Name: Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System
Uploaded: 2023-09-15T14:50:00
Description: Watch this Scientific Journal Video about Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System at JoVE.com

introducing human cells into a microfluidic device to investigate tumor progression in a vascularized micro-tumor system

Erstellung eines mikrofluidikbasierten vaskularisierten humanen Mikrotumormodells

Cancer remains a major health concern worldwide and is the second leading cause of death in the United States. For the year 2023 alone, the National Center for Health Statistics anticipates more than 1.9 million new cancer cases and over 600,000 cancer deaths occurring in the US1, highlighting the urgent need for effective treatment approaches. However, currently, only 5.1% of anti-cancer therapeutics entering clinical trials ultimately gain FDA approval. Failure of promising candidates to successfully progress through clinical trials can be partially attributed to the use of non-physiological model systems, such as 2D and spheroid cultures, during preclinical drug development2. These classical cancer models lack essential components of the tumor microenvironment, such as a stromal niche, associated immune cells, and perfused vasculature, which are key determinants of therapeutic resistance and disease progression. Thus, a new model system that better mimics the human in vivo tumor microenvironment is necessary to improve the clinical translation of preclinical findings.
The field of tissue engineering is rapidly advancing, providing improved methods for studying human diseases in laboratory settings. One significant development is the emergence of microphysiological systems (MPS), also known as organ chips or tissue chips, which are functional, miniaturized human organs capable of replicating healthy or diseased conditions3,4,5. Within this context, tumor chips, which are three-dimensional microfluidic-based in vitro human tumor models, have been developed for oncology research2,3,4,5,6,7,8,9,10,11,12,13. These advanced models incorporate biochemical and biophysical cues within a dynamic tumor microenvironment, enabling researchers to study tumor behavior and responses to treatments in a more physiologically relevant context. However, despite these advancements, few groups have successfully incorporated a living, functional vasculature, particularly one that self-patterns in response to physiologic flow3,4,5,6. The inclusion of a functional vascular network is crucial as it allows for modeling physical barriers that affect drug or cell delivery, cell homing to distinct microenvironments, and transendothelial migration of tumor, stromal, and immune cells. By including this feature, the tumor chip can better represent the complexities observed in the in vivo tumor microenvironment.
To address this unmet need, we have developed a novel drug-screening platform that enables micro-vessel networks to form within a microfluidic device8,9,10,11,12,13,14,15,16. This base organ chip platform, termed the vascularized micro-organ (VMO), can be adapted to virtually any organ system to replicate original tissue physiology for disease modeling, drug screening, and personalized medicine applications. VMOs are established by co-culturing endothelial colony-forming cell-derived endothelial cells (ECFC-EC), HUVEC or iPSC-EC (hereafter EC), and multiple stromal cells in the chamber, including normal human lung fibroblasts (NHLF), which remodel the matrix, and pericytes that wrap and stabilize the vessels. The VMO can also be established as a cancer model system by co-culturing tumor cells with the associated stroma to create a vascularized micro-tumor (VMT)8,9,10,11,12,13, or tumor chip, model. Through the co-culture of multiple cell types in a dynamic flow environment, perfused microvascular networks form de novo in the tissue chambers of the device, where vasculogenesis is closely regulated by interstitial flow rates14,15. Medium is driven through the microfluidic channels of the device by a hydrostatic pressure head that supplies the surrounding cells of the tissue chamber with nutrients exclusively through the micro-vessels, with a permeability coefficient of 1.2 x 10-7 cm/s, similar to what is seen for capillaries in vivo8.
The incorporation of self-organizing micro-vessels into the VMT model represents a significant breakthrough because it: 1) mimics the structure and function of vascularized tumor masses in vivo; 2) can model key steps of metastasis, including tumor-endothelial and stromal cell interactions; 3) establishes physiologically selective barriers for nutrient and drug delivery, improving pharmaceutical screening; and 4) allows direct assessment of drugs with anti-angiogenic and anti-metastatic capabilities. By replicating the in vivo delivery of nutrients, drugs, and immune cells in a complex 3D microenvironment, the VMO/VMT platform is a physiologically relevant model that can be used to perform drug screening and study cancer, vascular or organ-specific biology. Importantly, the VMT supports the growth of various types of tumors, including colon cancer, melanoma, breast cancer, glioblastoma, lung cancer, peritoneal carcinomatosis, ovarian cancer, and pancreatic cancer8,9,10,11,12,13. In addition to being low-cost, easily established, and arrayed for high throughput experiments, the microfluidic platform is fully optically compatible for real-time image analysis of tumor-stromal interactions and response to stimuli or therapeutics. Each cell type in the system is labeled with a different fluorescent marker to allow direct visualization and tracking of cell behavior throughout the entire experiment, creating a window into the dynamic tumor microenvironment. We have previously shown that the VMT more faithfully models in vivo tumor growth, architecture, heterogeneity, gene expression signatures, and drug responses than standard culture modalities10. Importantly, the VMT supports the growth and study of patient-derived cells, including cancer cells, which better models the pathology of the parent tumors than standard spheroid cultures and further advances personalized medicine efforts11. This manuscript outlines the methods for establishing the VMT, showcasing its utility for studying human cancers.

Autor im Rampenlicht: Schaffung humaner vaskularisierter Mikrotumoren als Modelle für die translationale Krebsforschung 

Etablierung eines physiologischen humanen vaskularisierten Mikrotumormodells für die Krebsforschung

Our tumor-on-a-chip platform was developed to physiologically model the growth of human vascularized micro tumors, and allows us to answer both basic and translational research questions. Questions that focus on basic tumor biology, drug efficacy, tumor responsiveness, underlying immunologic processes, potential immunotherapeutic intervention, and personalized therapy approaches. Effectively translating preclinical discoveries into potent cancer treatments hinges on faithfully replicating the disease state and model systems.This protocol outlines the establishment of the vascularized micro tumor, or VMT, to mimic the tumor microenvironment in vitro. The VMT faithfully reproduces key aspects of a human tumor, yielding clinically relevant findings. Our model allows us to perform physiologically relevant, translational human cancer research.Specifically, our protocol, in contrast to other models, incorporates dynamic flow conditions, enables the integration of immune cells and immunotherapy, and further allows us for the incorporation of personalized medicine approaches. We are employing the VMT model to propel personalized medicine initiatives forward in both preclinical and clinical arenas. This model deepens our understanding of microenvironmental factors in cell-cell interactions, influencing therapeutic resistance and cancer progression.Moreover, the VMT shows promise in discovering customized patient-specific treatments for enhanced clinical care.

Watch this Scientific Journal Video about Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System at JoVE.com

Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System  

Einbringen menschlicher Zellen in ein mikrofluidisches Gerät zur Untersuchung der Tumorprogression in einem vaskularisierten Mikrotumorsystem

Legen Sie zunächst die UV-sterilisierten Hochdurchsatzplatten und Thrombin-Aliquots in die Gewebekulturhaube ein. Die vorbereitete Zellfibrinmischung auf Eis legen, um die Gerinnung zu verlangsamen. Ziehen Sie mit einer P20-Pipette sechs Mikroliter der Zellfibrinmischung ab.Setzen Sie dann die Pipettenspitze vorsichtig in das Thrombin-Aliquot ein und mischen Sie zweimal, ohne Luftblasen einzubringen. Heben Sie die Hochdurchsatzplatte schräg an und stecken Sie die Pipettenspitze schnell in eine der Ladeöffnungen. Schieben Sie den Pipettenkolben in einer sanften und flüssigen Bewegung bis zum ersten Anschlag und injizieren Sie die Zellfibrinmischung in die Gewebekammer.Stellen Sie sicher, dass das Gel die Kammer vollständig durchquert. Legen Sie die Platte vorsichtig flach auf, ohne die Pipettenspitze zu entfernen oder die Pipette zu verschieben. Entfernen Sie die Pipettenspitze mit einer Hand vom P20 und lassen Sie sie im Ladeloch.Nach zwei Minuten entfernen Sie die Pipettenspitzen mit sanften Drehbewegungen aus den Ladeöffnungen und legen den Deckel auf die Platte. Inkubieren Sie die Platte für 15 bis 20 Minuten bei 37 Grad Celsius für die Gelpolymerisation. Platzieren Sie das mikrofluidische Gerät auf dem Mikroskoptisch, um die gleichmäßige Verteilung der Zellen in der Kammer ohne Luftblasen zu beobachten.Führen Sie die Pipettenspitze P20 in M1 oder M3 ein und stoßen Sie langsam vier Mikroliter Laminin aus, um die gesamte Deckplatte zu beschichten. Entfernen Sie die Pipettenspitze wie zuvor gezeigt und inkubieren Sie die Platte bei 37 Grad Celsius in 5%igem Kohlendioxid für 10 Minuten. Geben Sie 275 Mikroliter EGM-2-Komplettmedium in die entkoppelten Reservoirs der Wells, die sich in den Reihen A und B befinden.Stecken Sie die Pipettenspitze P200 in das Medieneinlassloch der Wells, die 275 Mikroliter EGM-2 enthalten, und stoßen Sie langsam 75 Mikroliter Medium aus, während Sie beobachten, wie das Medium durch den Kanal wandert und auf der anderen Seite nach oben sprudelt.Nachdem Sie die Pipettenspitze entfernt haben, schieben Sie das verbleibende Medium von der Spitze in das Medienreservoir. Fügen Sie 50 Mikroliter Medien hinzu, um die unteren Seitenvertiefungen in den Reihen G und H vollständig zu bedecken. Inkubieren Sie die Platten ein bis zwei Stunden lang, wie gezeigt. Identifizieren Sie unter dem Mikroskop alle Blasen in den Medienkanälen und entfernen Sie sie, indem Sie 75 Mikroliter Medien wieder in die Kanäle einführen.Prüfen Sie die Ein- oder Auslässe des Mediums mit bloßem Auge auf Luftblasen. Führen Sie dann die P200-Pipettenspitze in das Loch ein und ziehen Sie die Blase heraus, indem Sie den Kolben anheben. In vaskularisierten Mikroorganen oder VMOs verteilten sich die Endothelzellen zunächst gleichmäßig in der Gewebekammer, aber am zweiten Tag begannen sie, sich auszudehnen und zu leuchten.Am vierten Tag anastomosierten die Endothelzellen mit den äußeren mikrofluidischen Kanälen und bildeten ein kontinuierliches Gefäßnetzwerk. Die Funktion der engen vaskulären Barriere wurde durch die Perfusion von FITC-Dextran durch das mikrovaskuläre Netzwerk mit minimaler Leckage bestätigt. Die Perfusion von MDA-MB-231-Krebszellen in die VMOs führte zu deren Anhaftung an der Endothelauskleidung und anschließender Extravasation in den extrazellulären Raum, wobei innerhalb von 24 Stunden nach der Perfusion mehrere Mikrometastasen gebildet wurden.Durch mikroskopische Bildgebung im Zeitraffer wurde die Extravasation von T-Zellen in den extrazellulären Raum der VMOs über 45 Minuten beobachtet. In vaskularisierten Mikrotumoren mit voll ausgebildeten Gefäßen haften T-Zellen nach der Perfusion schnell an der Gefäßwand.

Research

JoVE Journal

Cancer Research

Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...