title: Medium perfusion enables engineering of compact and contractile cardiac tissue.
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.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139, USA.
authors.forename: Milica
authors.initials: M
authors.lastname: Radisic
authors.forename: Liming
authors.initials: L
authors.lastname: Yang
authors.forename: Jan
authors.initials: J
authors.lastname: Boublik
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Cohen
authors.forename: Robert
authors.initials: R
authors.lastname: Langer
authors.forename: Lisa E
authors.initials: LE
authors.lastname: Freed
authors.forename: Gordana
authors.initials: G
authors.lastname: Vunjak-Novakovic
mesh_terms: D000199:Actins / Q000032:analysis; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division; D002470:Cell Survival; D003094:Collagen; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012518:Sarcomeres / Q000648:ultrastructure; D014335:Tropomyosin / Q000032:analysis; D019210:Troponin I / Q000032:analysis
chemical_list: D000199:Actins; D014335:Tropomyosin; D019210:Troponin I; D003094:Collagen