Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels.

Abstract

We have develo ed a method for embedding cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H] The limited in vitro growth capacity of mammary epithelial cells in conventional primary culture in tissue culture dishes is a well-known phenomenon. The cells generally undergo a few rounds of division, but proliferation cannot be sustained nor can these cells be passaged. Mouse mammary epithelial cells cultured at low density become flattened and multinucleated, and rarely do they attain confluence (1, 2). Cells plated at high density are maintained well but little growth is ever achieved.Most studies to date dealing with proliferation of mammary epithelial cells in vitro have used established cell lines adapted to grow in conventional monolayer culture (3-6). Considerable effort is being devoted in several laboratories to analysis of hormone and drug sensitivity in these selected cell lines. An inherent limitation of this approach is that such lines may not be representative of the original cell population. Therefore, it is of considerable importance to improve the conditions for culture of primary cells. Our demonstration of differentiated function in mouse mammary cells cultured on floating collagen gels, as indicated by levels of casein (7), mammary tumor virus (8), and prolactin receptor (9), has prompted us to examine collagen as an appropriate substrate for growth. The most encouraging results were obtained when mammary cells were embedded within the collagen gel. The present study describes (10), and normal mammary gland from C3H/Crgl and BALB/cNIV/Crgl mice were dissociated by a modification of a previously described method (11,12). Briefly, finely minced mammary tissues were placed in 125-or 250-ml erlenmeyer flasks containing 0.1% collagenase (CLS III; 120-150 units/mg; Worthington) in Hanks' balanced salt solution (10 ml/g of tissue) and swirled on a gyratory water bath shaker (model G76, New Brunswick) at 120-150 rpm at 370C for approximately 90 min or until the suspensions were uniform without macroscopic lumps. The suspension was passed through Nitex cloth (mesh size, 150 ,um); the cells were collected by centrifugation at 80 X g for 5 min, and washed twice with Hanks' solution. The resulting preparation consisted mainly of small clumps of cells. Cell number was estimated by mixing 1 vol of cell suspension with 9 vol of 0.02% crystal violet in 0.1 M citric acid and counting stained nuclei in a hemocytometer. Dissociated human mammary epithelial cells were obtained from excised tissue (reduction mammoplasties and mastectomies) by modification of the above procedure and subsequent gradient centrifugation (details will be reported elsewhere).Culture Procedure. Collagen solution and ge...

Cell proliferation and milk protein gene expression in rabbit mammary cell cultures

“…The use of primary mammary cell cultures for the analysis of cell growth has been limited to studies of [aH]thymidine incorporation in short-term cultures, because of the inability to maintain mammary cells in long-term cultures. Due to recent developments in the cultivation of mammary cells using stromal collagen matrices (45), it became possible to maintain sustained growth and thus to determine the effect of various hormones and growth promoting factors by estimating the increase in cell number (20,32,46).…”

Fine structural aspects of follicle-like cavity formation from dispersed porcine thyroid cells cultured in a collagen substrate.

“…For instance, cells isolated from cavitary organs such as mammary epithelial cells (YANG et al, 1979), submandibular epithelial cells (YANG et al, 1982), Madin-Darby canine kidney (MDCK) cells (HALL et al, 1982), and capillary endothelial cells (MONTESANO et al, 1983) reconstitute cavitary structures when they are cultured in a three-dimensional collagen gel. As for the thyroid gland, CHAMBARD and his co-workers (1981) reported that thyroid follicular epithelial cells as well reconstitute the follicular structure when they are cultured in a collagen gel.…”

A Simple Method for Classification of Cell Death by Use of Thin Layer Collagen Gel for the Detection of Apoptosis and/or Necrosis after Cancer Chemotherapy

“…4) Further staining is possible, including immunohistochemical staining and TUNEL. 5) The use of collagen gel as an extracellular matrix mimics the condition in vivo, [24][25][26] except in the case of small cell lung cancer cells that become resistant to chemotherapeutic agents when they adhere to an extracellular matrix. 27) In cancer chemotherapy, induction of apoptosis in cancer tissue without affecting normal cells is a major objective, so a simple and reproducible method is required to identify apoptosis in experimental studies of cancer chemotherapy.…”