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

Préparation d’un modèle de micro-tumeur humaine vascularisée basé sur la microfluidique

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.

Pleins feux sur l’auteur : Créer des micro-tumeurs vascularisées humaines comme modèles pour la recherche translationnelle sur le cancer 

Établissement d’un modèle physiologique de micro-tumeurs vascularisées humaines pour la recherche sur le cancer

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  

Introduction de cellules humaines dans un dispositif microfluidique pour étudier la progression tumorale dans un système micro-tumoral vascularisé

Pour commencer, placez les plaques à haut débit stérilisées aux UV et les aliquotes de thrombine dans la hotte de culture tissulaire. Placez le mélange de fibrine cellulaire préparé sur de la glace pour ralentir la coagulation. À l’aide d’une pipette P20, prélever six microlitres du mélange de fibrine cellulaire.Placez ensuite délicatement l’embout de la pipette dans l’aliquote de thrombine et mélangez deux fois sans introduire de bulles d’air. Soulevez la plaque à haut débit en biais et insérez rapidement la pointe de la pipette dans l’un des orifices de chargement. Poussez le piston de la pipette jusqu’à la première butée dans un mouvement doux et fluide et injectez le mélange de fibrine cellulaire dans la chambre à tissus.Assurez-vous que le gel traverse entièrement la chambre. Placez délicatement la plaque à plat sans retirer la pointe de la pipette ni déplacer la pipette. Retirez l’embout de la pipette à l’aide d’une main du P20 et laissez-le dans le trou de l’orifice de chargement.Au bout de deux minutes, retirez les pointes de pipette des orifices de chargement en les tordant doucement et placez le couvercle sur la plaque. Incuber la plaque pendant 15 à 20 minutes à 37 degrés Celsius pour la polymérisation du gel. Placez le dispositif microfluidique sur la platine du microscope pour observer la répartition uniforme des cellules dans la chambre sans aucune bulle d’air.Insérez l’embout de pipette P20 dans M1 ou M3 et expulsez lentement quatre microlitres de laminine pour recouvrir l’ensemble du panneau supérieur. Retirez l’embout de la pipette comme indiqué précédemment et incubez la plaque à 37 degrés Celsius dans 5% de dioxyde de carbone pendant 10 minutes. Ajouter 275 microlitres de milieu complet EGM-2 dans les réservoirs non couplés des puits situés dans les rangées A et B.Insérez la pointe de la pipette P200 dans le trou d’entrée du milieu des puits contenant 275 microlitres d’EGM-2 et expulsez lentement 75 microlitres de milieu, en regardant le milieu voyager à travers le canal et bouillonner de l’autre côté.Après avoir retiré l’embout de la pipette, poussez le milieu restant de l’embout dans le réservoir de média. Ajouter 50 microlitres de substrat pour recouvrir entièrement les puits latéraux inférieurs des rangées G et H.Incuber les plaques pendant une à deux heures comme démontré. À l’aide d’un microscope, identifiez les bulles dans les canaux multimédias et éliminez-les en réintroduisant 75 microlitres de médias dans les canaux.Vérifiez à l’œil nu les entrées ou les sorties du fluide pour détecter la présence de bulles d’air. Insérez ensuite l’embout de pipette P200 dans le trou et retirez la bulle en soulevant le piston. Dans les micro-organes vascularisés ou VMO, les cellules endothéliales se sont d’abord réparties uniformément dans la chambre tissulaire, mais dès le deuxième jour, elles ont commencé à s’étirer et à s’illuminer.Au quatrième jour, les cellules endothéliales se sont anastomosées avec les canaux microfluidiques externes, formant un réseau vasculaire continu. La fonction de barrière vasculaire serrée a été confirmée par la perfusion de FITC-dextran à travers le réseau microvasculaire avec une fuite minimale. La perfusion de cellules cancéreuses MDA-MB-231 dans les VMO a entraîné leur adhérence à la muqueuse endothéliale et leur extravasation subséquente dans l’espace extracellulaire, formant de multiples micrométastases dans les 24 heures suivant la perfusion.Grâce à l’imagerie microscopique en accéléré, l’extravasation des lymphocytes T dans l’espace extracellulaire des VMO a été observée pendant 45 minutes. Dans les micro-tumeurs vascularisées avec des vaisseaux entièrement formés, les lymphocytes T ont rapidement adhéré à la paroi vasculaire lors de la perfusion.

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 ...