Laser-scanning in vivo confocal microscopy (IVCM) of the cornea is becoming an increasingly popular tool to examine the living human cornea with cellular-level detail in both healthy and pathologic states. Here, we describe the use of the IVCM technique to examine the processes of tissue healing and regeneration in the living human eye after biomaterial implantation. The regenerative response can be assessed by performing longitudinal IVCM imaging of a laboratory-made, cell-free biomaterial, after direct implantation into a pathologic eye as a primary alternative to human donor tissue transplantation.

The potential of cell therapy for the regeneration of diseased and damaged tissues is now widely -recognized. As a consequence there is a demand for the development of novel systems that can deliver cells to a particular location, maintaining viability, and then degrade at a predictable rate to release the cells into the surrounding tissues. Hydrogels have attracted much attention in this area, as the hydrogel structure provides an environment that is akin to that of the extracellular matrix. One widely investigated hydrogel is alginate, which has been used for cell encapsulation for more than 30 years. Alginate gels have the potential to be used as 3D cell culture systems and as prosthetic materials, both are applied to regeneration of the cornea. Here, we describe an alginate-based process that has been used for encapsulation of mammalian cells including corneal cells, with high levels of viability, and which allows subsequent retrieval of cell cultures for further characterization.

The injured or otherwise damaged cornea is healed by limbal stem cells (LSC). If the limbus where LSC reside is also damaged or nonfunctional, the cornea cannot heal properly and this defect leads to impaired vision that can result in blindness. The only way to treat total LSC deficiency is by transplantation of limbal tissue or a transfer of LSC. Recently, mesenchymal stem cells (MSC) have been shown as another promising source of stem cells for corneal healing and regeneration. Here, we describe a protocol for the use of polyamide 6/12 nanofiber scaffolds for the growth of MSC and LSC, and for their transfer onto a mechanically damaged ocular surface in the experimental mouse model.

In delivering tissues to the body, both natural and synthetic materials have been used. Currently, a natural membrane, the human amniotic membrane (AM), is used to deliver limbal epithelial cells (LEC) to the cornea. AM presents inherent problems with structural variation and requires extensive serological screening before use. Therefore alternatives are required to improve the predictability in clinical outcomes and economic costs associated with the use of this biological substrate. In this chapter, we describe the development of an alternative, structurally simple, synthetic biodegradable electrospun scaffold based on poly(lactide-co-glycolide) (PLGA: materials used in dissolvable sutures) to replace AM.

Fibroin extracted from silkworm cocoon silk provides an intriguing and potentially important biomaterial for corneal reconstruction. In this chapter we outline our methods for producing a composite of two fibroin-based materials that support the cocultivation of human limbal epithelial (HLE) cells and human limbal stromal (HLS) cells. The resulting tissue substitute consists of a stratified epithelium overlying a three-dimensional arrangement of extracellular matrix components (principally "degummed" fibroin fibers) and mesenchymal stromal cells. This tissue substitute is currently being evaluated as a tool for reconstructing the corneal limbus and corneal epithelium.

Human recombinant collagen can be cross-linked with a variety of chemical cross-linking agents. Cross-linking methods can be tuned to confer collagen-based scaffolds with specific physical properties, improved antigenicity and thermal stability without impeding the ability of the material to integrate into the surrounding tissue and to promote regeneration. Here, we describe a method to cross-link human recombinant collagen using a water soluble carbodiimide. Carbodiimides are referred to as zero-length cross-linking agents as they are not incorporated into the final cross-link and thus pose minimal risk with respect to cytotoxicity. The resulting collagen-based scaffold possesses properties comparable to that of the human cornea and is thus suitable for use as a corneal substitute.

Collagen has excellent biocompatibility, is biodegradable, and possesses low immunogenicity. Therefore, this protein is a very suitable substrate for the formation of a corneal scaffold for therapeutic use. The highly hydrated nature of conventional collagen gels, however, results in a gel that is structurally weak and difficult to manipulate. In this chapter, we describe a novel method to cultivate limbal epithelial cells (LEC) on a compressed collagen scaffold. The compressed collagen scaffold can be rapidly constructed using a cell-independent process, which produces dense and mechanically strong collagen constructs with controllable microscale features.We embedded corneal keratocytes in a collagen gel, which we subsequently compressed and coated with laminin. The resulting construct supported the physiological morphology and stratification of LEC. The expression of a specific marker for differentiated LEC, cytokeratin 3 (CK3), and a marker for undifferentiated LEC, cytokeratin 14 (CK14), were similar in LEC expanded on both the compressed collagen construct and the leading conventional scaffold, denuded amniotic membrane (AM). We therefore demonstrate that a laminin-coated, compressed collagen gel containing keratocytes can support LEC expansion, stratification, and differentiation to a degree that is comparable to denuded AM. Our novel compressed collagen/keratocyte construct has potential for use as a tissue-engineered artificial cornea.

The cornea is the transparent front part of the eye and comprises three distinct cell layers. One of these cell layers is a self-renewing epithelium long believed to harbor a resident stem cell population. The location and characteristics of corneal epithelial stem cells have now been confirmed by several research groups, and these cells are currently applied therapeutically. The corneal stroma and endothelium are largely quiescent after infancy, and until recently they were not considered to undergo self-renewal or to maintain stem cells. This view was overturned during the last two decades. At present, cell populations with characteristics of adult stem cells are routinely isolated and characterized from the limbal stroma and the corneal -endothelium. This chapter describes methods for isolation and culture of limbal stromal cells and corneal endothelial cells.

The transplantation of cultured limbal epithelial cells (LEC) has since its first application in 1997 emerged as a promising technique for treating limbal stem cell deficiency. The culture methods hitherto used vary with respect to preparation of the harvested tissue, choice of culture medium, culture time, culture substrates, and supplementary techniques. In this chapter, we describe a procedure for establishing human LEC cultures using a feeder-free explant culture technique with human amniotic membrane (AM) as the culture substrate.

The unambiguous identification of limbal epithelial stem cells is currently a major challenge in corneal stem cell biology. Specific molecular markers which characterize these cells are lacking. At present, the best strategy for identification of limbal epithelial stem cells is to investigate a variety of putative markers for these cells in a differentiated (cytokeratin (CK) 3: CK3, integrin α6), undifferentiated (CK14), and naive state (∆Np63α, ATP-binding cassette subfamily G member 2 (ABCG2), integrin α9, Notch-1), alongside functional assays which indicate their stemness. The focus of this chapter is to highlight advances in the Western blotting technique for quantitative assessment of corneal epithelial cell markers, and the use of this technique for investigation of a range of different protein markers which identify limbal epithelial stem cells.