Name: Preparing a Vascularized Micro-Tumor System Using Human Cells for Studying Tumors and Drug Responses
Uploaded: 2023-09-15T14:50:00
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preparing a vascularized micro-tumor system using human cells for studying tumors and drug responses 

הכנת מודל מיקרו-גידולי וסקולרי אנושי מבוסס מיקרופלואידיקה

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.

מחבר זרקור: יצירת מיקרו-גידולים וסקולריים אנושיים כמודלים לחקר סרטן תרגומי 

הקמת מודל פיזיולוגי למיקרו-גידולים וסקולריים אנושיים לחקר הסרטן

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 Preparing a Vascularized Micro-Tumor System Using Human Cells for Studying Tumors and Drug Responses at JoVE.com

Preparing a Vascularized Micro-Tumor System Using Human Cells for Studying Tumors and Drug Responses   

הכנת מערכת מיקרו-גידולים וסקולרית באמצעות תאים אנושיים לחקר גידולים ותגובות לתרופות 

כדי להתחיל, מקם את מגיב הדיסוציאציה של תאי HBSS EGM-2 ו- DMEM באמבט מים של 37 מעלות צלזיוס למשך 10 עד 15 דקות. הפשירו תרומבין ולמינין למשך הלילה בטמפרטורה של 4 מעלות צלזיוס ופיברינוגן בטמפרטורת החדר. הוסף 1.5 מיקרוליטר של טרומבין לתוך צינור מיקרוצנטריפוגה 500 מיקרוליטר.הכניסו לייבוש פלטות UV מעוקרות בעלות תפוקה גבוהה למשך 30 דקות כדי להסיר אוויר הכלוא במיקרופלואידיקה. הניחו את בקבוק T75 מתחת למיקרוסקופ בהגדלה של פי 4 כדי לאשר את מפגש התאים ואת יעילות הטרנסדוקציה. לשטוף את התאים פעמיים עם חמישה מיליליטר של HBSS.לאחר מכן, להוסיף מיליליטר אחד של מגיב דיסוציאציה לבקבוק לדגור ב 37 מעלות צלזיוס עם 5% פחמן דו חמצני במשך דקה עד שתי דקות. טפחו בעדינות על הבקבוק באמצעות כף היד והתבוננו מתחת למיקרוסקופ שכל התאים הורמו. לשטוף את התאים עם תשעה מיליליטר של מדיה מתאימה ולאסוף השעיה לצינור חרוטי 15 מיליליטר.מיד להסיר aliquot קטן ולספור את התאים. צנטריפוגה את מתלה התא ב 300G וארבע מעלות צלזיוס למשך שלוש עד חמש דקות. לאחר מכן, הסר את supernatant ו resuspend את הגלולה במדיה המתאימה על קרח.באמצעות המשוואה הנתונה, חשב את מספר התאים הדרושים. השהה מחדש את התאים בריכוז של 1 כפול 10 עד 6 תאים למיליליטר, וחשב את נפח התאים הדרוש באמצעות המשוואה הבאה. צנטריפוגה את הנפח הנדרש של תרחיף התא כפי שהודגם.לאחר מכן, בנפח מחושב של פיברינוגן, להשעות מחדש את הגלולה על קרח.

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Research

JoVE Journal

Cancer Research

Preparing a Vascularized Micro-Tumor System Using Human Cells for Studying Tumors and Drug Responses 

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 cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

To begin, place the HBSS cell dissociation reagent EGM-2 and DMEM in a 37 degree Celsius water bath for 10 to 15 minutes. Thaw thrombin and laminin overnight at 4 degrees Celsius and fibrinogen at room temperature. Add 1.5 microliters of thrombin into a 500 microliter microcentrifuge tube.Place a UV sterilized high-throughput plates into a desiccator for 30 minutes to remove air trapped in the microfluidics. Place the T75 flask under the microscope at 4x magnification to confirm cell confluency and transduction efficiency. Wash the cells twice with five milliliters of HBSS.Then, add one milliliter of dissociation reagent to the flask and incubate at 37 degrees Celsius with 5%carbon dioxide for one to two minutes. Gently tap the flask using the palm and observe under the microscope that all cells have been lifted. Wash the cells with nine milliliters of appropriate media and collect suspension to a 15-milliliter conical tube.Immediately remove a small aliquot and count the cells. Centrifuge the cell suspension at 300G and four degrees Celsius for three to five minutes. Then, remove the supernatant and resuspend the pellet in appropriate media on ice.Using the given equation, calculate the number of cells needed. Resuspend the cells at a concentration of 1 times 10 to the 6 cells per milliliter, and calculate the volume of cells needed using the following equation. Centrifuge the required volume of cell suspension as demonstrated.Then, in a calculated volume of fibrinogen, resuspend the pellet on ice.

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.