Isolation and Culture of Normal Rat Mammary Epithelial Cells

Darcy, Kathleen M.; Zangani, Danilo; Lee, Ping-Ping H.; Ip, Margot M.

doi:10.1007/978-1-4615-4295-7_16

Cited by 13 publications

(18 citation statements)

References 13 publications

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“…Incubation times of approximately 14 h in a humidified CO 2 incubator at 378C were utilized. After digestion, cells were passed through stainless mesh (pore size: 77 lm) to remove undigested tissues or clumps, and were suspended in a culture medium, which consisted of DMEM/F-12 supplemented with 10% FBS and other factors (insulin, apotransferrin, prolactin, mouse EGF, progesterone, hydrocortisone and ascorbic acid) as specified by Darcy et al (8). Cells were inoculated into 10 cm culture dishes at a density of 1 to 4 3 10 6 cells with 10 ml of medium.…”

Section: Methodsmentioning

confidence: 99%

“…Because mammary tumorigenesis in mice has been indicated to involve mammary tumor virus (7), which is not relevant in humans, rats are frequently utilized for such investigations. In addition, culture methods of rat mammary epithelial cells are well established (8), and rats would provide data as the third species after humans and mice in regard to investigation of the refractory nature of hematolymphoid cells in recording radiation damage when irradiated as a fetus.…”

Section: Introductionmentioning

confidence: 99%

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Prediction of Clonal Chromosome Aberration Frequency in Human Blood Lymphocytes

et al. 2004

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Prediction of Clonal Chromosome Aberration Frequency in Human Blood Lymphocytes

et al. 2004

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“…Tumor epithelial organoids were isolated from mammary tumors generated in MMTV/neu (NDL2-5) FVB transgenic mice according to the protocol developed by our laboratory (21), with the following modifications: digestion time was reduced to 2.75 h by placing the digestion flasks in a 37°C shaking water bath; the collagenase and dispase solutions were each 1% (w/v). The organoids were embedded within an Engelbreth Holmes Swarm sarcoma-derived reconstituted basement membrane (as described previously; ref.…”

Section: Isolation and Primary Culture Of Mammary Tumor Organoidsmentioning

confidence: 99%

“…The organoids were embedded within an Engelbreth Holmes Swarm sarcoma-derived reconstituted basement membrane (as described previously; ref. 21) and cultured in a serum-free medium consisting of phenol red-free DMEM-F12 (50:50, v/v) with 10 ng/mL of epidermal growth factor, 10 μg/mL of insulin, 1 μg/mL of progesterone, 1 μg/mL of hydrocortisone, 1 μg/mL of prolactin, 5 μg/mL of transferrin, 5 μM of ascorbic acid, 1 mg/mL of fatty acid-free bovine serum albumin, and 50 μg/mL of gentamicin, with or without recombinant mouse TNF (100 ng/mL; Biosource International). Ovine prolactin (NIDDK-oPRL-21) was obtained from Dr. A.F.…”

Section: Isolation and Primary Culture Of Mammary Tumor Organoidsmentioning

confidence: 99%

Tumor necrosis factor deficiency inhibits mammary tumorigenesis and a tumor necrosis factor neutralizing antibody decreases mammary tumor growth in neu/erbB2 transgenic mice

Warren

Shoemaker

Shealy

et al. 2009

Molecular Cancer Therapeutics

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Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine that is synthesized and secreted by cells of the immune system, as well as by certain epithelia and stroma. Based on our previous studies demonstrating TNF-stimulated proliferation of normal and malignant mammary epithelial cells, we hypothesized that TNF might promote the growth of breast cancer in vivo. To test this, we generated bigenic mice that overexpressed activated neu/erbB2 in the mammary epithelium and whose TNF status was wild-type, heterozygous, or null. Mammary tumorigenesis was significantly decreased in TNF−/− mice (n = 30) compared with that in TNF+/+ mice (n = 27), with a palpable tumor incidence of 10.0% and 44.4%, and palpable tumors/mouse of 0.10 ± 0.06 and 0.67 ± 0.17, respectively. Tumorigenesis in the heterozygous group fell between that in the TNF +/+ and TNF−/− groups, but was not significantly different from either of the homozygous groups. The decreased tumor development in the TNF−/− mice was associated with a decreased proliferative index in the lobular and ductal mammary epithelium. To further investigate the role of TNF in breast cancer, mammary tumor-bearing mice whose tumors overexpressed wild-type neu/ erbB2 were treated with a TNF-neutralizing antibody or a control antibody for 4 weeks (n = 20/group). Mammary tumor growth was significantly inhibited in mice treated with the anti-TNF antibody compared with the control antibody. Together, these data show a stimulatory role for TNF in the growth of breast tumors and suggest that TNF antagonists may be effective in a subset of patients with breast cancer.

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“…Primary mammary epithelial cells were prepared from mild collagenase and dispase digestion, as described (Darcy et al, 2000). Differential trypsinization was performed to separate MECs from LECs.…”

Section: Rotary Culturesmentioning

confidence: 99%

Slit2 and netrin 1 act synergistically as adhesive cues to generate tubular bi-layers during ductal morphogenesis

et al. 2006

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Development of many organs, including the mammary gland, involves ductal morphogenesis. Mammary ducts are bi-layered tubular structures comprising an outer layer of cap/myoepithelial cells (MECs) and an inner layer of luminal epithelial cells (LECs). Slit2 is expressed by cells in both layers, with secreted SLIT2 broadly distributed throughout the epithelial compartment. By contrast, Robo1 is expressed specifically by cap/MECs. Loss-of-function mutations in Slit2 and Robo1 yield similar phenotypes, characterized by disorganized end buds (EBs) reminiscent of those present in Ntn1 -/-glands, suggesting that SLIT2 and NTN1 function in concert during mammary development. Analysis of Slit2 -/-;Ntn1 -/-glands demonstrates an enhanced phenotype that extends through the ducts and is characterized by separated cell layers and occluded lumens. Aggregation assays show that Slit2-/-cells, in contrast to wild-type cells, do not form bi-layered organoids, a defect rescued by addition of SLIT2. NTN1 has no effect alone, but synergistically enhances this rescue. Thus, our data establish a novel role for SLIT2 as an adhesive cue, acting in parallel with NTN1 to generate cell boundaries along ducts during bi-layered tube formation.

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Isolation and Culture of Normal Rat Mammary Epithelial Cells

Cited by 13 publications

References 13 publications

Prediction of Clonal Chromosome Aberration Frequency in Human Blood Lymphocytes

Prediction of Clonal Chromosome Aberration Frequency in Human Blood Lymphocytes

Tumor necrosis factor deficiency inhibits mammary tumorigenesis and a tumor necrosis factor neutralizing antibody decreases mammary tumor growth in neu/erbB2 transgenic mice

Slit2 and netrin 1 act synergistically as adhesive cues to generate tubular bi-layers during ductal morphogenesis

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