scholarly journals Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells.

1975 ◽  
Vol 66 (2) ◽  
pp. 316-332 ◽  
Author(s):  
P B Pickett ◽  
D R Pitelka ◽  
S T Hamamoto ◽  
D S Misfeldt
Keyword(s):  
Primary Cultures ◽  
Freeze Fracture ◽  
Culture Conditions ◽  
Pavement Cells ◽  
Thin Sections ◽  
Occluding Junctions ◽  
Bulk Medium ◽  
Mammary Gland Cells ◽  
Junction Structure

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.

Transport properties of cultured branchial epithelia from freshwater rainbow trout: a novel preparation with mitochondria-rich cells

2000 ◽  
Vol 203 (10) ◽  
pp. 1523-1537 ◽  
Author(s):  
M. Fletcher ◽  
S.P. Kelly ◽  
P. Part ◽  
M.J. O'Donnell ◽  
C.M. Wood
Keyword(s):  
Rainbow Trout ◽  
Flux Ratio ◽  
Culture Conditions ◽  
Chloride Cells ◽  
Pavement Cells ◽  
Ratio Criterion ◽  
Peg 4000 ◽  
Tight Epithelia ◽  
Gill Filaments

A new double-seeded insert (DSI) technique is described for culture of branchial epithelial preparations from freshwater rainbow trout on filter supports. DSI epithelia contain both pavement cells and mitochondria-rich (MR) cells (15.7+/−2.5 % of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the ‘external environment’ and exhibit ultrastructural characteristics similar to those found in the ‘chloride cells’ of freshwater fish gills. After 6–9 days in culture with Leibovitz's L-15 medium on both surfaces (symmetrical conditions), transepithelial resistance (TER) stabilized at values as high as 34 k capomega cm(2), indicative of electrically ‘tight’ epithelia. The density of MR cells, the surface area of their clusters and transepithelial potential (TEP; up to +8 mV basolateral positive, mean +1.9+/−0.2 mV) were all positively correlated with TER. In contrast, preparations cultured using an earlier single-seeded insert (SSI) technique contained only pavement cells and exhibited a negligible TEP under symmetrical conditions. Na(+)/K(+)-ATPase activities of DSI preparations were comparable with those in gill filaments, but did not differ from those of SSI epithelia. Replacement of the apical medium with fresh water to mimic the in vivo situation (asymmetrical conditions) induced a negative TEP (−6 to −15 mV) and increased permeability to the paracellular marker PEG-4000. Under symmetrical conditions, unidirectional Na(+) and Cl(−) fluxes were in balance, and there was no active transport by the Ussing flux ratio criterion. Under asymmetrical conditions, there were large effluxes, small influxes and evidence for active Cl(−) uptake and Na(+) extrusion. Unidirectional Ca(2+) fluxes were only 0.5-1.0 % of Na(+) and Cl(−) fluxes; active net Ca(2+) uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater ‘chloride cells’.

Cultured branchial epithelia from freshwater fish gills

1997 ◽  
Vol 200 (6) ◽  
pp. 1047-1059 ◽  
Author(s):  
C M Wood ◽  
P Pärt
Keyword(s):  
Primary Cultures ◽  
Transepithelial Resistance ◽  
Apical Surface ◽  
Pavement Cells ◽  
Cell Layers ◽  
Cultured Epithelium ◽  
Net Fluxes ◽  
Foetal Bovine Serum ◽  
Tight Epithelium

We have developed a method for the primary culture of gill epithelial cells from freshwater rainbow trout on permeable supports, polyethylene terephthalate membranes ('filter inserts'). Primary cultures of gill cells (6-9 days in Leibowitz L-15 culture medium plus foetal bovine serum and glutamine) are trypsinized and the cells seeded onto the inserts. After 6 days of growth with L-15 medium on both surfaces (approximately isotonic to trout plasma), the cells form a tight epithelium as judged from a progressive rise in transepithelial resistance which reaches a stable plateau for a further 6 days, as long as L-15 exposure is continued on both surfaces. The cultured epithelium (approximately 8 µm thick) typically consists of 2-4 overlapping cell layers organized as in the lamellae in vivo, with large intercellular spaces, multiple desmosomes and putative tight junctions. The cells appear to be exclusively pavement-type cells with an apical surface glycocalyx, an abundance of rough endoplasmic reticulum, no selective DASPEI staining and relatively few mitochondria. Transepithelial resistance (approximately 3.5 k cm2), permeability to a paracellular marker (polyethylene glycol-4000; 0.17x10(-6) cm s-1) and unidirectional flux of Na+ and Cl- (approximately 300 nmol cm-2 h-1) all appear realistic because they compare well with in vivo values; net fluxes of Na+ and Cl- are zero. The preparation acidifies the apical medium, which accumulates a greater concentration of ammonia. Upon exposure to apical freshwater, resistance increases six- to elevenfold and a basolateral-negative transepithelial potential (TEP) develops as in vivo. These responses occur even when mannitol is used to prevent changes in apical osmotic pressure. Net Na+ and Cl- loss rates are low over the first 12 h (-125 nmol cm-2 h-1) but increase substantially by 48 h. The elevated resistance and negative TEP gradually attenuate but remain significantly higher than pre-exposure values after 48 h of apical freshwater exposure. The preparation may provide a valuable new tool for characterizing some of the mechanisms of active and passive ion transport in the pavement cells of the freshwater gill.

scholarly journals Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity.

1986 ◽  
Vol 102 (6) ◽  
pp. 2125-2136 ◽  
Author(s):  
J L Madara ◽  
D Barenberg ◽  
S Carlson
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Structure And Function ◽  
Cytochalasin D ◽  
Absorptive Cell ◽  
Absorptive Cells ◽  
Charge Selectivity ◽  
Occluding Junctions ◽  
And Function ◽  
Junction Structure

Intestinal absorptive cells may modulate both the structure and function of occluding junctions by a cytoskeleton dependent mechanism (Madara, J. L., 1983, J. Cell Biol., 97:125-136). To further examine the putative relationship between absorptive cell occluding junctions and the cytoskeleton, we assessed the effects of cytochalasin D (CD) on occluding junction function and structure in guinea pig ileum using ultrastructural and Ussing chamber techniques. Maximal decrements in transepithelial resistance and junctional charge selectivity were obtained with 10 micrograms/ml CD and the dose-response curves for these two functional parameters were highly similar. Analysis of simultaneous flux studies of sodium and the nonabsorbable extracellular tracer mannitol suggested that CD opened a transjunctional shunt and that this shunt could fully account for the increase in sodium permeability and thus the decrease in resistance. Structural studies including electron microscopy of detergent-extracted cytoskeletal preparations revealed that 10 micrograms/ml CD produced condensation of filamentous elements of the peri-junctional contractile ring and that this was associated with brush border contraction as assessed by scanning electron microscopy. Quantitative freeze-fracture studies revealed marked aberrations in absorptive cell occluding junction structure including diminished strand number, reduced strand-strand cross-linking, and failure of strands to impede the movement of intramembrane particles across them. In aggregate these studies show that CD-induced perturbation of the absorptive cell cytoskeleton results in production of a transepithelial shunt which is fully explained by a defect in the transjunctional pathway. Furthermore, substantial structural abnormalities in occluding junction structure accompany this response. Lastly, the abnormalities in occluding junction structure and function coincide with structural changes in and contraction of the peri-junctional actin-myosin ring. These data suggest that a functionally relevant association may exist between the cytoskeleton and the occluding junction of absorptive cells. We speculate that such an association may serve as a mechanism by which absorptive cells regulate paracellular transport.

scholarly journals Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures.

1982 ◽  
Vol 92 (1) ◽  
pp. 183-191 ◽  
Author(s):  
D M Larson ◽  
J D Sheridan
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
Tight Junctions ◽  
Lucifer Yellow ◽  
Primary Cultures ◽  
Freeze Fracture ◽  
Cell Transfer ◽  
Isolated Cells ◽  
Thin Sections ◽  
Cell To Cell Transfer

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.

scholarly journals Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus).

1980 ◽  
Vol 87 (2) ◽  
pp. 488-497 ◽  
Author(s):  
S A Ernst ◽  
W C Dodson ◽  
K J Karnaky
Keyword(s):  
Fundulus Heteroclitus ◽  
Short Circuit ◽  
Freeze Fracture ◽  
Short Circuit Current ◽  
Chloride Cells ◽  
Rectal Gland ◽  
Secretory Cells ◽  
Pavement Cells ◽  
Occluding Junctions ◽  
Mucosal Side

The structural features of the chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus) were examined by thin-section and freeze-fracture electron microscopy, with particular emphasis on the morphological appearance of occluding junctions. This epithelium is a flat sheet consisting predominantly of groups of mitochondriarich chloride cells with their apices associated to form apical crypts. These multicellular groups are interspersed in an otherwise continuous pavement cell epithelial lining. The epithelium may be mounted in Ussing-type chambers, which allow ready access to mucosal and serosal solutions and measurement of electrocal properties. The mean short-circuit current, potential difference (mucosal-side negative), and DC resistance for 19 opercular epithelia were, respectively, 120.0 +/- 18.2 microA/cm2, 12.3 +/- 1.7 mV, and 132.5 +/- 26.4 omega cm2. Short-circuit current, a direct measure of Cl- transport, was inhibited by ouabain (5 micron) when introduced on the serosal side, but not when applied to the mucosal side alone. Autoradiographic analysis of [3H]-ouabain-binding sites demonstrated that Na+,K+-ATPase was localized exclusively to basolateral membranes of chloride cells; pavement cells were unlabeled. Occluding junctions between adjacent chloride cells were remarkably shallow (20-25 nm), consisting of two parallel and juxtaposed junctional strands. Junctional interactions between pavement cells or between pavement cells and chloride cells were considerably more elaborate, extending 0.3-0.5 micron in depth and consisting of five or more interlocking junctional strands. Chloride cells at the lateral margins of crypts make simple junctional contacts with neighboring chloride cells and extensive junctions with contiguous pavement cells. Accordingly, in this heterogeneous epithelium, only junctions between Na+,K+-ATPase-rich chloride cells are shallow. Apical crypts may serve, therefore, as focal areas of high cation conductivity across the junctional route. This view is consistent with the electrical data showing that transmural resistance across the opercular eptihelium is low, and with recent studies demonstrating that transepithelial Na+ fluxes are passive. The simplicity of these junctions parallels that described recently for secretory cells of avian salt gland (Riddle and Ernst, 1979, J. Membr. Biol., 45:21-35) and elasmobranch rectal gland (Ernst et al., 1979, J. Cell Biol., 83:(2, Pt. 2):83 a[Abstr.]) and lends morphological support to the concept that paracellular ion permeation plays a central role in ouabain-sensitive transepithelial NaCl secretion.

scholarly journals Regulation of collagen production and collagen mRNA amounts in fibroblasts in response to culture conditions

1986 ◽  
Vol 239 (1) ◽  
pp. 179-183 ◽  
Author(s):  
S R Quinones ◽  
D S Neblock ◽  
R A Berg
Keyword(s):  
Type I Collagen ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Culture Conditions ◽  
Type I ◽  
Collagen Production ◽  
Tendon Cells ◽  
Chain Synthesis ◽  
Polypeptide Chains

Collagen synthesis and mRNA amounts for the alpha 1 and alpha 2 polypeptide chains of Type I collagen were measured in embryonic-chick tendons and in tendon cells both in suspension and in primary cultures. The percentage of protein production represented by collagen in suspension-cultured cells was initially the same as in the intact tendon; however, on an hourly basis, there was actually a steady decline in collagen production by suspended cells. Collagen production in primary cultures of chick tendon fibroblasts was decreased when compared with intact tendon, even though ascorbate-supplemented primary cultures were able to maintain higher rates of collagen production than were non-supplemented cultures. The amounts of mRNA for alpha 1(I) and alpha 2(I) polypeptide chains of collagen responded in similar fashions to different culture conditions and were compared with the amounts of mRNA for beta-actin. In primary cultures the available alpha 1 and alpha 2 collagen mRNAs support proportionately higher collagen production than in the intact tendon. However, the ratio of alpha 1/alpha 2 mRNA and polypeptide-chain synthesis did not remain 2:1, but increased with the concomitant production of Type I trimers composed of three alpha 1 chains. Removal of fibroblasts from their environment in vivo appears to alter the amounts of mRNA for alpha 1 and alpha 2 chains and to alter the utilization of those mRNAs for polypeptide synthesis.

L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture

1996 ◽  
Vol 271 (6) ◽  
pp. C2072-C2080 ◽  
Author(s):  
G. Nowak ◽  
R. G. Schnellmann
Keyword(s):  
Ascorbic Acid ◽  
Brush Border ◽  
Primary Cultures ◽  
Culture Conditions ◽  
Cellular Growth ◽  
O2 Consumption ◽  
Na Transport ◽  
Brush Border Enzyme ◽  
Stimulation Of

The addition of L-ascorbic acid 2-phosphate (AscP) to primary cultures of rabbit renal proximal tubular cells (RPTC) grown under improved culture conditions resulted in an extended growth phase and increased cellular density (1.3-fold increase in monolayer DNA and protein contents). AscP reduced glycolysis, increased net lactate consumption by 38%, and stimulated net glucose production by 47%. Basal O2 consumption increased by 39% in RPTC grown in the presence of AscP and was equivalent to that in freshly isolated proximal tubules. AscP increased ouabain-sensitive O2 consumption (81%) and Na(+)-K(+)-ATPase activity (2.5-fold), which suggested increased active Na+ transport. Addition of AscP increased Na(+)-dependent glucose uptake by 43% and brush-border enzyme marker activities by 46%. It is concluded that supplementation of media with AscP further improves RPTC culture conditions by promotion of cellular growth and stimulation of in vivo-like respiration, lactate utilization, and net glucose synthesis. These changes are accompanied by an increase in brush-border enzyme activities and stimulation of active Na+ transport and Na(+)-dependent glucose transport, which demonstrate an improved expression of brush-border membrane functions in RPTC.

scholarly journals Induction of differentiation in cultured rat and human podocytes.

1997 ◽  
Vol 8 (5) ◽  
pp. 697-705 ◽  
Author(s):  
P Mundel ◽  
J Reiser ◽  
W Kriz
Keyword(s):  
Primary Cultures ◽  
Growth Arrest ◽  
Culture Conditions ◽  
Phenotypic Changes ◽  
Differentiated Cells ◽  
Intermediate Phenotypes ◽  
Induction Of Differentiation ◽  
Cell Culture Conditions

Mature podocytes are highly differentiated cells that are unable to divide in vivo. During glomerulogenesis, podocytes develop from simple cuboidal cells into their adult phenotype, which is characterized by a complex pattern of processes. Cultivation of podocytes under standard conditions leads to dedifferentiation, including the loss of processes and of pp44, a marker of differentiated podocytes. In this study, the cell culture conditions for rat and human podocytes were modified by avoiding repeated subcultivation. This led to profound phenotypic changes in podocytes in vitro. The conversion of cobblestones into arborized cells was directly observed, and a series of intermediate phenotypes was documented. The cells converted within 3 wk from typical cobblestone appearance into individual arborized cells more closely resembling in vivo podocytes. Arborized cells were frequently binucleated and reached a size of up to 500 microns. Both cobblestone and arborized cells originated from podocytes, as evidenced by the expression of a podocyte-specific O-acetylated ganglioside and of the WT-1 protein. In contrast to primary cultures and early passages of cobblestones, a cloned rat podocyte cell line did not express WT-1 and could not be induced to differentiate into arborized cells. This finding indicates a role for WT-1 in maintaining differentiation of adult podocytes. The differentiation of arborized cells led to growth arrest and was reflected by the formation of processes and the expression of pp44 and desmin, which were never detected in cobblestones. It was concluded that partial differentiation of cultured podocytes can be achieved simply by avoiding repeated subcultivation, resulting in an arborized phenotype more closely reflecting in vivo podocytes.

Direct effect of insulin on albumin gene expression in primary cultures of rat hepatocytes

1985 ◽  
Vol 249 (5) ◽  
pp. E447-E453 ◽  
Author(s):  
K. E. Flaim ◽  
S. M. Hutson ◽  
C. E. Lloyd ◽  
J. M. Taylor ◽  
R. Shiman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Culture Conditions ◽  
Maximal Response ◽  
Maximal Effect ◽  
Albumin Secretion ◽  
Albumin Gene ◽  
A Cell

The purpose of this study was to identify a cell culture system in which the role of insulin in regulating albumin gene expression could be investigated. The system selected was rat hepatocytes maintained in primary culture in a chemically defined, serum-free medium. Under control conditions albumin secretion was nearly the same as the rate recorded in vivo and in perfused liver and was reasonably well maintained during 8 days of culture. Deletion of insulin from the culture medium for 3-6 days resulted in 40-60% reductions in albumin secretion. Furthermore, albumin secretion relative to the rate of total protein synthesis was reduced by approximately 50% as a result of insulin deficiency. Readdition of the hormone to insulin-deficient cultures restored secretion to the control rate. A maximal effect of insulin was observed within 3 days after readdition of the hormone, and a half-maximal response was obtained with a hormone concentration of approximately 3.0 nM. The relative abundance of albumin mRNA, as measured by solution hybridization using a complementary DNA probe, responded in a parallel fashion to the changes in albumin secretion. Thus rat hepatocytes maintained under appropriate culture conditions reflect the effects of diabetes and insulin treatment on albumin gene expression observed in vivo and provide an excellent model system in which to study the mechanism(s) of insulin action.

scholarly journals PRIMARY CULTURES OF EPITHELIAL CELLS FROM RAINBOW TROUT GILLS

1993 ◽  
Vol 175 (1) ◽  
pp. 219-232 ◽  
Author(s):  
P. Part ◽  
L. Norrgren ◽  
E. Bergstrom ◽  
P. Sjoberg
Keyword(s):  
Rainbow Trout ◽  
Epithelial Cells ◽  
Primary Cultures ◽  
Culture Conditions ◽  
Skin Extract ◽  
Respiratory Epithelial Cells ◽  
Attachment Efficiency ◽  
In Vivo Growth ◽  
Respiratory Epithelial

A method for obtaining primary cultures of epithelial cells from rainbow trout gills is described. The yield of cells from approximately 1.5 g wet mass of tissue was 218×106+/−12×106 cells with a viability defined by eosin exclusion of 80+/−6 %. Cells were seeded in culture dishes and grown in Leibowitz L-15 medium supplemented with 5 % foetal bovine serum. Attachment efficiency after 24 h was 35+/−6 %. The cells appeared confluent 10–12 days after seeding and exhibited surface structures similar to those seen on respiratory epithelial cells of trout gills in vivo. Growth rate, [3H]thymidine incorporation and attachment efficiency were used to evaluate culture conditions. Epidermal growth factor, insulin, transferrin, hydrocortisone, laminin and collagen did not improve growth and attachment. Similarly, coating the culture dishes with rat tail collagen, trout skin extract, laminin or a mixture of human basement membrane proteins (Matrigel) failed to improve attachment. It is concluded that the cells in culture are respiratory epithelial cells and that this culture system could provide a valuable new approach for studying the physiology of these cells.

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