What determines differentiation of urothelial umbrella cells?

Cytokeratins, uroplakins and the asymmetric unit membrane are biochemical and morphological markers of urothelial differentiation. The aim of our study was to follow the synthesis, subcellular distribution and supramolecular organization of differentiation markers, cytokeratins and uroplakins, during differentiation of umbrella cells of mouse bladder urothelium. Regenerating urothelium after destruction with cyclophosphamide was used to simulate de-novo differentiation of cells, which was followed from day 1 to day 14 after cyclophosphamide injection. Cytokeratin 7 and uroplakins co-localized in the subapical cytoplasm of superficial cells from the early stage of differentiation on. At early stages of superficial cell differentiation cytokeratin 7 was filamentary organized, and rare uroplakins were found on the membranes of relatively small cytoplasmic vesicles, which were grouped in clusters under the apical membrane. Later, cytokeratin 7 gradually reorganized into a continuous trajectorial network, and uroplakins became organized into plaques of asymmetric unit membrane, which formed fusiform vesicles. After insertion of fusiform vesicles into the apical plasma membrane, the surface acquired microridged appearance of umbrella cells. Cytokeratin 20 appeared as the last differentiation marker of umbrella cells. Cytokeratin 20 was incorporated into the pre-existing trajectorial cytokeratin network. These results indicate that differentiation of urothelial cells starts with the synthesis of differentiation-related proteins i.e., cytokeratins and uroplakins, and later with their specific organization. We consider that the umbrella cell has reached its final stage of differentiation when uroplakins form plaques of asymmetric unit membrane that are inserted into the apical plasma membrane and when cytokeratin 20 becomes included in a trajectorial cytokeratin network in the subapical area of cytoplasm.     

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation. The work was supported by the Ministry of Education and Sport, Government of Republic of Slovenia, Slovenia (grant no. 3311-04-831450).     

Antigenic and ultrastructural markers associated with urothelial cytodifferentiation in primary explant outgrowths of mouse bladder.

The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially-located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly-differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly-differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E-cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non-differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.     

Adenosine receptor expression and function in bladder uroepithelium

The uroepithelium of the bladder forms an impermeable barrier that is maintained in part by regulated membrane turnover in the outermost umbrella cell layer. Other than bladder filling, few physiological regulators of this process are known. Western blot analysis established that all four adenosine receptors (A₁, A_2a, A_2b, and A₃) are expressed in the uroepithelium. A₁ receptors were prominently localized to the apical membrane of the umbrella cell layer, whereas A_2a, A_2b, and A₃ receptors were localized intracellularly or on the basolateral membrane of umbrella cells and the plasma membrane of the underlying cell layers. Adenosine was released from the uroepithelium, which was potentiated 10-fold by stretching the tissue. Administration of adenosine to the serosal or mucosal surface of the uroepithelium led to increases in membrane capacitance (where 1 µF 1 cm² tissue area) of 30% or 24%, respectively, after 5 h. Although A₁, A_2a, and A₃ selective agonists all stimulated membrane capacitance after being administrated serosally, only the A₁ agonist caused large increases in capacitance after being administered mucosally. Adenosine receptor antagonists as well as adenosine deaminase had no effect on stretch-induced capacitance increases, but adenosine potentiated the effects of stretch. Treatment with U-73122, 2-aminoethoxydiphenylborate, or xestospongin C or incubation in calcium-free Krebs solution inhibited adenosine-induced increases in capacitance. These data indicate that the uroepithelium is a site of adenosine biosynthesis, that adenosine receptors are expressed in the uroepithelium, and that one function of these receptors may be to modulate exocytosis in umbrella cells. capacitance; exocytosis     

Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation

Using primary explant cultures of mouse bladder, the early response of the urothelium after superficial and full-thickness injuries was investigated. In such an in vitro wound healing model, explant surfaces with a mostly desquamated urothelial superficial layer represented superficial wounds, and the exposed lamina propria at the cut edges of the explants represented full-thickness wounds. The urothelial cell ultrastructure, the expression and subcellular distribution of the tight junctional protein occludin, and differentiation-related proteins CK 20, uroplakins, and actin were followed. Since singular terminally differentiated superficial cells remained on the urothelium after superficial injury (i.e., original superficial cells), we sought to determine their role during the urothelial wound-healing process. Ultrastructural and immunocytochemical studies have revealed that restored tight junctions are the earliest cellular event during the urothelial superficial and full-thickness wound-healing process. Occludin-containing tight junctions are developed before the new superficial cells are terminally differentiated. New insights into the urothelium wound-healing process were provided by demonstrating that the original superficial cells contribute to the urothelium wound healing by developing tight junctions with de novo differentiated superficial cells and by stretching, thus providing a large urothelial surface with asymmetric unit membrane plaques.     

Polyurethane membrane as an efficient immobilization carrier for high-density culture of rat hepatocytes in the fixed-bed reactor

A fixed-bed bioreactor with a polyurethane membrane (PUM) as a cell-supporting material was developed for high-density culture of rat hepatocytes. The PUM has a heterogeneous porous structure of micropores (pore size <100 microm) and macropores (pore size >100 microm) with a porosity of 90%. One important feature of a PUM is that the macropores have finger-like structures and their diameters gradually decrease from the upper to the lower layer of the PUM. Most rat hepatocytes were readily immobilized in the micropores of PUM. Immobilized cell densities of 1-3 x 10(7) cells/cm(3) PUM were achieved within 5 min by natural downflow of cell suspension and their immobilization efficiencies were more than 99%. Using a syringe pump, a cell density of 5 x 10(7) cells/cm(3) PUM was achieved with more than 96% immobilization efficiency. Perfusion cultures using this reactor were performed for 7 days without cell leakage. The optimal cell density for albumin secretion was between 2 x 10(7) and 3 x 10(7) cells/cm(3) PUM. Albumin secretion in the perfusion culture was maintained for a relatively long period of time when compared to that in the monolayer culture. The rate of albumin secretion in the perfusion culture was about 50% of that in monolayer culture. Hepatocytes immobilized in PUM were slightly aggregated, but they maintained spherical form individually even after 7 days of cultivation. The above results show that PUM is a promising cell-supporting material for efficient immobilization of high cell density of hepatocytes.     

Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.     

The uroepithelium: not just a passive barrier

The uroepithelium lines the inner surface of the renal pelvis, the ureters, and the urinary bladder, where it forms a tight barrier that allows for retention of urine, while preventing the unregulated movement of ions, solutes, and toxic metabolites across the epithelial barrier. In the case of the bladder, the permeability barrier must be maintained even as the organ undergoes cyclical changes in pressure as it fills and empties. Beyond furthering our understanding of barrier function, new analysis of the uroepithelium is providing information about how detergent-insoluble membrane/protein domains called plaques are formed at the apical plasma membrane of the surface umbrella cells, how mechanical stimuli such as pressure alter exocytic and endocytic traffic in epithelial cells such as umbrella cells, and how changes in pressure are communicated to the underlying nervous system.     

Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel dielectrophoresis-based device for the selective retention of viable cells in cell culture media

Cost-effective production of biopharmaceuticals on a large scale can be carried out by perfusion cultures of mammalian cells. One problem with this mode of operation for submerged free-cell cultures is the requirement for an efficient cell separation device located in the effluent stream. The present work investigates the potential for the development of a novel dielectrophoresis-based cell separator, capable of providing selective retention of viable cells in cell culture media, which are highly conductive. Predictions of the dielectrophoretic (DEP) response in culture media were first obtained through a series of DEP-levitation experiments. Subsequently, a prototype microelectrode "filter" was microfabricated and tested with C174 myeloma cell suspensions of density 1 x 10(6) cells/mL. The optimum frequency range for selective retention of viable cells was found in the range 5-15 MHz. A maximum separation efficiency of 98% was achieved at 10 MHz, with an applied peak-to-peak voltage of 30 V (maximum field strength of 10(5) V/m) and a flow rate of 30 mL/h which corresponds to a superficial velocity of 5.23 cm/h through the DEP-filter channels. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 239-250, 1997.     

Characterization of Sertoli cells cultured in the bicameral chamber system: relationship between formation of permeability barriers and polarized secretion of transferrin

Sertoli cells from immature rats (18 days old) were cultured on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Three types of cultures were obtained: 1) confluent monolayer cultures that formed a permeability barrier (impermeable), 2) confluent monolayer cultures that did not form a permeability barrier (permeable), and 3) subconfluent cultures (permeable). The relationships among fluid equilibrium, electrical resistance, and [3H]inulin transport between the apical and basal reservoirs of the chambers were examined. An impermeable confluent monolayer is defined when the cells of the Sertoli cell epithelial sheet are able to prevent hydrodynamic equilibration of fluid levels between the apical and basal reservoirs of a bicameral chamber. That is, a permeability barrier is present between the two sides of the chamber when fluid levels (volumes) do not change. In the impermeable confluent Sertoli cell monolayers, 7.5 +/- 0.6% of added [3H]inulin diffused across the monolayer during a 6-h collection period versus 13.7 +/- 0.5% in permeable cultures. Conversely, the electrical resistance was higher in the impermeable monolayers (41-71 ohm.cm2) than in the permeable layers (less than 33 ohm.cm2). A reciprocal linear relationship (Y = -4.68(X) + 91.50, r = 0.808) exists between inulin flux and electrical resistance, and this relationship is a function of cell density. Transferrin (Tf) was one of a few proteins detected in the basal medium of bicameral chambers, whereas most de novo synthesized proteins were secreted into the apical reservoir of the chamber. No significant differences in the total amount of Tf secreted by impermeable or permeable monolayers of Sertoli cells were observed. However, the Sertoli cell secretion ratios (apical/basal) of Tf during a 15-20-h collection period were 2.03 and 1.57 for impermeable monolayers plated at 2.4 x 10(6) and 3.6 x 10(6) cells/well, respectively, but less than 1.0 in permeable layers of cells. When fewer than 2 x 10(6) Sertoli cells were plated, the apical/basal polarity of Tf secretion declined to below 1 in a 24-h culture period, even though those chambers contained impermeable monolayers (recognized by the lack of hydrodynamic equilibrium). These results indicate that polarized secretion by Sertoli cells is dependent on (1) plating density and (2) formation of an impermeable epithelial sheet.     

Water permeability of asymmetric planar lipid bilayers: leaflets of different composition offer independent and additive resistances to permeation

To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:30176-30185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/P(AB) = 1/P(A) + 1/P(B) (Eq. l), where P(AB) is the permeability of a bilayer composed of leaflets A and B, P(A) is the permeability of leaflet A, and P(B) is the permeability of leaflet B. Using for the MDCK leaflet-specific liposomes gives an estimated value for the osmotic water permeability (P(f)) of 4.6 x 10(-4) cm/s (at 25 degrees C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:1711-1718). At 36 degrees C, P(f) was 3.44 +/- 0.35 x 10(-3) cm/s for symmetrical inner leaflet membranes and 3.40 +/- 0.34 x 10(-4) cm/s for symmetrical exofacial membranes. From, the estimated permeability of an asymmetric membrane is 6.2 x 10(-4) cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 +/- 0.7 x 10(-4) cm/s, which is within 10% of the calculated value. Direct experimental measurement of P(f) for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of.     

Multivesicular bodies in the transitional epithelium of the neonatal mouse urinary border.

The origin of late endosomes - multivesicular bodies (MVBs) in the superficial cells of 16 and 17 embryonic old transitional epithelium of mouse urinary bladder was studied by electron microscopy, lectin labelling and HRP tracing. Analysis of hexagonally structured membrane particles, WGA, and RCA I binding sites revealed structural similarity between plasmalemma, fusiform vesicles and multivesicular bodies. Early endosomes are lined by symmetric unit membrane as well as by asymmetric thickened membrane regions. Multivesicular bodies and fusiform vesicles have asymmetric unit membranes. MVBs may be derived from primary endosomes as well as from fusiform vesicles in the cytoplasm.     

Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment

A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number increased after 10 and 15 weeks of the diet. At the sites of superficial cell loss, regenerative simple hyperplasia developed. Within 5 weeks of NaSac removal, regeneration re-established normal differentiated urothelium. In order to follow urothelial differentiation during regeneration we studied the expression of uroplakins and cytokeratins by means of immunocytochemistry and immunohistochemistry, respectively. Normal urothelium was characterised by terminally differentiated superficial cells which expressed uroplakins in their luminal plasma membrane and cytokeratin 20 (CK20) in the cytoplasm. Basal and intermediate cells were CK20 negative and cytokeratin 17 (CK17) positive. In hyperplastic urothelium all cells synthesised CK17, but not CK20. Differentiation of the superficial layer was reflected in three successive cell types: cells with microvilli, cells with rounded microridges and those with a rigid-looking plasma membrane on the luminal surface. The cells with microvilli did not stain with anti-uroplakin antibody. When the synthesis of uroplakins was detected rounded microridges were formed. With the elevated expression of uroplakins the luminal plasma membrane becomes rigid-looking which is characteristic of asymmetric unit membrane of terminally differentiated cells. During differentiation, syn-thesis of CK17 ceased in superficial cells while the synthesis of CK20 started. These results indicate that during urothelial regeneration after NaSac treatment, specific superficial cell types develop in which the switch to uroplakin synthesis and transition from CK17 to CK20 synthesis are crucial events for terminal differentiation. Accepted: 19 August 1997     

Technological problems in cultivation of plant cells at high density. Reprinted from Biotechnology and Bioengineering, Vol. XXII, No. 6, Pages 1203-1218 (1981)

Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technological problems in the cultivation of the plant cells at high density were investigated. Using "shake" flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen in order to obtain high cell densities in shaken cultures, particle breakdown and damage to the largest cell aggregate group (above 1981 microm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K) for a model solid-liquid system (beta-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in suspension cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 x 10(-3) cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble dispersion). The most suitable bioreactor for culturing plant cells at high density was a jar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.     

Comparison of different bioreactor systems for the production of high titer retroviral vectors

Improved, human-based packaging cell lines allow the production of high-titer, RCR-free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled-up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10, 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2-5) x 10(6) PFU cm(-3) in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3- to 7-fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 x 10(7) PFU cm(-3) for the fixed bed and microcarrier systems, while the clump cultures produced only (2-5) x 10(5) PFU cm(-3). The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10-fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9-fold reduction of the seeding cell density, led to a 5-fold increased vector production rate accompanied by an average titer of 3 x 10(7) PFU cm(-3) (maximum titer (4-5) x 10(7) PFU cm(-3)) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 x 10(6) to 15 x 10(6) PFU cm(-3).     

Differentiation of epithelial cells in the urinary tract

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.W. Mello Jr. thanks FAPESP, São Paulo, Brazil for financial support.     

Perfluorocarbon-mediated aeration applied to recombinant protein production by virus-infected insect cells

Perfluorocarbon (PFC) was used as an oxygen carrier in the cultures of insect cells and virus-infected insect cells. The cell suspensions were placed on a planar layer of PFC, which was re-oxygenated in an outer aeration unit and continuously recirculated, and were agitated by two sets of impeller blades, lower one of which was set in such a way that the ridge of the blade touched the PFC layer. The maximum cell density attained in the PFC-mediated aeration culture was higher than that in surface aeration culture. On viral infection, a recombinant protein yield was significantly high in the PFC-mediated aeration culture as compared with that in the surface aeration culture, though the production was largely decreased by setting apart the lower set of the blade from the PFC-medium interface. These results showed that the PFC-mediated aeration would be a useful technique for insect cell/baculovirus expression system. Overall mass-transfer coefficient K(L) for oxygen was examined in both the PFC-mediated aeration and surface aeration systems, by using a flask whose dimensions were identical to those of spinner flasks used for the cultures. The K(L) value in the PFC-mediated system was 2.60x10(-3)cms(-1), 1.6 times higher than that in the surface aeration system, when impeller blades were positioned at PFC-medium and medium-air interfaces, respectively. However, the K(L) values in both the PFC-mediated and surface aeration systems were decreased and their differences were brought so close, as the blade was set apart from the interfaces. DO behavior in the cultures was well explained by the model calculation using the determined K(L) values and oxygen-consumption rates of viable cells. This calculation further suggested that crucial DO, under which recombinant protein productions were unsuccessful, was 0.24-0.5ppm (3-7%) in the insect cell/baculovirus expression system.     

Cultured porcine urinary bladder epithelial cells as a screening model for genotoxic effects of aromatic amines: characterisation and application of the cell culture model

Isolated epithelial cells from porcine urinary bladders were maintained in dividing long-term monolayer cultures, and were used as a model system for the urinary bladder in toxicological studies in vitro. To examine the state of differentiation during the culture period, the culture system was characterised morphologically by light and transmission electron microscopy and by immune fluorescence labelling with antibodies against cytokeratins 7,13 and pan. The cultured cells were identified as urothelial epithelium by their polarised structure, and by their expression of several uroepithelial specific morphological features, such as fusiform vesicles, tight junctions and an asymmetric apical cell membrane. Additionally, the cells were labelled with anti-cytokeratin 7,13 and pan antibodies, and negatively with anti-vimentin antibodies. The maintenance of suitable culture conditions was shown by the stable enzyme activities of (gamma-glutamyltranspeptidase, alkaline phosphatase and acid phosphatase over a culture period of 4 weeks. A good viability of the cultured cells under the chosen culture conditions was shown by the presence of low amounts of lactate dehydrogenase (< of = 5%) in the culture medium. The activities of the chosen marker enzymes for cell differentiation (gamma-glutamyltranspeptidase), lysosomes (acid phosphatase) and luminal membranes (alkaline phosphatase) were relatively stable over the observed culture period. Enzyme activities involved in metabolism of xenobiotics were determined, to define the ability for metabolism in cultured cells compared with bladder tissue in situ. Several constitutive phase I and II enzyme activities were found to be stable during the culture period, indicating that the cultured cells should be able to metabolise xenobiotics in a comparable manner to the urothelium in vivo. The cytotoxic effects of xenobiotics were investigated and IC50 values were determined by means of lactate dehydrogenase leakage and inhibition of neutral red uptake. The induction of sister chromatid exchanges was used as a parameter for the genotoxic effects of several xenobiotics. This cell culture system was found to be a very good screening system for the testing of substances that affect the bladder, especially aromatic amines.