Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.

G.S Figueiredo,P Rooney,F.C Figueiredo,H.S Mudhar,S.-P Wilshaw,C Paterson,C.J Connon,G Lepert,S Bojic,R.M Gouveia,M Lako

doi:10.1016/j.actbio.2017.07.041

Abstract

Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.

Highlights

Human amniotic membrane (HAM) has long been used in ophthalmic procedures to treat ocular surface diseases and burns, due to its ability to promote re-epithelialisation via growth factors and its basement membrane [1], and to inhibit fibrosis through suppression of TGFb signalling [2]
We cultured limbal epithelial stem cells (LSCs) on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation
We demonstrated that HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs

Summary

Introduction

Human amniotic membrane (HAM) has long been used in ophthalmic procedures to treat ocular surface diseases and burns, due to its ability to promote re-epithelialisation via growth factors (such as EGF, KGF and HGF) and its basement membrane [1], and to inhibit fibrosis through suppression of TGFb signalling [2]. Fresh HAM consists of an epithelial layer (devitalised by the freezing process), a stroma and a thick basement membrane. There has been a significant drive to better characterise HAM in order to develop a better substrate for the in vitro expansion of LSCs [13,15,16]. One such approach has been to decellularise HAM, as this process can remove all cellular, immunogenic components from a tissue, whilst preserving key extracellular matrix (ECM) components and the basement membrane, ensuring cell attachment and expansion. The improved cell proliferation has been attributed to better cell adhesion to the substrate compared to fresh HAM, due to exposure of the basement membrane, unobstructed by a devitalised epithelial cell layer

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