In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses
Summary
We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.
Abstract
We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.
Introduction
Endothelium, lining the inner surface of blood vessels, plays a pivotal role in maintaining vascular health. Endothelial cells (ECs) are central to regulating various cardiovascular functions, including vessel tone control, selective permeability, hemostasis, and mechanotransduction1,2. Research has firmly linked EC dysfunction to a primary role in atherosclerosis development. Notably, ECs encounter diverse mechanical forces at the interfaces where they interact with blood flow and underlying vessel tissues3,4. Several studies have associated EC dysfunction with abnormal changes in mechanical factors within the vascular environment, such as the fluid shear stress from blood flow and tissue rigidity5,6,7.
However, prior research has received limited attention in comprehending the combined effects of tissue rigidity and shear stress on EC function. To enhance the ability to translate research outcomes into effective treatments for atherosclerosis and other cardiovascular diseases, it is essential to improve the cellular models used in the field. Significant progress has been made in humanizing cellular models by employing human ECs and subjecting them to either shear stress or substrates with varying stiffness levels8,9,10. However, the adoption and refinement of cellular models that integrate dynamic flow environments with EC substrates possessing adjustable stiffness properties has progressed slowly. The challenge lies in devising non-swelling EC substrates to prevent alterations in flow parameters within the flow channel while also facilitating the cultivation of intact and well-adhered EC monolayers. An in vitro model capable of overcoming these obstacles could facilitate more effective investigations into how hypertension, aging, and flow conditions collaboratively influence EC mechanotransduction, vascular health, and, ultimately, the development of atherosclerosis. Various methods have been developed to apply shear stress on cells while controlling substrate stiffness, including rotating plates and microfluidic devices. In the rotating plate method, cells are placed between two plates and shear stress is applied through the rotational movement of the plates. This method is less complicated and provides a quick model; however, it suffers from spatial shear stress variation, with zero shear stress at the center and maximum shear stress at the periphery11.
On the other hand, microfluidic devices represent the new generation of tools with the ability to control substrate rigidity and flow conditions. These systems are suitable for mimicking microvasculatures under laminar flow conditions. However, studying atherosclerosis with such devices is impractical, as atherosclerosis occurs in large vessels with disturbed flow11. This paper aims to contribute to the critical research domain of EC studies by presenting a cost-effective system capable of examining the effects of varying stiffness levels in EC substrates under different flow conditions. The system integrates substrates with different stiffnesses to emulate pathological and physiological blood vessels. This protocol outlines the method for creating gelatin-based hydrogels with no swelling and stiffness levels of 5 kPa and 10 kPa, representing physiological and pathological stiffness, respectively. Additionally, the construction of a parallel-plate flow chamber capable of integrating these substrates is detailed. Computational fluid dynamics (CFD) was employed to evaluate shear stress and flow conditions. The preparation of hydrogels for EC culture and the execution of a 6 h flow experiment are described, followed by a discussion on post-experiment immunostaining.
Protocol
Representative Results
Discussion
The vascular system is a dynamic environment where various forces significantly influence cellular behavior. Studying biological events in cardiovascular diseases without considering these forces would be inaccurate. Thus, cellular models capable of emulating the vascular mechanical environment are crucial. Researchers have already made significant progress in highlighting the effect of these forces on cellular behavior11. However, to understand cell behavior under both pathological and physiologi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors extend their gratitude to Robert Egan for his assistance in fabricating the flow chamber. The authors thank Lucas McCauley for his help during the experiments. Additionally, they would like to acknowledge Northeastern University's Institute for Chemical Imaging of Living Systems (CILS) core facilities for granting access to confocal microscopes. The authors acknowledge the funding support provided by the National Institutes of Health (NIH 1R01EB027705 awarded to SB) and the National Science Foundation (NSF CAREER Awards: DMR 1847843 to SB and CMMI 1846962 to EE).
Materials
| (trimethoxysilyl)propyl methacrylate, tetramethylethylenediamine (TEMED) | Invitrogen | 15524-010 | Hydrogel Fabrication |
| 3-(Trimethoxysilyl)Propyl Methacrylate | Sigma-Aldrich | 440159 | Glass Salinization |
| 4’,6-diamidino-2-phenylindole (DAPI)-containing mounting media | Vector Laboratories | H-1200 | Immunostaining |
| Acetone | Thermo Fisher Scientifics | A18-4 | GelMA Synthesis |
| Alexa Fluor 555 Phalloidin | Cell Signaling Technology | 8953S | Immunostaining |
| Ammonium Persulfate (APS) | Bio-Rad | 1610700 | Hydrogel Fabrication |
| Clear Scratch- and UV-Resistant Cast Acrylic Sheet (45/64'') | McMaster-CARR | 8560K165 | Flow Chamber Fabrication |
| Confocal Microscope | Carl Zeiss Meditex AG | Zeiss LSM 800 | Immunostaining |
| Covidien Monoject Rigid Pack 60 mL Syringes without Needles | Fisher | 22-031-375 | Flow Experiment |
| EC growth kit | American Type Culture Collection (ATCC) | PCS-100-041 | Cell Culture |
| Ethanol 200 Proof | Decon Labs | 2701 | Glass Salinization |
| Gelatin Type A (300 bloom) from porcine skin | Sigma-Aldrich | G1890 | GelMA Synthesis |
| Glacial Acetic Acid | Thermo Fisher Scientifics | 9526-33 | Glass Salinization |
| High-Purity High-Temperature Silicone Rubber Sheet | McMaster-Carr | 87315K74 | Flow Chamber Fabrication |
| Human Umbilical Vein Endothelial Cells (HUVEC) | American Type Culture Collection (ATCC) | PSC-100-010 | Cell Culture |
| M3x30mm Machine Screws Hex Socket Round Head Screw 304 Stainless Steel Fasteners Bolts 20pcs | Uxcell | B07Q5RM2TP | Flow Chamber Fabrication |
| Masterflex L/S Digital Drive with Easy-Load® 3 Pump Head for Precision Tubing; 115/230 VAC | VWR | #MFLX77921-65 | Flow Experiment |
| Masterflex L/S Precision Pump Tubing, Puri-Flex, L/S 25; 25 ft | VWR | #MFLX96419-25 | Flow Experiment |
| Methacrylic Anhydride (MAH) | Sigma-Aldrich | 276685 | GelMA Synthesis |
| Paraformaldehyde | Thermo Fisher Scientifics | 043368.9M | Cell Culture |
| Phosphate-Buffered Saline (PBS) | Gibco | 14080-055 | General |
| Sodium Bicarbonate | Fisher Chemical | S233-3 | GelMA Synthesis |
| Sodium Carbonate | Fisher Chemical | S263-500 | GelMA Synthesis |
| SOLIDWORKS educational version | |||
| SOLIDWORKS Student Edition Desktop, 2023 | SolidWorks | N/A | Flow Chamber Design |
| Vascular Basal Medium | American Type Culture Collection (ATCC) | PCS-100-030 | Cell Culture |
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