Glycinergic transmission regulates dendrite size in organotypic culture

Interest in the potential adverse biological effects of exposure to power-frequency magnetic fields has centred on the possibility that these fields may influence tumour promotion, possibly by increasing the rate of cell proliferation. In order to investigate whether exposure to magnetic fields can indeed affect the rate of cell proliferation, normal human fibroblasts were serum starved overnight and then exposed to 50 Hz magnetic fields in a purpose-built facility. The rate of DNA synthesis was taken as a measure of cell proliferation, and was determined by following the incorporation of [3H]-thymidine into macromolecular material. The rate of DNA synthesis in exposed cells was compared with that in control cultures maintained in a standard CO2 incubator where they were exposed to background magnetic fields of < 200 nT. Positive controls were maintained in the same CO2 incubator, but were treated with human recombinant fibroblast growth factor to check that the cells were responsive to growth stimuli. Magnetic fields at 50 Hz and at a range of flux densities between 20 microT and 20 mT had no detectable effect on the rate of DNA synthesis by cells exposed for up to 30 h.     

Neuromuscular responses to pyridostigmine bromide in organotypic spinal cord-muscle culture

Pyridostigmine bromide (PB) promotes and then silences cholinergic muscle activity, and disrupts the junctional regions of muscle fibers and associated nerve terminals, in organotypic mouse spinal cord-muscle cultures continuously treated with low concentrations of the drug for up to 14 days. Spontaneous muscle activity is restored within 1 week of drug removal.     

Long-term culture of organotypic multicellular glioma spheroids: a good culture model for studying gliomas

Gliomas, as well as other solid tumours, contain tumour stroma composed of connective tissue, macrophages, capillaries and other non-cellular constituents. Therefore, a homogeneous culture of tumour cells alone, as is often used as a culture model for gliomas, is not ideal to study all aspects of gliomas. In the present study we describe an alternative culture model, i.e. organotypic multicellular spheroids (OMS), that histologically closely resembles the tumour in vivo. Glioma explants, obtained at surgery from five patients, were cultured on agarose to form OMS, which were cultured for up to 16 weeks. At regular intervals, OMS were fixed and histological and immunocytochemical analyses were carried out. The histology as well as the immunocytochemical characteristics of the OMS proved to be almost unchanged after a culture period of 16 weeks. In contrast to monolayer cultures, glial fibrillary acidic protein (GFAP) expression in the OMS is preserved after 16 weeks of culture. However, in OMS from three out of five patients, small GFAP-negative cells appeared in the outer cell layers between 1 and 2 weeks of culture. Furthermore, after about 6 weeks of culture, the capillaries disappeared from the OMS. After prolonged culture, tumour cell heterogeneity, the cellular composition, and the histology of the OMS still closely resembled the tumour in vivo. It is suggested that OMS provide a good long-term culture model for the study of gliomas.     

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

Secondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4-8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7-10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.     

A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays

Spatiotemporally coordinated activity of neural networks is crucial for brain functioning. To understand the basis of physiological information processing and pathological states, simultaneous multisite long-term recording is a prerequisite. In a multidisciplinary approach we developed a novel system of organotypically cultured rat hippocampal slices on a planar 60-microelectrode array (MEA). This biohybrid system allowed cultivation for 4 weeks. Methods known from semiconductor production were employed to fabricate and characterize the MEA. Simultaneous extracellular recording of local field potentials (LFPs) and spike activity at 60 sites under sterile conditions allowed the analysis of network activity with high spatiotemporal resolution. To our knowledge this is the first realization of hippocampus cultured organotypically on multi-microelectrode arrays for simultaneous recording and electrical stimulation. This biohybrid system promises to become a powerful tool for drug discovery and for the analysis of neural networks, of synaptic plasticity, and of pathophysiological conditions such as ischemia and epilepsy.     

High-density hybridoma perfusion culture. Limitation vs inhibition

Because our earlier work indicated a strong correlation between specific antibody productivity and cell density in perfusion culture, we conducted experiments to determine the optimum means of increasing cell density while maintaining high antibody productivity. The rates of medium supply and waste removal were varied to determine whether cell density was limited or inhibited, and whether a diffusable substance could be responsible for the correlation between antibody productivity and cell density. Nutrient supply was found to be a stronger determinant of cell density than waste removal; however, the rate of waste removal had a greater effect on cell growth at lower cell densities. Even at noninhibitory levels of ammonia and lactate, cellular metabolism was regulated to minimize their concentrations at lowered rates of waste removal. Separate step changes in glucose and glutamine resulted in increased cell density and antibody concentration. Specific antibody productivity increased following the step in glutamine, but not glucose. Both steps caused changes in cellular metabolism that prevented the levels of lactate and ammonia from reaching toxic levels.     

Tissue culture of middle ear epithelium using fibroblast-reorganized collagen gels

The middle ear epithelium of the guinea pig has been cultured on a modified floating collagen matrix. Fibroblasts harvested from abdominal skin dermis of allogenic animals were used to reorganize hydrated collagen gels into a dermal-like matrix. Within two days, the explants placed on the surface of these matrices showed proliferation of polygonal "outgrowth cells" which could be maintained for up to one month. During the first two weeks of the culture period, dividing cells were frequently identified. These cells were especially abundant in the marginal area of the explants and consisted of pseudostratified columnar cells. The reorganized collagen gel matrix was stable and did not dissolve even after the "outgrowth cells" became confluent. The present system should allow further elucidation of the growth and differentiation mechanisms of middle ear epithelial cells.     

A new organotypic culture method to study the actions of steroid hormones on the nervous system

A new organotypic culture method for growing slices of nervous system tissue, based on the use of hyaluronic acid as a growth supporting milieu, is described. This method allows cultures derived from either fetuses or newborns to grow and develop with markedly reduced amounts of added serum. Organotypic cultures from fetal rat hypothalamus were exposed to 17 beta estradiol and compared to control cultures exposed to the ethanol vehicle. When exposed to estradiol, cultures showed an outgrowth of thick nerve fibers that was accompanied by an elevation in the number of microtubules present in the neuronal processes, an increment in the number of synapses, and an increased morphological differentiation of synaptic terminals. Freeze-fracture analysis of neuronal membranes from estradiol-treated cultures revealed a significant increase in the number of exoendocytotic images and a decrease in the number of intramembranous particles. Estradiol's effects parallel those found in in vivo studies, indicating that hyaluronic acid-based organotypic cultures represent an appropriate model to study hormonal influences on the developing nervous system.     

Delayed protection by MK-801 and tetrodotoxin in a rat organotypic hippocampal culture model of ischemia

BACKGROUND AND PURPOSE: The hippocampus demonstrates a regional pattern of vulnerability to ischemic injury that depends on its characteristic differentiation and intrinsic connections. We now describe a model of ischemic injury using organotypic hippocampal culture, which preserves the anatomic differentiation of the hippocampus in long-term tissue culture. METHODS: Ischemic conditions were modeled by metabolic inhibition. Cultures were briefly exposed to potassium cyanide to block oxidative phosphorylation and 2-deoxyglucose to block glycolysis. The fluorescent dye propidium iodide was used to observe membrane damage in living cultures during recovery. RESULTS: 2-Deoxyglucose/potassium cyanide incubation resulted in dose-dependent, regionally selective neuronal injury in CA1 and the dentate hilus, which began slowly after 2 to 6 hours of recovery. Subsequent histological examination of cultures after 1 to 7 days of recovery demonstrated neuronal pyknosis that was correlated with the early, direct observation of membrane damage with propidium. Both propidium staining and histological degeneration were prevented by the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 when administered 30 minutes after the end of the exposure to 2-deoxyglucose and potassium cyanide. Tetrodotoxin, which blocks voltage-dependent sodium channels, had protective effects that were greatest during the period of 2-deoxyglucose and potassium cyanide incubation but also produced protection against the mildest conditions of metabolic inhibition when administered after 30 minutes of recovery. CONCLUSIONS: This in vitro model reproduced elements of the time course, regional vulnerability, and pharmacologic sensitivities of in vivo ischemic hippocampal injury. Inhibition of metabolism in organotypic culture provides a rapid, easily controlled injury and reproduces the in vitro pattern of hippocampal regional vulnerability to ischemia. It is the first in vitro model of ischemia to exhibit complete protection by delayed administration of an NMDA receptor antagonist during recovery from a brief insult. The protective effects of tetrodotoxin suggest that an early period of sodium entry into cells during and after ATP depletion may be responsible for the more prolonged period of toxic NMDA receptor activation.     

The effect of activated macrophage products on neurite growth in an organotypic culture of chick embryo sensory ganglia]

Murine peritoneal macrophages, activated by BCG vaccine, and human peripheral blood monocytes, activated by lipopolysaccharides, exerted neurite stimulating or neurite inhibiting effects in various periods of activation. The supernatants of these preparations were active in organotypic culture of chick embryo dorsal root ganglia. The inhibition of neurite growth on the 1st day of cultivation was followed by the neurite-stimulating effect. The fluctuation of neurite-inhibition and neurite-stimulation effect of macrophage supernatants suggest the availability of certain changes in cytokine composition in different periods of macrophage activation.     

Effect of human uremic serum and urea or creatinine containing serum on organotypic nervous tissue culture. Light microscopy studies

The mandibular glands of Kalotermes were examined in different castes. They show sexual dimorphism in the soldiers and primary reproductives, Moreover, in female soldiers and queens, mandibular gland cells contained numerous crystalline structures of mitochondrial origin. The role of these glands (secretion of saliva or pheromone) is discussed.     

Retroviral vectors efficiently transduce basal and secretory airway epithelial cells in vitro resulting in persistent gene expression in organotypic culture

Gene therapy of the lung requires the introduction and expression of a therapeutic gene in airway cells. Although retroviral vectors may be useful in this context, the ability of retroviruses to infect specific cell types in the airway is not known. In this study, we examined the ability of amphotropic recombinant retroviral vectors to transduce primary cultures of rabbit airway epithelial cell populations purified for basal or secretory cells. Transduction efficiencies in basal and secretory cell populations were found to be similar; about 27% after a single exposure to vector, and up to 77% after multiple exposures. The fate of genetically modified cells from the different populations was followed through terminal differentiation using organotypic cultures. The epithelium of the organotypic cultures generated from each population exhibited both pseudostratified and stratified morphology, produced mucin, and stained positively with antibodies specific for basal and ciliated cells. The mucociliary epithelium also showed co-localization of these phenotypic markers with the expression of the vector-encoded beta-galactosidase gene. We conclude that retroviruses can efficiently transduce primary cultures of basal and secretory cells, and that both of these cell types can be progenitor cells of the airway epithelium. In vivo delivery of a retroviral vector containing a human placental alkaline phosphatase gene resulted in expression of the heterologous gene in rabbit tracheal epithelial cells. However, transduction efficiency was low and occurred only in the wounded trachea.     

Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films

Thin films of 50:50 and 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 microm. The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line. Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls. The initial cell seeding density affected attachment, and the optimal value for 50:50 PLGA was 25000 cells cm(-2). After 7 days of in vitro culture, cell density on 50:50 and 75:25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS. The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology. Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions. These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.     

Regeneration of bronchial epithelium on chronic inflammatory changes under laser treatment

Investigation of structural and metabolic changes in 188 biopsy specimens from the epithelium of large bronchi of 76 patients with chronic lung disease was carried out. It was shown that endobronchial treatment by helium-neon laser irradiation induced the proliferative and metabolic processes in damaged epithelium, which after getting through the series of transitional forms restores its normal structure and differentiation into ciliated and goblet cells of a normal ultrastructure. The hyperemia, intensive diapedesis of leucocytes, the formation of leucocytic infiltrations and granulations develop in the lamina propria of the bronchial mucosa. Proliferative and metabolic activity of the endotheliocytes and stromal cells increases. Finally, delicate-fibrous connective tissue forms. The simultaneous reorganizations of the epithelium and underlying connective tissue is interpreted from the point of view of the concept of parenchymal-stromal interactions.     

Myelination in organotypic cultures of sympathetic ganglia

Explants of mouse superior cervical ganglion (SCG), co-cultured with dorsal spinal cord, were grown for up to 4 weeks in vitro. In such cultures, scattered internodes of peripheral nervous system (PNS) myelin were observed, apparently associated with SCG neurites. Although rare, the incidence of PNS myelination in this system might merit further experimentation to provide a model facilitating the evaluation of postganglionic sympathetic myelination, which in vivo may be both extensive and morphologically unusual.     

The hemodynamic changes during the intraperitoneal hyperthermic perfusion under induced hypothermia

We investigated the whole body oxygen consumption (VO2) and the hemodynamic changes during the intraperitoneal hyperthermic perfusion (IPHP), which was coupled with induced hypothermia to prevent the cerebral disorder. IPHP was carried out for 90-120 min with 45-47 degrees C perfusate after the operation. We induced hypothermia using the surface cooling method and the infusion of triflupromazin. In no patient, the pulmonary artery temperature (PAT) rose above 40 degrees C. In the IPHP, there was a significant correlation between VO2 and PAT. If PAT reached 42 degrees C during the IPHP, VO2 would increase to 130-140% of the value at 37-38 degrees C. This rise is smaller than that during the total body hyperthermia (TBH), in which VO2 at 42 degrees C reached 130-190% of the value at 38 degrees C. Heart rate increased in proportion to the rising rate of body temperature. During the IPHP, PAT sometimes rose remarkably about 8 degrees C (from 32 degrees C to 40 degrees C) with a marked rise in heart rate. This rising rate of PAT is greater than that of TBH, in which PAT rose about 4-5 degrees C (from 37-38 degrees C to 42 degrees C). We consider that IPHP is not applicable to the patients with ischemic heart disease. During the rise of PAT, other circulatory parameters related to IPHP, changed in the same direction as those related to TBH. The rate of change of these parameters related to IPHP was smaller than that of the TBH, because during the IPHP the highest PAT was lower than that during TBH.     

Markers for neuronal degeneration in organotypic slice cultures

This protocol describes ways of monitoring spontaneous or induced neuronal degeneration in organotypic brain slice cultures. Hippocampal cultures (4-week-old) are grown in normal serum-free control medium, or exposed to the neurotoxin trimethyltin (TMT) (0.5-100 microM) for 24 h or the excitotoxic glutamate agonist kainic acid (KA) (5-25 microM) for 48 h followed by 24 h or 48 h, respectively, in normal medium. Corticostriatal slice cultures (also 4-week-old) are exposed to KA (6-24 microM) for 48 h and normal medium for control. The resulting neurodegeneration is estimated by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux to the culture medium, (c) ordinary Nissl cell staining, (d) staining by the neurodegenerative marker Fluoro-Jade (FJ), (e) neuronal microtubule degeneration by immunohistochemical staining for microtubule-associated protein 2 (MAP2), and (f) Timm sulphide silver staining for heavy metal alterations. Both hippocampal and corticostriatal slice cultures show a dose- and time-dependent increase in PI uptake and LDH efflux after exposure to TMT and KA. The mean PI uptake and the LDH efflux into the medium correlate well for both types of cultures. Both TMT and KA exposed hippocampal cultures display in vivo patterns of differential neuronal vulnerability as evidenced by PI uptake, FJ staining and MAP2 immunostaining. Corticostriatal slice cultures exposed to a high dose of KA display extensive striatal and cortical degeneration in FJ staining as suggested by a high PI uptake. A change in Timm sulphide silver staining in deep central parts of some control cultures, corresponds to areas with loss of cells in cell staining, loss of MAP2 staining, PI uptake, and FJ staining. We conclude that organotypic brain slice cultures, in combination with appropriate markers in standardized protocols, represent feasible means for studies of excitotoxic and neurotoxic compounds.