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

Preparando um modelo de microtumor humano vascularizado baseado em microfluídica

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 em destaque: Criando microtumores vascularizados humanos como modelos para pesquisa translacional do câncer 

Estabelecendo um Modelo Fisiológico de Microtumor Vascularizado Humano para Pesquisa em Câncer

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  

Introdução de Células Humanas em um Dispositivo Microfluídico para Investigar a Progressão Tumoral em um Sistema Microtumoral Vascularizado

Para começar, coloque as placas de alto rendimento esterilizadas por UV e as alíquotas de trombina na capa de cultura de tecidos. Coloque a mistura de fibrina celular preparada no gelo para retardar a coagulação. Usando uma pipeta P20, desenhe seis microlitros da mistura de fibrina celular.Em seguida, coloque suavemente a ponta da pipeta na alíquota de trombina e misture duas vezes sem introduzir bolhas de ar. Levante a placa de alto rendimento em um ângulo e insira rapidamente a ponta da pipeta em uma das portas de carregamento. Empurre o êmbolo da pipeta para a primeira parada em um movimento suave e fluido e injete a mistura de fibrina celular na câmara de tecido.Certifique-se de que o gel atravessa totalmente a câmara. Coloque suavemente a placa plana sem remover a ponta da pipeta ou deslocar a pipeta. Retire a ponta da pipeta com uma mão do P20 e deixe-a no orifício da porta de carga.Após dois minutos, remova as pontas da pipeta com movimentos suaves de torção das portas de carga e coloque a tampa sobre a placa. Incubar a placa por 15 a 20 minutos a 37 graus Celsius para polimerização em gel. Coloque o dispositivo microfluídico no palco do microscópio para observar a distribuição uniforme das células por toda a câmara sem bolhas de ar.Insira a ponta da pipeta P20 em M1 ou M3 e expulse quatro microlitros de laminina lentamente para revestir todo o painel superior. Retire a ponta da pipeta como demonstrado anteriormente e incube a placa a 37 graus Celsius em 5% de dióxido de carbono por 10 minutos. Adicione 275 microlitros de meio completo EGM-2 aos reservatórios desacoplados dos poços localizados nas fileiras A e B.Insira a ponta da pipeta P200 no orifício de entrada do meio dos poços contendo 275 microlitros de EGM-2 e expulse lentamente 75 microlitros de meio, observando a mídia viajar pelo canal e borbulhar do outro lado.Depois de remover a ponta da pipeta, empurre o meio restante da ponta para o reservatório do meio. Adicione 50 microlitros de meio para cobrir totalmente os poços laterais baixos nas fileiras G e H.Incube as placas por uma a duas horas, conforme demonstrado. Sob um microscópio, identifique quaisquer bolhas nos canais de mídia e remova-as reintroduzindo 75 microlitros de mídia nos canais.Verifique as entradas ou saídas médias em busca de bolhas de ar a olho nu. Em seguida, insira a ponta da pipeta P200 no orifício e puxe a bolha para fora levantando o êmbolo. Em micro-órgãos vascularizados ou VMOs, as células endoteliais inicialmente se distribuíram uniformemente dentro da câmara tecidual, mas no segundo dia, começaram a se esticar e luminizar.No quarto dia, as células endoteliais anastomosaram-se com os canais microfluídicos externos, formando uma rede vascular contínua. A função de barreira vascular apertada foi confirmada pela perfusão de FITC-dextran através da rede microvascular com vazamento mínimo. A perfusão de células cancerosas MDA-MB-231 nos VMOs resultou em sua aderência ao revestimento endotelial e subsequente extravasamento para o espaço extracelular, formando múltiplas micrometástases dentro de 24 horas pós-perfusão.Através de imagens microscópicas de lapso de tempo, o extravasamento de células T para o espaço extracelular dos VMOs foi observado ao longo de 45 minutos. Em microtumores vascularizados com vasos totalmente formados, as células T aderiram rapidamente à parede vascular após a perfusão.

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 ...