yes / 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.
| Identifer | oai:union.ndltd.org:BRADFORD/oai:bradscholars.brad.ac.uk:10454/12944 |
| Date | 29 July 2017 |
| Creators | 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., Lako, M. |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Article, Published version |
| Rights | © 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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