Interaction of epithelial cells and fibroblasts in culture: Effect on glycosaminoglycan levels

Twelve- to fifteen-day chick embryo liver cells (epithelial) were cultured on top of confluent chick embryo fibroblasts to produce an in vitro model of an epithelial-mesenchymal interacting system. This cocultivation resulted in a marked increase in hyaluronic acid (HA) levels and a decrease in chondroitin sulfate (CS) levels, either in total or in proportion to HA, compared with the two cell types cultured separately. The liver cells cultured alone produced little or no detectable glycosaminoglycans (GAGs). Cocultivation of increasing numbers of liver cells with fibroblasts resulted in a progressive increase and decrease of HA and CS levels, respectively, and the combined effect of these changes was a progressive increase in the HA/CS ratio. Fibroblasts cultured in liver-cell-conditioned growth medium also showed increased levels of HA, but in contrast to cocultivation, an increase in CS and no change in the HA/CS ratio. Liver cells cultured in fibroblast-conditioned growth medium showed no changes in GAG level. This suggests that under conditions of cocultivation the fibroblasts alone could be responsible for the increased HA levels and that the decreased CS levels are a result of conditions produced by the close proximity of the two cell types. In vivo most connective tissues immediately adjacent to epithelial tissues are also characterized by a matrix rich in HA and these results support the concept that some epithelial tissues are able to modulate the GAG composition of adjacent connective tissues and thereby affect their immediate extracellular environment.     

Interaction of solid liposomes with epithelial cells

Binding of fluorescein-containing solid liposomes to cultured epithelial cells was studied. It was found that liposomes were preferentially attached to the contact-free lateral edges of epithelial cells but not to the upper surfaces of these cells and not to the edges forming lateral cell-cell contacts. This preferential binding to cell margins was characteristic only of the solid liposomes but not of the fluid ones. Binding of solid liposomes to the cells could be prevented and reversed by trypsin. Possible mechanisms of special adhesive properties of the surface of contact-free cell edges are discussed.     

Changes in myofibrils and cytoskeleton of neonatal hamster myocardial cells in culture: an immunofluorescence study

Myocardial cells in culture offer many possibilities for studying cellular and molecular biology of cardiac muscles. However, it is important to know how long these cells can be maintained in vitro without significant structural and biochemical changes. In this study, we have investigated the morphological changes of myofibril proteins and cytoskeletons by using immunofluorescent techniques in cultured neonatal hamster myocardial cells at different culture durations. Our results have demonstrated that these cultured cells still contain intact myofibrils and cytoskeletal proteins after 6 days in vitro incubation, however, the organization of some of these proteins is altered. The proteins most sensitive to these in vitro conditions are: myosin heavy chain, actin and desmin. The data indicate that the duration of the culture and the contractile activity of the myocardial cells in culture can influence organization of their contractile apparatus and cytoskeleton.     

An immunofluorescence study of neurofilament protein expression by developing hippocampal neurons in tissue culture

We have studied the development of intermediate filament proteins in the neurons found in hippocampal cell cultures using single and double label immunofluorescence with both monoclonal and polyclonal antibodies. Neurons in these cultures are known to differentiate in a manner similar to their counterparts in situ: in particular they develop axonal and dendritic processes which differ from each other in form, in ultrastructure, and in synaptic polarity. During the first days in culture, developing neurons could not be stained with antibodies against any of the neurofilament proteins, although many cells reacted with anti-vimentin. Later in the first week, antibody staining revealed clearly filamentous staining for the L (68 000 daltons) and the M (145 000 daltons) neurofilament subunits, though M reactivity was much stronger at this earlier stage of development. Some neurofilament positive profiles in many cells could also be stained with vimentin, though the vimentin immunoreactivity became progressively less pronounced during further development, and disappeared after about two weeks in culture. Also at about two weeks in vitro we noted the first appearance of neurofilament H protein (200 000 daltons) immunoreactivity, which was localized to a subset of long neurites which could be identified on morphological grounds as axons. These processes lacked staining for microtubule associated protein 2 (MAP2), a dendritic marker. They tended to be close to islands of glial cells, suggesting that H induction may require complex neuron-glial interactions. These results are consistent with the suggestion that H protein immunoreactivity is a marker for axonal outgrowth. In addition to obvious filamentous staining, we were able to localize neurofilament antigens to an interesting class of small ring-like structures, found increasingly frequently as the cultures aged. We also present evidence that tyrosinated alpha-tubulin is present both within dendrites and axons of neurons in these cultures.     

Interaction of enteropathogenicEscherichia coli with host epithelial cells

EnteropathogenicEscherichia coli (EPEC) causes severe diarrhea in young children. Upon infection, EPEC induces the assembly of highly organized pedestal-like actin structures in host epithelial cells. All the EPEC genes that are involved in inducing formation of actin pedestals are located in a unique 35 kbp chromosomal pathogenicity island, termed LEE. These genes include thesep genes that encode components of type III protein secretion system, and genes that encode proteins secreted by this system, theesp genes. This protein secretion system is activated upon contact with the host cell, resulting in increased secretion of Esp proteins. Some of these Esp proteins form the translocation apparatus while others are translocated into the cytoplasm of the host cell. Concerted activity of the LEE genes including theeae, esp and thesep genes is needed to trigger signal transduction in the host cell which results in formation of an actin pedestal. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

Plasminogen activating enzyme in cultured glioblastoma cells. An immunofluorescence study with monoclonal antibody

A monoclonal antibody against a 52,000 dalton human plasminogen activating enzyme (HPA52) was used for immunofluorescence staining of cultured glioblastoma cells. The fluorescence was located in the cytoplasm of the cells. A pronounced variation in the staining intensity was observed between the individual cells. The specificity of the fluorescent stain was supported by the findings that 1) no staining was obtained with a monoclonal antibody of the same subclass, but with irrelevant specificity (anti-2,4,6-trinitrophenyl); 2) adsorption with HPA52 purified to homogeneity removed the ability of anti-HPA52 to mediate staining; 3) the glioblastoma cells contained HPA52, as measured by enzymatic assay, while melanoma cells that were not stained did not contain HPA52 activity; 4) dexamethasone reduced both the enzymatically determined HPA52 content and the immunofluorescence in parallel, while progesterone affected none of these parameters; 5) we have previously found that culture fluid conditioned by the glioblastoma cells apart from HPA52 does not contain detectable amounts of any protein that binds to anti-HPA52. Several advantages of immunohistochemical detection of plasminogen activators compared with enzyme histochemical methods are discussed, among these that the immunohistochemical method distinguishes between plasminogen activators of different types.     

Differentiation of normal human mammary epithelial cells in culture: an ultrastructural study.

An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg++- dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. (2) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg++-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. (3) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were interconnected by a larger number of desmosomes with numerous pleomorphic microfilaments. The Mg++-dependent ATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days were prominent in the cells of the stratified layer. After 28 days in the culture, the cells began to round up and slough off the culture plate.     

Uterine epithelial cells in primary culture: a model system to study cell proliferation

Guinea-pig uterine glandular epithelial cells were grown in primary culture. During the 4-day initial culture period, a 6.7 fold increase in DNA synthesis and a doubling time of approximately 30 hours were observed. Then the cells were submitted to serum depletion (60 hours) and the quiescent cells were stimulated with 15% fetal calf serum (FCS). The control cells were submitted to 1% heated and dextran-coated charcoal stripped FCS. In stimulated cells, the DNA synthesis increased and peaked between the 12th and 24th hour. In these cells, c-fos mRNAs increased rapidly, within 30 min., peaked at 75 min. (ratio to the control = 2.5), and returned to basal level within 90 min. These results prove that uterine epithelial cells in primary culture are able to respond to unspecific mitogen by both rapid expression of c-fos gene and DNA synthesis, suggesting that this cell culture system will be useful in studying the growth regulation in endometrium.     

Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.     

Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells

The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.     

Tumor promoters: effects on proliferation and differentiation of cells in culture.

Tumor promoters enhance tumor formation when administered after an initiating action by a carcinogen. The phorbol diester class of tumor promoters has been shown to affect many biochemical and biological processes in mouse skin and cell culture. The effects of these compounds on the proliferation and differentiation of cells in culture are reviewed herein; the possible relation of these effects to the mechanism of tumor promotion are discussed.     

Electrostatic potentials of tumour promoters

Molecular-modelling studies and quantum-chemical calculations have been undertaken on a variety of structurally unrelated tumour promoters. The modelling studies reveal some broad areas of agreement in the associated electrostatic potentials for the molecules TPA, teleocidin, aplysiatoxin and ingenol. Azulene-type derivatives were used as models for inactive and active tumour promoters in the quantum chemical studies. The results obtained from these calculations of the electrostatic potentials show very little difference between active and inactive compounds, thus suggesting that other factors are of importance in these systems.     

An inhibitory effect of tumour promoters on human epithelial cell growth can be dissociated from an effect on junctional communication

Studies with rodent cells have indicated that the abilities of various tumour promoters to inhibit metabolic cooperation correlate with their potencies as mitogens. Here we have examined the effects of the most potent phorbol ester tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), on metabolic cooperation and growth of human epidermal cells transformed by SV40 (SVK14 cells). In this system, TPA inhibits Junctional communication and at the same concentration also inhibits growth in a reversible fashion. These effects appear to be mediated by binding of phorbol ester to a single class of high affinity binding site with a Kd similar to that reported for rodent cells (K_d = 20.9 nM at 4 °C). Further studies on the effects of phorbol esters on other human epithelial cell lines reveal that the inhibitory effects of TPA on growth and metabolic cooperation may be completely dissociated. Alternative mechanisms by which TPA may exert its growth-inhibitory effects are discussed.     

Interaction of cell culture with downstream purification: a case study

Separation of product from secreting mammalian cells in the culture broth means the transition from product generation to product isolation. This interface within a biotech production process has to perform a proper solid/liquid phase separation of the cell suspension to make the product containing fluid amenable for further purification. These subsequent steps require fluid with low occurence of contaminants in order to function properly. The goal of this study was to evaluate some economic and fast cell separation methods for the preparation of a product fluid ready for use in further ultrafiltration and chromatographic processes. We have performed experiments to test the usefulness of disc stack centrifuges and tangential flow microfiltration units at large scale. Both systems revealed outstanding prospects with regard to throughput and scale up properties. However, the centrificgation did not lead to a fluid sufficiently free of particles for direct ultrafiltration or chromatography. Thus, an additional filtration step was necessary. On the other hand microfiltration led to an acceptable quality of process fluid directly. By optimisation of process parameters an effective, reproducible and robust cell separation can be obtained. However, our experience has been that such optimal conditions are somewhat specific for a narrow range. Thus, even the equipment functioning well with one type of cell would possibly not perform as well with another cell or even with the same cell under conditions slightly different to the usual situation.