PC12 cells grown on cellulosic filters differentiate in response to NGF and exhibit a polarity not seen when they are grown on solid substrata

Summary Studies were performed with cellulosic filters and standard culture plates to compare methods of cell culture and differentiation of the cell line PC12, a clone originating from a rat pheochromocytoma. PC12 cells respond to nerve growth factor (NGF) by flattening of the cell body and subsequent extension of neurite-like processes. When PC12 cells are cultured in dishes without NGF, they have a diameter of approximately 3 to 7 μm and exhibit short processes of no longer than 3 to 5 μm. If PC12 cells are grown on a cellulosic filter they have the same average soma diameter and similar short processes extending laterally, but in addition have branching processes which extend as far as 10 to 15 μm into the filter substrate. When dish-cultured and filter-cultured cells are incubated with 50 ng/ml NGF they both exhibit differentiation-specific ultrastructural changes by 3 d of treatment. In the case of dish-cultured cells, large cytoplasmic processes exhibit an increase in the number of chromaffin cell-like secretory granules by 3 d of treatment. This characteristic is also demonstrated by filter-cultured cells, but the processes containing these granules are found concentrated within the cellulosic meshwork. Thus the timing of the NGF-elicited differentiation program is similar to both filter-cultured and dish-cultured cells, but the ultrastructural consequences are different. The filter-cultured PC12 cells exhibit a polarity not demonstrated by dish-cultured cells. Growing PC12 cells on cellulosic filters is a technique useful for “anchoring” neurons without the complication of the addition of extracellular matrix components. Filter-culture may represent a more in vivo-like method for studying neuronal growth and differentiation. This work was supported by grants S07RR07149-13 to R.V.B. and CA40929 to J.A.W. from the National Institutes of Health, Bethesda, MD.     

Effect of an exo-polysaccharide from the culture broth of Hericium erinaceus on enhancement of growth and differentiation of rat adrenal nerve cells

It was found that an exo-biopolymer (M.W. 1,000,000, molar ratio of 1.5:1.7:1.2:0.6:0.9, glucose:galactose:xylose:mannose:fructose, purity 99%) purified from the liquid culture broth of Hericium erinaceus mycelium enhanced the growth of rat adrenal nerve cells. The polymer also improved the extension of the neurites of PC12 cell. Its efficacy was found to be higher than those from known nerve growth factors such as Nerve Growth Factor (NGF) and Brain-Derived Nerve Factor (BDNF). The effect of two standards has not been observed above 0.1 (mg l⁻¹) of supplementation; however, the polymer did show the effect of cell growth and neurite extension at up to 1.0 (mg l⁻¹) of addition. While the polymer improved both cell growth and neurite extension, NGF and BDNF did only outgrowth of the neurites. Maximum cell density and length of the neurites were observed as 1.5×10⁵ (viable cells ml⁻¹) and 230 μm, respectively in adding 0.8 (mg l⁻¹) of the biopolymer for 8 days cultivation. The control growth was observed only as 1.2×10⁵ (viable cell ml⁻¹) of maximum cell density and 140 μm of maximum length, respectively. It was also confirmed that the polymer reacted with the nerve cells within 30 min after adding the sample, compared to 80 min in adding two other growth factors. Number of neurite-bearing cells remained relatively steady in adding the polymer even when the cell growth started to be decreased. It was interesting that the polymer effectively delayed apoptosis of PC12 cells by dramatically reducing the ratio of apoptotic cells to 20% from 50% of the control. This revised version was published online in September 2006 with corrections to the Cover Date.     

Artepillin C Derived from Propolis Induces Neurite Outgrowth in PC12m3 Cells via ERK and p38 MAPK Pathways

We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 μM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.     

Using PC12 cells to evaluate poly(caprolactone) and collagenous microcarriers for applications in nerve guide fabrication

Tissue-engineered nerve guides can provide mechanical support as well as chemical stimulation for nerve regeneration. PC12 cells were used to test the novel combination of poly(caprolactone) (PCL) and macroporous collagen-based microcarriers (CultiSphers) as an initial phase in the fabrication of multichanneled nerve guides. Laminin-coated PCL was an effective matrix for the attachment, proliferation, and viability of PC12 cells, relative to uncoated PCL. PC12 cells attached to laminin-coated PCL and extended neurites when cultured in the presence of nerve growth factor (NGF). PC12 cells attached and proliferated on CultiSphers and extended neurites in response to NGF. A novel PCL/CultiSpher composite material also supported PC12 attachment and proliferation and provides a potentially useful material for the fabrication of synthetic nerve guides.     

A surface acoustic wave sensor functionalized with a polypyrrole molecularly imprinted polymer for selective dopamine detection

A surface acoustic wave sensor operating at 104 MHz and functionalized with a polypyrrole molecularly imprinted polymer has been designed for selective detection of dopamine (DA). Optimization of pyrrole/DA ratio, polymerization and immersion times permitted to obtain a highly selective sensor, which has a sensitivity of 0.55°/mM (≈550 Hz/mM) and a detection limit of ≈ 10 nM. Morphology and related roughness parameters of molecularly imprinted polymer surfaces, before and after extraction of DA, as well as that of the non imprinted polymer were characterized by atomic force microscopy. The developed chemosensor selectively recognized dopamine over the structurally similar compound 4‐hydroxyphenethylamine (referred as tyramine), or ascorbic acid,which co‐exists with DA in body fluids at a much higher concentration. Selectivity tests were also carried out with dihydroxybenzene, for which an unexpected phase variation of order of 75% of the DA one was observed. Quantum chemical calculations, based on the density functional theory, were carried out to determine the nature of interactions between each analyte and the PPy matrix and the DA imprinted PPy polypyrrole sensing layer in order to account for the important phase variation observed during dihydroxybenzene injection. Copyright © 2015 John Wiley & Sons, Ltd.     

The generation of DPOAEs in the locust ear is contingent upon the sensory neurons

Tympanal organs of insects emit distortion-product otoacoustic emissions (DPOAEs) that are indicative of nonlinear ear mechanics. Our study sought (1) to define constraints of DPOAE generation in the ear of Locusta migratoria, and (2) to identify the sensory structures involved. We selectively destroyed the connection between the (peripheral) sensory ganglion and the tympanal attachment points of the “d-cell” dendrites; d-cells are most sensitive to sound frequencies above 12 kHz. This led to a decrease of DPOAEs that were evoked by f ₂ frequencies above 15 kHz (decrease of 15–40 dB; mean 28 dB; n = 12 organs). DPOAEs elicited by lower frequencies remained unchanged. Such frequency-specific changes following the exclusion of one scolopidial sub-population suggest that these auditory scolopidia are in fact the source of DPOAEs in insects. Electrical stimulation of the auditory nerve (with short current pulses of 4–10 μA or DC-currents of 0.5 μA) reversibly reduced DPOAEs by as much as 30 dB. We assume that retrograde electrical stimulation primarily affected the neuronal part of the scolopidia. Severing the auditory nerve from the central nervous system (CNS) did not alter the DPOAE amplitudes nor the effects of electrical stimulation.     

Change in striation density and systematics ofCocconeis scutellum var.Ornata (Bacillariophyceae)

Cocconeis scutellum var.ornata Grun. from three localities of Japan was studied. The striation density in 10 μm showed a marked tendency to increase with the decrease of the valve length in both raphe and rapheless valves, and this tendency did not vary with locality or environmental condition. The striation densities of rapheless valves were 4–6 in 10 μm for a valve length of 40μm, 4–6.5 for 30 μm, 6–9 for 20μm and 6.5–11 for 15μm. Those of raphe valves were 10–11 in 10μm for a valve length of 40μm, 10–12 for 30μm, 11–14.5 for 20μm and 12.5–17 for 15μm. According to the range of changing value in striation density obtained by the present study,C. scutellum var.schmidti Frenguelli andC. japonica Schmidt are identical withC. scutellum var.ornata. Dedicated to Prof. Munenao Kurogi on the occasion of his academic retirement. Culture experiment in the present study was undertaken at the Institute of Algological Research, Faculty of Science, Hokkaido University at Muroran.     

A Study of the Effects of Flux Density and Frequency of Pulsed Electromagnetic Field on Neurite Outgrowth in PC12 Cells

The aim of this study was to investigate the influence of pulsed electromagnetic fields with various flux densities and frequencies on neurite outgrowth in PC12 rat pheochromocytoma cells. We have studied the percentage of neurite-bearing cells, average length of neurites and directivity of neurite outgrowth in PC12 cells cultured for 96 hours in the presence of nerve growth factor (NGF). PC12 cells were exposed to 50 Hz pulsed electromagnetic fields with a flux density of 1.37 mT, 0.19 mT and 0.016 mT respectively. The field was generated through a Helmholtz coil pair housed in one incubator and the control samples were placed in another identical incubator. It was found that exposure to both a relatively high flux density (1.37 mT) and a medium flux density (0.19 mT) inhibited the percentage of neurite-bearing cells and promoted neurite length significantly. Exposure to high flux density (1.37 mT) also resulted in nearly 20% enhancement of neurite directivity along the field direction. However, exposure to low flux density field (0.016 mT) had no detectable effect on neurite outgrowth. We also studied the effect of frequency at the constant flux density of 1.37 mT. In the range from 1 ∼ 100 Hz, only 50 and 70 Hz pulse frequencies had significant effects on neurite outgrowth. Our study has shown that neurite outgrowth in PC12 cells is sensitive to flux density and frequency of pulsed electromagnetic field.     

Heparin Promotes Suspension Adaptation Process of CHO–TS28 Cells by Eliminating Cell Aggregation

While heparin has been shown to eliminate cell aggregation in suspension adaptations of insect and HEK293 cells for virus-based cell cultures, the role of heparin in long period serum-free suspension adaptation of the anchorage-dependent Chinese hamster ovary (CHO) cell lines remains inconclusive. In this paper, we explore the potential application of heparin in suspension adaptation of CHO cell line which produces an anti-human chimeric antibody cHAb18. Heparin showed a concentration-dependent inhibition of CHO–TS28 cell-to-cell adhesion, with a significant inhibitory effect occurring when the concentration exceeded 250 μg/ml (P < 0.001). Heparin also exhibited a cell aggregation elimination role at all concentrations (P < 0.001). Furthermore, heparin promoted cell growth and antibody secretion, with the highest cell density ((99.83 ± 12.21) × 10⁴ cells/ml, P = 0.034) and maximum antibody yield ((9.46 ± 0.94) mg/l, P < 0.001) both occurring at 250 μg/ml heparin. When agitated, cell aggregates were effectively dispersed by 250 μg/ml heparin and a single-cell suspension culture process was promoted. In suspension adapted CHO–TS28 cells, cell growth rates and specific antibody productivity were maintained; while antigen-binding activity improved slightly. Together, our results show that heparin may promote suspension adaptation of anchorage-depended CHO cells by resisting cell aggregation without reducing cell growth, antibody secretion, and antigen-binding activity.     

Vitamin B12, a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells

We previously isolated an analog to chlorophyll-related compounds, pheophytin a, from the marine brown alga Sargassum fulvellum and demonstrated that it is a neurodifferentiation compound. In the current study, we investigated the effects of the pheophytin a analog vitamin B₁₂ on PC12 cell differentiation. In the presence of a low level of nerve growth factor (10 ng ml⁻¹), vitamin B₁₂ demonstrated neurite outgrowth-promoting activity in PC12 cells. The effect was dose-dependent in the range of 6–100 μM. In the absence of nerve growth factor, vitamin B₁₂ did not promote differentiation. To investigate the mechanism for this effect, we conducted differentiation assays and western blot analysis with signal transduction inhibitors and found that vitamin B₁₂ did not promote PC12 cell differentiation in the presence of K252a or U0126 inhibitors. These results suggest that vitamin B₁₂ stimulates PC12 cell differentiation through enhancement of the mitogen-activated protein kinase signal transduction pathway, which is also induced by nerve growth factor. Thus, vitamin B₁₂ may be a good candidate for treatment of neurodegenerative diseases such as Alzheimer’s disease.     

Protective Effects of Piperine Against Corticosterone-Induced Neurotoxicity in PC12 Cells

Hyperactivation of the hypothalamic–pituitary–adrenal axis and the associated hippocampal atrophy were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Our previous studies have showed that treating mice with piperine produced antidepressant-like effect in animal models of behavioral despair. This study aimed to examine the protective effect of piperine treatment on corticosterone-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The results showed that piperine co-treatment revealed a differential effect on the cytotoxicity of corticosterone and had its maximum inhibitory effect at 1 μM. Piperine (1 μM) co-treatment also significantly decreased intracellular reactive oxygen species level, and enhanced superoxide dismutase activity and total glutathione level in corticosterone-treated PC12 cells. In addition, piperine (1 μM) co-treatment was found to reverse the decreased brain-derived neurotrophic factor (BDNF) mRNA level caused by corticosterone in PC12 cells. The results suggest that piperine exerts a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells, at least in part, via the inhibition of oxidative stress and the upregulation of BDNF mRNA expression. This neuroprotective effect may be one of the acting mechanisms accounts for the in vivo antidepressant activity of piperine.     

Protective Effect of <Emphasis Type="Italic">Nigella sativa</Emphasis> Extract and Thymoquinone on Serum/Glucose Deprivation-Induced PC12 Cells Death

The serum/glucose deprivation (SGD)-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Nigella sativa L. (family Ranunculaceae) and its active component thymoquinone (TQ) has been known as a source of antioxidants. In the present study, the protective effects of N. sativa and TQ on cell viability and reactive oxygen species (ROS) production in cultured PC12 cells were investigated under SGD conditions. PC12 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded overnight and then deprived of serum/glucose for 6 and 18 h. Cells were pretreated with different concentrations of N. sativa extract (15.62–250 μg/ml) and TQ (1.17–150 μM) for 2 h. Cell viability was quantitated by MTT assay. Intracellular ROS production was measured by flow cytometry using 2′,7′-dichlorofluorescin diacetate (DCF-DA) as a probe. SGD induced significant cells toxicity after 6, 18, or 24 h (P < 0.001). Pretreatment with N. sativa (15.62–250 μg/ml) and TQ (1.17–37.5 μM) reduced SGD-induced cytotoxicity in PC12 cells after 6 and 18 h. A significant increase in intracellular ROS production was seen following SGD (P < 0.001). N. sativa (250 μg/ml, P < 0.01) and TQ (2.34, 4.68, 9.37 μM, P < 0.01) pretreatment reversed the increased ROS production following ischemic insult. The experimental results suggest that N. sativa extract and TQ protects the PC12 cells against SGD-induced cytotoxicity via antioxidant mechanisms. Our findings might raise the possibility of potential therapeutic application of N. sativa extract and TQ for managing cerebral ischemic and neurodegenerative disorders.     

Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells

The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm² with 50 μM PC, and 17.0 and 23.0 nmol/h/cm² with 100 μM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 μM DHA-enriched PC, and to 25 and 30% with 50 and 100 μM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 μM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells. (Mol Cell Biochem xxx: 1–9, 2005)     

Electrical stimulation modulates osteoblast proliferation and bone protein production through heparin‐bioactivated conductive scaffolds

Electrical fields are known to interact with human cells. This principle has been explored to regulate cellular activities for bone tissue regeneration. In this work, Saos‐2 cells were cultured on conductive scaffolds made of biodegradable poly(L ‐lactide) and the heparin‐containing, electrically conducting polypyrrole (PPy/HE) to study their reaction to electrical stimulation (ES) mediated through such scaffolds. Both the duration and intensity of ES enhanced cell proliferation, generating a unique electrical intensity and temporal “window” within which osteoblast proliferation was upmodulated in contrast to the downmodulation or ineffectiveness in other ES regions. The favourable ES intensity (200 mV/mm) was further investigated in terms of the gene activation and protein production of two important osteoblast markers characterised by extracellular matrix maturation and mineralisation, that is alkaline phosphatase (ALP) and osteocalcin (OC). Both genes were found activated and the relevant protein production increased significantly following ES. In contrast, ES in the down‐modulation region (400 mV/mm) suppressed the production of both ALP and OC. This work demonstrated that important osteoblast markers can be modulated with specific ES parameters mediated through conductive polymer substrates, providing a unique strategy for bone tissue engineering. Bioelectromagnetics 34:189–199, 2013. © 2012 Wiley Periodicals, Inc.     

Deep-sea parasitic nematodes of the genus <Emphasis Type="Italic">Trophomera</Emphasis> Rubtsov et Platonova, 1974 (Benthimermithidae) from the Equatorial Atlantic,with the descriptions of two new species

Nematode females of the genus Trophomera (Benthimermithidae) from the collection of the Smithsonian’s National Museum of Natural History (Washington, DC, USA) were examined. Nematodes were collected in different parts of the Western Atlantic (Hatteras Abyssal Plain, Brazil Basin, and Argentina Basin) from depths of 467–5,223 m. Two new species are described. Body length of T. americana sp. n. is 3,250–4,470 μm; posterior end conical with rounded tip; cephalic setae about 3–4 μm long; trophosome consisting of several longitudinal rows of large cells; ovaries reflected; mature eggs 35 μm in diameter. Body length of T. longiovaris sp. n. is 7,870–15,400 μm; posterior end conical with rounded tip; cephalic sensilla 7 μm long; mouth opening vestigial, present as very narrow apical pore; pharynx devoid of internal lumen and muscular envelope; midgut represents a trophosome without internal lumen; trophosomal cells arranged in 3–4 longitudinal rows; rectum and anus vestigial; female reproductive system didelphic, amphidelphic, very long, occupying about 0.8 total body length; ovaries telogonic, outstretched; oviducts very long, repeatedly folded across body axis; proximal parts of oviducts being than distal ones, uterus distinctly formed. New finds of two known species, T. arnauidi and T. marionensis, are also recorded and described.     

A cytological observation of the fluid in the primo-nodes and vessels on the surfaces of mammalian internal organs

We report on the preliminary cytological observation of fluid in the primo-nodes and vessels on the surfaces of the internal organs of mammals. With some microsurgical procedures, we observed many cells and microcells that spread out of the nodes on the organ surfaces of rats and rabbits. These cells generally showed the following morphologies: (1) round or oval cells, 10 μm in size, with predominantly little cytoplasm; (2) cells with nuclei that exhibited a collapsed shape; (3) binucleated cells, 20 μm in size; (4) spherical granules, ranging 0.5–2.0 μm in size (primo-microcells); and (5) aggregations of such granules. These findings on the existence of cells with diverse morphologies in the fluid of primo-nodes and vessels could be evidence supporting the hypothesis that the anatomical basis of acupuncture meridians (i.e., primo-vascular system) may be a migration channel for various kind of cells.     

Enhancement of pheochromocytoma nerve cell growth by consecutive fractionization of <Emphasis Type="Italic">Angelica gigas</Emphasis> Nakai extracts

This work was to investigate the effect of flavonoids from Angelica gigas Nakai on the proliferation and differentiation of PC12 cells. Several solvents including hexane, chloroform, ethyl acetate, butanol and water consecutively partitioned. We determined the ethanol crude extract of Angelica gigas Nakai. The hexane fraction was shown to contain the highest number of flavonoids as follows; 21.48 mg/g and the composition of the flavonoids was as follows: 12.24 mg/g of quercetin, 4.39 mg/g of myricetin and 2.58 mg/g of catechin. In addition, this hexane fraction greatly increased both cell growth and outgrowth of the neurite, and whose effects were three times higher than those of the other fractions. The length of the neurites was measured as ca. 110 μm in adding 50 μg/mL of the hexane fraction, which was about the same as the case of adding 50 ng/mL of NGF as a positive control. This result indicates that the differentiation of PC12 cells by the addition of the hexane fraction was comparable to the case of adding NGF. The hexane fraction was also determined to prevent apoptosis of PC12 cells by suppressing DNA fragmentation. It is interesting that the mixture of three major flavonoids, quercetin, myricetin and catechin showed stronger activity on, both PC12 cell growth and neuritis outgrowth, than when adding each flavonoid alone. We believe this was due to the synergistic effects of the three flavonoids. The activities of these flavonoids from Angelica gigas Nakai are reported for the first time in this study.     

Fast and Local Electrochemical Monitoring of Noradrenaline Release from Sympathetic Terminals in Isolated Rat Tail Artery

Abstract: Noradrenaline release from sympathetic nerve terminals was evoked by electrical nerve stimulation of an isolated segment of rat tail artery. This release was recorded by a carbon fiber electrode combined with differential pulse amperometry. The active part of the electrode (one carbon fiber 8 μm in diameter and 50 μm in length) was placed in close contact with the arterial surface. The oxidation current appearing at +120 mV and corresponding to the local noradrenaline concentration at the electrode surface was recorded every 0.5 s. No oxidation current was detected under resting conditions, but electrical stimulation evoked an immediate increase in this current. This response was suppressed when tetrodotoxin was added to the perfusion medium and was enhanced when noradrenaline reuptake was inhibited by cocaine. The amplitude of the response was increased with increasing stimulation frequencies (2–25 Hz) and train lengths (1–16 pulses). Finally, the time resolution of the method (0.5 s) was good enough to show that noradrenaline release precedes the postsynaptic response, i.e., the electrically evoked contraction of the artery.     

聚吡咯薄膜上大鼠肝细胞生长的研究

Poypyrrole(Ppy)films were prepared at 1×10-3 mA/cm2 electropolymerization current density on indium-tin oxide(ITO)substrate. The Ppy films were wall-distributed, translucent, stable and insoluble. Moreover, they can be sterilized by steam disinfection. Rat hepatic cells were cultured on these films. The results show that Ppy films have good biocompatibility and they can accelerate cell growth under electrical stimulation. The cells on Ppy films reach the largest cell density earlier than the cells on tissue culture polystyrene(TCPS). Furthermore, rat hepatic cells can generate on Ppy films. The cells on Ppy films grow faster and enter logarithmic growth phase earlier than those on TCPS.     

Artificial biofilm model – a useful tool for biofilm research

For biofilm studies, artificial models can be very helpful in studying processes in hydrogels of defined composition and structure. Two different types of artificial biofilm models were developed. Homogeneous agarose beads (50–500 μm diameter) and porous beads (260 μm mean diameter) containing pores with diameters from 10 to 80 μm (28 μm on average) allowed the embedding of cells, particles and typical biofilm matrix components such as proteins and polysaccharides. The characterisation of the matrix structures and of the distribution of microorganisms was performed by confocal laser scanning microscopy. The physiological condition of the embedded bacteria was examined by redox activity (CTC-assay) and membrane integrity (Molecular Probes LIVE/DEAD-Kit). Approximately 35% of the immobilised cells (Pseudomonas aeruginosa SG81) were damaged due to the elevated temperature required for the embedding process. It was shown that the surviving cells were able to multiply when provided with nutrients. In the case of homogeneous agarose beads, cell growth only occurred near the bead surface, while substrate limitation prevented growth of more deeply embedded cells. In the porous hydrogel, cell division was observed across the entire matrix due to better mass transport. It could be shown that embedding in the artificial gel matrix provided protection of immobilized cells against toxic substances such as sodium hypochlorite (0.5 mg/l, 30 min) in comparison to suspended cells, as observed in other immobilized systems. Thus, the model is suited to simulate important biofilm matrix properties. Received: 21 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000