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Decellularisation Of Allogeneic Aortic And Pulmonary Valves Using Low Concentration SDS

Tayyebeh Vafaee1, Daniel Thomas1, Paul Rooney2, John Kearney2, Helen Berry3, Sotirios Korossis1, John Fisher1, Eileen Ingham1.
1University of Leeds, Leeds, United Kingdom, 2NHS Blood and Transplant Tissue Services, Liverpool, United Kingdom, 3Tissue Regenix Group plc, York, United Kingdom.


Objective: Over 200,000 patients benefit from aortic valve replacement annually worldwide. Cryopreserved allografts are the “gold standard” but they are subject to immunological responses leading to calcification and stenosis resulting in multiple reoperations. We have previously developed methods to remove the immunogenic cells from porcine cardiac valves whilst maintaining the biochemical and biomechanical properties of tissues, creating immunocompatible replacements (1, 2), which have been shown to regenerate in the pulmonary position of juvenile sheep. The aim of this project is to transfer this technology to human cardiac valves and produce biomechanically and immunologically compatible homograft scaffolds for heart valve replacement.
Methods: Eight human cryopreserved cardiac valves (5 aortic and 3 pulmonary) were treated consecutively with 10mM hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer containing protease inhibitors (0.1 % EDTA and 10 KIU aprotinin) each for 24 hours at 45ºC, and nuclease solution (RNase at 1 U.ml-1 and DNase at 50 U.ml-1). Histological analysis (H&E and Dapi) and DNA assay of acellular matrices using the DNeasy kit were performed to determine the quality of the process and to compare the DNA content of 4 different areas of the acellular roots with cryopreserved valves.
Results: Haematoxylin & eosin staining of decellularised tissues showed no evidence of any cell nuclei or cell remnants. DNA assay confirmed 94 - 99.6% removal of DNA from aortic and pulmonary valves [Table]
Conclusions: Allogeneic cardiac valves were successfully decellularised using low concentration SDS, whilst maintaining their gross histoarchitecture. Future work will investigate the effect of the treatment on the biomechanical behaviour of the allogeneic valvular scaffolds.
References:
1. Booth et al., J HVD 2002 Jul;11(4):457-62.
2. Korossis et al., J HVD 2002 Jul;11(4):463-7

The percentage reduction in DNA content following decellularisation
Cardiac valveswalljunctionleafletmuscle
Aortic98.2 %97.1%97.3%99.5%
Pulmonary97.6%94%96.5%98.3%


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