Author:
Sands Sandra S.,Meek William D.,Hayashi Jun,Ketchum Robert J.
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.
Publisher
Cambridge University Press (CUP)
Reference69 articles.
1. Turksen, K. , Opas, M. , Aubin, J.E. , & Kalnins, V.I. (1983).Microtubules, microfilaments and adhesion patterns indifferentiating chick retinal pigment epithelial (RPE) cells invitro.Exp Cell Res 147,379–391.
2. Watt, F.M. , Mattey, D.L. , & Garrod, D.R. (1984).Calcium-induced reorganization of desmosomal components in culturedhuman keratinocytes.J Cell Biol 99,2211–2215.
3. Zamansky, G.B. , Nguyen, U. , & Chou, I.N. (1991).An immunofluorescence study of the calcium-induced coordinatedreorganization of microfilaments, keratin intermediate filaments, andmicrotubules in cultured human epidermal keratinocytes.J Invest Dermatol 97,985–994.
4. Mattey, D.L. & Garrod, D.R. (1986).Calcium-induced desmosome formation in cultured kidney epithelialcells.J Cell Sci 85,95–111.
5. Odorico, J.S. , Barker, C.F. , Posselt, A.M. , & Naji, A. (1992).Induction of donor-specific tolerance to rat cardiac allografts byintrathymic inoculation of bone marrow.Surgery 112,370–376;discussion 376–377.
Cited by
2 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献