Abstract
Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions.Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR).Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture.Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.
Highlights
Three-dimensional (3D) culture is an alternative to classic cell culture using flat surfaces in flasks and dishes and can help bridge the gap between 2D culture and native tissue (Abbott, 2003; Bissell, 2017; Verjans et al, 2018)
Co-culture spheroids made of hiPSC-CMs and cardiac fibroblasts (CF) were produced in hanging drops, cultured for 10 days in GravityTrap multi-well plates and allowed to adhere to gelatine-coated glass coverslips in medium-filled 35 mm dishes for 2 days
In phase contrast light microscopy, spheroids made of hiPSC-CMs and CF appeared to have a smoother surface and to be more spherical than those made of hiPSC-CMs only, which might be caused by layers of protein deposition on the surface
Summary
Three-dimensional (3D) culture is an alternative to classic cell culture using flat surfaces in flasks and dishes and can help bridge the gap between 2D culture and native tissue (Abbott, 2003; Bissell, 2017; Verjans et al, 2018). A number of studies have made use of cardiac scaffold-free microtissues, called spheroids, for drug testing and toxicology, using a mix of several cell types such as rodent or human, primary- or hiPSC-derived cardiomyocytes, fibroblasts, stem cells, and endothelial cells (Garzoni et al, 2009). Adding multiple cell types might mimic the composition of the original tissue better, the role of the endothelial component is not very well-defined in spheroid models as the cells are less well-organized than in vivo. To address these issues, we decided to study two cell types, hiPSCderived human cardiomyocytes (hiPSC-CMs) and primary human cardiac fibroblasts (CF), comparing single-type 3D- and 2D-culture conditions. Human cell-derived models offer the possibility of drug testing in a framework of personalized medicine
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