Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

1993 ◽  
Vol 25 (6) ◽  
pp. 799-816 ◽  
Author(s):  
Rabih S. Talhouk ◽  
Ronald L. Neiswander ◽  
Floyd L. Schanbacher
Keyword(s):  
Functional Differentiation ◽  
Mammary Cells ◽  
Collagen Gels ◽  
Cells Cultured

Manipulation of the phenotype of immortalised rat hepatocytes by different culture configurations and by dimethyl sulphoxide

2000 ◽  
Vol 19 (5) ◽  
pp. 309-317 ◽  
Author(s):  
M I Grant ◽  
K Anderson ◽  
G McKay ◽  
M Wills ◽  
C Henderson ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Toxicity Testing ◽  
Normal Liver ◽  
Rat Hepatocytes ◽  
In Vitro Toxicity ◽  
Collagen Gels ◽  
Testosterone Metabolism ◽  
Glutathione S Transferases ◽  
Cells Cultured

The liver-specific phenotype of immortalised rat hepato-cytes is not irretrievably lost as they age in culture but can be manipulated by modifying the culture environment. Testosterone metabolism was used to investigate the profile of cytochrome P450 isoenzymes present in two immortalised cell lines, P9 and LQC, and in primary cultures of rat hepatocytes, cultured on collagen films, gels and double gel cultures (sandwich configuration). The extent of testosterone metabolism, and the range of metabolites produced, was increased in immortalised cells by the presence of collagen as a substratum film or gel but survival was poorer and the range of metabolites was reduced in sandwich culture. In contrast, testosterone metabolism was retained in primary hepatocytes in sandwich cultures at a higher level than in collagen film or gel cultures. Expression of alpha class glutathione-S-transferases (GSTs) increased and that of GSTP1 decreased (changes which indicate a recovery of normal liver GST phenotype) when the medium of immortalised cell cultures was supplemented with dimethyl sulph-oxide (DMSO). DMSO also improved ethoxyresorufin 0-deethylation (EROD) and testosterone metabolism in immortalised cells. It also markedly inhibited prolifera-tion, DNA, RNA and protein synthesis. Maximal testosterone metabolism was observed in immortalised cells cultured on collagen gels in the presence of l1o (v/v) DMSO. Development of a protocol for treating immortalised liver cells cultured on collagen gels with DMSO to switch between proliferation and differentiation may provide a convenient system expres-sing the xenobiotic metabolising enzymes required for in vitro toxicity testing.

Metabolism of 7,12-dimethylbenz[a]anthracene by mouse mammary epithelial cells cultured serum-free inside collagen gels

1988 ◽  
Vol 40 (2) ◽  
pp. 123-132 ◽  
Author(s):  
Raphael C. Guzman ◽  
Rebecca C. Osborn ◽  
Jack C. Bartley ◽  
Satyabrata Nandi
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Collagen Gels ◽  
Serum Free ◽  
Cells Cultured

scholarly journals Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum

1990 ◽  
Vol 268 (1) ◽  
pp. 225-230 ◽  
Author(s):  
A E Canfield ◽  
R P Boot-Handford ◽  
A M Schor
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Cell Shape ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Gels ◽  
Cells Cultured

Endothelial cells plated on the surface of a two-dimensional substratum (gelatin-coated dishes, dishes coated with native type I collagen or collagen gels) form a cobblestone monolayer at confluence, whereas cells plated within a three-dimensional gel matrix elongate into a sprouting morphology and self-associate into tube-like structures. In this study, we have compared the synthesis of thrombospondin by quiescent endothelial cells displaying (a) the same morphological phenotype (cobblestone) on different substrata (gelatin and collagen) and (b) different morphological phenotypes (cobblestone and sprouting) on the same substratum (collagen). We demonstrate that thrombospondin is a major biosynthetic product of confluent, quiescent cells cultured on dishes coated with either gelatin or collagen, and that the synthesis of this protein is markedly decreased when cells are plated on or in three-dimensional collagen gels. Moreover, we demonstrate that cells plated in gel (sprouting) secrete less thrombospondin than do cells plated on the gel surface (cobblestone). The regulation of thrombospondin synthesis is reversible and occurs at the level of transcription, as steady-state mRNA levels for thrombospondin decrease in a manner comparable with the levels of protein secreted by these cells. We also show that mRNA levels for laminin B2 chains are increased when cells are cultured on and in collagen gels compared with on gelatin-coated dishes, suggesting that the syntheses of thrombospondin and laminin are regulated by different mechanisms. When cells are cultured on gelatin- or collagen-coated dishes, thrombospondin gene expression is directly proportional to the proliferative state of the cultures. By contrast, the synthesis of thrombospondin by cells cultured on collagen gels remains at equally low levels whether they are labelled when they are sparse and rapidly proliferating or when they are confluent and quiescent. Fibronectin synthesis was found to increase with increasing confluency of the cells plated on all three substrata. These results demonstrate that thrombospondin gene expression is modulated by cell shape, cell proliferation and the nature of the substratum used for cell culture.

Variation of transferrin mRNA concentration in the rabbit mammary gland during the pregnancy–lactation–weaning cycle and in cultured mammary cells. A comparison with the other major milk protein mRNAs

1994 ◽  
Vol 130 (5) ◽  
pp. 522-529 ◽  
Author(s):  
C Puissant ◽  
M Bayat-Sarmadi ◽  
E Devinoy ◽  
L-M Houdebine
Keyword(s):  
Extracellular Matrix ◽  
Mammary Gland ◽  
Milk Protein ◽  
The Other ◽  
Mammary Cells ◽  
High Concentration ◽  
Dot Blot ◽  
Mrna Concentration ◽  
Lactogenic Hormones ◽  
Cells Cultured

Puissant C, Bayat-Sarmadi M, Devinoy E, Houdebine L-M. Variation of transferrin mRNA concentration in the rabbit mammary gland during the pregnancy–lactation–weaning cycle and in cultured mammary cells. A comparison with the other major milk protein mRNAs. Eur J Endocrinol 1994;130:522–9. ISSN 0804–4643 The concentration of transferrin mRNA was evaluated during pregnancy and lactation in rabbit mammary gland and liver using northern blot and dot blot assays. Transferrin mRNA was present in the virgin rabbit mammary gland and its concentration increased as pregnancy proceeded, with a major enhancement after day 15. A high concentration was reached 3 days after parturition, with no additional increase during lactation and with a marked decline after weaning. During the same period, the concentration of transferrin mRNA showed only a very weak variation in liver. This mRNA was six times more abundant in mammary gland than in liver of lactating rabbit. The accumulation of transferrin mRNA in the mammary gland was concomitant with the accumulation of αs1-, β-, kcasein and WAP (whey acidic protein) mRNAs. The concentration of glyceraldehyde 3-phosphate dehydrogenase mRNA, taken as a non-inducible control mRNA, declined progressively during pregnancy to reach its lower level in lactation. These observations suggest that casein, WAP and transferrin mRNAs are subjected to a similar control mechanism in vivo, at least in the second half of pregnancy and during lactation. Experiments carried out in vitro using isolated rabbit epithelial mammary cells cultured on collagen I gel indicated that transferrin mRNA was abundant and only weakly inducible by the lactogenic hormones insulin, cortisol and prolactin, as opposed to caseins and WAP mRNAs. R5020, an analogue of progesterone, inhibited at most very slightly the accumulation of αs1-casein mRNA in the presence of prolactin and it did not reduce the expression of transferrin gene. The mammary cells cultured on a plastic support contained much less transferrin mRNA than those maintained on collagen gel or on EHS (Engelbreth–Holm–Swarm) extracellular matrix independently of any hormonal stimulation. These data suggest that although caseins, WAP and transferrin mRNAs have parallel variations during the pregnancy–lactation–weaning cycle, they are subjected to different mechanisms of regulation at the molecular level. The accumulation of the mRNAs for caseins and WAP is positively regulated by lactogenic hormones and by the presence of the extracellular matrix, whereas the accumulation of transferrin mRNA is positively regulated essentially by the presence of the matrix. The fact that the levels of all the mRNAs studied here are increased simultaneously as progesterone starts declining suggests that the steroid controls the action of a factor, possibly the presence of the extracellular matrix, that regulates the expression of all the milk protein genes. L-M Houdebine, Unité de Differenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cédex, France

scholarly journals Expression of extracellular matrix components is regulated by substratum.

1990 ◽  
Vol 110 (4) ◽  
pp. 1405-1415 ◽  
Author(s):  
C H Streuli ◽  
M J Bissell
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Type I Collagen ◽  
Basement Membranes ◽  
Type I ◽  
Collagen Gels ◽  
Extracellular Matrix Components ◽  
Matrix Components ◽  
Cells Cultured

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.

scholarly journals Alignment of Skeletal Muscle Cells Cultured in Collagen Gel by Mechanical and Electrical Stimulation

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Takara Tanaka ◽  
Noriko Hattori-Aramaki ◽  
Ayano Sunohara ◽  
Keisuke Okabe ◽  
Yoshiaki Sakamoto ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Tissue Engineering ◽  
Electrical Stimulation ◽  
Collagen Gel ◽  
Muscle Cells ◽  
Mechanical Force ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Collagen Gels ◽  
Cells Cultured

For in vitro tissue engineering of skeletal muscle, alignment and fusion of the cultured skeletal muscle cells are required. Although the successful alignment of skeletal muscle cells cultured in collagen gel has been reported using a mechanical force, other means of aligning cultured skeletal muscle cells have not been described. However, skeletal muscle cells cultured in a two-dimensional dish have been reported to align in a uniform direction when electrically stimulated. The purpose of this study is to determine if skeletal muscle cells cultured three-dimensionally in collagen gels can be aligned by an electrical load. By adding direct current to cells of the C2C12 skeletal muscle cell line cultured in collagen gel, it was possible to align C2C12 cells in a similar direction. However, the ratio of alignment was better when mechanical force was used as the means of alignment. Thus for tissue engineering of skeletal muscle cells, electrical stimulation may be useful as a supplementary method.

scholarly journals Alteration of collagen-dependent adhesion, motility, and morphogenesis by the expression of antisense alpha 2 integrin mRNA in mammary cells

1995 ◽  
Vol 108 (2) ◽  
pp. 595-607 ◽  
Author(s):  
P.J. Keely ◽  
A.M. Fong ◽  
M.M. Zutter ◽  
S.A. Santoro
Keyword(s):  
Cell Surface ◽  
Three Dimensional ◽  
Cellular Growth ◽  
Laminin Receptor ◽  
Breast Carcinoma Cell Line ◽  
Integrin Expression ◽  
Mammary Cells ◽  
Collagen Gels ◽  
T47d Cells ◽  
Beta 1 Integrin

Although integrins are known to mediate adhesive binding of cells to the extracellular matrix, their role in mediating cellular growth, morphology, and differentiation is less clear. To determine more directly the role of the alpha 2 beta 1 integrin, a collagen and laminin receptor, in mediating the collagen-dependent differentiation of mammary cells, we reduced expression of the integrin by the well differentiated human breast carcinoma cell line, T47D, by stably expressing alpha 2 integrin antisense mRNA. Flow cytometry demonstrated that the antisense-expressing clones had levels of alpha 2 beta 1 integrin on their surfaces that were decreased by 30–70%. Adhesion of antisense-expressing clones to both collagens I and IV was decreased relative to controls in a manner that correlated with the level of cell surface alpha 2 beta 1 integrin expression. Adhesion to fibronectin and laminin were not affected. Motility across collagen-coated filters in haptotaxis assays was increased for only those clones that exhibited intermediate levels of adhesion to collagen, suggesting that an intermediate density of cell-surface alpha 2 beta 1 integrin optimally supports cell motility. When cultured in three-dimensional collagen gels, T47D cells organized in a manner suggestive of a glandular epithelium. In contrast, antisense-expressing clones with decreased alpha 2 beta 1 integrin were not able to organize in three-dimensional collagen gels. The growth rate of T47D cells was reduced when the cells were cultured in three-dimensional collagen gels. Unlike adhesion, motility, and morphogenesis, growth rates were unaffected by reduction of alpha 2 beta 1 integrin expression. Our results suggest that adhesive interactions mediated by a critical level of surface alpha 2 beta 1 integrin expression are key determinants of the collagen-dependent morphogenetic capacity of mammary epithelial cells.

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