Medium perfusion enables engineering of compact and contractile cardiac tissue.

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.

[1]  D. Mann,et al.  Blocking the Endogenous Increase in HSP 72 Increases Susceptibility to Hypoxia and Reoxygenation in Isolated Adult Feline Cardiocytes , 1996 .

[2]  R Langer,et al.  Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies. , 2001, American journal of physiology. Heart and circulatory physiology.

[3]  P. V. van Wachem,et al.  Cardiac tissue engineering: characteristics of in unison contracting two- and three-dimensional neonatal rat ventricle cell (co)-cultures. , 2002, Biomaterials.

[4]  A Haverich,et al.  In vitro engineering of heart muscle: artificial myocardial tissue. , 2002, The Journal of thoracic and cardiovascular surgery.

[5]  F. Accurso,et al.  Impaired clearance of apoptotic cells from cystic fibrosis airways. , 2002, Chest.

[6]  P. Arthur,et al.  Hibernation in Noncontracting Mammalian Cardiomyocytes , 2000, Circulation.

[7]  S R Gonda,et al.  Cardiac organogenesis in vitro: reestablishment of three-dimensional tissue architecture by dissociated neonatal rat ventricular cells. , 1999, Tissue engineering.

[8]  D. Mann,et al.  Blocking the endogenous increase in HSP 72 increases susceptibility to hypoxia and reoxygenation in isolated adult feline cardiocytes. , 1997, Circulation.

[9]  Milica Radisic,et al.  High-density seeding of myocyte cells for cardiac tissue engineering. , 2003, Biotechnology and bioengineering.

[10]  P. Athias,et al.  Oxygen and substrate deprivation on isolated rat cardiac myocytes: temporal relationship between electromechanical and biochemical consequences. , 1990, Canadian journal of physiology and pharmacology.

[11]  B. Schumacher,et al.  Images in cardiovascular medicine. Fistulous communication between coronary sinus and left atrium. , 2002, Circulation.

[12]  Thomas Eschenhagen,et al.  Three‐dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  H. Lodish.  Molecular Cell Biology , 1986 .

[14]  R. Virmani,et al.  Myocardial protection of contractile function after global ischemia by physiologic estrogen replacement in the ovariectomized rat. , 1997, Journal of molecular and cellular cardiology.

[15]  Thomas Eschenhagen,et al.  Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[16]  Andreas Hess,et al.  Cardiac Grafting of Engineered Heart Tissue in Syngenic Rats , 2002, Circulation.

[17]  Gordana Vunjak-Novakovic,et al.  Effects of oxygen on engineered cardiac muscle. , 2002, Biotechnology and bioengineering.

[18]  W. Zimmermann,et al.  Tissue Engineering of a Differentiated Cardiac Muscle Construct , 2002, Circulation research.

[19]  R. Voll,et al.  Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. , 1998, Arthritis and rheumatism.

[20]  F J Schoen,et al.  Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization. , 1999, Biotechnology and bioengineering.

[21]  M. Lotz,et al.  Mechanisms of chondrocyte apoptosis. , 1999, Osteoarthritis and cartilage.

[22]  J. Leor,et al.  Bioengineered Cardiac Grafts: A New Approach to Repair the Infarcted Myocardium? , 2000, Circulation.

[23]  Gordana Vunjak-Novakovic,et al.  Perfusion improves tissue architecture of engineered cardiac muscle. , 2002, Tissue engineering.

[24]  S. Baksh,et al.  Apoptotic Cells Induce Migration of Phagocytes via Caspase-3-Mediated Release of a Lipid Attraction Signal , 2003, Cell.

[25]  D L Eckberg,et al.  Mathematical treatment of autonomic oscillations. , 1999, Circulation.

[26]  Mitsuo Umezu,et al.  Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Cell Culture Surfaces , 2002, Circulation research.

[27]  J. Curtis,et al.  Deficient In Vitro and In Vivo Phagocytosis of Apoptotic T Cells by Resident Murine Alveolar Macrophages1 , 2000, The Journal of Immunology.

[28]  T. Vanden Hoek,et al.  Reperfusion injury on cardiac myocytes after simulated ischemia. , 1996, The American journal of physiology.

[29]  R. Weisel,et al.  Survival and function of bioengineered cardiac grafts. , 1999, Circulation.

[30]  Payam Akhyari,et al.  Mechanical Stretch Regimen Enhances the Formation of Bioengineered Autologous Cardiac Muscle Grafts , 2002, Circulation.

[31]  R J Cohen,et al.  Cardiac muscle tissue engineering : toward an in vitro model for electrophysiological studies , 1999 .

[32]  K. Brown,et al.  Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. , 2002, The Journal of clinical investigation.

[33]  F. Di Lisa,et al.  Overexpression of the stress‐protein Grp94 reduces cardiomyocyte necrosis due to calcium overload and simulated ischemia , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[34]  R. Weisel,et al.  Construction of a bioengineered cardiac graft. , 2000, The Journal of thoracic and cardiovascular surgery.

[35]  U. Brunk,et al.  Ceroid/Lipofuscin-loaded human fibroblasts show increased susceptibility to oxidative stress , 1999, Experimental Gerontology.

[36]  J. Burt,et al.  Uncoupling of cardiac cells by fatty acids: structure-activity relationships. , 1991, The American journal of physiology.

[37]  S. Hart,et al.  Role of macrophage CD44 in the disposal of inflammatory cell corpses. , 2002, Clinical science.

[38]  Akif Uzman,et al.  Molecular Cell Biology (4th edition): Harvey Lodish, Arnold Berk, S. Lawrence Zipursky, Paul Matsudaira, David Baltimore and James Darnell; Freeman & Co., New York, NY, 2000, 1084 pp., list price $102.25, ISBN 0-7167-3136-3 , 2001 .

[39]  R. Fournier.  Basic Transport Phenomena In Biomedical Engineering , 1998 .

[40]  W. Zimmermann,et al.  Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. , 2000, Biotechnology and bioengineering.