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 cancer research and therapeutic development. 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.
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.
1. Design and fabrication
Figure 1. Microfluidic platform design. (A) The schematic of the platform assembly shows the PDMS feature layer with 12 device units bonded to a bottomless 96-well plate and sealed with a thin transparent polymer membrane. Each device unit occupies a column of wells on the plate. The single device unit outlined in red is shown with details in (B). (B) Schematic of one device unit shows a single tissue chamber positioned within one well of the 96-well plate and two loading ports with inlet and outlet (L1-L2) hole punched to allow cell-matrix mix to be introduced. Medium inlets and outlets (M1-M2, M3-M4) are hole-punched and positioned within wells that serve as media reservoirs. Different volumes of media establish a hydrostatic pressure gradient across the tissue chamber via decoupled microfluidic channels. The pressure regulator (PR) unit serves as a gel burst valve to increase ease of loading. Note that the device is 200 µm deep, and the tissue chamber is 2 mm x 6 mm. Please click here to view a larger version of this figure.
2. Preparations prior to loading
3. Loading of samples
NOTE: Loading is time-sensitive and should be completed from start (cell lifting) to finish (addition of media to devices) within about 1.5-1.75 h to ensure optimal results. Each step is noted with a suggested timer to help keep the user on track.
Figure 2. Schematic of device loading. (A) Using a P20 pipette, cell/fibrin mix is introduced into the tissue chamber of each device unit via one of the loading ports. (B) Brightfield micrograph shows a microfluidic device loaded EC, fibroblasts, and cancer cells to form a VMT. Scale bar = 500 µm. (C) Fluorescence micrograph of the device in B showing EC in red, tumor in cyan, and fibroblasts in blue. (D) The schematic shows the addition of medium into the reservoirs, with 350 µL on the high side and 50 µL on the low side to generate the hydrostatic pressure head. (E) Day 2 of VMT culture shows fibroblasts and EC beginning to stretch out to form the vascular network. Scale bar = 200 µm. Please click here to view a larger version of this figure.
4. Device maintenance and experimental applications
Figure 3. Preparing platform for immunostaining. (A) Schematic of fully assembled device platform with membrane layer on top. To remove the membrane, carefully pull each corner of the outer layer down in a steady, gentle motion. (B) Once the membrane layer is removed completely, use a blade, scalpel, or knife to cut rectangles around the tissue chamber of each device unit, taking care not to cut into the tissue itself. A spatula can then be wedged under each rectangle to dislodge it from the plate and place each unit into a single well of a 24-well plate with PBS for staining. Please click here to view a larger version of this figure.
Following the protocols outlined here, VMOs and VMTs were established using commercially purchased EC, NHLF, and, for VMT, the triple-negative breast cancer cell line MDA-MB-231. Established VMOs were also perfused with cancer cells to mimic metastasis. In each model, by day 5 of co-culture, a vascular network self-assembles in response to gravity-driven flow across the tissue chamber, serving as a conduit for in vivo like delivery of nutrients, therapeutics, and cancer or immune cells to the stromal niche (Figure 4). VMOs were first established by introducing mCherry-labeled EC into the tissue chamber, as shown in Figure 4A (day 0 of culture), with an even distribution of cells. On day 2 of VMO culture, EC begin to stretch out and lumenize (Figure 4B), and by day 4, EC have anastomosed with the outer microfluidic channels and form a continuous vascular network (Figure 4C). After the vasculature formed anastomoses and lined the outer channels, VMO tissue was perfused with 70 kD FITC-dextran to confirm vascular patency (Figure 4D). FITC-dextran was introduced into the media reservoir with the highest hydrostatic pressure and allowed to perfuse across the tissue chamber via micro-vessels from the high-pressure side to the low-pressure side, as indicated by the arrows. In the VMO, FITC-dextran fully perfused the microvascular network within 15 min with minimal vascular leak, confirming tight vascular barrier function (Figure 4E). MDA-MB-231 cells were then perfused into VMO, where cells adhered to the endothelial lining (Figure 4F) and extravasated into the extra-vascular space within 24 h post-perfusion, forming multiple micro-metastases within the tissue chamber (Figure 4G). Time-lapse microscopic fluorescent images were taken every 50 ms with 4x and 10x air objectives on an inverted confocal microscope to observe cancer cells perfusing through the microvasculature in real-time (Supplementary Video 1, Supplementary Video 2).
In the VMO, T cells can be seen extravasating into the extracellular space over the course of 45 min (Figure 4H-I). Time-lapse fluorescent micrographs were taken on a confocal microscope to acquire a z-stack of 150 µm depth every 15 min to observe T cell extravasation in real time (Supplementary Video 3). As shown in Figure 4J, MDA-MB-231 VMT with fully formed, non-leaky vessels were perfused with T cells (yellow), many of which rapidly adhered to the vascular wall (arrowheads; Figure 4K, Supplementary Video 4, Supplementary Video 5). These results, in addition to prior studies8,9,10,11,12,13,14,15, demonstrate the utility of the VMO and VMT platforms for immunology and immune-oncology research, respectively.
Figure 4. Representative results for MDA-MB-231 VMT and VMO. (A) VMO on day 0 immediately after loading cells into the tissue chamber. EC are shown in red. Scale bar = 500 µm. (B) By day 2 of VMO culture, EC begin to stretch in response to flow. (C) VMO day 4 shows that the vascular network is anastomosed with the outer microfluidic channels, and vessels are nearly mature. (D) VMO networks are fully perfused and patent on day 5 of culture. Vessels shown in red, 70 kD FITC-dextran green. The direction of flow is indicated by arrows. Scale bar = 500 µm. (E) Zoom view of perfused VMO. Scale bar = 100 µm. (F) MDA-MB-231 (cyan) is perfused through the same VMO network shown in E, and at time 0, cancer cells have adhered to the endothelial vessel lining (arrowheads). Scale bar = 100 µm. (G) By 24 h, MDA-MB-231 cells have extravasated into the extracellular space, establishing multiple micro-metastases within the vascular niche. Scale bar = 100 µm. (H) Time-lapse confocal fluorescent microscopy reveals T cell extravasation through a micro-vessel within the VMO (I) over the course of 45 min. Arrowheads denote areas of extravasation. Scale bar = 50 µm. (J) Triple-negative breast cancer cell line MDA-MB-231 is established in the VMT and perfused on day 5. Scale bar = 500 µm. The vascular network shows minimal leak at 15 min post-perfusion (inset, scale bar = 100 µm). (K) MDA-MB-231 VMT (same as in B) is perfused with T cells (yellow), with multiple areas of T cell adherence to the vascular wall (arrowheads). Scale bar = 500 µm. Please click here to view a larger version of this figure.
Supplementary Video 1. Perfusion of ovarian cancer cells in the VMO. Time-lapse fluorescent microscopy of COV362 cells (cyan) perfused through a vascular network (red) and imaged at 4x objective every 50 ms for 1 min. Please click here to download of this Video.
Supplementary Video 2. Perfusion of triple-negative breast cancer cells in the VMO. Time-lapse fluorescent microscopy of MDA-MB-231 cells (cyan) perfused through a vascular network (red) and imaged at 10x objective every 50 ms for 30 s. Please click here to download of this Video.
Supplementary Video 3. T cell perfusion of VMO. Time-lapse confocal fluorescent microscopy captured the process of T cell extravasation through a micro-vessel within the VMO over a 45 min duration. Z-stack images were obtained every 15 min, with a step size of 2 µm and a depth of 150 µm. The vessel is red, T cells are yellow. Please click here to download of this Video.
Supplementary Video 4. T cell perfusion of VMT. Time-lapse fluorescent microscopy of T cells perfused through MDA-MB-231 VMT at 4x objective. Images were acquired every 50 ms for 30 s. T cells are shown in yellow, MDA-MB-231 in cyan, and vessels/EC in red. Please click here to download of this Video.
Supplementary Video 5. Zoom view of T cell-VMT perfusion. Magnified 10x view of MDA-MB-231 VMT perfused with T cells (from Supplementary Video 4). Images were acquired every 50 ms for 30 s. T cells are shown in yellow, MDA-MB-231 in cyan, and vessels/EC in red. Please click here to download of this Video.
Nearly every tissue in the body receives nutrients and oxygen through the vasculature, making it a critical component for realistic disease modeling and drug screening in vitro. Moreover, several malignancies and disease states are defined by vascular endothelial dysfunction and hyperpermeability3. Notably, in cancer, tumor-associated vasculature is often ill-perfused, disrupted, and leaky, thus acting as a barrier to therapeutic and immune cell delivery to the tumor. Furthermore, vasculature serves as a conduit through which cancer cells can metastasize to seed distant tissues and facilitates cell-cell communications that dampen the immune response while further promoting cancer cell growth and dissemination. These phenomena highlight the crucial role the vascular niche plays in therapeutic resistance and cancer progression and the need to accurately model the tumor microenvironment during preclinical study. Yet standard in vitro model systems fail to include appropriate stromal and vascular components or incorporate dynamic flow conditions. To address these shortcomings in current model systems, methods to establish a well-characterized microphysiological system that supports the formation of a living, perfused human micro-tumor (VMT) for physiologic oncology research were presented. Importantly, the VMT models key characteristics of aberrant tumor-associated vessels and tumor-stromal interactions, making it ideal for biomimetic disease modeling and therapeutic efficacy testing10.
For ease of use, the platform does not require any external pumps or valves and, owing to the 96-well plate format can be adapted to standard culture equipment and workflows. Further, different device iterations to address distinct biological questions and established tissue- and patient-specific compartments have been validated8,9,10,11,12,13,14,15,16,17. While the platform can be adapted to virtually any organ- or tissue-specific use by integrating various cell types, cells must be tested first for growth and vasculogenic capacity within the VMO/VMT at varying cell concentrations to determine the optimal seeding density and co-culture conditions. To establish the vasculature, human endothelial colony-forming cell-derived endothelial cells (ECFC-EC) can be purchased commercially or freshly isolated from cord blood by selecting CD31+ cells. Human umbilical vein endothelial cells (HUVEC) can also be used to establish vasculature within the VMO/VMT and can either be purchased commercially or freshly isolated from umbilical cords. Additionally, induced pluripotent stem cell-derived endothelial cells (iPSC-EC) have been successfully tested in the platform, opening the possibility of a completely autologous system18. Commercially derived fibroblasts (standard, normal human lung fibroblasts for their vasculogenic potential) work well in the VMO/VMT, and some primary-derived stromal cell populations can be incorporated or substituted as well. Primary-derived tumors can be introduced into the VMT as single cells, spheroids, organoids, or tumor chunks. Matrix composition can be modified according to experimental needs, including spiking with collagens, laminin, fibronectin, or even decellularized tissue matrices19.
The protocol includes several critical steps where special care is essential to avoid common issues (Figure 5). During loading, ensure homogeneous mixing of the cell/fibrin slurry by careful pipetting and smooth introduction into the tissue chamber (Figure 5A). Apply proper pressure to expel the gel completely into the chamber to prevent partial loading (Figure 5B). Visualizing the cell/fibrin slurry transversing the entire tissue chamber is necessary to ensure complete chamber filling and can be facilitated by placing a gloved finger behind the device unit. Avoid pressing too hard on the micropipette plunger to prevent bursting the cell/fibrin mix into the microfluidic channels (Figure 5C). Care must be taken not to introduce air bubbles during pipetting to prevent interference with tissue development and downstream applications (Figure 5D). Proper mixing and loading speed are crucial to avoid areas of inconsistent clotting (Figure 5E) while removing the pipette tip prematurely may also disrupt the gel in the chamber (Figure 5F). Practice loadings are recommended to familiarize users with the timed element of the procedure and the loading step. Further, properly introducing laminin into the microfluidic channels is crucial for EC migration, anastomosis with outer channels, and the formation of a continuous, perfusable network for nutrient delivery. Incomplete or absent channel lining will lead to poor perfusion results and unusable VMO/VMT.
Figure 5. Common mistakes with loading. (A) Microfluidic device unit properly loaded without defects. (B) Cell/fibrin mix was not introduced completely into the chamber, resulting in partial loading. (C) Too much pressure applied during loading, resulting in gel bursting into the microfluidic channel, blocking flow. (D) Air bubbles introduced into the cell/fibrin mix within the chamber while pipetting. (E) Improper mixing of the cell/fibrin mix or slow loading causing inconsistencies in clotting. (F) Removing the pipette tip from the loading port before the gel has sufficiently set will result in disruption of the cell/fibrin mix in the tissue chamber. Please click here to view a larger version of this figure.
Robust and standardized workflows for analysis are crucial in VMO/VMT studies, as they generate substantial amounts of imaging data. Image processing and analytical methods for VMO/VMT have been described previously8,9,10,11,12,13. For tumor quantitative analysis, the fluorescent intensity in the color channel representing the tumor cells is measured using open-source software like ImageJ/Fiji (National Institute of Health)20 or CellProfiler (Broad Institute)21. The threshold of tumor micrographic images is set to select the fluorescent tumor region, and the mean fluorescence intensity is measured within that region. The tumor's total fluorescent intensity is calculated as the product of the fluorescent area and its mean intensity, normalized to baseline values (pre-treatment) to obtain the fold change in tumor growth per device over the experimental period. Regarding vessel quantitative analysis, AngioTool (National Cancer Institute)22, ImageJ/Fiji macro scripts, or MATLAB software, such as REAVER23, can be used to quantify total vessel length, number of endpoints, number of junctions, average vessel length, vessel diameter, mean lacunarity, and vessel percentage area. Machine learning algorithms can be integrated into workflows for automatic analysis of vascular images to identify compounds that effectively disrupt the vasculature24. Perfusion images are analyzed by measuring the change in fluorescence intensity within regions of the extracellular space and calculating the permeability coefficient10. Finite element simulations of intraluminal flow inside a microvascular network can be performed using COMSOL Multiphysics25. Implementation of standardized analytical methods is critical for extracting meaningful insights from the vast amount of data generated in VMT studies.
The protocol outlined here will allow the user to leverage the VMO/VMT platform to study many aspects of tumor biology, including tumor growth/progression, tumor metastasis, intra-tumor T cell dynamics, and tumor response to chemotherapy and anti-angiogenic treatment. To enable physiologically relevant immuno-oncology studies, it was demonstrated how freshly isolated T cells perfuse through the microvasculature, extravasate across the endothelial cell barrier, and migrate into the tissue construct. Time-lapse microscopic confocal imaging was presented as a tool to view spatially random, temporally rapid events, including T cell extravasation, that are not readily visualized with other model systems. In addition, we have previously tested multiple types of antineoplastic drugs in the VMT, including chemotherapeutics, small molecule/tyrosine kinase inhibitors, monoclonal antibodies (such as anti-PD1 and bevacizumab), anti-angiogenic compounds, and vascular stabilizing agents, underscoring how the platform can be used to test different classes of drugs targeting both the tumor and the associated stroma8,9,10,11,12,13. Effluent can be collected from the platform and analyzed for various cytokines as well as exosomes. In future studies, the VMT platform can be used to assess the sensitivity of tumor cells to T-cell-mediated attack at the individual patient level. In conclusion, the VMT is a flexible and powerful platform and one that is ideal for studying tumor biology, where remodeling of the vascular and stromal components is key to tumor progression.
The authors have nothing to disclose.
We thank members of Dr. Christopher Hughes' lab for their valued input into the procedures described, as well as our collaborators in Dr. Abraham Lee's lab for their assistance with platform design and fabrication. This work was supported by the following grants: UG3/UH3 TR002137, R61/R33 HL154307, 1R01CA244571, 1R01 HL149748, U54 CA217378 (CCWH) and TL1 TR001415 and W81XWH2110393 (SJH).
Fabrication | |||
(3-Mercaptopropyl)trimethoxysilane, 95% | Sigma-Aldrich | 175617-100G | |
Greiner Bio-One μClear Bottom 96-well Polystyrene Microplates | Greiner Bio-One | 655096 | |
Methanol ≥99.8% ACS | VWR Chemicals BDH | BDH1135-1LP | |
MILTEX Sterile Disposable Biopsy Punch with Plunger, 1mm diameter, | Integra Miltex | 33-31AA-P/25 | |
PDMS membrane | PAX Industries | HT-6240 | |
Plasma Cleaner PDC-001 | Harrick Plasma | N/A | |
Smooth-Cast 385 | Smooth-On | N/A | |
SP Bel-Art Lab Companion Clear Polycarbonate Cabinet Style Vacuum Desiccator | Bel-Art | F42400-4031 | |
Standard Lids with Condensation Rings, 96-well plate | VWR | 82050-827 | |
SYLGARD 184 Silicone Elastomer Kit (PDMS) | Dow | 4019862 | |
Cell culture/Loading | |||
BioTek Lionheart FX Automated Microscope | Agilent | CYT5MFAW | |
CELLvo Human Endothelial Progenitor Cells | StemBioSys | N/A | |
Collagen I, rat tail | Enzo Life Sciences | ||
Collagenase from Clostridium histolyticum (type 4) | Sigma-Aldrich | C5138 | |
Corning Hank’s Balanced Salt Solution, 1X without calcium and magnesium | Corning | 21-021-CV | |
Corning DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate | Corning | 10013CV | |
DAPI | Sigma-Aldrich | D9542 | |
DPBS, no calcium, no magnesium | Gibco | 14190144 | |
EGM-2 Endothelial Cell Growth Medium-2 BulletKit | Lonza | CC-3162 | |
Fibrinogen from bovine plasma | Neta Scientific | SIAL-341573 | |
Fibronectin human plasma | Sigma-Aldrich | F0895 | |
Fluorescein isothiocyanate–dextran (70kDa) | Sigma-Aldrich | FD70S-1G | |
Gelatin from porcine skin | Sigma-Aldrich | G1890 | |
Hyaluronidase from sheep testes (type 4) | Sigma-Aldrich | H6254 | |
Laminin Mouse Protein | Gibco | 23017015 | |
Leica TCS SP8 | Leica | N/A | |
MDA-MB-231 | ATCC | HTB-26 | |
NHLF – Normal Human Lung Fibroblasts | Lonza | CC-2512 | |
Nikon Eclipse Ti | Nikon | N/A | |
Paraformaldehyde 4% in 0.1M Phosphate BufferSaline, pH 7.4 | Electron Microscopy Sciences | 15735-90-1L | |
PBMCs – Peripheral blood mononuclear cells | Lonza | CC-2702 | |
PBS, pH 7.4 | Gibco | 10010049 | |
Premium Grade Fetal Bovine Serum (FBS), Heat Inactivated | Avantor Seradigm | 97068-091 | |
ProLong Gold Antifade Mountant | Invitrogen | P10144 | |
Quick-RNA Microprep Kit | Zymo Research | R1051 | |
Thrombin from bovine plasma | Sigma-Aldrich | T4648 | |
Triton X-100 (Electrophoresis), | Fisher BioReagents | BP151-100 | |
TrypLE Express Enzyme (1X), phenol red | Gibco | 12605028 | |
Trypsin-EDTA (0.05%), phenol red | Gibco | 25300062 | |
Vasculife | Lifeline Cell Technology | LL-0003 |