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

Preparazione di un modello di microtumore umano vascolarizzato basato sulla microfluidica

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.

Autore in primo piano: Creazione di microtumori vascolarizzati umani come modelli per la ricerca traslazionale sul cancro 

Creazione di un modello fisiologico di microtumore vascolarizzato umano per la ricerca sul cancro

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  

Introduzione di cellule umane in un dispositivo microfluidico per studiare la progressione del tumore in un sistema microtumorale vascolarizzato

Per iniziare, posizionare le piastre ad alto rendimento sterilizzate con raggi UV e le aliquote di trombina nella cappa per coltura tissutale. Mettere la miscela di fibrina cellulare preparata sul ghiaccio per rallentare la coagulazione. Utilizzando una pipetta P20, prelevare sei microlitri della miscela di fibrina cellulare.Quindi posizionare delicatamente il puntale della pipetta nell'aliquota di trombina e mescolare due volte senza introdurre bolle d'aria. Sollevare la piastra ad alta produttività inclinandola e inserire rapidamente il puntale della pipetta in una delle porte di caricamento. Spingere lo stantuffo della pipetta fino al primo arresto con un movimento fluido e regolare e iniettare la miscela di fibrina cellulare nella camera del tessuto.Assicurarsi che il gel attraversi completamente la camera. Posizionare delicatamente la piastra in piano senza rimuovere il puntale della pipetta o spostare la pipetta. Rimuovere il puntale della pipetta con una mano dal P20 e lasciarlo nel foro della porta di caricamento.Dopo due minuti, rimuovere i puntali delle pipette con delicati movimenti di torsione dalle porte di caricamento e posizionare il coperchio sulla piastra. Incubare la piastra per 15-20 minuti a 37 gradi Celsius per la polimerizzazione del gel. Posizionare il dispositivo microfluidico sul tavolino del microscopio per osservare la distribuzione uniforme delle cellule in tutta la camera senza bolle d'aria.Inserire il puntale della pipetta P20 in M1 o M3 ed espellere lentamente quattro microlitri di laminina per rivestire l'intero pannello superiore. Rimuovere il puntale della pipetta come mostrato in precedenza e incubare la piastra a 37 gradi Celsius in anidride carbonica al 5% per 10 minuti. Aggiungere 275 microlitri di terreno completo EGM-2 ai serbatoi disaccoppiati dei pozzetti situati nelle file A e B.Inserire il puntale della pipetta P200 nel foro di ingresso del terreno di coltura contenente 275 microlitri di EGM-2 ed espellere lentamente 75 microlitri di terreno, osservando il terreno viaggiare attraverso il canale e gorgogliare dall'altro lato.Dopo aver rimosso il puntale della pipetta, spingere il terreno rimanente dal puntale nel serbatoio del terreno. Aggiungere 50 microlitri di terreno per coprire interamente i pozzetti laterali inferiori nelle file G e H.Incubare le piastre per una o due ore come dimostrato. Al microscopio, identificare eventuali bolle nei canali dei supporti e rimuoverle reintroducendo 75 microlitri di materiale nei canali.Controllare gli ingressi o le uscite del fluido per le bolle d'aria ad occhio nudo. Quindi inserire il puntale della pipetta P200 nel foro ed estrarre la bolla sollevando lo stantuffo. Nei microorgani vascolarizzati o VMO, le cellule endoteliali inizialmente si distribuivano uniformemente all'interno della camera tissutale, ma dal secondo giorno hanno iniziato ad allungarsi e a illuminarsi.Entro il quarto giorno, le cellule endoteliali si sono anastomosizzate con i canali microfluidici esterni, formando una rete vascolare continua. La stretta funzione di barriera vascolare è stata confermata dalla perfusione del destrano FITC attraverso la rete microvascolare con perdite minime. La perfusione di cellule tumorali MDA-MB-231 nei VMO ha provocato la loro adesione al rivestimento endoteliale e il successivo stravaso nello spazio extracellulare, formando micrometastasi multiple entro 24 ore dalla perfusione.Attraverso l'imaging microscopico time-lapse, è stato osservato lo stravaso di cellule T nello spazio extracellulare dei VMO per oltre 45 minuti. Nei microtumori vascolarizzati con vasi completamente formati, le cellule T hanno aderito rapidamente alla parete vascolare dopo la perfusione.

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 ...