Serum-free primary culture of normal mouse mammary epithelial and stromal cells

Summary An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells, fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In contrast, fibroblast-enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone. In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells. These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard to hormone and growth factor regulation of their growth and differentiation.     

Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Serum-free growth of adult human prostatic epithelial cells

Summary Proliferation of adult human prostatic epithelial cells in serum-free medium occurs upon the addition of cholera toxin, epidermal growth factor, pituitary extract, and hydrocortisone to basal medium PFMR-4A. Insulin and selenium enhance proliferation and permit growth at lower cell densities. Reducing the level of calcium in the medium dramatically alters morphology and also seems to increase proliferation. Mortal strains of cells derived from normal central or peripheral zone, benign hyperplasia, or cancer respond similarly to growth factors and calcium, but two populations of cancer cells which have been long-lived and may be immortal lines behave differently. GKC-CA cells require serum proteins or high levels of pituitary extract for optimal growth, and neither GKC-CA cells or cells of another cancer line, WB-CA, proliferate well in medium containing reduced levels of calcium. These observations may, however, be a reflection of attachment phenomena rather than of growth responses per se. Growth of cells in serum-free medium has allowed definitive studies of the effects of androgens, and regardless of cell type no response to androgens of prostate epithelial cells under any experimental conditions has been seen.     

Growth of normal human mammary cells in culture

Summary Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.     

奶山羊乳腺上皮细胞的分离、培养及鉴定

应用组织块培养法高密度培养、连续传代法建立西农萨能奶山羊乳腺上皮细胞体外培养体系,通过生长曲线绘制、核型分析、免疫荧光染色 (角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白)、油红染色及β-酪蛋白基因的RT-PCR分析进行培养细胞鉴定。实验结果表明细胞生长曲线为典型的S型,染色体数目众数为60,细胞角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白表达均呈阳性,油红染色后可见细胞质内的脂滴,且细胞表达酪蛋白mRNA。说明运用本方法培养的细胞为正常的乳腺上皮细胞,并具有一定的泌乳功能。     

Serum-free primary culture of human normal mammary epithelial cells in collagen gel matrix

Collagen gel matrix has been used successfully to promote sustained growth of human normal mammary epithelial cells in primary culture using serum-containing medium supplemented with hormones and growth factors (Nandi et al., 1932). Sustained growth can now be accomplished in a serum-free medium consisting of a 1:1 mixture of Ham's F12 and DMEM supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, cortisol, and BSA. Human normal mammary epithelial cells derived from reduction mammoplasties can be routinely propagated in this serum-free medium. The extent of growth and the resulting three-dimensional outgrowths in this serum-free medium, using the collagen gel matrix system, are comparable to those seen in serum-containing medium. This is the first demonstration of sustained growth of human normal mammary epithelial cells in serum-free primary culture.     

A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells

Summary Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free “attachment medium” was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation. This research was sponsored by the National Cancer Institute, DHHS, under Contract NO1-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.     

Cholera toxin stimulation of human mammary epithelial cells in culture

Summary Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system. This work was supported by USPHS Grant CA-24844 from the National Cancer Institute and Grant CD-61B from the American Cancer Society.     

Serum-free culture of stromal and functionally polarized epithelial cells of guinea-pig endometrium: a potential model for the study of epithelial-stromal paracrine interactions.

Stromal and glandular epithelial (GE) cells were isolated from guinea-pig endometrium and growth to near confluency (6-8 days) in primary culture on plastic surfaces in a serum-supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5-7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell-PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter-cultured GE monolayers were polarized morphologically, and displayed epithelial-specific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory proteins after labelling with [35S]-methionine and analysis by polyacrylamide gel electrophoresis. The filter-cultured GE monolayers allowed identification of the proteins released vectorially in the apical or the basal secretory compartment, thus demonstrating the functional polarization of GE cells in this bicameral culture system. Within the defined conditions of this culture system, the paracrine factors released by the two endometrial cell populations as well as the interplay of stromal-epithelial interactions and ovarian hormones could be investigated.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Defined medium for primary culture de novo of adult rat alveolar epithelial cells

Summary Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions. In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high levels, peaking at 1.76±0.14 KΩ-cm² by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71±0.35 μA/cm². With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope. These observations indicate that it is feasible to culture alveolar epithelial cell monolayers under completely defined serum-free conditions de novo. This culture system should prove useful for identifying soluble growth factors and matrix substances that modulate alveolar epithelial cell biological properties.     

Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors

Summary A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mamary glands of immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's medium-F12 culture medium containing 10 μg/ml insulin, 1 μg/ml prolactin, 1 μg/ml progesterone, 1 μg/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 μg/ml transferrin, and 5 μM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like projections, stellate colonies, and two-and three-dimensional web units. Optimal proliferation required insulin, prolactin, progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane. In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this model should prove to be an improtant tool to study the hormonal regulation of the growth and development of rat mammary cells. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Functional differentiation and primary metabolism of mouse mammary epithelial cells in extended-batch and hollow-fiber culture

Growth, expression of functional differentiation (as characterized by synthesis and secretion of milk proteins), and primary metabolism were studied for a mouse mammary epithelial cell line, COMMA-1D, in extended-batch and hollow-fiber reactor cultures. Batch cultures were performed on Costar polycarbonate membrane inserts, allowing basal and apical exposure to medium. Protein production was induced in both batch and hollow-fiber cultures in hormonesupplemented medium. In batch cultures, high levels of protein production and secretion were maintained for 18 days. Once differentiation was induced, the rate of deinduction was low, even in medium containing epidermal growth factor (EGF) and serum; cells continued to express and secrete proteins for at least 12 days after prolactin and hydrocortisone were removed. Cells in both batch and hollow-fiber cultures were highly glycolytic and exhibited low rates of glutaminolysis. In batch culture on membrane inserts, cells showed polarized metabolism between the apical and basal side, maintaining significant gradients of glucose and lactate. Medium hormonal composition and subsequent differentiation affected both glucose uptake and lactate yield for COMMA-1D in batch culture. (c) 1992 John Wiley & Sons, Inc.     

Cultured proliferating rat mammary epithelial cells

Summary Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with³H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. This work was supported by the Veterans Administration, Washington, DC, and by CA grant 05388 from the U.S. Public Health Service, Washington, DC.     

Comparative growth of normal and malignant mouse mammary epithelium cultured serum-free on a biomatrix from preadipocytes

Summary Growth of normal and malignant mouse mammary epithelial cells (MMEC) on a biomatrix of substrate-attached material from 3T3-L1 preadipocytes was evaluated to devise culture conditions that are suitable for transformation studies but do not involve embedding cells in a gel. The biomatrix was prepared as described by Levine and Stockdale (18), and serum-free medium contained bovine serum albumin, insulin, progesterone, prolactin, and linoleic acid. Each cell type produced a distinctive pattern of colony architecture in this culture system. Cells from virgin mice (vMMEC) usually formed elaborate, three-dimensional structures resembling ducts and alveoli; cells from pregnant mice (pMMEC) grew as flat monolayers; and tumor cells grew in multilayered clusters. Cell growth was monitored by an assay for succinate dehydrogenase. Similar growth rates were found through Day 8 in cultures of vMMEC and D2 carcinoma cells. Growth of vMMEC slowed thereafter, whereas tumor cells typically continued growing through Day 14 to 18. Increase in cell number during 18 days in culture was 3-, 7-, 9-, and 11-fold for cells from pregnant and virgin mice, BALB/cfC3H and D2 carcinomas, respectively. The percent cells in S phase on Day 2 of culture was 9% for pMMEC, 4 to 11% for BALB/cfC3H tumor cells, 20% for vMMEC, and 24% for D2 tumor cells. Thus, this culture system promotes extended growth of MMEC and offers several advantages over embedding cells in a collagen gel. It may therefore be applicable to in vitro transformation studies with MMEC. Supported by research grant CA-32937 from the National Institutes of Health, Bethesda, MD, to BBA.     

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors

A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8 Mouse myeloma cell line P3-X63-Ag8.653 - BME Basal Medium Eagle - BSA Bovine Serum Albumin - DMEM Dulbecco's Modified Eagle's Medium - EDTA Ethylenediaminete-traacetic Acid - e-PC Phosphatidyl choline from egg yolk - FCS Fetal Calf Serum - FGF Fibroblast Growth Factor - GHL Glycyl-histidyl-lysine - HDL High Density Lipoprotein - HPL Human Plasma Lipid - IF 1:1 mixture of IMDM and Ham's F12 - IMDM Iscove's Modified Dulbecco's medium - LDL Low Density Lipoprotein - NS1 Mouse myeloma cell line NSI-1-Ag4-1 - PBS Phosphate Buffered Saline - s-PC Phosphatidylcholine from soy beans - s-PE Phosphatidylethanolamine from soy beans - s-lecithin lecithin from soy beans     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.