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.
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.
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.
1. Synthesis of GelMA
2. Glass salinization
NOTE: Attaching hydrogels to glass slides provides a flat and even surface, facilitating handling and ensuring stability under flow-derived shear stress. Functionalizing the glass with 3-(trimethoxysilyl)propyl methacrylate is necessary to enhance surface properties and enable the covalent attachment of hydrogels during the polymerization process.
3. Hydrogel preparation
4. Coating hydrogels
5. Seeding cells on the substrates
6. Flow chamber fabrication
NOTE: The approach for designing the flow chamber is cost-effective and requires minimal expertise for fabrication and utilization.
7. Run uniform laminar flow
8. Immunostaining setup for confocal microscopy with high magnification
NOTE: To increase study efficiency, a method was developed for immunostaining small portions of hydrogels, enabling the examination of multiple biological targets in a single sample.
Figure 1 depicts the experimental setup, outlining the process of GelMA synthesis through a methacrylation reaction. The resulting product was then used to fabricate the hydrogel substrate, onto which ECs were seeded. Subsequently, the cells were introduced into the flow chamber for a 6 h flow experiment at 12 dyne/cm2.
1H NMR spectroscopy was used to assess the success of the methacrylation reaction (Figure 2A). The presence of a methyl group at 1.9 ppm and a vinylic peak between 5.4-5.6 ppm in GelMA confirmed successful methacrylation. Additionally, the decrease in the lysine peak at 3 ppm in GelMA indicates the consumption of lysine residues, which are replaced with methacrylate residues12,13,16. The stiffness of GelMA hydrogels was evaluated using a compression test, which showed that the compression moduli increased with GelMA concentrations (Figure 2B). Hydrogels composed of 4% and 8% (w/v) GelMA were used to mimic physiological (5 kPa) and pathological (10 kPa) matrix stiffnesses, respectively8.
The flow chamber was engineered to be cost-effective and for easy sterilization, by utilizing acrylic polymer that is UV-resistant. Its transparency facilitates real-time monitoring of hydrogels and flow conditions during experiments. Designed with three distinct layers, the chamber minimizes the risk of hydrogel damage during loading or unloading: the bottom plate provides a sturdy base, the middle layer offers lateral support for the hydrogels, and the top plate, along the gasket, creates the clearance necessary for fluid flow (Figure 3A). Computational simulations were conducted using CFD to assess flow conditions and shear stress within the chamber. The following equation – shear stress = 0.0558 x flow rate – calculated the applied shear stress to the cells based on the flow rate as an input (Figure 3B). Notably, changes in material properties, such as stiffness, did not alter shear stress in the simulations. To count for differences in the hydrogel size in the final experimental setup, the hydrogels were intentionally sized slightly smaller in the computational model. A 0.5 mm gap was created between one side of the hydrogels and the chamber's middle plate walls, perpendicular to the flow direction. This configuration allowed for the analysis of shear stress effects in these gaps. While irregularities in shear stress were observed at gap locations (Figure 3B), their impact was confined to a small area adjacent to gaps, with the remaining hydrogel surface experiencing uniform shear stress (Figure 3C). These insights suggest discarding cells from the edges of hydrogels to minimize the potential impact of turbulent regions. It is worth mentioning that higher shear stress, up to 15 dyne/cm², was experimentally applied to ECs seeded on 5 kPa and 10 kPa hydrogels with no leakage in the device (data not included). However, increasing shear stress further could potentially result in cell detachment and hydrogel failure, emphasizing the need for careful optimization of experimental conditions.
For seeding cells to form a monolayer, it is crucial to use a higher cell density than in traditional cultures. Low seeding density has been shown to hinder the monolayer formation on softer hydrogels8. Additionally, pre-coating hydrogels with gelatin before cell seeding enhances initial cell attachment and spreading on softer hydrogels. However, it is important to note that the beneficial effect of this coating is temporary, as it primarily facilitates the initial interaction between the cells and the substrate.
Figure 4 demonstrates how stiffness and shear stress influence the formation of actin fibers. Under shear stress, thicker stress fibers formed, suggesting a stronger attachment to the surface. In softer samples, there were more peripheral actin fibers, which are indicators of physiological conditions. However, in ECs on stiffer substrates, the presence of stronger stress fibers and fewer peripheral fibers could potentially lead to EC dysfunction17. This data confirms the effectiveness of the presented system in modulating EC behavior.
Figure 1: Overview of the current study. (A) GelMA synthesis. Gelatin was chemically modified to gelatin methacrylate (GelMA) through a reaction between gelatin and methacrylic anhydride (MAH) at 55 °C. The product was then precipitated in acetone and dried under a vacuum. (B) Hydrogel fabrication. The cover glass was prepared by attaching spacers. Then, the modified glass was attached to the cover glass. The cover glass was placed on the mold, with the spacers providing the desired clearance between the modified glass and the bottom of the mold. The GelMA solution containing initiators was added to the opening between the modified glass and the mold, polymerizing to form a hydrogel covalently bound to the modified glass. (C) Flow experiment. The resulting hydrogel was used to seed ECs. After forming a monolayer, the cells underwent a 6 h flow experiment at a shear stress of 12 dyne/cm². This figure was created with BioRender.com. Please click here to view a larger version of this figure.
Figure 2: 1H-NMR spectra for gelatin and GelMA pre-polymerization. (A) Refer to dashed-lined boxes for relevant peaks. Methacryloyl peaks (i.e., vinylic and methyl groups) appeared after the chemical modification of gelatin, while the lysine group was used to quantify the degree of substitution following the chemical reaction18. (B) Young's modulus of the hydrogels was measured by a compression test, and 4% (w/v) GelMA hydrogels were considered as physiological substrates, and 10% (w/v) was considered as pathological substrate (n=4, mean ± SEM). This figure has been modified from8. Please click here to view a larger version of this figure.
Figure 3: Parallel-plates flow chamber design and computational simulation. (A) Three separate plates were used to reduce the possibility of damaging the hydrogels during loading or unloading; where the bottom plate provided a backing surface, the middle surface offered lateral support for the hydrogels, and the top plate and gasket formed the clearance for the fluid to flow. (B) The flow chamber underwent computational simulations11. When the flow rate is 215 mL/min, the shear stress along the drawn line is approximately 12 dyne/cm2, representing the physiological shear stress. (C) The influence of the 0.5 mm gap is confined to a small area adjacent to the gap. The remaining surface of the hydrogel experiences uniform shear stress. Please click here to view a larger version of this figure.
Figure 4: Shear stress and stiff hydrogel increase stress fiber formation. More peripheral actin fibers are formed in ECs on 5 kPa samples under flow. Stronger stress fibers were formed when the cells were exposed to shear stress on 10 kPa hydrogels, showcasing the model's effectiveness on EC's behavior. Arrows indicate peripheral actin, and the asterisks indicate stressed fibers. Scale bar= 10 µm (Blue: DAPI, Red: Actin Fibers). Please click here to view a larger version of this figure.
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 physiological conditions in the human body, it is essential to develop more precise models that more closely resemble the blood vessel's environment. Therefore, we aimed to develop a system that more accurately replicates the blood vessel environment while maintaining ease of access and user-friendliness.
The model can apply controlled flow-derived shear stress to human cells on substrates with varying stiffness levels, producing conditions closer to physiological realities compared to existing models. GelMA was synthesized and utilized in this model to meet the following criteria: 1) tunable mechanical properties, 2) non-swelling behavior, 3) cell compatibility and adhesion, and 4) the capability of embedding vascular cells to model the blood vessels more accurately. The adjustability of mechanical properties was achieved by varying the biopolymer concentration8 to mimic physiological and pathological conditions. The second criterion was the non-swelling behavior. It is crucial to have a non-swelling substrate to maintain consistent flow chamber dimensions, related flow conditions, and shear stress on the cells. GelMA with a high degree of methacrylation demonstrated non-swelling properties, preserving the hydrogel's shape and surface smoothness throughout the experiment8. Importantly, the concentration and stiffness did not affect the swelling behavior, which simplified the model by eliminating the need for separate adjustments for each experimental group. The third criterion was cell adhesion, as proper attachment is necessary to prevent cell detachment and preserve monolayer integrity. GelMA provided cell adhesion, thereby reducing the need for additional steps to conjugate cell-adhesive molecules to the substrate, which is essential for many biopolymers. Furthermore, GelMA's capability for cell encapsulation was considered, although it was not directly tested in this study. The cell encapsulation potential has indications for supporting 3D cell culture and integrating layers of cells, such as vascular smooth muscle cells or pericytes, to enhance the model's accuracy19. In addition, the synthesis of GelMA is cost-effective and requires minimal equipment, making it an excellent candidate as a biomaterial for substrate fabrication20,21,22.
The parallel-plate flow chamber is commonly used to apply shear stress to cells, but it has traditionally only been used with glass coverslips or rigid materials. However, such materials lack physiological relevance23. In contrast, microfluidic devices have introduced more geometric complexity and softer substrates by utilizing polymer-based materials. However, these devices often cannot control the flow regime accurately, and their small dimensions limit their capacity to studying only a small number of cells, limiting experimental outcomes11. The proposed device combines the benefits of both systems by integrating endothelial cell monolayer seeded hydrogels with a flow chamber applying precisely controlled shear stress.
The device has demonstrated the capability to integrate both flow-derived and solid-derived mechanical forces. When a shear stress of 12 dyne/cm2 was applied for 6 h, a formation of cytosolic stress fibers was observed, contrasting with the predominance of peripheral actin in the softer substrate group. This is in line with many reports showing fewer stress fibers formed when ECs are cultured on softer surfaces24,25,26,27. On the other hand, laminar flow could result in prominent stress fiber formation. It has been shown that cytoskeletal response to flow conditions starts within 1 h of exposure to the flow but requires a remarkably longer time to complete reorganization28,29,30. The peripheral actin network is essential for various EC functions, including cell-cell adhesion and barrier functionality17. Upregulating this network in a healthy experimental group in comparison to the pathologic group with extensive stress fibers approves the device's successful modeling of healthy and diseased conditions.
One drawback of this device is the potential for damage to the hydrogels, which could disrupt the flow and diminish the success rate of experiments. This issue primarily arises from initial defects in the hydrogels, which, under shear stress, may worsen, leading to the detachment of the sample and partial flow obstruction. Therefore, the sample preparation steps, including polymerization, equilibration, and cutting, should be conducted carefully to prevent any additional damage to the samples. Another challenge in this system is achieving and maintaining the integrity of the monolayer. While coating the hydrogels with gelatin can improve initial cell attachment, our previous work showed that this coating does not affect cell proliferation8. Therefore, to enhance monolayer formation, especially considering that cell proliferation is slower on softer hydrogels31, increasing the seeding density is beneficial. Additionally, the cells may detach due to the shear stress induced by fluid flow. Hence, it is crucial to gradually increase the flow rate, allowing the cells sufficient time to adapt to the new environmental conditions.
In conclusion, the device represents a significant advancement in simulating the vascular environment more accurately due to its ability to simultaneously simulate both fluid-derived and solid-derived mechanical forces. It offers a comprehensive platform for studying EC behavior under various physiological and pathological conditions. This versatility makes it a valuable tool for advancing our understanding of vascular biology and disease progression. This model can contribute to a variety of research studies, including mechanobiology, atherosclerosis, cancer metastasis development, vascular tissue engineering and angiogenesis, and drug delivery and screening.
The authors have nothing to disclose.
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).
(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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