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Gary Hin-Fai Yam, Nur Zahirah Binte M Yusoff, Jodhbir S Mehta; A Comprehensive Comparison of Decellularization Methods for Human Corneal Stromal Lenticules to Yield Bioscaffold for Keratocyte Implantation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1946.
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© ARVO (1962-2015); The Authors (2016-present)
Decellularization of corneal stromal tissue can remove immunogenic cells prior to transplantation. As yet there is no standard technique to perform this. Here, we optimized the decellularization of stromal lenticules (SL) to maintain transparency and preserve collagen stromal matrix architecture, aiming to provide a suitable bioscaffold for implanting corneal stromal keratocytes [CSK].
Research-grade cadaveric corneal tissues (Lions Eye Institute, FL) were ablated for lamellar cut by using a Femto LDV Z6 (Zeimer) femtosecond laser with thickness of 70-150μm. A total of 14 protocols to decellularize SL were tested, including hyperosmolar treatment (1.5M sodium chloride [NaCl]), detergents (0.1-1% Triton X-100 [Tx], 0.1-1% sodium dodecylsulfate [SDS]), nucleases and their combinations. Treated SL samples were examined for changes of thickness and light transmittance (wavelength 380-780nm), transmission electron microscopy and histochemistry for matrix architecture and protein content (periodic acid-Schiff for glycoproteins, alcian blue for proteoglycans [PGs] and picrosirius red for collagen) and immunofluoresecence using phalloidin and 4',6-diamidino-2-phenylindole for cellular and nucleic acid residues. CSK viability was studied after seeding to SL decellularized by the optimized protocol.
In quadruplicate assays, human SL treated with 1.5M NaCl, Tx or 0.1% SDS had the thickness and transmittance comparable to control SL. However, NaCl and Tx groups showed cellular and DNA residues, low collagen (both 41%) and PG content (34% and 40%). In contrast, SDS treatment gave the highest PG (58%) and collagen (55%) content, negligible cellular debris, and reticular stromal fibril pattern similar to control SL. CSK seeded to SDS-treated SL maintained viable and displayed keratocyte markers. SL treated with NaCl and nucleases (0.5-10U/ml DNase and RNase, from different sources) had inconsistent results and loosening of fibrillar structures from SL, hence deemed unsuitable for cell seeding.
Thin human SL decellularized by 0.1% SDS provided a viable bioscaffold for CSK implantation in stromal tissue engineering.
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