Date Published: October 01, 2017
Author(s): G.S. Figueiredo, S. Bojic, P. Rooney, S.-P. Wilshaw, C.J. Connon, R.M. Gouveia, C. Paterson, G. Lepert, H.S. Mudhar, F.C. Figueiredo, M. Lako.
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 variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or γ-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 γ-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ΔNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and γ-irradiation did not significantly affect the LSC phenotype, 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 γ-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. 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.
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 , and to inhibit fibrosis through suppression of TGFβ signalling . Fresh HAM consists of an epithelial layer (devitalised by the freezing process), a stroma and a thick basement membrane. The basement membrane and extracellular matrix components of HAM, when used as a substrate, have shown similar properties to conjunctival and corneal epithelium , .
All experimental protocols were previously approved by Newcastle University and research was conducted in accordance with the tenets of the Declaration of Helsinki.
A confluent limbal epithelial monolayer of cells was successfully established on 14 of the 16 explant cultures, on all four HAM substrate preparations (fresh HAM, HAM decellularised with NaOH, HAM decellularised with SDS and γ-irradiated HAM decellularised with SDS). A confluent monolayer was not established in one SDS decellularised HAM culture, and one fresh HAM culture. Fig. 1 shows haematoxylin and eosin stained sections of fresh HAM (A) and decellularised HAM (B). There was no subjective difference in HAM transparency after decellularisation and/or γ-irradiation. All HAM were freshly prepared by NHSBT and provided frozen in the same way.
The difference in rates of limbal explant outgrowth was determined by two-way ANOVA. The difference between the groups in terms of the expression of ΔNp63, ABCG2, Ki67, CK12 and CK13 by qRT-PCR, as well as of the Brillouin frequency shift measurements, was analysed by Kruskal-Wallis non-parametric tests. The difference in expression of ΔNp63, ABCG2, Ki67, MUC5AC, CK12 and CK13 by immunocytochemistry was calculated by one-way ANOVA relative to the control group (fresh HAM), with Bonferroni post hoc analysis. Data was analysed using a statistical software package (Prism 6, Graphpad Software Inc, USA). Statistical significance was determined by p < 0.05. In the last couple of decades, there have been major leaps forward in tissue engineering to repair or replace tissue function or whole organs using cells, biomaterials alone or in combination . Decellularisation processes were designed to produce a biological scaffold onto which stem cells could be seeded that reduced the risk of immune rejection and disease transmission. This is particularly pertinent in our phase II clinical trial of patients with unilateral total limbal stem cell deficiency (TLSCD) treated with ex vivo expanded autologous limbal stem cell transplant (ALSCT) where the HAM is the only allogeneic product used in the manufacture of LSCs. As part of a process to refine our culture method, where we previously refined our culture medium , we wanted to minimise the risk of a host response by using decellularised HAM and minimise the risk of disease transmission by using γ-irradiated HAM and sought to determine whether using a decellularised and/or γ-irradiated substrate would have a detrimental effect on our product. Similarly to previous reports, we have demonstrated that ex vivo expansion of limbal epithelial cells using the explant culture system occurs at a faster rate on decellularised HAM compared with fresh HAM. Using Brillouin spectro-microscopy, we have shown that the frequency shifts of HAM tissues are, even after the process of decellularisation or sterilisation, similar to that of the human corneal limbus. As such, the mechanical properties of HAM tissues showed to be suitable for LSC maintenance. Moreover, LSC differentiation does not appear to be influenced primarily by HAM substrate stiffness; however HAM stiffness did appear to affect cell migration rate. This leads us to conclude that SDS decellularised HAM may be more efficacious as a substrate for the ex vivo expansion of limbal epithelial cells for use in clinical trials, and in particular, the use of a γ-irradiated decellularised HAM allows the user to start the manufacturing process with a sterile substrate, making it potentially safer. Source: http://doi.org/10.1016/j.actbio.2017.07.041