A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Summary

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

Abstract

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

Introduction

Vascular calcification (VC) is an independent risk factor for cardiovascular morbidity and mortality^1,2,3. Long considered a passive chemical process of ectopic mineral deposition, it now appears a modifiable tissue healing response involving the active contribution of various cells including activated vascular smooth muscle cells (hVSMC) as a driver of the disease^4,5. In vivo VC can be measured by multislice CT scans as an assessment of atherosclerotic burden⁶

Protocol

1. Cell seeding, maintenance, and calcification induction

1. For culturing primary cells, use a laminar airflow cabinet, gloves, and sterile equipment. Disinfect hands and workspace before and after conducting any work. Treat all primary cells and culture media as a potential biohazard, unless proven otherwise. Preferably autoclave surplus cells and media before disposal. Do not chemically inactivate and autoclave since this will liberate toxic fumes.
2. Culture hVSMC on uncoated cell culture plates.
3. Routinely maintain hVSMC in growth medium consisting of M199 medium supplemented with 10%-20% FBS and 1% Pen/Strep and incubate....

Results

The outcome includes original images of HOECHST-stained nuclei, RFP-labeled calcification, and brightfield images. Different stages of calcification ranging from low (Figure 2) to high (Figure 3) may be detected and analyzed. Calcification can usually be spotted as black speckles using light microscopy (Figure 2D and Figure 3B, arrows indicate calcification), which are useful for primary assessment.......

Discussion

In this manuscript, we describe a semi-automated method for in vitro calcification determination. For this method, three critical steps of hVSMC calcification should be optimized. First, cellular density is critical for hVSMC calcification development. Low densities of hVSMCs will result in slow or no calcification and cell death due to the lack of cell-to-cell contact and the stress that is induced under calcifying conditions²¹. High cellular densities result in over-confluency, after wh.......

Materials

Name	Company	Catalog Number	Comments
Calcium chloride, 93%, anhydrous	Thermo Fisher Scientific	349615000
Costar 6-well Clear TC-treated well plates	Corning	3516
Cytation 3 System	BioTek, Abcoude, The Netherlands
Fetal Bovine Serum	Merck	F7524-100ML
Fetuin-A-Alexa Fluor-546	Prepared in-house
Gen5 Software v3.10	BioTek
Gibco Medium 199	Thermo Fisher Scientific	11150059
Hoechst 33342, Trihydrochloride	Thermo Fisher Scientific	H3570
PBS (10X), pH 7.4	Thermo Fisher Scientific	70011044
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher Scientific	25300062