Defining the optimal cryoprotectant and concentration for cryopreservation of limbal stem cells.

Limbal stem cell (LSC) deficiency causes progressive loss of vision but may be treated by transplant of autologous LSCs. Cryopreservation has the potential to indefinitely extend the lifespan of LSCs allowing re-transplant in case of graft failure. In this study, we aimed to identify the optimal cryoprotectant and cryoprotectant concentration for LSC cultures. Suspension cultures derived from cadaveric corneoscleral rims were cooled to 4 °C with Me2SO, propylene glycol or ethylene glycol at a concentration of 5%, 10% or 15%. Cell tolerance was measured in terms of membrane integrity, colony-forming efficiency and alamarBlue® reduction. Increasing cryoprotectant concentration above 5% reduced membrane integrity, metabolism and colony-forming efficiency. Cryoprotectant choice did not significantly influence these characteristics. Cells demonstrating Side Population were maintained after cryopreservation with 5% propylene glycol in vapour phase liquid nitrogen for 1 week, indicating that cryopreservation of LSCs with relatively low cryoprotectant concentration (5%) has promise in low-temperature eye banking.

Human limbal mesenchymal stem cells express ABCB5 and can grow on amniotic membrane.

AIM: To isolate and characterize limbal mesenchymal stem cells (LMSCs) from human corneoscleral rings. MATERIALS & METHODS: Cells were isolated from corneoscleral rings and cultured in a mesenchymal stem cell (MSC)-selective media and examined for differentiation, phenotyping and characterization. RESULTS: LMSCs were capable of trilineage differentiation, adhered to tissue culture plastic, expressed HLA class I and cell surface antigens associated with human MSC while having no/low expression of HLA class II and negative hematopoietic lineage markers. They were capable for CXCL12-mediated cellular migration. LMSCs adhered, proliferated on amniotic membrane and expressed the common putative limbal stem cell markers. CONCLUSION: Limbal-derived MSC exhibited plasticity, could maintain limbal markers expression and demonstrated viable growth on amniotic membrane.

Investigating the biological response of human mesenchymal stem cells to titanium surfaces.

BACKGROUND: We have investigated the behaviour of a newly characterised population of haemarthrosis fluid-derived human mesenchymal stem cells (HF-hMSCs) with titanium (Ti) surfaces. METHODS: HF-hMSCs were seeded onto round cannulated interference (RCI; Smith and Nephew) screws or control Ti discs and cultured under pro-osteogenic conditions. RESULTS: Electron microscopy showed the attachment and spreading of HF-hMSCs across both Ti surfaces during the early stages of osteogenic culture; however, cells were exclusively localised to the basal regions within the vertex of the Ti screws. In the later stages of culture, an osteoid matrix was deposited on the Ti surfaces with progressive culture expansion and matrix deposition up the sides and the top of the Ti Screws. Quantification of cellular content revealed a significantly higher number of cells within the Ti screw cultures; however, there was no difference in the cellular health. Conversely, alizarin red staining used as both a qualitative and quantitative measure of matrix calcification was significantly increased in Ti disc cultures compared to those of Ti screws. CONCLUSIONS: Our results suggest that the gross topography of the metal implant is able to create microenvironment niches that have an influence on cellular behaviour. These results have implications for the design of advanced tissue engineering strategies that seek to use cellular material to enhance biological remodelling and healing following tissue reconstruction.

The limbal epithelium of the eye--a review of limbal stem cell biology, disease and treatment.

The limbus is a narrow band of tissue that encircles the cornea, the transparent 'window' into the eye. The outermost layer of the cornea is the epithelium, which is necessary for clear vision. The limbus acts as a 'reservoir' for limbal stem cells which maintain and regenerate the corneal epithelium. It also functions as a barrier to the conjunctiva and its blood vessels. Limbal stem cell deficiency is a general term for diseases which are characterised by the impairment of the limbus, limbal stem cells and their ability to replenish the corneal epithelium through proliferation and differentiation. Consequently, sufferers experience chronic pain and progressive blindness. This paper will highlight the salient milestones of limbal stem cell biology and potential future treatments for limbal stem cell deficiency.

The culture and transplantation of human limbal stem cells.

The cornea is the clear front of the eye and its surface is composed of an epithelium. This is renewed by stem cells located at the limbus, which encircles the periphery of the cornea. These limbal stem cells become lost or deficient in the blinding disease of limbal stem cell deficiency. In this review article, we discuss the historical perspective in managing limbal stem cell deficiency as well as describing the more contemporary treatment options, and in particular the culture and transplantation of human limbal stem cells. This treatment was first proposed 13 years ago and many case series have been presented to date showing promising outcomes of this technique. However, challenges still remain in treating the debilitating disease of limbal stem cell deficiency. Here we discuss some of the questions, which remain to be answered in this field.

Culture conditions for primary human limbal epithelial cells.

The cornea at the front of the eye is covered by an epithelium. This epithelium is maintained by stem cells located at the periphery of the cornea, in a region known as the limbus. Because this region harbors the stem cells for the corneal epithelium, the so-called limbal stem cells, its culture provides considerable interest. Limbal epithelial culture is used for two main reasons. The first is to further our understanding of limbal stem-cell biology. The second is for the culture expansion of limbal stem cells for transplantation purposes in patients with limbal stem-cell deficiency. However, considerable variations in the culture methods for limbal epithelium exist. These include culture media, sera used in the culture, use of 3T3 fibroblasts or amniotic membrane or both, the culture of whole pieces of limbal tissue or enzymatically digested tissue, and the use of airlifting.