Springer protocols feed by Molecular Medicine 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Encapsulation and Culture of Mammalian Cells Including Corneal Cells in Alginate Hydrogels
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 h...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
The Growth and Delivery of Mesenchymal and Limbal Stem Cells Using Copolymer Polyamide 6/12 Nanofiber Scaffolds
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 mecha...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
Cultivation of Limbal Epithelial Cells on Electrospun Poly (lactide-co-glycolide) Scaffolds for Delivery to the Cornea
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 p...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
Fabrication of a Corneal-Limbal Tissue Substitute Using Silk Fibroin
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...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
Fabrication of a Human Recombinant Collagen-Based Corneal Substitute Using Carbodiimide Chemistry
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 cy...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
The Culture of Limbal Stromal Cells and Corneal Endothelial Cells
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 w...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
The Culture of Limbal Epithelial 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. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
Limbal Epithelial Stem Cell Identification Using Immunoblotting Analysis
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 chapt...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
Enrichment of Human Corneal Epithelial Stem/Progenitor Cells by Magnetic Bead Sorting Using SSEA4 as a Negative Marker
The use of a specific antibody bound to magnetic beads to isolate subpopulations of cells is an efficient and simple technique that allows for the subsequent study of different cell populations. One important use of this isolation technique is the purification of stem cells from a mixed cell population. In this protocol, we describe a method to purify human corneal epithelial stem/progenitor cells or limbal stem cells (LSC), using stage-specific embryonic antigen-4 (SSEA4) as a negative surface marker. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
The Use of Bromodeoxyuridine Incorporation Assays to Assess Corneal Stem Cell Proliferation
Bromodeoxyuridine (BrdU) incorporation assays have long been used to detect DNA synthesis in vivo and in vitro. The key principle of this method is that BrdU incorporated as a thymidine analog into nuclear DNA represents a label that can be tracked using antibody probes. In this chapter, we describe BrdU incorporation into limbal stem cells. The colorimetric reaction produced by this assay can be detected by immunohistochemistry, and using appropriate controls, it can be used for determination of proliferating properties of restricted progenitor cells derived from the cornea. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
Clonal Analysis of Limbal Epithelial Stem Cell Populations
While convincing data clearly suggest the presence of stem cells in the basal limbal epithelium in vivo, testing the proliferation, self-renewal, and differentiation capacity of stem cells relies on the development of methodologies that allow for their isolation and extensive propagation in vitro. Clonal analysis involving differentiation between short-lived transient cell clones and long-lived stem cell clones is an invaluable technique to identify stem cells in vitro, and allows cells to be expanded over multiple passages. This chapter describes a protocol for the isolation, expansion, and clonal analysis of limbal epith...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
The Artificial Cornea
Human corneal transplantation to date suffers from the shortage of good-quality donor tissue, and in some conditions, allografting is contraindicated. A range of artificial replacements to donor allograft corneas have been developed. These range from keratoprostheses (KPro) that replace basic corneal functions of light transmission and protection to regenerative medicine strategies for regenerating one or more layers of the human cornea. This chapter reviews the advances made in developing artificial corneas or more accurately, artificial alternatives to donor allograft corneas for ocular application. (Source: Springer pro...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
Limbal Epithelial Cell Therapy: Past, Present, and Future
The cornea, the clear window at the front of the eye, transmits light to the retina to enable vision. The corneal surface is renewed by stem cells located at the peripheral limbal region. These cells can be destroyed by a number of factors, including chemical burns, infections, and autoimmune diseases, which result in limbal stem cell deficiency (LSCD), a condition that can lead to blindness. Established therapy for LSCD based on ex vivo expanded limbal epithelial cells is currently at a stage of refinement. Therapy for LSCD is also rapidly evolving to include alternative cell types and clinical approaches as treatment mod...
Source: Springer protocols feed by Molecular Medicine - June 4, 2013 Category: Molecular Biology Source Type: news
Cell Isolation Through Whole-Liver Perfusion and Preparation of Hepatocytes for Cytochrome P45 Analysis
Activity of cytochrome p450 (CYP450) enzyme is used to measure the ability of hepatocytes to metabolize pharmaceutical compounds. When determining functionality of hepatocytes, the cells may be induced in order to determine metabolic activity after drug induction. Hepatocytes, whether in suspension or plated, are used in the pharmaceutical industry as a surrogate to assess in vivo drug metabolism. Within this chapter, isolation of hepatocytes from whole-liver tissue and subsequent preparation for CYP450 istotype 3A4 activity is discussed. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - April 11, 2013 Category: Molecular Biology Source Type: news
Isolation of Pulsatile Cell Bodies from Esophageal Tissue
Pulsatile cell bodies, three-dimensional cell clusters with satellite streaming cells, can be isolated from esophageal tissue. One of the key features of these clusters is that they pulsate at rhythmic rates and demonstrate contractility under several in vitro conditions. Their ability to pulsate appears to be due to the presence of interstitial cells of Cajal (ICC), which mediate signal transmission from nerve to muscle cells. As predicted, the cells comprising these clusters express phenotypic and genotypic markers characteristic of smooth and skeletal muscle, neuronal, and epithelial cells. Because of the critical ...
Source: Springer protocols feed by Molecular Medicine - April 11, 2013 Category: Molecular Biology Source Type: news
