James Richards

Method for culturing mammary epithelial cells in a rat tail collagen gel matrix

Mammary glands are enzymatically dissociated and the resulting tissue digest enriched for epithelial cells by isopycnic banding on a density gradient of Percoll. The cells are embedded within a rat tail collagen gel matrix and fed with the appropriate medium. Growth and differentiation are superior in such a system when compared to culture on plastic, using identical media.

Maintenance of normal rat mammary epithelial cells by insulin and insulin-like growth factor 1☆

Normal rat mammary epithelial cells were cultured within a rat tail collagen gel matrix formed under improved conditions for controlling pH and osmolarity. Under these conditions, growth can be maintained for up to 3 weeks with a 10- to 15-fold increase in cell number. The cells grow in response to prolactin, progesterone, epidermal growth factor, and cholera toxin, in a medium of DME: Hams F12 supplemented with BSA and insulin at 10 micrograms/ml. When the insulin concentration was reduced to more physiological levels (10 ng/ml) the cells did not grow. However, at these more physiological concentrations it could be shown that insulin had a concentration-dependent effect on the maintenance of the cells with an optimum concentration around 25 ng/ml. The cells could be maintained in hormone-supplemented medium with low levels of insulin in a quiescent state for up to 14 days. The high levels of insulin needed for optimal growth could be replaced by insulin-like growth factor 1 (IGF-1) at much lower concentrations (25-50 ng/ml). The superphysiological level of insulin required for optimum growth is probably due to its acting weakly through an IGF-1-mediated growth-promoting mechanism. Insulins effect on cell maintenance occurs at physiological levels and may better reflect its role in mammary cell growth.

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

SummaryMammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Hams F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture.

Response of end bud cells from immature rat mammary gland to hormones when cultured in collagen gel

End buds from 4- to 5-week-old rat mammary glands were isolated and cultured within a rat tail tendon collagen gel matrix. Media containing equine serum or porcine serum and cholera toxin promoted growth, but not the production of casein or thioesterase II, nor did they induce a state of differentiation as assessed by cell ultrastructure. Medium supplemented with only 5% porcine serum, insulin and cholera toxin did not support growth or differentiation. However, when prolactin, estradiol, progesterone and hydrocortisone were added to this medium, growth was stimulated greatly and a differentiated state was induced as assessed by the production of casein and thioesterase and by the appearance of a highly secretory ultrastructure.

Effects of antioxidants and reduced oxygen tension on rat mammary epithelial cells in culture

SummaryFree radical damage has the potential to significantly affect the behavior of cells in culture. In this study the effects of antioxidants (superoxide dismutase, catalase, and vitamin E) and lowered oxygen tension (1% oxygen) on primary culture of rat mammary epithelial cells were examined. Rat mammary epithelial cells were dissociated in collagenase with or without the addition of antioxidants and low oxygen tension, then cultured for 10 d in rat-tail collagen gel matrix and fed with Dulbecco’s modified Eagle’sF12 medium supplemented with various hormones and growth factors. Growth potential of the mammary cells was enhanced when antioxidants and low oxygen tension were used, alone or in combination, during the cell dissociation period. Using antioxidants and low oxygen tension during the culture period failed to improve growth potential regardless whether cells were dissociated in standard conditions or with antioxidants and low oxygen tension. The use of antioxidants and low oxygen tension during the cell dissociation period also reduced the degree of keratinization of the cells after 10 d of culture. Using antioxidants and low oxygen tension during the cell culture period did not further reduce keratinization if antioxidants and low oxygen tension were used during the dissociation period, but were effective in reducing keratinization if cells were dissociated in standard condition. In this system, antioxidants and low oxygen tension reduced lipid peroxidation during the cell dissociation period. An iron chelator, desferal, can also reduce lipid peroxidation and enhance growth when used during cell dissociation, suggesting the enhanced growth potential by the addition of antioxidants and low oxygen to be due to the reduction of lipid peroxidation.

The use of thioesterase II as a rat mammary epithelial cell-specific marker.

SummaryThe avidin-biotin-peroxidase complex immunoperoxidase technique was employed to determine the intercellular distribution of thioesterase II in rat mammary glands. This enzyme is responsible for shifting the product specificity of the fatty-acid synthetase enzyme complex from long to medium chain fatty acids. Thioesterase II was found exclusively in the cells lining the lumen of the ductal and alveolar structures in glands from mature virgin (150 days old) and pregnant rats. The ductal cell staining intensity was considerably less than that of the alveolar cells in the mature virgin rat glands. No immunoreactive thioesterase II was found in the stromal, adipose, vascular, or myoepithelial components of the gland in the developmental stages examined. In the glands from immature virgin rats (40–45 days old) thioesterase II was again found only in the epithelial cells lining the lumen of the ductal and end-bud structures although this layer was usually more than one cell thick.Quantitative determination of thioesterase II activity in cytosol preparations revealed similar levels in mammary fragments from enzymatically-dissociated glands obtained from mature virgins and in end buds derived from immature virgins, but somewhat higher levels in mammary structures derived from late-pregnant animals. These immunohistological and biochemical results demonstrate thioesterase IIs usefulness as a mammary epithelial cell-specific marker.