Developing regenerative therapies targeting cardiomyocytes using organotypic slice culture from human adult ventricular myocardium
Authors: Lodrini AM , Bogunovic N , Kruithof BP , Smits A , De Vries AA , Goumans MJ 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): Regenerative Medicine Across Borders (RegMedXB) Foundation

Introduction
Cardiovascular diseases (CVDs) are the number one cause of mortality among non-communicable diseases. Two mechanisms of cardiac remodelling after injury have been hypothesized. In the early phases, remodelling is caused by cardiomyocytes (CMs) death, while later it is due to the attempts at reconstruction from the surviving myocardium. Due to the inability of cardiomyocytes to divide, the adult mammalian heart has negligible endogenous regenerative capacity, and the injured myocardium heals through formation of a scar, while surviving CMs become hypertrophic. These mechanisms can lead to progressive left ventricular dilatation, loss of contractility and transition to heart failure. With significant effort from the research community, new therapies to treat cardiac injury are being investigated, with particular attention to regenerative cellular therapies.

Purpose
The aim of this study is to devise therapies able to target CMs to eventually replenish the heart of contractile units by inducing CMs proliferation.

Methods
Atrial appendage or ventricle wall samples were derived from the surgical waste material of adult patients who underwent heart surgery for heart valve disease and/or Morrow myectomy. Cardiac-resident mesenchymal progenitor cells and endothelial cells were derived and amplified from the atrial samples, while organotypic cardiac slices (thickness 300 um) were obtained by cutting the ventricular samples with a vibratome and then cultured at a liquid-air interface. Functionality was proven by viability staining and biochemical assays. Cells and slices were treated with compounds aimed to improve cell health (dexamethasone or SB-431542) and/or vectors carrying reporters (Fiber-modified HAdV vectors or nanoparticles enveloped in a lipid membrane).

Results
Human myocardial slices were viable up to 7 days in culture without electrical or mechanical stimulation. During this time in control conditions there was collagen deposition and onset of fibrosis. Treatment with dexamethasone (100 nM) prevented loss of collagen structure and activation of markers of cardiac remodelling. The specific inhibition of the remodelling marker Smad-3 with SB-431542 didn’t have any evident effect on the viability and structural integrity of the slices. Vectors HadV-5 and HadV-11 had highly efficient transduction in monolayers of human cells peaking around 48h, but low efficiency in myocardial slices. Nanoparticles had efficient transduction in monolayers of cells and myocardial slices, but shorter particle lifespan (&lt;48h).

Conclusions
We established a quick and simple method for the preparation of vital tissue slices from human adult ventricular myocardium as well as their preservation in culture. This model represents a novel platform for testing vectors targeting CMs in a 3-D environment, highlighting the differences in transduction efficiency when compared to standard monolayer culture techniques.