Medium perfusion enables engineering of compact and contractile cardiac tissue

TLDR

The authors hypothesized that functional cardiac constructs with physiological cell densities could be engineered in vitro by mimicking convective‑diffusive oxygen transport and tested this by designing a culture system that continuously supplies oxygen during seeding and maturation. They cultured neonatal rat cardiomyocytes at high density in Matrigel on collagen sponges with interstitial medium flow, assessing cell number, viability, metabolism, contractile protein expression, and electrical contractile response, and compared the constructs to orbitally mixed dishes, isolated ventricles, and freshly isolated cardiomyocytes. Interstitial perfusion yielded a physiologically dense, aerobic, viable, and differentiated cell population, higher cell viability and S‑phase fraction, and synchronous, low‑threshold contraction that recovered after gap‑junction blockade, whereas dish‑grown constructs showed a thin viable layer, anaerobic core, arrhythmic contraction, and failure to recover MCR.

Abstract

We hypothesized that functional constructs with physiological cell densities can be engineered in vitro by mimicking convective-diffusive oxygen transport normally present in vivo. To test this hypothesis, we designed an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation and characterized in detail construct metabolism, structure, and function. Neonatal rat cardiomyocytes suspended in Matrigel were cultured on collagen sponges at a high initial density (1.35 x 10(8) cells/cm(3)) for 7 days with interstitial flow of medium; constructs cultured in orbitally mixed dishes, neonatal rat ventricles, and freshly isolated cardiomyocytes served as controls. Constructs were assessed at timed intervals with respect to cell number, distribution, viability, metabolic activity, cell cycle, presence of contractile proteins (sarcomeric alpha-actin, troponin I, and tropomyosin), and contractile function in response to electrical stimulation [excitation threshold (ET), maximum capture rate (MCR), response to a gap junctional blocker]. Interstitial flow of culture medium through the central 5-mm-diameter x 1.5-mm-thick region resulted in a physiological density of viable and differentiated, aerobically metabolizing cells, whereas dish culture resulted in constructs with only a 100- to 200-microm-thick surface layer containing viable and differentiated but anaerobically metabolizing cells around an acellular interior. Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase compared with dish-grown constructs. In response to electrical stimulation, perfused constructs contracted synchronously, had lower ETs, and recovered their baseline function levels of ET and MCR after treatment with a gap junctional blocker; dish-grown constructs exhibited arrhythmic contractile patterns and failed to recover their baseline MCR levels.