2008 Nature Publishing Group http www nature com naturemedicine TECHNICAL REPORTS Perfusion decellularized matrix using nature s platform to engineer a bioartificial heart Harald C Ott1 Thomas S Matthiesen2 Saik Kia Goh2 Lauren D Black3 Stefan M Kren2 Theoden I Netoff3 Doris A Taylor2 4 About 3 000 individuals in the United States are awaiting a donor heart worldwide 22 million individuals are living with heart failure A bioartificial heart is a theoretical alternative to transplantation or mechanical left ventricular support Generating a bioartificial heart requires engineering of cardiac architecture appropriate cellular constituents and pump function We decellularized hearts by coronary perfusion with detergents preserved the underlying extracellular matrix and produced an acellular perfusable vascular architecture competent acellular valves and intact chamber geometry To mimic cardiac cell composition we reseeded these constructs with cardiac or endothelial cells To establish function we maintained eight constructs for up to 28 d by coronary perfusion in a bioreactor that simulated cardiac physiology By day 4 we observed macroscopic contractions By day 8 under physiological load and electrical stimulation constructs could generate pump function equivalent to about 2 of adult or 25 of 16 week fetal heart function in a modified working heart preparation In the United States alone nearly 5 million people live with heart failure and about 550 000 new cases are diagnosed annually Heart transplantation remains the definitive treatment for end stage heart failure but the supply of donor organs is limited Once a heart is transplanted individuals face lifelong immunosuppression and often trade heart failure for hypertension diabetes and renal failure1 The creation of a bioartificial heart could theoretically solve these problems Attempts to engineer heart tissue have involved numerous approaches2 Engineered contractile rings and sheets have been transplanted into small animals and have improved ventricular function3 5 The creation of thick 4100 200 mm cardiac patches has been limited by an inability to create the geometry necessary to support the high oxygen and energy demands of cardiomyocytes at a depth greater than B100 mm from the surface2 6 The use of channeled cardiac extracellular matrix ECM constructs oxygen carriers and stacked cardiac sheets4 7 8 to improve thickness has reinforced the direct relationship between perfusion and graft size or cell density6 9 To create a whole heart scaffold with intact three dimensional geometry and vasculature we attempted to decellularize cadaveric hearts by coronary perfusion with detergents which have been shown to generate acellular scaffolds for less complex tissues by direct immersion10 14 We then repopulated decellularized rat hearts with neonatal cardiac cells or rat aortic endothelial cells and cultured these recellularized constructs under simulated physiological conditions for organ maturation15 Ultimately chronic coronary perfusion pulsatile left ventricular load and synchronized left ventricular stimulation led to the formation of contractile myocardium that performed stroke work RESULTS Perfusion decellularization of cadaveric hearts To develop a valid perfusion decellularization protocol we carried out antegrade coronary perfusion of 140 cadaveric rat hearts on a modified Langendorff apparatus and compared the degree of decellularization that is removal of DNA and intracellular structural proteins that resulted from the use of three detergent solutions Fig 1 The use of SDS Fig 1c f gave better results than did polyethylene glycol PEG Fig 1a d Triton X100 Fig 1b e or enzyme based protocols data not shown for full removal of cellular constituents Antegrade coronary SDS perfusion over 12 h Fig 1c yielded a fully decellularized construct Histological evaluation revealed no remaining nuclei or contractile elements Fig 1g DNA content decreased to less than 4 of that in cadaveric heart Supplementary Fig 1 online whereas the glycosaminoglycan content was unchanged After perfusion with Triton X100 ref 16 and washing SDS levels in the decellularized myocardium could not be differentiated from zero in a quantitative assay Supplementary Fig 1 Properties of the decellularized construct Collagens I and III laminin and fibronectin Fig 2a remained within the thinned decellularized heart matrix The fiber composition weaves struts and coils and orientation of the myocardial ECM were preserved whereas cardiac cells were removed Fig 2b resulting in compressed constructs Within the retained ventricular ECM we saw intact vascular basal membranes without endothelial or 1Department of Surgery Massachusetts General Hospital Harvard Medical School 55 Fruit Street Boston Massachusetts 02114 USA 2Center for Cardiovascular Repair University of Minnesota 312 Church Street Southeast 7 105A NHH Minneapolis Minnesota 55455 USA 3Department of Biomedical Engineering University of Minnesota 312 Church Street Southeast 7 NHH Minneapolis Minnesota 55455 USA 4Department of Integrative Biology and Physiology University of Minnesota 6 125 Jackson Hall 312 Church Street Southeast Minneapolis Minnesota 55455 USA Correspondence should be addressed to D A T dataylor umn edu Received 29 May 2007 accepted 18 October 2007 published online 13 January 2008 doi 10 1038 nm1684 NATURE MEDICINE VOLUME 14 NUMBER 2 FEBRUARY 2008 213 TECHNICAL REPORTS a RA LA LV 1 PEG in deionized water 77 4 mm Hg 20 C b e Ao RA RV LA LV 1 Triton X 100 in deionized water 77 4 mm Hg 20 C c f Ao RA RV LA LV DAPI MHC Cadaveric heart Decellularized heart DAPI s Actin g DAPI MHC 1 SDS in deionized water 77 4 mm Hg 20 C DAPI s Actin 2008 Nature Publishing Group http www nature com naturemedicine RV Figure 1 Perfusion decellularization of whole rat hearts a c Photographs of cadaveric rat hearts mounted on a Langendorff apparatus Ao aorta LA left atrium LV left ventricle RA right atrium RV right ventricle Retrograde perfusion of cadaveric rat heart using PEG a Triton X 100 b or SDS c over 12 h The heart becomes more translucent as cellular material is washed out from the right ventricle then the atria and finally the left ventricle d e Corresponding H E staining of thin sections from LV of rat hearts perfused with PEG d or Triton X 100 e showing incomplete decellularization Hearts treated with PEG or Triton X 100 retained nuclei and myofibers Scale bars 200 mm f H E staining of thin section of SDS treated
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