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E. M. Bueno-Hoyos, N. Saeidi, J. W. Ruberti; In vitro Characterization of Corneal Stromal Fibroblast Cell and Matrix Morphology in Response to Bovine Serum and Insulin-Transferrin-Selenium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4041. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To identify culture medium formulations conducive to secretion of organized matrix by bovine corneal stromal fibroblasts (BCF).
4th passage BCF seeded on glass surfaces at 3×105 cells/cm2 were cultivated with DMEM+50 mg/L ascorbate+10 ng/ml b-FGF+1% antibiotic supplemented with 0-10% FBS (fetal bovine serum) and 0-1%ITS (insulin transferrin selenium), and monitored every 3-4 days via differential interference contrast microscopy (Nikon TE2000U) with a 60×1.4 NA oil-immersion objective (Nikon, Melville, NY). 28-day cultures were slam frozen onto a copper block at -194ºC (Delaware Diamond Knives, Dover, DE), etched in a customized CFE-40 freeze fracture/freeze etch apparatus (Cressington Scientific, Watford, UK), low-angle rotary-shadowed with Pt/C and backed with carbon at 90º angle. Replicas were cleaned in bleach and observed via electron microscopy (JEOL JEM-1000). Additionally, in situ immunofluorescence for α-smooth muscle actin (SMA) and collagen I was performed. At least 4 independent experiments were carried out, and Student’s T-test with equal variances was used to determine significance (p<0.05).
Cell survival, confluence, stratification and ‘clumping’ (contraction and pulling of the cell sheet into clumps) were observed only in FBS-supplemented groups. Cultures with 1%FBS+1%ITS exhibited stratification and aligned matrix fibers on day 3. These cultures were the thickest, reaching 36±1 µm on day 21. Clumping was observed on day 17 (n=2). Cells produced aligned intracellular α-SMA and extracellular collagen I. In contrast, 21-day cells on 1%FBS were 33±8% as thick as those on 1% FBS+1% ITS. Unaligned matrix fibers appeared on day 7, clumping occurred between days 10 and 14 (n=5), and there was disorganized intracellular α-SMA and little extracellular collagen I. Cultures on 10% FBS were 44±6% as thick as the 1%FBS+1%ITS group on day 21; showed disorganized matrix fibers on day 3, clumping on day 17 (n=1), splotches of extracellular or cell-process-localized α-SMA and a dense mesh of disorganized collagen I.
Factors present in FBS and ITS play an important role in BCF stratification, differentiation and organized matrix secretion. Myofibroblastic markers and behaviors occur more often at decreased FBS concentration, and can be countered with ITS. To the authors’ knowledge, this study is the first on high resolution microscopy of the in vitro evolution of stromal-like matrix secretion under defined medium formulations.
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