Background: Three dimensional (3D) acinar cultures from primary mammary epithelial cells have been used as in vitro strategies to study mammary growth and differentiation. Primary epithelial cells cultured on a reconstituted basement membrane matrix are able to form functional alveolar-like multicellular structures with a luminal compartment and to secrete milk proteins in a vectorial manner. However, the validation of 3D acinar cultures for elucidating functional mammary development requires the study of the hormone dependence of growth and differentiation of acini in vitro. Aim: The aim of this study was to characterize the hormone dependence of growth and functional differentiation of acinar cultures derived from the pregnant gland, with emphasis on insulin. Methods: Acini are dissociated by collagenase digestion of #4 and 5 mammary glands from 14 day pregnant CD1 mice. In some experiments organoids were treated with trypsin and filtered through a 70 um cell strainer to obtain single cells. Organoids or single cells are grown embedded in 100% Matrigel in the following growth medium: DMEM-F12 with insulin (concentrations tested: 870 nM, 100 nM, 50 nM), EGF (5 ng/ml), Prolactin (PRL; 3 ug/ml), hydrocortisone (HC; 1 ug/ml) and fetal bovine serum (FBS; 5%). After one week in culture, the EGF and serum are removed to obtain differentiation medium. After another week, acini are fixed, embedded in paraffin and sectioned. Immunohistochemistry is carried out using primary antibodies for casein and ADPH. Results: In standard culture in differentiation medium, staining for ADPH and casein revealed CLD formation and movement of casein from the cytoplasm to the lumen. Removal of PRL from the differentiation medium resulted in smaller CLD and reduced casein, while removal of HC resulted in casein remaining in the cytoplasm. Insulin was required to support the organization of functional acini from mammary organoids so the omission of insulin resulted in more disordered assemblies showing incomplete lumen formation. Also, the multicellular structures formed from organoids were smaller and fewer in number. Moreover, insulin was required to promote acinar formation starting with single cells. Many acini in good shape were detected with insulin at 870 nM (supraphysiological concentration used in in vitro mammary cultures). A good response was obtained with insulin at 100 and 50 nM as well. Conclusions: Taken together these data demonstrate that 3D acinar cultures respond to hormones as observed in other mammary preparations. They also suggest that this in vitro model will be useful to elucidate signaling pathways utilized by hormones to regulate mammary growth and differentiation in pregnancy. In addition, these results provide evidence that insulin is a critical hormone that supports the organization of mammary epithelial cells in alveolar-like structures, capable of functional differentiation.
Three dimensional acinar cultures of mammary epithelium as an in vitro model for studying hormonal mechanisms in the mammary gland / C. Pecorini, P. Webb, M.C. Neville. ((Intervento presentato al convegno Mammary Gland Biology : GRC Gordon Research Conferences tenutosi a Lucca nel 2010.
Three dimensional acinar cultures of mammary epithelium as an in vitro model for studying hormonal mechanisms in the mammary gland
C. PecoriniPrimo
;
2010
Abstract
Background: Three dimensional (3D) acinar cultures from primary mammary epithelial cells have been used as in vitro strategies to study mammary growth and differentiation. Primary epithelial cells cultured on a reconstituted basement membrane matrix are able to form functional alveolar-like multicellular structures with a luminal compartment and to secrete milk proteins in a vectorial manner. However, the validation of 3D acinar cultures for elucidating functional mammary development requires the study of the hormone dependence of growth and differentiation of acini in vitro. Aim: The aim of this study was to characterize the hormone dependence of growth and functional differentiation of acinar cultures derived from the pregnant gland, with emphasis on insulin. Methods: Acini are dissociated by collagenase digestion of #4 and 5 mammary glands from 14 day pregnant CD1 mice. In some experiments organoids were treated with trypsin and filtered through a 70 um cell strainer to obtain single cells. Organoids or single cells are grown embedded in 100% Matrigel in the following growth medium: DMEM-F12 with insulin (concentrations tested: 870 nM, 100 nM, 50 nM), EGF (5 ng/ml), Prolactin (PRL; 3 ug/ml), hydrocortisone (HC; 1 ug/ml) and fetal bovine serum (FBS; 5%). After one week in culture, the EGF and serum are removed to obtain differentiation medium. After another week, acini are fixed, embedded in paraffin and sectioned. Immunohistochemistry is carried out using primary antibodies for casein and ADPH. Results: In standard culture in differentiation medium, staining for ADPH and casein revealed CLD formation and movement of casein from the cytoplasm to the lumen. Removal of PRL from the differentiation medium resulted in smaller CLD and reduced casein, while removal of HC resulted in casein remaining in the cytoplasm. Insulin was required to support the organization of functional acini from mammary organoids so the omission of insulin resulted in more disordered assemblies showing incomplete lumen formation. Also, the multicellular structures formed from organoids were smaller and fewer in number. Moreover, insulin was required to promote acinar formation starting with single cells. Many acini in good shape were detected with insulin at 870 nM (supraphysiological concentration used in in vitro mammary cultures). A good response was obtained with insulin at 100 and 50 nM as well. Conclusions: Taken together these data demonstrate that 3D acinar cultures respond to hormones as observed in other mammary preparations. They also suggest that this in vitro model will be useful to elucidate signaling pathways utilized by hormones to regulate mammary growth and differentiation in pregnancy. In addition, these results provide evidence that insulin is a critical hormone that supports the organization of mammary epithelial cells in alveolar-like structures, capable of functional differentiation.Pubblicazioni consigliate
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