introducing human cells into a microfluidic device to investigate tumor progression in a vascularized micro-tumor system

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

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.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

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  

To begin, place the UV-sterilized high-throughput plates and thrombin aliquots into the tissue culture hood. Place the prepared cell fibrin mix on ice to slow clotting. Using a P20 pipette, draw six microliters of the cell fibrin mix.Then gently place the pipette tip into the thrombin aliquot and mix twice without introducing air bubbles. Lift the high-throughput plate at an angle and quickly insert the pipette tip into one of the loading ports. Push the pipette plunger to the first stop in a smooth and fluid motion and inject the cell fibrin mix into the tissue chamber.Ensure the gel crosses entirely through the chamber. Gently place the plate flat without removing the pipette tip or displacing the pipette. Remove the pipette tip using a hand from the P20 and leave it in the loading port hole.After two minutes, remove the pipette tips with gentle twisting motions from the loading ports and place the lid on the plate. Incubate the plate for 15 to 20 minutes at 37 degrees Celsius for gel polymerization. Place the microfluidic device on the microscope stage to observe the even distribution of cells throughout the chamber without any air bubbles.Insert the P20 pipette tip into M1 or M3 and expel four microliters of laminin slowly to coat the entire top panel. Remove the pipette tip as demonstrated previously and incubate the plate at 37 degrees Celsius in 5%carbon dioxide for 10 minutes. Add 275 microliters of EGM-2 complete media to the uncoupled reservoirs of wells located in rows A and B.Insert the P200 pipette tip into the media inlet hole of the wells containing 275 microliters of EGM-2 and slowly expel 75 microliters of medium, watching the media travel through the channel and bubble up on the other side.After removing the pipette tip, push the remaining media from the tip into the media reservoir. Add 50 microliters of media to entirely cover the low side wells in rows G and H.Incubate the plates for one to two hours as demonstrated. Under a microscope, identify any bubbles in the media channels and remove them by reintroducing 75 microliters of media into the channels.Check medium inlets or outlets for air bubbles using the naked eye. Then insert the P200 pipette tip into the hole and pull the bubble out by lifting the plunger. In vascularized micro-organs or VMOs, endothelial cells initially distributed evenly within the tissue chamber but by day two, they began to stretch out and luminize.By day four, the endothelial cells anastomosed with the outer microfluidic channels, forming a continuous vascular network. The tight vascular barrier function was confirmed by perfusing FITC-dextran through the microvascular network with minimal leakage. Perfusing MDA-MB-231 cancer cells into the VMOs resulted in their adherence to the endothelial lining and subsequent extravasation into the extracellular space, forming multiple micrometastases within 24 hours post-perfusion.Through time-lapse microscopic imaging, the extravasation of T-cells into the extracellular space of the VMOs was observed over 45 minutes. In vascularized micro-tumors with fully-formed vessels, T-cells rapidly adhered to the vascular wall upon perfusion.

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Cancer Research

92 次观看.  University of California, Irvine. 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?...