Cell and Tissue Banking: International Journal for Banking, Engineering and Transplantation of Cells and Tissues, Dec, 2012, Vol.13(4), p.663(9)
Byline: Kelvin G. M. Brockbank (1,2,3), Katja Schenke-Layland (4,5,6), Elizabeth D. Greene (1), Zhenzhen Chen (1), Olaf Fritze (7), Martina Schleicher (7), Renate Kaulitz (8), Iris Riemann (9), Falko Fend (10), Johannes M. Albes (11), Ulrich A. Stock (7), Milan Lisy (7) Keywords: Cryopreservation; Heart valve; Allogeneic; Transplantation Abstract: The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6 mol/l cryoprotectant (4.65, 4.65, and 3.31 mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at -80degC. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4 mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-free tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P 〈 0.05). After 3 and 6 months in vivo valve function was determined by two-dimensional echo-Doppler and at 7 months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P = 0.0403, P = 0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P 〈 0.05) and infiltrating CD3+ T-cells (P 〈 0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at -80degC avoids ice formation, tissue-glass cracking and preserves extracellular matrix integrity resulting in minimal inflammation and improved hemodynamics in allogeneic juvenile sheep. Author Affiliation: (1) Cell and Tissue Systems, Inc., 2231 Technical Parkway, Suite A, North Charleston, SC, 29406, USA (2) Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA (3) Deparment of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA (4) Cardiovascular Research Laboratories, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA (5) Deparment of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), Stuttgart, Germany (6) Inter-University Centre for Medical Technology Stuttgart-Tubingen (IZST), Eberhard Karls University Tubingen, Tubingen, Germany (7) Deparment of Thoracic, Cardiac and Vascular Surgery, University Hospital Tuebingen, Tuebingen, Germany (8) Deparment of Pediatric Cardiology, University Hospital Tuebingen, Tuebingen, Germany (9) Fraunhofer Institute of Biomedical Technology, St. Ingbert, Germany (10) Deparment of General Pathologie and Pathological Anatomy, University Hospital Tuebingen, Tuebingen, Germany (11) Deparment of Cardiac Surgery, Heart Centre Brandenburg, Bernau, Germany Article History: Registration Date: 19/12/2011 Received Date: 01/06/2011 Accepted Date: 19/12/2011 Online Date: 03/01/2012 Article note: This paper received an Outstanding Abstract Award and was presented orally at the 2009 American Association of Tissue Banks Annual Meeting in Las Vegas, Nevada.
Tissue Engineering -- Analysis ; T Cells -- Analysis ; Propylene Glycol -- Analysis ; Pamphlets -- Analysis ; Fluorescence -- Analysis ; Transplantation -- Analysis
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