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...

Cultured on floating collagen membranes in the presence of lactogenic hormones, dissociated normal mammary epithelial cells from prelactating mice acquire the ultrastructural and biochemical characteristics of differentiated mammary secretory cells in vivo. The cells on floating collagen membranes in medium containing insulin alone have sparse secretory organelles, and a small amount of casein can be detected in these cells with a sensitive radioimmunoassay. These cells resemble counterpart cells in early-pregnant mice. When the cells are exposed to insulin, cortisol, and prolactin, the secretory apparatus is elaborated and significant increases in intracellular and extracellular casein are observed. In this environment, the intracellular casein content is generally four to eight times greater than in freshly dissociated cells or cells cultured in insulin alone. The amount of casein secreted into the medium by floating-collagen-membrane cultures in the three hormones is from 25 to 200 times greater than that secreted by cultures in insulin alone. Cells cultured on plastic substrates in either hormone combination fail to show any increase in intracellular or extracellular casein. On floating collagen membranes, the cells differentiate in response to hormones as they do in vivo and in organ culture. This cell-culture system provides an opportunity to study direct effects of environmental factors on mammary differentiation at the cellular level. Preparation of Floating Collagen Membranes. The collagen solution and collagen gels were prepared according to the method of Michalopoulos and Pitot (11) as modified by us (7).Eighteen-twenty hours after the cells were seeded onto collagen-gel-coated dishes, the gels were removed from the plastic substrate by rimming the gels with a scalpel blade and gently

cDNA clones for L-SF, the precursor of a low-molecular-weight subunit (ZI-3) of the inner layer of the Oryzias latipes egg envelope were isolated from Lambda ZAP cDNA libraries constructed from the poly(A)+ RNA of the liver of spawning female fish and estrogen-treated male fish. Among them, a clone, L-SF41, is 1473 bp long and contains an open reading frame encoding a signal peptide of 19 amino acids and L-SF protein of 420 amino acids. L-SF protein seems to be glycosylated, judging from the result of the glycanase digestion. L-SF protein contains a domain similar to ZP-domains in ZP3 of some mammalian species. Northern blot analysis employing XhoI-SmaI fragments of the cloned cDNA as probes revealed that expression of the L-SF gene occurred exclusively in the livers of spawning female fish and estrogen-treated male fish and that there was no mRNA encoding L-SF in the ovary of the spawning female fish.

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