Autologous cultivated limbal epithelial transplantation using a xeno-free explant culture technique was effective in long-term restoration of corneal epithelial stability and improvement of vision in eyes with ocular surface burns.

Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes approximately 2 weeks to establish a confluent monolayer from which approximately 3 x 10(6) cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.

Allogeneic cultivated limbal epithelial transplantation, followed by PK when needed, can successfully restore the ocular surface and improve vision in patients with corneal blindness due to bilateral LSCD.

BackgroundHyperglycemia is a significant risk factor for diabetic retinopathy and induces increased inflammatory responses and retinal leukostasis, as well as vascular damage. Although there is an increasing amount of evidence that miRNA may be involved in the regulation in the pathology of diabetic retinopathy, the mechanisms by which miRNA mediate cellular responses to control onset and progression of diabetic retinopathy are still unclear. The purpose of our study was to investigate the hypothesis that miR-15a/16 inhibit pro-inflammatory signaling to reduce retinal leukostasis.MethodsWe generated conditional knockout mice in which miR-15a/16 are eliminated in vascular endothelial cells. For the in vitro work, human retinal endothelial cells (REC) were cultured in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC in high glucose with miRNA mimic (hsa-miR-15a-5p, hsa-miR-16-5p). Statistical analyses were done using unpaired Student t test with two-tailed p value. p < 0.05 was considered significant. Data are presented as mean ± SEM.ResultsWe demonstrated that high glucose conditions decreased expression of miR-15a/16 in cultured REC. Overexpression of miR-15a/16 with the mimic significantly decreased pro-inflammatory signaling of IL-1β, TNFα, and NF-κB in REC. In vivo data demonstrated that the loss of miR-15a/16 in vascular cells led to increased retinal leukostasis and CD45 levels, together with upregulated levels of IL-1β, TNFα, and NF-κB.ConclusionsThe data indicate that miR-15a/16 play significant roles in reducing retinal leukostasis, potentially through inhibition of inflammatory cellular signaling. Therefore, we suggest that miR-15a/16 offer a novel potential target for the inhibition of inflammatory mediators in diabetic retinopathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0771-8) contains supplementary material, which is available to authorized users.

Limbal tissues can be cultured on various types of scaffolds to create a sheet of limbal-corneal epithelium for research as well as clinical transplantation. An optically clear, biocompatible, biomimetic scaffold would be an ideal replacement graft for transplanting limbal stem cells. In this study, we evaluated the physical and culture characteristics of the recombinant human cross-linked collagen scaffold (RHC-III scaffold) and compared it with denuded human amniotic membrane (HAM). Optical/mechanical properties and microbial susceptibility were measured for the scaffolds. With the approval of the institutional review board, 2 mm fresh human limbal tissues were cultured on 2.5 x 2.5 cm(2) scaffolds in a medium containing autologous serum in a feeder cell-free submerged system. The cultured cell systems were characterized by morphology and immunohistochemistry for putative stem cells and differentiated cell markers. The refractive index (RI) and tensile strength of the RHC-III scaffold were comparable to human cornea, with delayed in vitro degradation compared to HAM. RHC-III scaffolds were 10-fold less susceptible to microbial growth. Cultures were initiated on day 1, expanded to form a monolayer by day 3 and covered the entire growth surface in 10 days. Stratified epithelium on the scaffolds was visualized by transmission electron microscopy. The cultured cells showed p63 and ABCG2 positivity in the basal layer and were immunoreactive for cytokeratin K3 and K12 in the suprabasal layers. RHC-III scaffold supports and retains the growth and stemness of limbal stem cells, in addition to resembling human cornea; thus, it could be a good replacement scaffold for growing cells for clinical transplantation.