title: A Novel Organ Culture Model to Quantify Collagen Remodeling in Tree Shrew Sclera.
abstract: Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 μL and (iv) 200 μL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.
authors.affiliation: Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Sarah
authors.initials: S
authors.lastname: Baldivia
authors.affiliation: Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Alexander
authors.initials: A
authors.lastname: Levy
authors.affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Shylaja
authors.initials: S
authors.lastname: Hegde
authors.affiliation: Department of Biomedical Engineering and Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, The Netherlands.
authors.forename: Stijn J A
authors.initials: SJ
authors.lastname: Aper
authors.affiliation: Department of Biomedical Engineering and Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
authors.affiliation: Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Rafael
authors.identifier: http://orcid.org/0000-0001-9539-4070
authors.initials: R
authors.lastname: Grytz
mesh_terms: D000818:Animals; D019149:Bioreactors; D002470:Cell Survival; D003094:Collagen / Q000378:metabolism*; D016895:Culture Media, Serum-Free / Q000737:chemistry*; D009216:Myopia / Q000378:metabolism; D009924:Organ Culture Techniques / Q000295:instrumentation*; D012029:Refraction, Ocular; D012590:Sclera / Q000166:cytology* / Q000378:metabolism; D014418:Tupaiidae
chemical_list: D016895:Culture Media, Serum-Free; D003094:Collagen