Amniotic membrane (AM) transplantation is increasingly used in ophthalmological and dermatological surgeries to promote re-epithelialization and wound healing. Biologically active cells in the epithelial and stromal layers deliver growth factors and cytokines with anti-inflammatory, anti-bacterial, anti-immunogenic and anti-fibrotic properties. In this work, confocal microscopy was used to show that our cryopreservation protocol for AM yielded viable cells in both the stromal and epithelial layers with favorable post-transplant outcome. AM was obtained from Caesarean-section placenta, processed into allograft pieces of different sizes (3 cm × 3 cm, 5 cm × 5 cm, and 10 cm × 10 cm) and cryopreserved in 10 % dimethyl sulfoxide using non-linear controlled rate freezing. Post-thaw cell viability in the entire piece of AM and in the stromal and epithelial cell layers was assessed using a dual fluorescent nuclear dye and compared to hypothermically stored AM, while surveys from surgical end-users provided information on post-transplant patient outcomes. There was no significant statistical difference in the cell viability in the entire piece, epithelial and stromal layers regardless of the size of allograft piece (p = 0.092, 0.188 and 0.581, respectively), and in the entire piece and stromal layer of hypothermically stored versus cryopreserved AM (p = 0.054 and 0.646, respectively). Surgical end-user feedback (n = 49) indicated that 16.3 % of AM allografts were excellent and 61.2 % were satisfactory. These results support the expanded clinical use of different sizes of cryopreserved AM allografts and address the issue of orientation of the AM during transplant for the treatment of dermatological defects and ocular surface disorders.
Amniotic membrane Stroma Epithelium Cryobiology Cryopreservation Ocular surgery Dermal surgery Tissue transplantation
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This work was partially funded by the Canadian Institutes of Health Research (CIHR MOP 86492, OGBF INO 126778, INO 131572, and MOP 133684). J. A. W. Elliott holds a Canada Research Chair in Thermodynamics. The authors would like to thank Luciana Da Silveira Cavalcante for her assistance with statistical analysis.
Cooke M, Tan E, Mandrycky C et al (2014) Comparison of cryopreserved amniotic membrane and umbilical cord tissue with dehydrated amniotic membrane/chorion tissue. J Wound Care 23:465–476CrossRefPubMedGoogle Scholar
Davis JS (1910) Skin transplantation with a review of 550 cases at the Johns Hopkins Hospital. Johns Hopkins Hosp Reports 15:307–396Google Scholar
Füst A, Pállinger E, Stündl A et al (2012) Both freshly prepared and frozen-stored amniotic membrane cells express the complement inhibitor CD59. Sci World J 2012:815615. doi:10.1100/2012/815615CrossRefGoogle Scholar
Hopkinson A, McIntosh RS, Shanmuganathan V et al (2006) Proteomic analysis of amniotic membrane prepared for human transplantation: characterization of proteins and clinical implications. J Proteome Res 5:2226–2235. doi:10.1021/pr050425qCrossRefPubMedGoogle Scholar
Kim EY, Lee K-B, Kim MK (2014) The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy. Biochem Mol Biol Reports 47:135–140Google Scholar
Kobayashi A, Sugiyama K, Li W, Tseng S (2008) In vivo laser confocal microscopy findings of cryopreserved and fresh human amniotic membrane. Ophthalmic Surg Lasers Imaging 39:312–318CrossRefPubMedGoogle Scholar
Koizumi N, Inatomi T, Sotozono C et al (2000) Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 20:173–177CrossRefPubMedGoogle Scholar
Koizumi N, Rigby H, Fullwood N et al (2006) Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells. Graefe’s Arch Clin Exp Ophthalmol 245:123–134CrossRefGoogle Scholar
Kruse FE, Joussen AM, Rohrschneider K et al (2000) Cryopreserved human amniotic membrane for ocular surface reconstruction. Graefe’s Arch Clin Exp Ophthalmol 238:68–75CrossRefGoogle Scholar
Micera A, Jirsova K, Normando EM et al (2014) Molecular and biochemical expression of TLRs in human amniotic membrane: a comparative study of fresh and cryopreserved specimens. Graefe’s Arch Clin Exp Ophthalmol 252:267–274. doi:10.1007/s00417-013-2540-zCrossRefGoogle Scholar
Niknejad H, Peirovi H, Jorjani M et al (2008) Properties of the amniotic membrane for potential use in tissue engineering. Eur Cell Mater 15:88–99PubMedGoogle Scholar