Pulsatile perfusion bioreactor for cardiac tissue engineering

Abstract

Cardiovascular disease is the number one cause of mortality in North America. Cardiac tissue engineering aims to engineer a contractile patch of physiological thickness to use in surgical repair of diseased heart tissue. We previously reported that perfusion of engineered cardiac constructs resulted in improved tissue assembly. Because heart tissues respond to mechanical stimuli in vitro and experience rhythmic mechanical forces during contraction in vivo, we hypothesized that provision of pulsatile interstitial medium flow to an engineered cardiac patch would result in enhanced tissue assembly by way of mechanical conditioning and improved mass transport. Thus, we constructed a novel perfusion bioreactor capable of providing pulsatile fluid flow at physiologically relevant shear stresses and flow rates. Pulsatile perfusion (PP) was achieved by incorporation of a normally closed solenoid pinch valve into the perfusion loop and was carried out at a frequency of 1 Hz and a flow rate of 1.50 mL/min (PP) or 0.32 mL/min (PP-LF). Nonpulsatile flow at 1.50 mL/min (NP) or 0.32 mL/min (NP-LF) served as controls. Static controls were cultivated in well plates. The main experimental groups were seeded with cells enriched for cardiomyocytes by one preplating step (64% cardiac Troponin I+, 34% prolyl-4-hydroxylase+), whereas pure cardiac fibroblasts and cells enriched for cardiomyocytes by two preplating steps (81% cardiac Troponin I+, 16% prolyl-4-hydroxylase+) served as controls. Cultivation under pulsatile flow had beneficial effects on contractile properties. Specifically, the excitation threshold was significantly lower in the PP condition (pulsatile perfusion at 1.50 mL/min) than in the Static control, and the contraction amplitude was the highest; whereas high maximum capture rate was observed for the PP-LF conditions (pulsatile perfusion at 0.32 mL/min). The enhanced hypertrophy index observed for the PP-LF group was consistent with the highest cellular length and diameter in this group. Within the same cultivation groups (Static, NP-LF, PP-LF, PP, and NP) there were no significant differences in the diameter between fibroblasts and cardiomyocytes, although cardiomyocytes were significantly more elongated than fibroblasts under PP-LF conditions. Cultivation of control cell populations resulted in noncontractile constructs when cardiac fibroblasts were used (as expected) and no overall improvement in functional properties when two steps of preplating were used to enrich for cardiomyocytes in comparison with only one step of preplating.

Influence of substrate stiffness on the phenotype of heart cells

“…8). In Figure 8, cells of which only the nuclei stain could be smooth muscle cells, present in the native heart cell isolates at 3-4% after one pre-plating step (Brown et al, 2008;Naito et al, 2006).…”

“…Briefly, a solution of 10 mM CFDA and 2.5 mg/ mL PI in PBS was applied to the cells for 40 min at 378C. For immunostaining, cells were fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature and stained for cardiac troponin I to identify CM and vimentin to identify nonmyocytes, according to protocols we have described (Brown et al, 2008;Radisic et al, 2008a). Primary antibodies were polyclonal rabbit anti-cardiac troponin I (Chemicon, Etobicoke, ON, 1:150) and Cy3-conjugated mouse monoclonal anti-vimentin (Sigma, 1:50).…”

Endothelial cells guided by immobilized gradients of vascular endothelial growth factor on porous collagen scaffolds

“…Improved cell viability was observed [30,31]. Our lab has designed a similar bioreactor that mimics the in vivo-like convective-diffusive oxygen transport, by providing a pulsatile flow through a cultured construct in a bio-reactor [28,32]. Cells are seeded on porous collagen scaffolds containing interconnected network of channels, which enhance diffusion and also act as a precursor to blood vessel development, and cultured in the bio-reactor.…”

Stem cell therapy for cardiac disease

“…Neonatal CMs within cardiac tissue patches cultivated under pulsatile fluid flow exhibited highly ordered sarcomeres, intercalated discs, and better contractile properties than those cultivated in nonpulsatile medium flow (85,86). Application of electrical stimulation during cultivation of neonatal rat cardiac constructs enhanced ultrastructural differentiation and augmented contractile behavior as compared with nonstimulated constructs (87,88).…”