Spatiotemporal tracking of cells in tissue-engineered cardiac organoids

Abstract

Cardiac tissue engineering aims to create myocardial patches for repair of defective or damaged native heart muscle. The inclusion of non-myocytes in engineered cardiac tissues has been shown to improve the properties of cardiac tissue compared to tissues engineered from enriched populations of myocytes alone. While attempts to mix non-myocytes (fibroblasts, endothelial cells) with cardiomyocytes have been made, very little is understood about how the tissue properties are affected by varying the respective ratios of the three cell types and how these cells assemble into functional tissues with time. The goal of this study was to investigate the effects of modulating the ratios of the three cell types as well as to spatially and temporally track cardiac tricultures of cells. Primary neonatal cardiac fibroblasts and D4T endothelial cells were incubated in 5µM of CellTracker™ Green dye and CellTracker™ Red dye respectively while neonatal cardiomyocytes were labeled with 20µg/mL of DAPI. The non-myocytes were seeded either sequentially (Pre-culture) or simultaneously (Tri-culture) in Matrigel-coated microchannels and allowed to form organoids, as in our previous studies. We also varied the seeding percentage of cardiomyocytes while keeping the total cell number constant in an attempt to improve the functional properties of the organoids. Organoids were imaged on days 1 and 4. Endothelial cells were seen to aggregate into clusters when Simultaneously Tri-cultured with myocytes and fibroblasts, while Pre-cultures contained elongated cells. Functional properties of organoids were improved by increasing the seeding percentage of enriched cardiomyocytes from 40% to 80%.

Biofabrication enables efficient interrogation and optimization of sequential culture of endothelial cells, fibroblasts and cardiomyocytes for formation of vascular cords in cardiac tissue engineering

“…The two EC densities at the lower end of the spectrum (8% and 15%) were chosen because they were in agreement with seeding densities previously reported to support cord formation (Levenberg et al 2002, Matsumoto et al 2007, Nor et al 1999, Wright et al 2002). The value of 31% ECs served as an effective control for comparison to our previous studies (Iyer et al 2009b). …”

“…In another example using hESC-derived CMs, Murry and colleagues (Stevens et al 2009a, 2009b) created scaffold-less tissues, and Levenberg and colleagues (Caspi et al 2007, Lesman et al 2010) created tissues on porous scaffolds using CMs, ECs and mesenchymal cells such as FBs. Their work collectively points to the fact that integration of the engineered cardiac tissue with the host myocardium was enhanced in the case of tri-culture (Iyer et al 2009b, Lesman et al 2010). In those cases, the primitive vessels in the engineered tissue functionally integrated with the host coronary vasculature and enabled graft survival.…”

“…For sequential preculture organoids (supplemental figure 1(A) available at stacks.iop.org/BF/4/035002/mmedia), the total cell number seeded was 2 × 10 5 cells per PEG disc as in our previous studies (Iyer et al 2009a, 2009b). ECs were seeded first (to enable cord formation), followed by seeding of FBs 24 h later (to enable cord stabilization through ECM deposition), and enriched CMs 48 h later (to form cardiac tissue).…”

“…ECs were seeded first (to enable cord formation), followed by seeding of FBs 24 h later (to enable cord stabilization through ECM deposition), and enriched CMs 48 h later (to form cardiac tissue). The fraction of CMs was fixed at 60%, as determined to be favorable for organoid formation in our previous studies (Iyer et al 2009b). The fractions of ECs and FBs in the remaining 40% of non-myocytes were varied, to form three experimental groups: ( i ) 8% ECs + 32% FBs, ( ii ) 15% ECs + 25% FBs and ( iii ) 31% of ECs + 9% of FBs.…”

Applications of fabricated micro- and nanostructures in biomedicine

“…47 Other researchers have cultured heart muscle cells (cardiomyocytes) with non-muscle cell types in matrigel-coated microchannels. 48 Compared to the simultaneous seeding of different cell types, it was found that seeding non-muscle cells to form organoids, followed by seeding of muscle cells, yielded more elongated and densely arranged cells. This led to cardiac tissues with better functionality.…”