Optimized Preservation of Extracellular Matrix in Cardiac Tissues: Implications for Long- Term Graft Durability Katja Schenke-Layland, PhD, Jiansong Xie,

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Optimized Preservation of Extracellular Matrix in Cardiac Tissues: Implications for Long- Term Graft Durability Katja Schenke-Layland, PhD, Jiansong Xie, MD, PhD, Sepideh Heydarkhan-Hagvall, PhD, Sarah F. Hamm-Alvarez, PhD, Ulrich A. Stock, MD, Kelvin G.M. Brockbank, PhD, W. Robb MacLellan, MD The Annals of Thoracic Surgery Volume 83, Issue 5, Pages 1641-1650 (May 2007) DOI: 10.1016/j.athoracsur.2006.12.005 Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Representative light-micrographs of fresh (A, D, G), thawed vitrified (B, E, H), and thawed frozen cryopreserved (C, F, I) aortic cardiac tissues. (A–C) Movat-pentachrome stained cross-sections of cardiac muscle; (D–F) Hart’s stained slides of aortic vascular tissue; (G–I): of the inflow side (v, ventricularis), the inner layer (s, spongiosa), and the valve outflow side (f, fibrosa) of aortic heart valve leaflets. (A through F scale bar equals 200 μm; G through I scale bar equals 50 μm.) The Annals of Thoracic Surgery 2007 83, 1641-1650DOI: (10.1016/j.athoracsur.2006.12.005) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Electron micrographs of representative parts of the ventricularis layer of fresh (A), vitrified (B), and frozen cryopreserved (C) pulmonary heart valve leaflets, demonstrating elastic fibers (EF), including microfibrills (MF), and collagenous fibers (CF). Notice that within vitrified as well as frozen cryopreserved tissues almost no microfibrillar components could be detected. The Annals of Thoracic Surgery 2007 83, 1641-1650DOI: (10.1016/j.athoracsur.2006.12.005) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Multiphoton-induced autofluorescence imaging and second-harmonic generation (SHG) signal profiling of the collagen-rich outflow (A,B; E,F; I,J) and the predominantly elastin-containing inflow side (C,D; G,H; K,L) of representative areas of fresh (A–D), vitrified (E–H) and frozen cryopreserved (I–L) aortic leaflets. The graphs represent peak SHG intensities of collagen-containing structures in the corresponding lambda stack overlay images, denoted by the red cross (B,D; F,H; J,L). The SHG imaging demonstrates the substantial ultrastructural deterioration and disintegration of most collagenous structures in cryopreserved tissues (J, L). Sufficient SHG signals were only inducible in fresh and vitrified tissue structures (B,D; F,H). Compared with fresh and vitrified tissues (A,C; E,G), just a few elastic fibers are detectable within cryopreserved leaflets (I, K). Living cells were only found within fresh and some of the vitrified leaflet tissues (white arrows, E). The Annals of Thoracic Surgery 2007 83, 1641-1650DOI: (10.1016/j.athoracsur.2006.12.005) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Multiphoton imaging (A,C; E,G; I,K) and second-harmonic generation profiling (B,D; F,H; J,L) of aortic (A,B; E,F; I,J) and pulmonary (C,D; G,H; K,L) cardiac muscle specimens. Collagenous structures (red: 840 nm), elastic fibers and cells (green: 760 nm) are clearly visible within fresh (A–D) and vitrified (E–H) tissues. Only weak autofluorescence signals are detectable in frozen cryopreserved cardiac muscle (I–L). The Annals of Thoracic Surgery 2007 83, 1641-1650DOI: (10.1016/j.athoracsur.2006.12.005) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Two-photon images of aortic (A, E, I) and pulmonary (C, G, K) trunk regions induced with wavelengths of 760 nm (green: elastic fibers, cells) and 840 nm (red: collagen). In comparison with the fresh controls (A, C), vascular vitrified structures (E, G) are well preserved, whereas the ECM of frozen cryopreserved tissues (I, K) appears to be damaged and not well organized. The SHG signal detection of the collagenous fibers of aortic (B, F, J) and pulmonary (D, H, L) trunk regions by spectral fingerprinting reveals a dramatic loss of signal intensities, especially within the pulmonary frozen cryopreserved specimens (L) when compared with fresh (D) tissues. The Annals of Thoracic Surgery 2007 83, 1641-1650DOI: (10.1016/j.athoracsur.2006.12.005) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions