title: Two Bioactive Molecular Weight Fractions of a Conditioned Medium Enhance RPE Cell Survival on Age-Related Macular Degeneration and Aged Bruch's Membrane.
abstract: OBJECTIVE
  To characterize molecular weight fractions of bovine corneal endothelial cell conditioned medium (CM) supporting retinal pigment epithelium (RPE) cell survival on aged and age-related macular degeneration (AMD) Bruch's membrane.
  
  
  METHODS
  CM was subject to size separation using centrifugal filters. Retentate and filtrate fractions were tested for bioactivity by analyzing RPE survival on submacular Bruch's membrane of aged and AMD donor eyes and behavior on collagen I-coated tissue culture wells. Protein and peptide composition of active fractions was determined by mass spectrometry.
  
  
  RESULTS
  Two bioactive fractions, 3-kDa filtrate and a 10-50-kDa fraction, were necessary for RPE survival on aged and AMD Bruch's membrane. The 3-kDa filtrate, but not the 10-50-kDa fraction, supported RPE growth on collagen 1-coated tissue culture plates. Mass spectrometry of the 10-50-kDa fraction identified 175 extracellular proteins, including growth factors and extracellular matrix molecules. Transforming growth factor (TGF)β-2 was identified as unique to active CM. Peptides representing 29 unique proteins were identified in the 3-KDa filtrate.
  
  
  CONCLUSIONS
  These results indicate there is a minimum of two bioactive molecules in CM, one found in the 3-kDa filtrate and one in the 10-50-kDa fraction, and that bioactive molecules in both fractions must be present to ensure RPE survival on Bruch's membrane. Mass spectrometry analysis suggested proteins to test in future studies to identify proteins that may contribute to CM bioactivity.
  
  
  CONCLUSIONS
  Results of this study are the first steps in development of an adjunct to cell-based therapy to ensure cell transplant survival and functionality in AMD patients.
authors.affiliation: Institute of Ophthalmology and Visual Science Rutgers, New Jersey Medical School, Newark, NJ, USA.
authors.forename: Ilene K
authors.initials: IK
authors.lastname: Sugino
authors.affiliation: Institute of Ophthalmology and Visual Science Rutgers, New Jersey Medical School, Newark, NJ, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Sun
authors.affiliation: Institute of Ophthalmology and Visual Science Rutgers, New Jersey Medical School, Newark, NJ, USA.
authors.forename: Carola
authors.initials: C
authors.lastname: Springer
authors.affiliation: Institute of Ophthalmology and Visual Science Rutgers, New Jersey Medical School, Newark, NJ, USA.
authors.forename: Noounanong
authors.initials: N
authors.lastname: Cheewatrakoolpong
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Advanced Proteomics Research, Neuroproteomics Core Facility, Rutgers, New Jersey Medical School, Newark, NJ, USA.
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Advanced Proteomics Research, Neuroproteomics Core Facility, Rutgers, New Jersey Medical School, Newark, NJ, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.affiliation: Institute of Ophthalmology and Visual Science Rutgers, New Jersey Medical School, Newark, NJ, USA.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Zarbin
keywords: Bruch's membrane; age-related macular degeneration; cell survival; cell transplantation; retinal pigment epithelium