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Majlinda Lako, Sanja Bojic, Paul Rooney, Stacey Wilshaw, Hardeep Mudhar, Adam Meeney, Francisco C Figueiredo, Gustavo Figueiredo; Expansion of limbal epithelial stem cells on cryopreserved fresh and decellularized human amniotic membrane. Invest. Ophthalmol. Vis. Sci. 2016;57(12):896.
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© ARVO (1962-2015); The Authors (2016-present)
Human amniotic membrane (HAM) is not a defined substrate but is considered the best for expansion of limbal epithelial stem cells (SC) in vitro. Decellularization with SDS removes all immunogenic, cellular components from HAM whilst preserving key components of extracellular matrix. Decellularization creates a consistent, defined substrate which does not elicit an adverse immunological response in vivo. We aim to investigate the differences in growth rate and cell phenotype of limbal explants cultured on fresh and decellularized HAM
Limbal explant cultures were established on four different cryopreserved HAM substrates (n=4/group) 1) Fresh HAM; 2) 0.5M NaOH treated HAM; 3) HAM decellularized with 0.5% SDS and 4) γ-irradiated SDS decellularized HAM. Upon reaching >90% confluency 1 culture from each group was examined histologically and immunostained for ΔNp63, ABCG2, CK12, CK13, Ki67. In the remaining explant cultures cells were separated from the HAM by trypsinization; 500 cells from each group were used in colony forming efficiency assays and 100000 cells from each group were used in qPCR (p63, ABCG2, CK12, CK13, Ki67; n=3/group).
Explant cultures grew fastest on γ-irradiated SDS decellularized HAM>non-γ-irradiated SDS decellularized HAM>NaOH treated HAM>fresh HAM (p=0.02). Immunostaining showed explants cultured on fresh HAM and NaOH treated HAM expressed similar levels of Ki67 (fresh <5% of all cells, NaOH treated 10%), ABCG2 (fresh 50%, NaOH treated 70%), ΔNp63 (both 60%) and CK12 (fresh 5%, NaOH treated 10%). Explants cultured on SDS decellularized HAM had higher expressions of Ki67 (non-γ-irradiated 25%, γ-irradiated 40%), ABCG2 (non-γ-irradiated 100%, γ-irradiated 90%), p63 (both 80%) and did not express CK12 or CK13. These results were supported by qPCR which showed lower levels of CK12 and CK13 and higher levels of p63 in SDS decellularized HAM (p>0.05).
Limbal explant cultures grew significantly faster on SDS decellularized HAM but there was no significant difference in growth rate or preservation of SC properties between explants grown on γ-irradiated or non-γ-irradiated decellularized HAM. Cells grown on these substrates showed higher levels of putative epithelial SC markers and were less likely to differentiate. Thus, SDS decellularized HAM is more efficacious for use in clinical trials of ex vivo expanded LSC transplantation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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