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Amniotic membrane transplantation is an established therapeutic and biological adjunct for several clinical situations, including treatment of diabetic foot ulcers and ocular surface disease. However, poorly standardized and validated clinical preparation and storage procedures can render the final product highly variable and an unpredictable biomaterial. We have therefore developed a novel, standardized method for processing and dry-preserving amniotic membrane, minimizing biochemical, compositional, and structure damage to produce a potentially superior membrane suitable for clinical use. The intellectual property associated with this methodology was patented by the University of Nottingham and licensed to NuVision® Biotherapies which formed the basis of the Tereo® manufacturing process which is used to manufacture Omnigen®.Tissue engineering by self-assembly allows for the formation of living tissue substitutes, using the cells' innate capability to produce and deposit tissue-specific extracellular matrix. However, in order to develop extracellular matrix-rich implantable devices, prolonged culture time is required in traditionally utilized dilute ex vivo microenvironments. Macromolecular crowding, by imitating the in vivo tissue density, dramatically accelerates biological processes, resulting in enhanced and accelerated extracellular matrix deposition. Herein, we describe the ex vivo formation of corneal stromal-like assemblies using human corneal fibroblasts and macromolecular crowding.Tissue engineering is a flourishing field of regenerative medicine that allows the reconstruction of various tissues of our body, including the cornea. In addition to addressing the growing need for organ transplants, such tissue-engineered substitutes may also serve as good in vitro models for fundamental and preclinical studies. Recent progress in the field of corneal tissue engineering has led to the development of new technologies allowing the reconstruction of a human bi-lamellar cornea. One unique feature of this model is the complete absence of exogenous material. Indeed, these human corneal equivalents are exclusively composed of untransformed human corneal fibroblasts (hCFs) entangled in their own extracellular matrix, as well as untransformed human corneal epithelial cells (hCECs), both of which isolated from donor corneas. The reconstructed human bi-lamellar cornea thereby exhibits a well-organized stroma as well as a well-differentiated epithelium. This chapter describes the methods used for the isolation and culture of hCFs, the production and assembly of hCFs stromal sheets, the seeding of hCECs, and the maturation of the tissue-engineered cornea.Gene delivery approaches using adeno-associated virus (AAV) vectors are currently the preferred method for human gene therapy applications and have demonstrated success in clinical trials for a diverse set of diseases including retinal blindness. To date, no clinical trials using AAV gene therapy in the anterior eye have been initiated; however, corneal gene delivery appears to be an attractive approach for treating both corneal and ocular surface diseases. Multiple preclinical studies by our lab and others have demonstrated efficient AAV vector-mediated gene delivery to the cornea for immunomodulation, anti-vascularization, and enzyme supplementation. Interestingly, the route of AAV vector administration and nuances such as administered volume influence vector tropism and transduction efficiency. In this chapter, a detailed protocol for AAV vector production and specific approaches for AAV-mediated gene transfer to the cornea via subconjunctival and intrastromal injections are described.CRISPR/Cas9 gene editing holds the promise of sequence-specific alteration of the genome to achieve therapeutic benefit in the treated tissue. Cas9 is an RNA-guided nuclease in which the sequence of the RNA can be altered to match the desired target. However, care must be taken in target choice and RNA guide design to ensure both maximum on-target and minimum off-target activity. learn more The cornea is an ideal tissue for gene therapy due to its small surface area, accessibility, immune privilege, avascularity, and ease of visualization. Herein, we describe the design, testing, and delivery of Cas9 and guide RNAs to target genes expressed in the cornea.The cornea is the outermost transparent and refractive barrier surface of the eye necessary for vision. Development of the cornea involves the coordinated production of extracellular matrix, epithelial differentiation, and endothelial cell expansion to produce a highly transparent tissue. Here we describe the production of multilayered three-dimensional organoids from human-induced pluripotent stem cells. These organoids have the potential for multiple downstream applications which are currently unattainable using traditional in vitro techniques.Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.The cultivation of corneal-limbal cells in vitro represents an excellent means to generate models to study cornea function and disease processes. These in vitro expanded cornea-limbal epithelial cell cultures are rich in stem cells for cornea, and hence can be used as a cell therapy for cornea-limbal deficiency. This chapter details the primary culture of these cornea-limbal cells, which can be used as model for further studies of the cornea surface.The corneal endothelium forms a leaky barrier between the corneal stroma and the aqueous humor of the anterior chamber. This cell monolayer maintains the corneal stroma in a state of relative dehydration, a process called deturgescence, which is required in order to obtain corneal stromal transparency. Endothelial dysfunctions lead to visual impairment that ultimately can only be treated surgically via the corneal transplantation of a functional endothelium. Shortages of corneas suitable for transplantation has motivated research toward new alternatives involving in vitro corneal endothelial cell (CEC) expansion.This chapter describes current methods that allow isolate and culture CECs. In brief, Descemet membrane is peeled out of the cornea and digested in order to obtain CECs. Cells are then seeded and cultured.
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