Medium calcium concentration determines keratin intermediate filament density and distribution in immortalized cultured thymic epithelial cells (TECs)

Sandra S. Sands, William D. Meek, Jun Hayashi, Robert J. Ketchum

    Research output: Contribution to journalArticle

    1 Citation (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)283-292
    Number of pages10
    JournalMicroscopy and Microanalysis
    Volume11
    Issue number4
    DOIs
    StatePublished - 1 Aug 2005

    Fingerprint

    keratins
    Keratin
    calcium
    Calcium
    filaments
    cultured cells
    insulin
    Insulin
    culture techniques
    cholera
    Cells
    Thymus
    Tissue culture
    calves
    spindles
    antibiotics
    Antibiotics
    serums
    rats
    Epithelial Cells

    Keywords

    • Calcium
    • Cells
    • Culture
    • Epithelial
    • Intermediate filament
    • Keratin
    • Thymic

    Cite this

    @article{b5f90863d5504910b744d70be769f169,
    title = "Medium calcium concentration determines keratin intermediate filament density and distribution in immortalized cultured thymic epithelial cells (TECs)",
    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 = "Calcium, Cells, Culture, Epithelial, Intermediate filament, Keratin, Thymic",
    author = "Sands, {Sandra S.} and Meek, {William D.} and Jun Hayashi and Ketchum, {Robert J.}",
    year = "2005",
    month = "8",
    day = "1",
    doi = "10.1017/S1431927605050282",
    language = "English",
    volume = "11",
    pages = "283--292",
    journal = "Microscopy and Microanalysis",
    issn = "1431-9276",
    publisher = "Cambridge University Press",
    number = "4",

    }

    Medium calcium concentration determines keratin intermediate filament density and distribution in immortalized cultured thymic epithelial cells (TECs). / Sands, Sandra S.; Meek, William D.; Hayashi, Jun; Ketchum, Robert J.

    In: Microscopy and Microanalysis, Vol. 11, No. 4, 01.08.2005, p. 283-292.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Medium calcium concentration determines keratin intermediate filament density and distribution in immortalized cultured thymic epithelial cells (TECs)

    AU - Sands, Sandra S.

    AU - Meek, William D.

    AU - Hayashi, Jun

    AU - Ketchum, Robert J.

    PY - 2005/8/1

    Y1 - 2005/8/1

    N2 - 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.

    AB - 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.

    KW - Calcium

    KW - Cells

    KW - Culture

    KW - Epithelial

    KW - Intermediate filament

    KW - Keratin

    KW - Thymic

    UR - http://www.scopus.com/inward/record.url?scp=23844458421&partnerID=8YFLogxK

    U2 - 10.1017/S1431927605050282

    DO - 10.1017/S1431927605050282

    M3 - Article

    C2 - 16079012

    AN - SCOPUS:23844458421

    VL - 11

    SP - 283

    EP - 292

    JO - Microscopy and Microanalysis

    JF - Microscopy and Microanalysis

    SN - 1431-9276

    IS - 4

    ER -