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

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 0.5M 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. STATEMENT OF SIGNIFICANCE: 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.

Single dipole evanescently coupled to a multimode waveguide.

We consider a single dipole evanescently coupled to a cylindrical multimode waveguide. The emission rate into the waveguide is calculated as a function of the waveguide diameter and the dipole orientations, and the result is confirmed by finite-difference-time-domain simulations. We show that as the guide radius increases, the coupling to a given mode decreases but new decay channels to higher order modes open up to increase the density of states. This study gives insight for designing waveguide-based single photon sources that exploit superposition of transverse modes.

Demonstration of UV-written waveguides, Bragg gratings and cavities at 780 nm, and an original experimental measurement of group delay.

We present direct UV-written waveguides and Bragg gratings operating at 780 nm. By combining two gratings into a Fabry-Perot cavity we have devised and implemented a novel and practical method of measuring the group delay of Bragg gratings.