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Comparison of the collagenous products synthesized in culture by pig aortic endothelial and smooth-muscle cells. Variability in endothelial-cell cultures.

ABSTRACT: In contrast with smooth-muscle cells from the same tissue, endothelial cells from pig aorta were found to exhibit in culture considerable variability in the pattern of collagen synthesis between one isolation of cells and the next. Synthesis varied from largely collagen type I to virtually all type III in the absence of type I but with small amounts still of collagens types IV and V, to, in one instance, synthesis basically of only type V. Synthesis usually by these cells of collagen predominantly of the interstitial type (I and III) rather than, as might be expected, that from basement membrane (type IV) was not attributable to the influence of subculture. All four collagen types were deposited in the cell layer to an increased extent in primary compared with secondary cultures of either smooth muscle or endothelial origin. Endothelial cells appeared sometimes to synthesize a large-Mr collagenous entity that might conceivably be related to 'short-chain' collagen. In addition, small-Mr hydroxyproline-containing peptides were detected that might reflect rapid collagen(s) turnover in endothelial cultures.

SUBMITTER: Sankey EA

PROVIDER: S-EPMC1153302 | biostudies-other | 1984 Feb

REPOSITORIES: biostudies-other

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Project description:BackgroundGlobal shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.ResultsEstablished primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.ConclusionsOur results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

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Comparison of the collagenous products synthesized in culture by pig aortic endothelial and smooth-muscle cells. Variability in endothelial-cell cultures.

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