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

Preparación de un modelo de microtumor humano vascularizado basado en 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 destacado: Creación de microtumores vascularizados humanos como modelos para la investigación traslacional del cáncer 

Establecimiento de un modelo fisiológico de microtumor vascularizado humano para la investigación del cáncer

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  

Introducción de células humanas en un dispositivo microfluídico para investigar la progresión tumoral en un sistema microtumoral vascularizado

Para empezar, coloque las placas de alto rendimiento esterilizadas por UV y las alícuotas de trombina en la campana de cultivo de tejidos. Coloque la mezcla de fibrina celular preparada en hielo para retrasar la coagulación. Con una pipeta P20, extraiga seis microlitros de la mezcla de fibrina celular.A continuación, coloque suavemente la punta de la pipeta en la alícuota de trombina y mezcle dos veces sin introducir burbujas de aire. Levante la placa de alto rendimiento en ángulo e inserte rápidamente la punta de la pipeta en uno de los puertos de carga. Empuje el émbolo de la pipeta hasta el primer tope con un movimiento suave y fluido e inyecte la mezcla de fibrina celular en la cámara de tejido.Asegúrese de que el gel atraviese completamente la cámara. Coloque suavemente la placa plana sin quitar la punta de la pipeta ni desplazarla. Retire la punta de la pipeta con una mano del P20 y déjela en el orificio del puerto de carga.Después de dos minutos, retire las puntas de pipeta con suaves movimientos giratorios de los puertos de carga y coloque la tapa en la placa. Incubar la placa durante 15 a 20 minutos a 37 grados centígrados para la polimerización en gel. Coloque el dispositivo microfluídico en la platina del microscopio para observar la distribución uniforme de las células por toda la cámara sin burbujas de aire.Inserte la punta de pipeta P20 en M1 o M3 y expulse lentamente cuatro microlitros de laminina para recubrir todo el panel superior. Retire la punta de la pipeta como se ha demostrado anteriormente e incube la placa a 37 grados centígrados con un 5 % de dióxido de carbono durante 10 minutos. Agregue 275 microlitros de medios completos EGM-2 a los depósitos desacoplados de los pocillos ubicados en las filas A y B. Inserte la punta de pipeta P200 en el orificio de entrada de medios de los pocillos que contienen 275 microlitros de EGM-2 y expulse lentamente 75 microlitros de medio, observando cómo el medio viaja a través del canal y burbujea por el otro lado.Después de retirar la punta de la pipeta, empuje el medio restante de la punta hacia el depósito del medio. Agregue 50 microlitros de medio para cubrir completamente los pocillos del lado bajo en las filas G y H. Incube las placas durante una o dos horas como se demuestra. Bajo un microscopio, identifique cualquier burbuja en los canales de medios y elimínelas reintroduciendo 75 microlitros de medios en los canales.Compruebe a simple vista las entradas o salidas medianas en busca de burbujas de aire. A continuación, inserte la punta de la pipeta P200 en el orificio y extraiga la burbuja levantando el émbolo. En los microórganos vascularizados o VMO, las células endoteliales inicialmente se distribuyeron uniformemente dentro de la cámara de tejido, pero al segundo día, comenzaron a estirarse y luminizarse.Al cuarto día, las células endoteliales se anastomosaron con los canales microfluídicos externos, formando una red vascular continua. La función de barrera vascular ajustada se confirmó mediante la perfusión de FITC-dextrano a través de la red microvascular con fugas mínimas. La perfusión de células cancerosas MDA-MB-231 en las VMO dio lugar a su adherencia al revestimiento endotelial y a la posterior extravasación al espacio extracelular, formando múltiples micrometástasis dentro de las 24 horas posteriores a la perfusión.A través de imágenes microscópicas de lapso de tiempo, se observó la extravasación de las células T en el espacio extracelular de los VMO durante 45 minutos. En los microtumores vascularizados con vasos completamente formados, las células T se adhirieron rápidamente a la pared vascular tras la perfusión.

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 ...