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 mechanotransduction^1,2. 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 tissues^3,4. Several studies have associated EC dysfun....

Protocol

1. Synthesis of GelMA

1. Prepare a 0.2 M solution of anhydrous sodium carbonate and a 0.2 M solution of sodium bicarbonate.
2. Mix 46 mL of sodium bicarbonate solution with 15 mL of sodium carbonate solution and add 139 mL of deionized (DI) water. Adjust the pH to 9.5 using 0.1 M NaOH and HCl if necessary.
3. Add 10 g of type A, 300-bloom gelatin from porcine skin to 100 mL of carbonate-bicarbonate buffer at a concentration of 10% w/v.
4. Dissolve the gelatin using a 55 .......

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.......

Materials

Name	Company	Catalog Number	Comments
(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