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

Förberedelse av en mikrofluidikbaserad vaskulariserad human mikrotumörmodell

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.

Författare i fokus: Skapa humana vaskulariserade mikrotumörer som modeller för translationell cancerforskning 

Etablering av en fysiologisk human vaskulariserad mikrotumörmodell för cancerforskning

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  

Introduktion av mänskliga celler i en mikrofluidisk enhet för att undersöka tumörprogression i ett vaskulariserat mikrotumörsystem

Till att börja med, placera de UV-steriliserade plattorna med hög genomströmning och trombinalikvoter i vävnadsodlingskåpan. Placera den beredda cellfibrinblandningen på is för att bromsa koagulationen. Använd en P20-pipett och dra sex mikroliter av cellfibrinblandningen.Placera sedan försiktigt pipettspetsen i trombinalikvoten och blanda två gånger utan att införa luftbubblor. Lyft plattan med hög genomströmning i en vinkel och för snabbt in pipettspetsen i en av laddningsportarna. Skjut pipettkolven till första stoppet i en jämn och flytande rörelse och injicera cellfibrinblandningen i vävnadskammaren.Se till att gelen passerar helt genom kammaren. Placera försiktigt plattan platt utan att ta bort pipettspetsen eller förskjuta pipetten. Ta bort pipettspetsen med en hand från P20 och lämna den i laddningsporthålet.Efter två minuter tar du bort pipettspetsarna med försiktiga vridande rörelser från laddningsportarna och lägger locket på plattan. Inkubera plattan i 15 till 20 minuter vid 37 grader Celsius för gelpolymerisation. Placera mikrofluidikanordningen på mikroskopet stage för att observera den jämna fördelningen av celler i hela kammaren utan några luftbubblor.Sätt in P20-pipettspetsen i M1 eller M3 och spruta ut fyra mikroliter laminin långsamt för att täcka hela topppanelen. Ta bort pipettspetsen som visats tidigare och inkubera plattan vid 37 grader Celsius i 5 % koldioxid i 10 minuter. Tillsätt 275 mikroliter EGM-2 komplett media till de okopplade reservoarerna i brunnarna i raderna A och B. Sätt in P200-pipettspetsen i medieinloppshålet på brunnarna som innehåller 275 mikroliter EGM-2 och spruta långsamt ut 75 mikroliter medium, se hur mediet färdas genom kanalen och bubblar upp på andra sidan.När du har tagit bort pipettspetsen trycker du in det återstående mediet från spetsen i mediebehållaren. Tillsätt 50 mikroliter media för att helt täcka de låga sidobrunnarna i raderna G och H. Inkubera plattorna i en till två timmar enligt bilden. Identifiera eventuella bubblor i mediekanalerna under ett mikroskop och ta bort dem genom att återinföra 75 mikroliter media i kanalerna.Kontrollera mediets inlopp eller utlopp för luftbubblor med blotta ögat. Sätt sedan in pipettspetsen P200 i hålet och dra ut bubblan genom att lyfta kolven. I vaskulariserade mikroorgan eller VMO:er fördelade sig endotelcellerna till en början jämnt i vävnadskammaren, men på dag två började de sträcka ut sig och lysa upp.På dag fyra anastomoserade endotelcellerna med de yttre mikrofluidiska kanalerna och bildade ett kontinuerligt vaskulärt nätverk. Den täta vaskulära barriärfunktionen bekräftades genom att perfusion av FITC-dextran genom det mikrovaskulära nätverket med minimalt läckage. Infusion av MDA-MB-231-cancerceller i VMO:erna resulterade i att de vidhäftade till endotelfodret och efterföljande extravasering i det extracellulära utrymmet, vilket bildade flera mikrometastaser inom 24 timmar efter perfusion.Genom time-lapse mikroskopisk avbildning observerades extravasering av T-celler i det extracellulära utrymmet i VMO:erna under 45 minuter. I vaskulariserade mikrotumörer med fullt utvecklade kärl fäste T-cellerna snabbt vid kärlväggen vid 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 ...