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

制备基于微流体的血管化人类微肿瘤模型

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.

作者聚焦：创建人类血管化微肿瘤作为转化癌症研究的模型 

建立用于癌症研究的生理性人血管化微肿瘤模型

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  

将人类细胞引入微流控装置以研究血管化微肿瘤系统中的肿瘤进展

首先，将紫外线灭菌的高通量平板和凝血酶等分试样放入组织培养罩中。将准备好的细胞纤维蛋白混合物放在冰上以减缓凝血。使用 P20 移液器，抽取 6 微升细胞纤维蛋白混合物。然后轻轻地将移液器吸头放入凝血酶等分试样中，混合两次，不要引入气泡。以一定角度抬起高通量板，然后快速将移液器吸头插入其中一个加载端口。以平稳流畅的运动将移液器柱塞推到第一个停止点，然后将细胞纤维蛋白混合物注射到组织室中。确保凝胶完全穿过腔室。轻轻地将板平放，不要取下移液器吸头或移位移液器。用手从 P20 上取下移液器吸头，并将其留在装载端口孔中。两分钟后，轻轻扭转移液器吸头，从装载端口取出移液器吸头，然后将盖子放在板上。将板在 37 摄氏度下孵育 15 至 20 分钟以进行凝胶聚合。将微流控装置放在显微镜载物台上，观察细胞在整个腔室中的均匀分布，没有任何气泡。将 P20 移液器吸头插入 M1 或 M3 中，然后缓慢排出 4 微升层粘连蛋白以覆盖整个顶板。如前所述取出移液器吸头，并将板在 37 摄氏度的 5% 二氧化碳中孵育 10 分钟。将 275 μL 的 EGM-2 完全培养基加入位于 A 行和 B 行的井的未偶联储液器中，将 P200 移液器吸头插入含有 275 μL EGM-2 的孔的介质入口孔中，缓慢排出 75 μL 培养基，观察培养基通过通道并在另一侧冒泡。取下移液器吸头后，将吸头中剩余的培养基推入培养基储液器。加入 50 μL 培养基以完全覆盖 G 行和 H 行中的低侧孔，如图所示将板孵育一到两个小时。在显微镜下，识别培养基通道中的任何气泡，并通过将 75 微升培养基重新引入通道中来去除它们。用肉眼检查介质入口或出口是否有气泡。然后将 P200 移液器吸头插入孔中，并通过抬起柱塞将气泡拉出。在血管化微器官或VMO中，内皮细胞最初均匀分布在组织室内，但到第二天，它们开始伸展并发光。到第四天，内皮细胞与外部微流体通道吻合，形成连续的血管网络。通过微血管网络灌注 FITC-葡聚糖，以最小的渗漏证实了紧密的血管屏障功能。将 MDA-MB-231 癌细胞灌注到 VMO 中导致它们粘附在内皮内膜上，随后外渗到细胞外空间，在灌注后 24 小时内形成多个微转移。通过延时显微成像，观察到 T 细胞外渗到 VMO 的细胞外空间超过 45 分钟。在血管完全形成的血管化微肿瘤中，T细胞在灌注后迅速粘附在血管壁上。

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 ...