited mostly subdiffusive motion, especially near filopodia and boutons in mature dissociated hippocampal neurons. We found that axonal ß-actin mRNA was likely to colocalize with actin patches (APs), regions that have a high density of filamentous actin (F-actin) and are known to have a role in branch initiation. Moreover, simultaneous imaging of F-actin and axonal ß-actin mRNA in live neurons revealed that moving ß-actin mRNA tended to be docked in the APs. Our findings reveal that axonal ß-actin mRNA localization is facilitated by actin networks and suggest that localized ß-actin mRNA plays a potential role in axon branch formation.

Engineering Organ-on-a-Chip to Accelerate Translational Research.

We guide the use of organ-on-chip technology in tissue engineering applications. Organ-on-chip technology is a form of microengineered cell culture platform that elaborates the in-vivo like organ or tissue microenvironments. The organ-on-chip platform consists of microfluidic channels, cell culture chambers, and stimulus sources that emulate the in-vivo microenvironment. These platforms are typically engraved into an oxygen-permeable transparent material. Fabrication of these materials requires the use of microfabrication strategies, including soft lithography, 3D printing, and injection molding. Here we provide an overview of what is an organ-on-chip platform, where it can be used, what it is composed of, how it can be fabricated, and how it can be operated. In connection with this topic, we also introduce an overview of the recent applications, where different organs are modeled on the microscale using this technology.

All-in-one microfluidic design to integrate vascularized tumor spheroid into high-throughput platform.

The development of a scalable and highly reproducible in vitro tumor microenvironment (TME) platform still sheds light on new insights into cancer metastasis mechanisms and anticancer therapeutic strategies. Here, we present an all-in-one injection molded plastic array three-dimensional culture platform (All-in-One-IMPACT) that integrates vascularized tumor spheroids for highly reproducible, high-throughput experimentation. This device allows the formation of self-assembled cell spheroids on a chip by applying the hanging drop method to the cell culture channel. Then, when the hydrogel containing endothelial cells and fibroblasts is injected, the spheroid inside the droplet can be patterned together in three dimensions along the culture channel. In just two steps above, we can build a vascularized TME within a defined area. This process does not require specialized user skill and minimizes error-inducing steps, enabling both reproducibility and high throughput of the experiment. We have successfully demonstrated the process, from spheroid formation to tumor vascularization, using patient-derived cancer cells (PDCs) as well as various cancer cell lines. Furthermore, we performed combination therapies with Taxol (paclitaxel) and Avastin (bevacizumab), which are used in standard care for metastatic cancer. The All-in-One IMPACT is a powerful tool for establishing various anticancer treatment strategies through the development of a complex TME for use in high-throughput experiments.

BBB-on-a-Chip: Modeling Functional Human Blood-Brain Barrier by Mimicking 3D Brain Angiogenesis Using Microfluidic Chip.

Organ-on-a-chip enables human cell-based 3D tissue culture, which recapitulates the physiological structure and function of the tissue. In terms of the blood-brain barrier (BBB) modeling, the 3D structure of the vessel is essential for studying the cellular interactions among BBB composing cells and investigating the barrier function. Here, we describe a BBB-on-a-chip model with 3D perfusable human vasculature tri-cultured with pericytes and astrocytes. The culture method is based on mimicking angiogenic sprouting since the barrier formation is parallel with angiogenesis during the developmental process. This microfluidic-based 3D tri-culture system enables the comparative study on how surrounding BBB-related cells affect brain angiogenic sprouting. Moreover, the engineered perfusable vasculature is eligible for quantitative analysis on barrier function such as efflux transport system. We expect the BBB-on-a-chip could be used to enhance understanding BBB-related pathologies as well as the drug modulating barrier function of BBB.

Advances in 3D Vascularized Tumor-on-a-Chip Technology.

Tumors disrupt the normal homeostasis of human body as they proliferate in abnormal speed. For constant proliferation, tumors recruit new blood vessels transporting nutrients and oxygen. Immune system simultaneously recruits lymphatic vessels to induce the death of tumor cells. Hence, understanding tumor dynamics are important to developing anti-cancer therapies. Tumor-on-a-chip technology can be applied to identify the structural and functional units of tumors and tumor microenvironments with high reproducibility and reliability, monitoring the development and pathophysiology of tumors, and predicting drug effectiveness. Herein, we explore the ability of tumor-on-a-chip technology to mimic angiogenic and lymphangiogenic tumor microenvironments of organs. Microfluidic systems allow elaborate manipulation of the development and status of cancer. Therefore, they can be used to validate the effects of various drug combinations, specify them, and assess the factors that influence cancer treatment. We discuss the mechanisms of action of several drugs for cancer treatment in terms of tumor growth and progression involving angiogenesis and lymphangiogenesis. Moreover, we present future applications of emerging tumor-on-a-chip technology for drug development and cancer therapy.

Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymphatic Expansion.

BACKGROUND: Mutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. METHODS: Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. RESULTS: Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. CONCLUSIONS: Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.

Perfusable micro-vascularized 3D tissue array for high-throughput vascular phenotypic screening.

Microfluidic organ-on-a-chip technologies have enabled construction of biomimetic physiologically and pathologically relevant models. This paper describes an injection molded microfluidic platform that utilizes a novel sequential edge-guided patterning method based on spontaneous capillary flow to realize three-dimensional co-culture models and form an array of micro-vascularized tissues (28 per 1 × 2-inch slide format). The MicroVascular Injection-Molded Plastic Array 3D Culture (MV-IMPACT) platform is fabricated by injection molding, resulting in devices that are reliable and easy to use. By patterning hydrogels containing human umbilical endothelial cells and fibroblasts in close proximity and allowing them to form vasculogenic networks, an array of perfusable vascularized micro-tissues can be formed in a highly efficient manner. The high-throughput generation of angiogenic sprouts was quantified and their uniformity was characterized. Due to its compact design (half the size of a 96-well microtiter plate), it requires small amount of reagents and cells per device. In addition, the device design is compatible with a high content imaging machine such as Yokogawa CQ-1. Furthermore, we demonstrated the potential of our platform for high-throughput phenotypic screening by testing the effect of DAPT, a chemical known to affect angiogenesis. The MV-IMPACT represent a significant improvement over our previous PDMS-based devices in terms of molding 3D co-culture conditions at much higher throughput with added reliability and robustness in obtaining vascular micro-tissues and will provide a platform for developing applications in drug screening and development.

Vascularization of iNSC spheroid in a 3D spheroid-on-a-chip platform enhances neural maturation.

In vitro platforms for studying the human brain have been developed, and brain organoids derived from stem cells have been studied. However, current organoid models lack three-dimensional (3D) vascular networks, limiting organoid proliferation, differentiation, and apoptosis. In this study, we created a 3D model of vascularized spheroid cells using an injection-molded microfluidic chip. We cocultured spheroids derived from induced neural stem cells (iNSCs) with perfusable blood vessels. Gene expression analysis and immunostaining revealed that the vascular network greatly enhanced spheroid differentiation and reduced apoptosis. This platform can be used to further study the functional and structural interactions between blood vessels and neural spheroids, and ultimately to simulate brain development and disease.

High-Throughput 3D In Vitro Tumor Vasculature Model for Real-Time Monitoring of Immune Cell Infiltration and Cytotoxicity.

Recent advances in anticancer therapy have shown dramatic improvements in clinical outcomes, and adoptive cell therapy has emerged as a type of immunotherapy that can modulate immune responses by transferring engineered immune cells. However, a small percentage of responders and their toxicity remain as challenges. Three-dimensional (3D) in vitro models of the tumor microenvironment (TME) have the potential to provide a platform for assessing and predicting responses to therapy. This paper describes an in vitro 3D tumor model that incorporates clusters of colorectal cancer (CRC) cells around perfusable vascular networks to validate immune-cell-mediated cytotoxicity against cancer cells. The platform is based on an injection-molded 3D co-culture model and composed of 28 microwells where separate identical vascularized cancer models can be formed. It allows robust hydrogel patterning for 3D culture that enables high-throughput experimentation. The uniformity of the devices resulted in reproducible experiments that allowed 10× more experiments to be performed when compared to conventional polydimethylsiloxane (PDMS)-based microfluidic devices. To demonstrate its capability, primary natural killer (NK) cells were introduced into the vascularized tumor network, and their activities were monitored using live-cell imaging. Extravasation, migration, and cytotoxic activity against six types of CRC cell lines were tested and compared. The consensus molecular subtypes (CMS) of CRC with distinct immune responses resulted in the highest NK cell cytotoxicity against CMS1 cancer cells. These results show the potential of our vascularized tumor model for understanding various steps involved in the immune response for the assessment of adoptive cell therapy.