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Alvena Kureshi, Rosemary Drake, Julie Daniels; Development Of A Reference Standard And Potency Assay For The Clinical Production Of Plastic Compressed Collagen Tissue Equivalents For Limbal Epithelial Cell Culture. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1817.
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© ARVO (1962-2015); The Authors (2016-present)
Plastic compressed collagen tissue equivalents, for ocular surface reconstruction, must be manufactured to exacting quality standards (Good Manufacturing Practice) prior to patient transplantation. Our aim was to develop a Reference Standard and a Potency Assay that could be used to demonstrate reproducibility of production and epithelial cell function respectively.
Our novel RAFT (Real Architecture for 3D Tissues) technology, based upon plastic compression of cell-seeded collagen gels (Brown et al., 2005), was used to prepare ocular surface tissue equivalents (Levis et al., 2010). In order to reproducibly control fluid loss during compression of the gels and provide a means of transporting the constructs, bespoke cassettes were designed. Briefly, constructs populated with human limbal fibroblasts were compressed, seeded with human limbal epithelial cells (LECs) and cultured for 19 days within the cassettes. The constructs were assessed with light microscopy, histology, and immunohistochemistry to characterize the LEC phenotype. Parameters included cell morphology and p63α (putative stem cell marker) production. To test potency, constructs were subjected to a 1mm stripe defect on the epithelial surface using a corneal Algerbrush. Re-epithelialisation of the defect was quantified over time with light microscopy and correlated with histology.
RAFT constructs were reproducibly prepared using cassettes. LECs achieved confluence and formed patches of multilayered epithelium after 19 days of culture on the surface. The basal LECs were small in size with characteristic cobblestone morphology. These cells also expressed p63α, which is indicative of poorly, differentiated cells with a high proliferative capacity, suggesting they are ‘stem-like’. RAFT constructs supported re-epithelialisation of wound defects demonstrating potency with respect to epithelial cell function. Not surprisingly, the rate of re-epithelialisation was variable between different biological cell donors. Histological comparison of the constructs before and after wounding confirmed that re-epithelialisation had occurred with the onset of cell multi-layering that is required to regenerate full epithelium.
The RAFT process has been modified to include the use of bespoke cassettes that take into account manufacturing and regulatory requirements for delivery of a cell therapy to a patient. RAFT constructs repeatedly demonstrated the capacity to re-epithelialise a wound defect over time thus also providing an in vitro model of wound healing.
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