A Cell Culture Model of Resistance Arteries

Summary

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

Abstract

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 µm²), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 µm pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

Introduction

In small diameter resistance arteries, a thin internal elastic lamina (IEL) separates endothelial (EC) and smooth muscle cells (SMC). The cells can project through holes in this elastic matrix and make direct cytoplasmic connections via gap junction channels^1,2. This unique structure is termed the myoendothelial junction (MEJ). The MEJ is a small (approximately 0.5 µm x 0.5 µm, depending on the vascular bed), cellular projection composed predominantly of endothelial cells but may originate from smooth muscle as well^3,4. A number of investigat....

Protocol

1. Plating Cells for the VCCC

1. Culture large 225 cm² flasks of EC and SMC under sterile conditions at 37 °C, until 70-90% confluent. Ensure that more EC than SMC are used; if using primary human EC and SMC, typically 3 large flasks of EC to 1 large flask of SMC.
   1. Use primary cells at lower passages and do not use past passage 10. Choose the culture media based upon cells to be co-cultured. For primary human coronary artery EC, use EBM-2 EC basal media with EGM2 MV growth factors. For primary human coronary SMC, use SmBM SMC basal media with SmGM-2 growth factors. Prior to use with cultured cells, add growth factor....

Results

The filter inserts used for the VCCC have small, 0.4 µm holes. Figure 1A, an en face view of the VCCC filter inset, shows the pores in which the MEJ can form in vitro. The schematic depicts the smooth muscle cells plated on the lower side of the filter one day before the endothelial cells are plated on the opposite side (Figure 1B). Once the cells are plated, the EC, MEJ, and SMC fractions can be isolated three .......

Discussion

The VCCC has a number of advantages in terms of recapitulating MEJs in vitro, but there are points of discussion when determining if the VCCC can be utilized for specific application. For example, if different cell types are to be used with this model, it will need to be optimized. We have used primary human cells but it is possible to isolate cells from bovine aortas²⁴, or wildtype or knockout mice, and use them in the VCCC^1,5

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 mm diameter, 0.4 µm Transwell Polyester membrane filter insert	Corning	3450	This is one 6 well plate of inserts
225 cm flask	Corning	431082
6 well plates (without filter inserts)	Corning	3506
Primary human coronary artery endothelial cells	Lonza	CC-2585
Primary human coronary artery smooth muscle cells	Lonza	CC-2583
EBM-2 EC Basal media	Lonza	CC-3156
EC growth factors (EGM2 MV Single quots)	Lonza	CC-4147	Add to basal media before use
SmBM SMC basal media	Lonza	CC-3181
SMC growth factors (SmGM-2 SingleQuot)	Lonza	CC-4149	Add to basal media before use
0.5% Trypsin-EDTA 10X	Gibco	15400-054	Dilute to 2X with sterile dPBS
Hemocytometer	VWR	15170-208
large petri dishes for SMC Plating (150mm)	Corning	353025
Bovine gelatin	Sigma	G-9382
Human fibronectin	Corning	356008	5µg/ml of fibronectin and store at -20
10x Hanks' Buffered salt solution (HBSS)	Gibco	14065-056	Dilute to 1x with sterile water
10x Dulbecco's Phosphate buffered saline (dPBS)	Gibco	14200-075	Dilute to 1x with sterile water
Disposable Curved Scalpel (30mm cutting edge) with handle	Amazon	MDS15210
Forceps	Fisher	17-467-201
Cell lifter	Corning	3008
Cell scraper	BD Falcon	353085
50 mL conical tube	Corning	352070
10 mm petri dishes	Falcon	35-1008
Rneasy mini kit	Qiagen	74104
RNA lysis reagent	Qiagen	79306
Paraformaldehyde	Sigma	158127	Make 4% solution with dPBS
70% ethanol	Fisher	04-355-305
Cavicide disinfectant	Fisher	131000
RIPA protein lysis buffer	Sigma	R0278
Sonic dismembranator	Fisher	Model 50
1x PBS for harvesting EC/SMC and immunocytochemistry	Gibco	10010023	does not need to be sterile
Name	Company	Catalog Number	Comments
Embedding/Sectioning/Staining
Paraffin	Fisher	P31-500
Automated tissue processer	Excelsior	ES
Embedding station + cold plate	Leica	HistoCore Arcadia
Tissue Tek Accu-Edge Microtome blade	VWR	25608-961 or 25608-964
Microscope slides	Thermo Fisher	22-230-900
Coverslips	Thermo Fisher	12-548-5E
Prolong Gold mounting medium	Thermo Fisher	P36931
Name	Company	Catalog Number	Comments
Other Solutions			**Perform all steps in sterile conditions if using the solution on live cells
Sterile dPBS			Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2µm filter). Final concentration: 1X
sterile HBSS			Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2µm filter). Final concentration: 1X
Fibronectin stock solution			Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed Final concentration: 100µg/mL
Fibronectin to coat SMC side of VCCC			thaw one aliquot and add 1mL of fibronectin (100µg/mL) to 19mLs of 1X HBSS to reach a final concentration of 5µg/mL. Final concentration: 5µg/mL
2x Trypsin EDTA solution			Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) . Final concentration: 2X
Blocking/antibody solution			(9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum)
Bovine gelatin to coat EC VCCC			Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions. Final concentration: 0.50%
1x PBS for harvesting cells			does not need to be sterile