Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Summary

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

Abstract

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

Introduction

Endothelial cell (EC) dysfunction is a key player in saphenous vein graft failure^1,2,3,4. The sustained increase of shear stress and cyclic stretch induces the proinflammatory phenotype of human saphenous vein endothelial cells (hSVECs)^3,4,5,6. The underlying molecular pathways are still not fully understood, and standardized protocols for in vitro studies may leverage the efforts for novel insights in the....

Protocol

Unused segments of saphenous veins were obtained from patients undergoing aortocoronary bypass surgery at the Heart Institute (InCor), University of São Paulo Medical School. All individuals gave informed consent to participate in the study, which was reviewed and approved by the local ethics committee.

1. Isolation, culture, and characterization of primary human saphenous vein endothelial cells (hSVECs)

1. Preparation
   1. Autoclave a pair of straight or curved forceps and tissue scissors (7-8 cm).
   2. Prepare sterile gelatin. Mix 0.1 g (0.1% w/v) or 3 g (3% w/v) of porcine skin gelatin with 100....

Results

Typically, adhered ECs can be observed 3-4 days after extraction. hSVECs initially form clusters of cells and display a typical "cobblestone" morphology (Figure 1B). They express the EC markers CD31 (Figure 1C,D) and VE-cadherin (Figure 1D). hSVECs can be easily propagated on a non-coated treated cell culture dish, and they retain the endothelial phenotype in culture up to eight passages.

Discussion

The saphenous vein segment should have be least 2 cm to successfully isolate hSVECs. Small segments are difficult to handle and tie the ends of the vessel to maintain the collagenase solution to isolate the cells. The reduced luminal surface area does not yield sufficient cells to expand the culture. To minimize the risk of contamination with non-ECs, the manipulation of the saphenous vein segment needs to be very gentle during the entire procedure. It is important to be careful when introducing the pipette tips into the.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-0.02% EDTA solution	Gibco	25200072
15 µ slide I 0.4 Luer	Ibidi	80176
4',6-Diamidino-2-Phenylindole, Dilactate (DAPI)	Thermo Fisher Scientific	D3571
6-wells equibiaxial loading station of 25 mm	Flexcell International Corporation	LS-3000B25.VJW
8-well chamber slide with removable well	Thermo Fisher Scientific	154453
Acetic Acid (Glacial)	Millipore	100063
Acrylic sheet 1 cm thick	Plexiglass
Anti-CD31 antibody	Abcam	ab24590
Anti-CD31, FITC antibody	Thermo Fisher Scientific	MHCD3101
Anti-VE-cadherin antibody	Cell Signaling	2500
Bioflex plates collagen I	Flexcell International Corporation	BF3001C
Bovine serum albumin solution	Sigma-Aldrich	A8412
Cotton suture EP 3.5 15 x 45 cm	Brasuture	AP524
Cyclophilin forward primer	Thermo Fisher Scientific	Custom designed
Cyclophilin reverse primer	Thermo Fisher Scientific	Custom designed
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D4540
EBM-2 basal medium	Lonza	CC3156
EGM-2 SingleQuots supplements	Lonza	CC4176
Fetal bovine serum (FBS)	Thermo Fisher Scientific	2657-029
Flexcell FX-5000 tension system	Flexcell International Corporation	FX-5000T
Fluoromount aqueous mounting medium	Sigma-Aldrich	F4680
Gelatin from porcine skin	Sigma-Aldrich	G2500
Glycerol	Sigma-Aldrich	G5516
Goat anti-Mouse IgG Alexa Fluor 488	Thermo Fisher Scientific	A11001
Goat anti-Rabbit IgG Alexa Fluor 488	Thermo Fisher Scientific	A11008
Heparin sodium from porcine intestinal mucosa 5000 IU/mL	Blau Farmacêutica	SKU 68027
Ibidi pump system (Pump + Fluidic Unit)	Ibidi	10902
KLF2 forward primer	Thermo Fisher Scientific	Custom designed
KLF2 reverse primer	Thermo Fisher Scientific	Custom designed
KLF4 forward primer	Thermo Fisher Scientific	Custom designed
KLF4 reverse primer	Thermo Fisher Scientific	Custom designed
NOA 280 nitric oxide analyzer	Sievers Instruments	NOA-280i-1
NOS3 forward primer	Thermo Fisher Scientific	Custom designed
NOS3 reverse primer	Thermo Fisher Scientific	Custom designed
Paraformaldehyde (PFA)	Sigma-Aldrich	158127
Perfusion set 15 cm, ID 1.6 mm, red, 10 mL reservoirs	Ibidi	10962
Phalloidin - Alexa Fluor 488	Thermo Fisher Scientific	A12379
Phalloidin - Alexa Fluor 568	Thermo Fisher Scientific	A12380
Phosphate buffered saline (PBS), pH 7.4	Thermo Fisher Scientific	10010031
Potassium Iodide	Sigma-Aldrich	221945
QuanTitec SYBR green PCR kit	Qiagen	204143
QuantStudio 12K flex platform	Applied Biosystems	4471087
RNeasy micro kit	Quiagen	74004
Slide glass (24 mm x 60 mm)	Knittel Glass	VD12460Y1D.01
Sodium nitrite	Sigma-Aldrich	31443
SuperScript IV first-strand synthesis system	Thermo Fisher Scientific	18091200
Triton X-100	Sigma-Aldrich	T8787
Trypan blue stain 0.4%	Gibco	15250-061
Type II collagenase from Clostridium histolyticum	Sigma-Aldrich	C6885