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Medium Calcium Concentration Determines Keratin Intermediate Filament Density and Distribution in Immortalized Cultured Thymic Epithelial Cells (TECs)

Published online by Cambridge University Press:  07 July 2005

Sandra S. Sands ,
William D. Meek ,
Jun Hayashi  and
Robert J. Ketchum
Sandra S. Sands
Affiliation:
Oklahoma State University Center for Health Sciences (OSU-CHS), College of Osteopathic Medicine, Department of Anatomy and Cell Biology, 1111 W. 17th Street, Tulsa, OK 74107, USA
William D. Meek
Affiliation:
Oklahoma State University Center for Health Sciences (OSU-CHS), College of Osteopathic Medicine, Department of Anatomy and Cell Biology, 1111 W. 17th Street, Tulsa, OK 74107, USA
Jun Hayashi
Affiliation:
University of Maryland–Baltimore, School of Pharmacy, Baltimore, MD 21201, USA
Robert J. Ketchum
Affiliation:
Oklahoma State University Center for Health Sciences (OSU-CHS), College of Osteopathic Medicine, Department of Anatomy and Cell Biology, 1111 W. 17th Street, Tulsa, OK 74107, USA
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Abstract

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4–5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37°C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

Keywords

thymicepithelialcellsculturecalciumkeratinintermediate filament
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 11 , Issue 4 , August 2005 , pp. 283 - 292
Copyright
© 2005 Microscopy Society of America

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