Uncoated Dishes Research Articles

Evidence for distinct fast and slow myogenic cell lineages in human foetal skeletal muscle.

To analyse the myogenic cell lineages in human foetal skeletal muscle, muscle cell cultures were prepared from different foetal stages of development. The in vitro muscle cell phenotype was defined by staining the myotubes with antibodies to fast and slow skeletal muscle type myosin heavy chains using immunoperoxidase or double immunofluorescence procedures. The antibodies to fast skeletal muscle myosin heavy chains stained nearly all myotubes dark in cell cultures prepared from quadriceps muscles at 10-18 weeks of gestation. The antibodies to slow skeletal muscle myosin heavy chains, in contrast, stained only 10-40% of the myotubes very dark. The remaining myotubes were further subdivided into two populations, one of which was unstained while the other stained with variable intensity for slow myosin heavy chain. The slow myosin heavy chain staining was not influenced by the nature of the substratum used to culture these cells, although the growth of muscle cell cultures was greatly improved on matrigel-coated dishes. The presence of both slow and fast myosin heavy chains was detected even when myotubes were grown on uncoated petri dishes. The myotube diversity was further investigated by analysing the clonal populations of human foetal skeletal muscle cells in vitro. When cultured at clonal densities, two types of myogenic clones were identified by their differential staining with antibodies to slow myosin heavy chain. As was the case with the high density muscle cell cultures, virtually all myotubes in both groups of clones stained with antibodies to fast myosin heavy chains. Antibodies to slow myosin heavy chains stained nearly all myotubes dark in one group of myogenic clones, but only a subset of the myotubes stained dark for slow myosin heavy chain in the second group of clones. The proportion of slow myosin heavy chain positive myotubes in this group varied in different clones. The myogenic diversity was thus apparent in both high density and clonal human muscle cell cultures, and myogenic cells retained their ability to modify their muscle cell phenotype.

Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment

This paper presents a new and gentle method to separate Schwann cells from fibroblasts obtained from foetal rat dorsal root ganglia (DRG). The method exploits the different growth and adhesion characteristics of fibroblasts and Schwann cells under different experimental conditions such that antiproliferative (cytotoxic) drugs or time-consuming centrifugation is not needed. Standard procedures were used to obtain mixed cultures of Schwann cells, fibroblasts and neurons. After about 5 days further purification of the cells was achieved by exploiting the different responses of Schwann cells and fibroblasts to a temperature shock. Cooling the cells with cold phosphate-buffered saline (PBS), followed by pipetting cold medium directly on top of the cells (`cold jet'), resulted in specific detachment of Schwann cells and neurons, whereas fibroblasts remained securely attached. Schwann cells attached to the surface of new, uncoated culture dishes whereas neurons did not. Two cycles of the cold jet procedure resulted in nearly pure (98–100%) cultures of Schwann cells. Besides being gentle, this method is easy and fast, and because cytotoxic drugs are not used, it does not affect cell survival negatively.

Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium.

Several models for preparing and isolating human and animal gallbladder epithelial cells, including low-grade gallbladder carcinoma cells, as well as proposed systems for culturing these isolated epithelial cells are reviewed here. Several reports concerning tissue culture of the gallbladder are also reviewed. The cell culture systems are divided into monolayer cell culture on collagen-coated or uncoated culture dishes or other culture substrate and three-dimensional cell culture in collagen gel. To prepare and isolate gallbladder epithelial cells, digestion of the gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of mucosal explants are applied. In monolayer cell culture, most of the specific biological features of isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages, though quantitative and objective analyses of the pathophysiology of cultured cells and their secretory substances can be performed. Tissue culture using explants of the gallbladder has mainly been used for physiological studies of the gallbladder, such as investigating the transport of water and electrolytes. In this tissue culture system, quantitative assessment is difficult, though the original and specific biological and histological characteristics of the gallbladder are retained. Three-dimensional collagen gel culture could be an ideal model combining monolayer cell culture and tissue culture systems, and create controllable conditions or environments when several biologically active substances, such as growth factors, proinflammatory cytokines and adhesion molecules, are added to the culture medium. Advantages and shortcomings of individual cultivation models are discussed, and selecting the culture model most appropriate to the purpose of the study will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases such as cholelithiasis.

Expression of phenotype- and proliferation-related genes in rat aortic smooth muscle cells in primary culture.

After endothelial injury, smooth muscle cells (SMCs) in the arterial media are modified from a contractile to a sympathetic phenotype. This process includes a prominent structural reorganization and makes the cells able to migrate into the intima, divide, and secrete extracellular matrix components. A similar change occurs in culture and then in vitro system has been established as a useful model in which to study the control of SMC differentiation. The purpose of this study was to analyze the expression of a number of phenotype- and proliferation-related genes in vascular SMCs during the first week in primary culture. SMCs were enzymatically isolated from rat aorta and seeded on substrates of fibronectin (an adhesive plasma protein) and laminin-collagen type IV (two major basement membrane proteins) in a serum-free medium or in uncoated dishes in a serum-containing medium. Total RNA was isolated from the cells after different times of culture and analyzed by Northern blotting for expression of specific gene transcripts. In part, expression of the corresponding proteins was also explored by Western blotting and indirect immunofluorescence microscopy. The results indicate that the proto-oncogenes c-fos, c-jun and c-ets-1 were already activated during the isolation of the cells and then continued to be strongly expressed for a few days. Especially in the serum-free groups, there was also early activation of the genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2). In parallel, an increased expression of the genes for two extracellular matrix components was observed, with an early rise in osteopontin mRNA and a later rise in collagen type I mRNA. At the end of the test period, the corresponding proteins were deposited around the cells in a fibrillar pattern. Among the matrix receptors investigated, the beta 1 integrin subunit showed a high and persistent expression, whereas the alpha 5 and alpha 1 integrin subunits showed lower and more variable mRNA level. In support of the existence of an autocrine or paracrine platelet-derived growth factor (PDGF) loop, an early rise in expression of the PDGF A-chain gene and a subsequent rise in expression of the PDGF alpha-receptor gene were noted. It is proposed that the coordinated shift in gene expression here described to take place in connection with the phenotypic modulation of vascular SMCs in primary culture is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.

Different enzyme activities in coronary capillary endothelial cells.

Differential distributions of alkaline phosphatase (AP) and dipeptydylpeptidase IV (DPPIV) were studied in coronary microvascular endothelial cells. Endothelial cells were obtained by the perfusion of coronary vessels with 0.1% trypsin PBS solution and cultured in uncoated culture dishes. Staining of cultured endothelial cells with AP- and DPPIV-sensitive reagents revealed blue or red staining, respectively. Most colonies showed cells of only one color, blue or red, even at the fifth passage. AP-sensitive cells, which were originally elongated, shortened and widened, proliferating to form monolayer colonies of cobble stone-like cells. AP-stainability became weak with repeated passages. DPPIV-sensitive endothelial cells remained elongated even after repeated passages. The cell shape and stainability seemed to be coupled and maintained through the five passages studied.

Adhesion Proteins Increase Cellular Attachment, Follicle-Stimulating Hormone Receptors, and Progesterone Production in Cultured Porcine Granulosa Cells

We sought to determine the influence of different constituents of the extracellular matrix on porcine granulosa cell function by assessing cellular attachment, cellular morphology, follicle-stimulating hormone (FSH) receptors, and progesterone production. Cells from immature porcine ovarian follicles were cultured for up to 6 days in serum-free medium containing porcine FSH (pFSH, 10 ng/ml) in culture dishes either uncoated or coated with one of the following adhesion proteins: gelatin (1 mg/cm2), fibronectin (1 microgram/cm2), laminin (1 microgram/cm2), type I collagen (10 micrograms/cm2), or type IV collagen (7.8 micrograms/cm2). Fibronectin, laminin, type I collagen, and type IV collagen increased cellular attachment significantly (P < 0.05). All adhesion proteins except gelatin influenced cellular morphology. Cells cultured on laminin or type IV collagen formed dense clusters of rounded cells. Cells cultured in dishes coated with each adhesion protein except gelatin had higher 125I-pFSH binding per cell than cells cultured in uncoated dishes, with increases of 7- to 12-fold over control (P < 0.05). All adhesion proteins increased progesterone production, ranging from 10- to 50-fold over control (P < 0.05). In summary, not only did adhesion proteins increase attachment to the dishes but they also increased FSH receptors and differentiated function (progesterone production) of granulosa cells from immature porcine ovarian follicles.

Influence of extracellular matrices on ganglioside pattern of two hepatoma cell lines with different adhesive properties

A cell culture model was developed to investigate the involvement of gangliosides in cell-matrix adhesion. Two cell lines with different adhesive properties derived from solid Morris hepatoma 7777 were established. Cultured in horse serum-containing medium, the adhesive cell line (MH 7777 A) adheres and spreads on uncoated culture dishes, whereas the revertant cell line (MH 7777 A>N) does not adhere and grows in suspension. The adhesiveness of both cell lines is dependent on the coating protein used (none, bovine serum albumin, fibronectin or collagen I) and the horse serum concentration in the culture medium. Both cell lines, although of the same origin, differed in their ganglioside composition. The most abundant ganglioside of both MH 777 A and MH 7777 A>N cell lines was fucosyl-G M1, 0.78 and 0.72 μg per mg cellular protein, respectively. The G M3 and G D1a content of MH 7777 A>N cells was significantly higher than that of MH 7777 A cells. Furthermore, a matrix-dependency of the ganglioside pattern of both cell lines was demonstrated.

Concanavalin A Promotes Regeneration of Processes of Isolated Ganglion Cells from the Adult Newt Retina

Abstract Retinal cells dissociated from adult newt eyes were seeded onto plastic dishes which were coated with various cell adhesion substances. They were cultured in Leibovitz's L-15 medium with no serum. Concanavalin A (ConA) and poly-L-lysine (PLL) caused relatively good cell attachment, regardless of retinal cell types, in comparison with uncoated dishes. On the other hand, extracellular matrix (ECM) components such as collagen, fibronectin and laminin tended to inhibit the cell attachment. In both ConA- and PLL-coated dishes, ganglion cells survived for longer periods than other retinal cells and extended neurites with time in culture. The outgrowth of neurites by ganglion cells was found to be promoted to a far greater degree by ConA than PLL. Branching of neurites also appeared to occur more frequently on ConA. From these observations, ConA may be an useful tool for investigating retinal regeneration in vitro.

Development of surface photochemical modification method for micropatterning of cultured cells

This article reports the development of micropatterning technology of cultured cells by precise surface regional modification via photochemical fixation of phenyl azido-derivatized polymers on polymer surfaces. Photoreactive polymers prepared in this study included poly(N,N-dimethylacrylamide-co-3-azidostyrene), bis-4-azidobenzamide-polyethylene glycol, and poly(styrene-co-3-azidostyrene). The photochemical fixation of these photoreactive polymers consisted of three steps: 1) coating of a photoreactive polymer on a material surface, 2) ultraviolet irradiation through a photomask, and 3) removal of nonreacted polymer by a solvent. Electron spectroscopy for chemical analysis and water contact angle measurement were employed for surface characterization. Two different types of regionally modified surfaces were prepared; one was a hydrophilic polymer regionally fixed on a tissue culture dish and the other was a hydrophobic polymer regionally fixed on poly(vinyl alcohol) (PVA). Photochemical surface microfabrication permits mu-order dimensional precision, which was verified by the micropatterned tissue formation of bovine aorta endothelial cells (ECs) when ECs were seeded on these surfaces. ECs adhered, spread, and confluently proliferated only on uncoated tissue culture dish surfaces or hydrophobic regions on PVA. Thus, the regionally differentiated cell adhesional regions were created by photochemically driven surface microprocessing.

Matrix Modulates Uptake of Calcium Oxalate Crystals and Cell Growth of Renal Epithelial Cells

Handling of urinary crystals by renal epithelial and medullary interstitial cells may play an important role in the pathogenesis of renal stones and associated renal scarring. We examined the effects of calcium oxalate monohydrate (CaOM) crystals on the proliferative activity of renal tubular cells (opossum kidney) and renal medullary interstitial cells in culture. We also studied the impact of altered extracellular matrix on cell proliferation as well as on uptake of crystals by epithelial cells. Epithelial cells incubated with CaOM showed greater (p < 0.05) growth when compared with untreated cells (control, 10.5 +/- 1.1 versus CaOM, 16.6 +/- 1.6 x 10(6) cells per dish). the CaOM crystal-cell interaction also enhanced proliferation of interstitial cells (at 48 hours, control, 3.3 +/- 0.2 versus CaOM, 4.2 +/- 0.1 x 10(6) cells per dish, p < 0.02; at 72 hours, control, 3.6 +/- 0.3 versus 5.5 +/- 0.4 x 10(6) cells per dish, p < 0.01). Collagen, a constituent of extracellular matrix, inhibited (p < 0.01) proliferation of epithelial cells. Semiconfluent epithelial cells grown on collagen gels showed greater (p < 0.01) uptake of 45CaOM crystals when compared with uptake by cells grown on either uncoated (control) or albumin-coated plastic dishes (control, 979.9 +/- 51.1, albumin, 876.4 +/- 28.3, collagen gel, 1502.5 +/- 103.8 cpm per well). Epithelial cells grown to confluence on collagen gels also showed enhanced (p < 0.05) uptake of 45CaOM crystals. Reflectance microscopy as well as ultrastructural studies revealed intracellular localization of CaOM crystals. These results indicate that CaOM crystals stimulate the growth of both epithelial and interstitial cells. Enhanced growth of interstitial cells may also lead to increased synthesis of extracellular matrix. The latter may further modulate crystal uptake as well as cell growth of adjacent epithelial cells. These findings may be important in the development of nephrolithiasis and associated interstitial scarring.

Laminin promotes differentiation, adhesion and proliferation of cell cultures derived from human acoustic nerve schwannoma.

The influence of laminin on cell cultures derived from unilateral acoustic nerve schwannomas was investigated. Cell cultures were initiated from 12 schwannomas, removed via the enlarged middle cranial fossa approach. Tumor tissue was dispersed by collagenase treatment and cells seeded in uncoated or laminin-coated culture dishes. Confluent cultures were immunocytochemically characterized with antibodies against S-100, CD 68, factor VIII-related antigen and type IV collagen. Cell adhesion in response to different doses of laminin was evaluated with an electronic cell counter. The effect of laminin on cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxy-uridine (BRDU) into cellular DNA. Cells cultured on laminin as substrate appeared more differentiated with long, fusiform, cytoplasmic processes. Cultured cells stained positive for S-100, not for factor VIII-related antigen or CD 68. Only cells cultured on laminin deposited a dense extracellular network of type IV collagen. When laminin was added to the culture medium, cell attachment and proliferation was stimulated in a dose dependent manner. Maximal stimulation of both was observed with a laminin concentration of 50 micrograms/ml, which induced a nearly 2-fold increase in cell attachment and an approximately 66% increase in DNA content. Since laminin is a major component of the extracellular matrix in schwannomas, the possibility exists that laminin is also mitogenic for human neoplastic Schwann cells in situ.

Formation of connections between cultured identified neurones from the pleural ganglion of the pteropod mollusc Clione limacina

A cluster of electrically interconnected neurosecretory cells (the ‘white cells’) involved in the control of reproductive behavior was identified in the right pleural ganglion of the marine mollusc, Clione limacina. Pleural ganglia also contain large neurons (PL1 and PL2) having no connections with each other and with the white cells. Most isolated white cells put into the simple unconditioned medium (50% L-15) adhered to the bottom of uncoated dishes and demonstrated neurite outgrowth for 7–10 days. If growing processes overlapped, the white cells formed electrical connections with each other, but they formed no connections with the PL1 and PL2 neurons. It is concluded that in the case which was under study cellular intrinsic properties were sufficient for the formation of ‘correct’ connections between neurones.

Vascular smooth muscle cells grown on Matrigel. A model of the contractile phenotype with decreased activation of mitogen-activated protein kinase.

Vascular smooth muscle cells have been shown to exist in two phenotypic states which have been designated proliferative and contractile. The properties of rat aortic vascular smooth muscle cells grown on Matrigel were compared with cells grown on untreated plastic culture dishes. Cells grown on Matrigel manifested at least four important properties characteristic of the contractile phenotype as compared with cells grown on plastic. The cells grown on Matrigel had altered morphology similar to in vivo studies of contractile vascular smooth muscle. The cells had a low proliferative index, showed enhanced levels of the smooth muscle isoform of alpha-actin, and had an enhanced contractile response to the vasoconstrictor arginine vasopressin. All of these changes were maintained through at least five passages and could be reversed by replating cells grown on Matrigel back to uncoated plastic dishes. Changes in post-receptor signaling pathways which could account for the altered physiologic responses of the cells were investigated. Cells grown on Matrigel showed no alterations in agonist-induced mobilization of intracellular Ca2+ or agonist-stimulated cAMP levels. However, stimulation of mitogen-activated protein kinase (MAP kinase) by both vasoconstrictors and growth factors was inhibited by 50% in cells grown on Matrigel as compared with plastic. This decrease in agonist-induced MAP kinase was associated with a decrease in the levels of both p42 and p44 MAP kinase protein and a decrease in tyrosine phosphorylation of both isoforms in cells grown on Matrigel. Alterations in MAP kinase activation can account at least in part for the observed physiologic responses of contractile vascular smooth muscle. Growth of vascular smooth muscle cells on Matrigel represents a useful model to examine phenotypic-dependent alterations in post-receptor signaling.

Keratinocytes Are Involved in Regulating the Developmental Changes in the Proliferative Activity of Mouse Epidermal Melanoblasts in Serum-Free Culture

When epidermal cell suspensions derived from 0.5-, 2.5-, and 4.5-day-old mice were plated onto uncoated polystyrene dishes and cultured with serum-free medium supplemented with dibutyryl cyclic adenosine 3′,5′-monophosphate and basic fibroblast growth factor, melanoblasts proliferated dramatically around keratinocyte colonies and after 12-14 days pure and enriched cultures of melanoblasts (ca. 75%) and melanocytes (ca. 25%) were obtained. In contrast, when epidermal cell suspensions derived from 7.5-, 20.5-, and 60.5-day-old mice were cultured similarly, keratinocytes failed to attach to the dish and melanoblasts did not proliferate at all. However, when epidermal cell suspensions of older mice were plated onto type I collagen-coated dishes and cultured similarly, keratinocytes attached well to the dish and melanoblasts proliferated dramatically around keratinocyte colonies. Moreover, pure melanoblasts and melanocytes derived from primary cultures of young and old mice could be subcultured on collagen-coated dishes with the medium in the presence of secondary keratinocytes that were subcultured from a pure population of primary keratinocytes. No differences were observed in the proliferative activity of secondary melanoblasts between young and old mice. These results suggest that keratinocytes are involved in regulating the proliferation of mouse epidermal melanoblasts and that the developmental changes in the proliferative activity of epidermal melanoblasts in culture are due to the developmental changes in the substrate attachment and proliferation of keratinocytes, rather than to intrinsic changes in melanoblasts.

Establishment and immunological characterization of cultured human gallbladder epithelial cells

Biliary epithelial cells are a primary site of damage in liver allograft rejection and in immunologically mediated diseases such as primary biliary cirrhosis. Human leukocyte antigens and adhesion molecules on the biliary epithelium are associated with T-lymphocytic binding, recognition and destruction. To investigate relevant cellular immunological mechanisms under standard conditions, we have established an in vitro model using human gallbladder epithelial cells. Although not directly affected in these aberrations, gallbladder epithelial cells are excellent objects for immunological investigations. More than 10(8) highly purified cells were isolated and cultured longer than 6 wk in confluent monolayers. Cell growth was routinely established on uncoated plastic culture dishes, and serum-free media could be applied for immunological experiments. Cell characterization was performed by means of specific monoclonal antibodies typical for biliary epithelial cells. Cytokeratins 1 through 8, 18 and 19 and human epithelial cell antibody 125 always showed strong positive staining. Antigen patterns were examined before and after treatment with interferon-gamma by use of immunohistochemical staining methods. Untreated human gallbladder epithelial cells expressed human leukocyte class I antigens but few or no class II antigens. After stimulation with interferon-gamma induction of human leukocyte antigen-DR, -DP and -DQ was detected. In addition, intercellular adhesion molecule 1 was induced on these gallbladder epithelial cells. Therefore an immunological competence similar to that of biliary epithelial cells could be demonstrated. In vitro cell cultures of gallbladder epithelial cells offer a promising tool for subsequent investigations concerning intrahepatic biliary epithelial cells and their interactions with cells of the immune system.

Glucose Transporter-mediated Dynamic Attachment of Erythrocytes onto a Reducing Glucose-carrying Polystyrene.

Erythrocytes, which carry type-1 glucose transporters (GLUT-1) in their plasma membrane, were found to attach specifically and strongly to dishes which had been coated with a polystyrene derivative (PVG) substituted with reducing glucose moieties. The concave erythrocytes took teardrop shapes, stood on the rim, and swayed with gentle disturbance in the medium. In contrast, erythrocytes attached by nonspecific hydrophobic interaction on uncoated polystyrene dishes assumed fiat morphology and remained unperturbed by movements in the medium. The attachment to PVG substratum was suppressed by (1) GLUT-1 inhibitors such as phloretin, phloridzin, and cytochalasin B, and (2) PVG molecules in solution, and (3) at 4°C, suggesting that the specific recognition of high density of glucose on PVG and the dynamic movement of GLUT-1 are essential for the attachment. This novel combination of glucose transporter-carrying cells and reducing glucose-carrying polymer may lead to biomaterials with a wide range of applications.

Senescence of hepatocytes isolated from rainbow trout (Oncorhynchus mykiss) in primary culture

In order to evaluate the suitability of isolated fish hepatocytes for toxicological studies, hepatocytes were isolated from rainbow trout (Oncorhynchus mykiss) by liver collagenase perfusion. Isolated hepatocytes were investigated for seven days in primary culture on uncoated petri dishes using light and electron microscopy. Viability of isolated hepatocytes, as estimated from trypan blue exclusion, declined from >90% at the beginning of the incubation to ⩽80% after eight days of primary culture. Survival of hepatocytes was best at an incubation temperature of 14°C, and addition of fetal calf serum failed to improve cell performance. Freshly isolated hepatocytes appeared as solitary spherical cells with numerous microvilli at the outer surface. Except for a 30% reduction in cell size due to stress-induced glycogen reduction, the ultrastructure of freshly isolated hepatocytes closely resembled that of rainbow trout hepatocytes in vivo, which is characterized by distinct cytoplasmic segregation into a perinuclear portion containing rough endoplasmic reticulum (RER) arranged in extensive stacks, mitochondria, peroxisomes and the peribiliary complex (dictyosomes, lysosomes), and large peripheral glycogen fields occasionally interspersed with small lipid inclusions. Within one day of culture, about 60–80% of the isolated hepatocytes sedimented to form a monolayer attached to the culture dishes, whereas up to 20% remained in suspension forming hepatocyte aggregates. Cell adhesion was weak, and during prolonged culture increasing amounts of cell detached, whereas the floating cell accumulations grew to aggregates of more than 100 cells. Cell viability and ultrastructure was similar in monolayers and spheroids, and only from the fifth day in culture, did hepatocytes in the centre of floating aggregates become necrotic. From day 1 to day 5, hepatocytes in primary culture displayed only minor cytological alterations. Excellent cytoplasmic compartmentation, restoration of hepatocytic glycogen stores, high secretion rates of very low density lipoproteins by dictyosomes, establishment of cell-to-cell contacts, restitution of cellular polarity and the epithelial character of the cells, as well as formation of bile canaliculi documented recovery of the hepatocytes in primary culture. From day 5 in culture, an increasing number of cells detached from the substratum, and cell senescence was indicated by a marked increase in ultrastructural heterogeneity, with progressive vesiculation and fractionation of the RER, transformation of RER stacks into huge membrane whorls, aggregation and proliferation of peroxisomes and SER, lack of dictyosomal VLDL production, drastic accumulation of lysosomes, myelinated bodies and autophagic vacuoles, as well as successive exhaustion of cellular glycogen deposits. Whereas with conventional methods for assessing cell viability, most hepatocytes appeared intact for up to ten days in culture, cytological investigations revealed severe deterioration of cellular integrity from day 7. However, for incubation periods of up to five days, isolated rainbow trout hepatocytes can be recommended as an excellent model for physiological and toxicological studies.

Role of extracellular matrix molecules in the development of the sodium current in quail mesencephalic neural crest cells.

The occurrence of the voltage-dependent sodium current has been studied in developing neurons from quail mesencephalic neural crest on different substrates, using the whole-cell patch clamp technique. Explants from 9–12 somite embryos were cultured on dishes coated with type I collagen, fibronectin, laminin or on plastic dishes in a chemically defined medium. After 18 h of culture the sodium current was observed in 70% of the neurons tested, and at 24 h some of these neurons were able to generate an action potential. After 18–25 h cells grown on fibronectinor collagen I-coated dishes showed a significantly higher occurrence of the sodium current (83% and 84% respectively) as compared to cells grown on uncoated plastic dishes (51%). Moreover, in the presence of fibronectin, the current density of the sodium current was more than doubled in comparison with cells grown on other substrates.

Collagen increases the synthesis of membrane‐associated proteoglycans produced by sertoli cells

Sertoli cells in culture produce two isoforms of proteoglycans which are found in the culture medium and associated with the cell membrane. The amount of both types of proteoglycans increased when Sertoli cells were plated on type I collagen-coated dishes as compared to uncoated dishes. The effect is due to an increase in the synthesis of proteoglycans rather than a diminished rate of degradation of these molecules. The collagen substrate also affects the distribution of these macromolecules; an increase in the amount of membrane-associated proteoglycans occurs at the expense of the proteoglycans released to the culture medium.

Complex cytokine modulation of a continuous line of mink lung epithelial cells (Mv1Lu).

The continuous mink lung epithelial cell line Mv1Lu has proven to be a sensitive reporter line in the bioassay for purified TGF-beta, exhibiting a sigmoid-shaped concentration-response relationship with an EC50 of 12 pM (0.3 ng/mL). Maximal inhibition of Mv1Lu cells generates a 75-95% decrement in the number of adherent cells. However, this bioassay is not specific for TGF-beta as originally claimed. Mv1Lu cells are sensitive to other cytokines and substances found in complex biological fluids. In this study the effects of other biological response modifiers in this assay were tested and several were found to have important growth modulatory capacities that confound the quantitation of TGF-beta. EGF, TGF-alpha, fibronectin, and IGF-I all induce Mv1Lu cell proliferation. In contrast, neither PDGF (-AA, -AB, -BB) nor endotoxin (< or = 10 micrograms/mL) affect Mv1Lu cell number. TGF-beta and TNF-alpha at high concentrations (> or = 10 ng/mL) are the only cytokines examined that inhibit Mv1Lu proliferation. TGF-beta decreases final cell number both by preventing mitosis and by inhibition of adherence of cells to the uncoated dish. Several strategies are suggested to assure the specificity of this otherwise convenient bioassay for TGF-beta.