[Development of novel cell culture systems utilizing the advantages of collagen vitrigel membrane].

The white of an egg, rendered opaque by boiling, can be converted into a thin, transparent and rigid material like glass by evaporating the moisture. This phenomenon is known as the vitrification of heat-denatured proteins. We applied vitrification technology to a collagen gel and converted it into a rigid glass-like material. We attempted to rehydrate the glass-like material and succeeded in preparing a novel stable state of collagen gel that was a thin and transparent membrane with excellent gel strength and protein permeability. We called it "collagen vitrigel" because it was produced from the vitrification process of a traditional hydrogel. Further, a framework-embedded collagen vitrigel membrane that can be easily turned inside out with tweezers was prepared by inserting a nylon membrane ring in the collagen sol prior to the gelation, thereby allowing the membrane to function as a removable cell culture substratum. Different types of anchorage-dependent cells could be cultured on both surfaces of the substratum by the manipulation of two-dimensional cultures, and consequently a three-dimensional crosstalk model with paracrine effects from each cell type was reconstructed. Also, the collagen vitrigel membrane containing a bioactive molecule provided a drug delivery system (DDS) with sustainable release. In this review, we summarize the recent progress of applied studies using the collagen vitrigel membrane as follows: a corneal model for eye irritant and permeability tests, a skin model for sensitization test, a renal glomerular model for evaluating blood filtration, an endometrial model for developing a new treatment and a DDS of hepatocyte growth factor for improving liver disorder.

Integration and application of vitrified collagen in multilayered microfluidic devices for corneal microtissue culture.

This paper describes the fabrication and application of microfluidic devices containing collagen vitrigel (CV) used as both a functional and sacrificial cell growth substrate for the development of corneal microtissue patches. Within the device, vacuum fixation of the CV in a dehydrated state enables quick integration with standard multilayer soft lithographic techniques, while on-chip rehydration results in a gel-like collagen substrate for microfluidic cell culture. Fluidic connectivity to both the apical and basal side of the CV permits bilayered culture of epithelium and supporting stromal cell layers. In addition, microfluidic introduction of a collagenase etching media enables sacrificial degradation of the supporting CV membrane for development of barrier tissue constructs containing minimal synthetic substrate. The utility of this platform was evaluated by miniaturizing the standard transepithelial permeability (TEP) assay in order to measure the integrity of an array of corneal tissue micropatches.

A tale of two tissues: stem cells in cartilage and corneal tissue engineering.

Laboratory investigations of stems cells in regenerative medicine have generated considerable interest within recent years, however some of this excitement is yet to be matched in the clinical arena. Two fields that are well poised to make significant clinical impact in the coming years are those of cartilage and corneal regeneration. In the case of cornea, it is widely acknowledged that corneal epithelium is derived from an adult stem cell type resident within the cornea. These cells, known as limbal stem cells (LSC's), have been widely investigated for their ex-vivo culture and subsequent transplantation efficacy, with some techniques already enjoying limited clinical application. Thus far however, only preliminary evidence currently exists to suggest that there is a population of adult stem cells which gives rise to stromal keratocytes or to the corneal endothelium. A handful of reports have discussed studies in which non-LSC adult stem cells such as mesenchymal stems cells (MSCs) or embryonic stem cells (ESCs) are being applied to corneal regeneration. Though adult stem cells have been shown to exist in articular cartilage, they have proven elusive, which corroborates the limited ability of this tissue to self-repair. Rather, MSCs, ESCs as well as adipose-derived, periosteum-derived, muscle-derived and synovium-derived stem cells (ADCs, PDCs, MDCs and SDCs respectively) are being extensively explored for cartilage regeneration. This review discusses emerging trends in the applications of both adult and embryonic stem cells to cartilage and corneal regeneration, with an emphasis on those techniques that have been applied clinically or which show significant potential for clinical translation.

Collagen Vitrigel membranes for the in vitro reconstruction of separate corneal epithelial, stromal, and endothelial cell layers.

The goal of this study was to evaluate the potential suitability of collagen Vitrigel (CV) membrane as a substrate for the separate reconstruction of the three main cellular layers of the cornea. Limbal explants, keratocytes, and endothelial cells were cultured on transparent membranes made of type I collagen. The resulting cell sheets were evaluated using RT-PCR, in addition to light and electron microscopy. Tensile testing was also performed to examine the mechanical properties of CV. Limbal explant cultures resulted in partially stratified epithelial sheets with upregulation of the putative stem cell marker p63. Keratocytes cultured in serum on CV exhibited stellate morphology along with a marked increase in expression of corneal crystallin ALDH and keratocan, (a keratan sulphate proteoglycan: KSPG), compared to identical cultures on tissue culture plastic. Endothelial cells formed dense monolayers with uniform cell size, tight intercellular junctions, and expression of voltage-dependent anion channels VDAC2 and VDAC3, chloride channel protein CLCN2, and sodium bicarbonate transporter NBC1. Epithelial and endothelial cells exhibited adhesive structures (desmosomes and hemidesmosomes) and evidence of apical specialization (microplicae), while endothelial cells also produced a Descemet's membrane-like basal lamina. CV was found to possess ultimate tensile strengths of 6.8 +/- 1.5 MPa when hydrated and 28.6 +/- 7.0 MPa when dry. Taken together, these results indicate that CV holds promise as a substrate for corneal reconstruction.

Decellularization of bovine corneas for tissue engineering applications.

Scaffolds derived from processed tissues offer viable alternatives to synthetic polymers as biological scaffolds for regenerative medicine. Tissue-derived scaffolds provide an extracellular matrix (ECM) as the starting material for wound healing and the functional reconstruction of tissues, offering a potentially valuable approach for the replacement of damaged or missing tissues. Additionally, acellular tissue may provide a natural microenvironment for host-cell migration and the induction of stem cell differentiation to contribute to tissue regeneration. There are a number of processing methods that aim to stabilize and provide an immunologically inert tissue scaffold. Furthermore, these tissue-processing methods can often be applied to xenogenic transplants because the essential components of the ECM are often maintained between species. In this study, we applied several tissue-processing protocols to the cornea in order to obtain a decellularized cornea matrix that maintained the clarity and mechanical properties of the native tissue. Histology, mechanical testing and electron microscopy techniques were used to assess the cell extraction process and the organization of the remaining ECM. In vitro cell seeding experiments confirmed the processed corneas' biocompatibility.