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E. M. Bueno-Hoyos, N. Saeidi, J. W. Ruberti; Medium Formulation More Effective Than Initial Cell Density at Increasing Thickness and Organized Matrix Secretion in Corneal Stromal Constructs. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6225.
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© ARVO (1962-2015); The Authors (2016-present)
To optimize in vitro stratification of, and organized extracellular matrix secretion by bovine corneal stromal fibroblasts (BCF).
4-5th passage BCF seeded on tissue culture inserts at densities of 2.2×105 (D1) or 1.1×106 (D2) cells/cm2 were cultivated with DMEM+50 mg/L Vitamin C+10 ng/ml basic fibroblastic growth factor+1% antibiotic plus either 10% fetal bovine serum (FBS) or 1% FBS+1% insulin-transferrin selenium (ITS). 4 week cell-matrix constructs were scrutinized with differential interference contrast (DIC) or transmission electron (TEM) microscopy. DIC was performed in a Nikon TE2000U with a 60×1.4 NA oil-immersion objective (Nikon, Melville, NY). TEM samples went through immersion in 50% Karnovsky’s for 1 hour, 2% osmium tetroxide, serial ethanol dehydration, epon-araldite embedding, thin-sectioning, staining with uranyl acetate and lead citrate in methanol, and viewing on a JEOL JEM-1000. Results are average ± standard error of at least 4 replicates analyzed via Student’s t-test for significant differences (p<0.05).
D1 and D2 constructs cultivated with FBS were 7.2±0.5 and 12.8±2.2 µm thick (p=0.02), and contained 1.7±0.2 and 2.6±0.3 layers of cells, respectively (p=0.03). D1 constructs were 5.6-fold thicker (p<0.0001) and contained 3-fold more cell layers (p<0.0001) in ITS vs. FBS media. Similarly, D2 constructs were 3.5-fold thicker (p<0.0001) and contained 2.4-fold more cell layers (p<0.0001) in ITS vs. FBS media. Dense, aligned matrix fibers were observed via DIC and TEM of constructs cultivated with ITS. Cultivation in ITS strikingly increased matrix density in D1 (and to a lesser degree in D2) constructs compared with FBS. Seeding cell density did not appreciably affect matrix density or alignment in ITS medium.
Factors present in FBS and ITS have contrasting effects on the stratification, differentiation and secretion of organized matrix of BCF, with ITS yielding better results. The effects of these factors were greater than those of the seeding cell density in the present study.
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