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Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells
Figueiredo, G.S. Bojic, S. Rooney, P. Wilshaw, Stacy-Paul Connon, C.J. Gouveia, R.M. Paterson, C. Lepert, G. Mudhar, H.S. Figueiredo, F.C.
Figueiredo, G.S.
Bojic, S.
Rooney, P.
Wilshaw, Stacy-Paul
Connon, C.J.
Gouveia, R.M.
Paterson, C.
Lepert, G.
Mudhar, H.S.
Figueiredo, F.C.
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Publication Date
2017
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© 2017 Acta Materialia Inc. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
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28/07/2017
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Abstract
The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variabil-ity and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmis-sion, we sought to determine if decellularisation and/or c-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without c-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, DNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and c-irradiation did not significantly affect the LSC phe-notype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p < 0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a c-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer.
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Figueiredo GS, Bojic S, Rooney P, Wilshaw S-P, Connon CJ, et al (2017) Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells. Acta Biomaterialia. 61: 124-133.
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Article