Isolation and culture of microvascular endothelial cells from the primate corpus luteum

Lane K. Christenson, Richard Stouffer

    Research output: Contribution to journalArticle

    42 Citations (Scopus)

    Abstract

    Endothelial cells have common as well as specialized roles in different tissues and organs; as such, the abundant endothelial cells in the corpus luteum (> 50% of total cell population) could have unique activities necessary for luteal function. Our objective was to establish a method for isolating a pure population of endothelial cells from the primate corpus luteum and to determine the basic conditions for in vitro culture. Corpora lutea collected from rhesus monkeys throughout the luteal phase of the menstrual cycle were minced and enzymatically dispersed into single cell suspensions. Endothelial cells were isolated from the remaining cells (i.e., steroidogenic, fibroblastic, etc.) utilizing magnetic beads labeled with a lectin, Ulex europaeus agglutinin-1 (UEA-1), which binds a sugar found only on primate endothelial cells. After exposure to a magnetic field, UEA-1- negative (-) cells were decanted from the pelleted UEA-1-positive (+) cells; to remove beads from the UEA-1 (+) cells, excess sugar was applied. After optimization of the bead-to-cell ratio, the UEA-1 (+) group contained a population of cells 8-12 μm in diameter (typical endothelial cell size) and 93% of all cells in the UEA-1 (+) group stained positive for the specific endothelial cell marker, platelet/endothelial cell adhesion molecule-1. Cultured UEA-1 (+) cells produced low levels of progesterone and were unresponsive to hCG (100 ng/ml). In contrast, cultured UEA-1 (-) and mixed (unsorted) cells produced high basal levels of progesterone and exhibited a > 3-fold increase in response to hCG treatment. Preliminary experiments comparing different culture media and matrices demonstrated that 1) cell proliferation was unaffected by type of medium (i.e., Dulbecco's Modified Eagle Medium [DMEM]/F12 or McCoy's 5A); 2) the presence of serum was essential in the absence of added growth factors, and 3) extracellular matrix had a profound effect on cell proliferation. UEA-1 (+) cells exhibited a dose-dependent increase in cell proliferation in response to vascular endothelial growth factor (VEGF) in the presence and absence of fetal calf serum. In the absence of serum, VEGF stimulated proliferation of UEA-1 (+) cells plated on fibronectin but not collagen I, whereas in the presence of 10% fetal calf serum, both matrices supported VEGF-induced mitogenesis. These studies provide for the first time an efficient, reliable method for isolating primate luteal endothelial cells and describe in vitro culture conditions for subsequent studies examining luteal endothelial cell function and regulation.

    Original languageEnglish (US)
    Pages (from-to)1397-1404
    Number of pages8
    JournalBiology of Reproduction
    Volume55
    Issue number6
    StatePublished - Dec 1996

    Fingerprint

    Corpus Luteum
    Primates
    Endothelial Cells
    Vascular Endothelial Growth Factor A
    Luteal Cells
    Cell Proliferation
    Serum
    Progesterone
    Ulex europaeus lectins
    CD31 Antigens
    Population
    Eagles
    Luteal Phase
    Magnetic Fields
    Macaca mulatta
    Cell Size
    Fibronectins
    Lectins
    Extracellular Matrix
    Culture Media

    ASJC Scopus subject areas

    • Cell Biology
    • Developmental Biology
    • Embryology

    Cite this

    Isolation and culture of microvascular endothelial cells from the primate corpus luteum. / Christenson, Lane K.; Stouffer, Richard.

    In: Biology of Reproduction, Vol. 55, No. 6, 12.1996, p. 1397-1404.

    Research output: Contribution to journalArticle

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