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

미세유체역학(Microfluidics) 기반 혈관화된 인간 미세종양 모델 준비

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.

저자 스포트라이트: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research (중개 암 연구를 위한 모델로서의 인간 혈관 미세 종양 생성) 

암 연구를 위한 생리학적 인간 혈관화 미세종양 모델 확립

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  

혈관화된 미세종양계의 종양 진행을 조사하기 위해 미세유체 장치에 인간 세포 도입

먼저 UV 멸균 고처리량 플레이트와 트롬빈 분취액을 조직 배양 후드에 넣습니다. 준비된 세포 피브린 혼합물을 얼음 위에 올려 응고를 늦춥니다. P20 피펫을 사용하여 6마이크로리터의 세포 피브린 혼합물을 그립니다.그런 다음 피펫 팁을 트롬빈 분취액에 부드럽게 넣고 기포가 생기지 않도록 두 번 혼합합니다. 고처리량 플레이트를 비스듬히 들어 올리고 피펫 팁을 로딩 포트 중 하나에 빠르게 삽입합니다. 피펫 플런저를 부드럽고 유동적인 동작으로 첫 번째 스톱까지 밀고 세포 피브린 혼합물을 조직 챔버에 주입합니다.겔이 챔버를 완전히 통과하는지 확인합니다. 피펫 팁을 제거하거나 피펫을 옮기지 않고 플레이트를 평평하게 부드럽게 놓습니다. P20에서 손으로 피펫 팁을 제거하고 로딩 포트 구멍에 그대로 둡니다.2분 후, 로딩 포트에서 부드럽게 비틀어 피펫 팁을 제거하고 플레이트 위에 뚜껑을 놓습니다. 겔 중합을 위해 섭씨 37도에서 15-20분 동안 플레이트를 배양합니다. 미세유체 장치를 현미경 스테이지에 놓아 기포 없이 챔버 전체에 세포가 고르게 분포하는 것을 관찰합니다.P20 피펫 팁을 M1 또는 M3에 삽입하고 4마이크로리터의 라미닌을 천천히 배출하여 전체 상단 패널을 코팅합니다. 앞서 설명한 대로 피펫 팁을 제거하고 플레이트를 섭씨 37도에서 5% 이산화탄소에서 10분 동안 배양합니다. 275 마이크로리터의 EGM-2 전체 배지를 A열과 B열에 위치한 웰의 결합되지 않은 저장소에 추가합니다.P200 피펫 팁을 275마이크로리터의 EGM-2가 들어 있는 웰의 배지 입구 구멍에 삽입하고 75마이크로리터의 배지를 천천히 배출하여 매체가 채널을 통해 이동하고 반대쪽에서 거품이 생기는 것을 확인합니다.피펫 팁을 제거한 후 팁에 남아 있는 미디어를 미디어 저장소로 밀어 넣습니다. 50 마이크로 리터의 배지를 추가하여 G 및 H행의 낮은 측면 웰을 완전히 덮고 설명된대로 플레이트를 1-2 시간 동안 배양합니다. 현미경으로 매체 채널의 기포를 식별하고 75마이크로리터의 매체를 채널에 다시 주입하여 기포를 제거합니다.육안으로 중간 흡입구 또는 배출구에 기포가 있는지 확인하십시오. 그런 다음 P200 피펫 팁을 구멍에 삽입하고 플런저를 들어 올려 기포를 빼냅니다. 혈관화된 미세 장기 또는 VMO에서 내피 세포는 처음에는 조직 챔버 내에 고르게 분포되어 있지만 2일째에는 늘어나고 발광하기 시작했습니다.넷째 날, 내피 세포는 외부 미세유체 채널과 문합되어 연속적인 혈관 네트워크를 형성했습니다. 미세한 혈관 네트워크를 통해 FITC-덱스트란을 관류하여 누출을 최소화함으로써 단단한 혈관 장벽 기능을 확인했습니다. MDA-MB-231 암세포를 VMO에 관류하면 내피 내막에 부착되어 세포외 공간으로 유출되어 관류 후 24시간 이내에 여러 개의 미세전이가 형성되었습니다.타임 랩스 현미경 이미징을 통해 T 세포가 VMO의 세포 외 공간으로 유출되는 것이 45분 이상 관찰되었습니다. 혈관이 완전히 형성된 혈관화된 미세종양에서 T세포는 관류 시 혈관벽에 빠르게 부착되었습니다.

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 ...