A novel human donor cornea preservation cocktail incorporating a thermo-reversible gelation polymer (TGP), enhancing the corneal endothelial cell density maintenance and explant culture of corneal limbal cells

Purpose McCarey-Kaufman’s (MK) medium and Optisol-GS medium are the most commonly employed media for human donor corneal preservation. In this study, we evaluated the preservation efficacy of discarded human donor corneas using a Thermo-reversible gelation polymer (TGP) added to these two media. Methods Thirteen human corneal buttons collected from deceased donors, which were otherwise discarded due to low endothelial cell density (ECD) were used. They were stored in four groups: MK medium, MK medium with TGP, Optisol-GS and Optisol-GS with TGP at 4 °C for 96 h. Slit lamp examination and specular microscopy were performed. Corneal limbal tissues from these corneas were then cultured using explant methodology one with and the other without TGP scaffold, for 21 days. Results MK + TGP and Optisol-GS + TGP preserved corneas better than without TGP, which was observed by maintenance of ECD which was significantly higher in Optisol-GS + TGP than MK + TGP (p-value = 0.000478) and corneal thickness remaining the same for 96 h. Viable corneal epithelial cells could be grown from the corneas stored only in MK + TGP and Optisol-GS + TGP. During culture, the TGP scaffold helped maintain the native epithelial phenotype and progenitor/stem cell growth was confirmed by RT-PCR characterization. Conclusion TGP reconstituted with MK and Optisol—GS media yields better preservation of human corneal buttons in terms of relatively higher ECD maintenance and better in vitro culture outcome of corneal limbal tissue. This method has the potential to become a standard donor corneal transportation-preservation methodology and it can also be extended to other tissue or organ transportation upon further validation.

Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium

ObjectiveTo detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method.MethodsSeven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16 S and 18 S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments.ResultsIn both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples.ConclusionMetagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turn-around time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.