Development of a neurotoxicity test-system, using human post-mitotic, astrocytic and neuronal cell lines in co-culture

Astrocytes are essential for neuronal function and survival, so both cell types were included in a human neurotoxicity test-system to assess the protective effects of astrocytes on neurons, compared with a culture of neurons alone. The human NT2.D1 cell line was differentiated to form either a co-culture of post-mitotic NT2.N neuronal (TUJ1, NF68 and NSE positive) and NT2.A astrocytic (GFAP positive) cells (∼2:1 NT2.A:NT2.N), or an NT2.N mono-culture. Cultures were exposed to human toxins, for 4 h at sub-cytotoxic concentrations, in order to compare levels of compromised cell function and thus evidence of an astrocytic protective effect. Functional endpoints examined included assays for cellular energy (ATP) and glutathione (GSH) levels, generation of hydrogen peroxide (H2O2) and caspase-3 activation. Generally, the NT2.N/A co-culture was more resistant to toxicity, maintaining superior ATP and GSH levels and sustaining smaller significant increases in H2O2 levels compared with neurons alone. However, the pure neuronal culture showed a significantly lower level of caspase activation. These data suggest that besides their support for neurons through maintenance of ATP and GSH and control of H2O2 levels, following exposure to some substances, astrocytes may promote an apoptotic mode of cell death. Thus, it appears the use of astrocytes in an in vitro predictive neurotoxicity test-system may be more relevant to human CNS structure and function than neuronal cells alone. © 2007 Elsevier Ltd. All rights reserved.

Apoptosis induction in gastric mucous cells in vitro:lesser potency of Helicobacter pylori than Escherichia coli lipopolysaccharide, but positive interaction with ibuprofen

Non-steroidal anti-inflammatory drugs (NSAIDs) cause peptic ulcer disease, but whether they interact with Helicobacter pylori to promote damage is controversial. Moreover, the reported induction of apoptosis in gastric cells by H. pylori lipopolysaccharide (LPS) (10-9 g /ml) contrasts with studies showing low immunological potency of this LPS. Therefore, the effects of LPS from H. pylori NCTC 11637 and Escherichia coli 0111:B4 on apoptosis in a primary culture of guinea-pig gastric mucous cells were investigated in the presence and absence of the NSAID, ibuprofen. Cell loss was estimated by a crystal violet assay, and apoptosis determined from caspase activity and from condensation and fragmentation of nuclei. Exposure to E. coli LPS for 24 h caused cell loss and enhanced apoptotic activity at concentrations ≥ 10-9 g/ml, but similar effects were only obtained with H. pylori LPS at concentrations ≥10-6 g/ml. Although ibuprofen (250 μM) caused cell loss and apoptosis, addition of either E. coli or H. pylori LPSs further enhanced these effects. In conclusion, LPS and ibuprofen interact to enhance gastric cell loss and apoptosis. In such interactions, E. coli LPS is more potent than that of H. pylori. The low potency of H. pylori LPS may contribute to a chronic low-grade gastritis that can be enhanced by the use of NSAIDs. © W. S. Maney & Son Ltd.

High permeability of the anionic form restricts accumulation of indomethacin by cultured gastric surface epithelial cells exposed to low apical pH

The 'ion-trapping' hypothesis suggests that the intracellular concentration of acidic non-steroidal anti-inflammatory drugs in gastric epithelial cells could be much higher than in the gastric lumen, and that such accumulation could contribute to their gastrotoxicity. Our aim was to examine the effect of the pH of the apical medium on the apical to basal transfer of the acidic drug indomethacin (pK a 4.5) across a gastric mucous epithelial cell monolayer, and to determine whether indomethacin accumulated in cells exposed to a low apical pH. Guinea-pig gastric mucous epithelial cells were grown on porous membrane culture inserts (Transwells®) for 72 h. Transfer and accumulation of [ 14C] indomethacin were assessed by scintillation counting. Transfer of [ 3H]mannitol and measurement of trans-epithelial electrical resistance were used to assess integrity of the monolayer. Distribution of [ 14C] urea was used to estimate the intracellular volume of the monolayer. The monolayer was not disrupted by exposure of the apical face to media of pH ≥ 3, or by indomethacin. Transfer of indomethacin (12 μM) to the basal medium increased with decreasing apical medium pH. The apparent permeability of the undissociated acid was estimated to be five times that of the anion. The intracellular concentration of indomethacin was respectively 5.3, 4.1 and 4.3 times that in the apical medium at pH 5.5, 4.5 and 3.0. In conclusion, this study represents the first direct demonstration that indomethacin accumulates in gastric epithelial cells exposed to low apical pH. However, accumulation of indomethacin was moderate and the predictions of the ion-trapping hypothesis were not met, probably due to the substantial permeability of anionic indomethacin across membranes. © 2006 Elsevier B.V. All rights reserved.

Human intervertebral disc cells promote nerve growth over substrata of human intervertebral disc aggrecan

Study Design. Coculture assays of the migration and interaction of human intervertebral disc cells and chick sensory nerves on alternate substrata of collagen and aggrecan. Objective. To examine the effects of aggrecan on disc cell migration, how disc cells and sensory nerves interact, and whether disc cells affect previously reported inhibitory effects of aggrecan on sensory nerve growth. Summary of Background Data. Human intervertebral disc aggrecan is inhibitory to sensory nerve growth in vitro, suggesting that a loss of aggrecan from the disc may have a role in the increased innervation seen in disc degeneration. Endothelial cells that appear to co-migrate with nerves into degenerated intervertebral disc express neurotrophic factors, but the effects of disc cells on nerve growth are not known. Methods. Human disc cells were seeded onto tissue culture plates that had been coated with type I collagen and human intervertebral disc aggrecan. Explants of chick dorsal root ganglions (DRGs) were subsequently added to the plates and sensory neurite outgrowth stimulated by the addition of nerve growth factor. Time-lapse video and fluorescence microscopy were used to examine the migration and interaction of the disc cells and sensory neurites, in the context of the different matrix substrata. The effects of disc cell conditioned medium on nerve growth were also examined. Results. Disc cells spread and migrated on collagen until they encountered the aggrecan substrata, where some cells, but not all, were repelled. In coculture, DRG neurites extended onto the collagen/disc cells until they encountered the aggrecan, where, like the disc cells, many were repelled. However, in the presence of disc cells, some neurites were able to cross onto this normally inhibitory substratum. The number of neurite crossings onto aggrecan correlated significantly with the number of disc cells present on the aggrecan. In control experiments using DRG alone, all extending neurites were repelled at the collagen/aggrecan border. Conditioned medium from disc cell cultures stimulated DRG neurite outgrowth on collagen but did not increase neurite crossing onto aggrecan substrata. Conclusions. Human disc cells migrate across aggrecan substrata that are repellent to sensory DRG neurites. Disc cells synthesize neurotrophic factors in vitro that promote neurite outgrowth. Furthermore, the presence of disc cells in coculture with DRG partially abrogates the inhibitory effects of aggrecan on nerve growth. These findings have important implications for the regulation of nerve growth into the intervertebral disc, but whether disc cells promote nerve growth in vivo remains to be determined.

Vascular endothelial growth factor promotes physical wound repair and is anti-apoptotic in primary distal lung epithelial and A549 cells

Objective: There is evidence to suggest a beneficial role for growth factors, including vascular endothelial growth factor (VEGF), in tissue repair and proliferation after injury within the lung. Whether this effect is mediated predominantly by actions on endothelial cells or epithelial cells is unknown. This study tested the hypothesis that VEGF acts as an autocrine trophic factor for human adult alveolar epithelial cells and that under situations of pro-apoptotic stress, VEGF reduces cell death. Design: In vitro cell culture study looking at the effects of 0.03% H2O2 on both A549 and primary distal lung epithelial cells.Measurement and Main Results: Primary adult human distal lung epithelial cells express both the soluble and membrane-associated VEGF isoforms and VEGF receptors 1 and 2. At physiologically relevant doses, soluble VEGF isoforms stimulate wound repair and have a proliferative action. Specific receptor ligands confirmed that this effect was mediated by VEGF receptor 1. In addition to proliferation, we demonstrate that VEGF reduces A549 and distal lung epithelial cell apoptosis when administered after 0.03% H2O2 injury. This effect occurs due to reduced caspase-3 activation and is phosphatidylinositol 3′–kinase dependent. Conclusion: In addition to its known effects on endothelial cells, VEGF acts as a growth and anti-apoptotic factor on alveolar epithelial cells. VEGF treatment may have potential as a rescue therapy for diseases associated with alveolar epithelial damage such as acute respiratory distress syndrome.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Disparate effects of non-steroidal anti-inflammatory drugs on apoptosis in guinea-pig gastric mucous cells: inhibition of basal apoptosis by diclofenac

Non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastrointestinal cancer cell lines. Similar actions on normal gastric epithelial cells could contribute to NSAID gastropathy. The present work therefore compared the actions of diclofenac, ibuprofen, indomethacin, and the cyclo-oxygenase-2 selective inhibitor, NS-398, on a primary culture of guinea-pig gastric mucous epithelial cells. Cell number was assessed by staining with crystal violet. Apoptotic activity was determined by condensation and fragmentation of nuclei and by assay of caspase-3-like activity. Necrosis was evaluated from release of cellular enzymes. Ibuprofen (250 μM for 24 h) promoted cell loss, and apoptosis, under both basal conditions and when apoptosis was increased by 25 μM N-Hexanoyl-D-sphingosine (C6-ceramide). Diclofenac (250 μM for 24 h) reduced the proportion of apoptotic nuclei from 5.2 to 2.1%, and caused inhibition of caspase-3-like activity, without causing necrosis under basal conditions. No such reduction in apoptotic activity was evident in the presence of 25 μM C6-ceramide. The inhibitory effect of diclofenac on basal caspase-3-like activity was also exhibited by the structurally similar mefenamic and flufenamic acids (1–250 μM), but not by niflumic acid. Inhibition of superoxide production by the cells increased caspase-3-like activity, but the inhibitory action of diclofenac on caspase activity remained. Diclofenac did not affect superoxide production. Diclofenac inhibited caspase-3-like activity in cell homogenates and also inhibited human recombinant caspase-3. In conclusion, NSAIDs vary in their effect on apoptotic activity in a primary culture of guinea-pig gastric mucous epithelial cells, and the inhibitory effect of diclofenac on basal apoptosis could involve an action on caspase activity.

Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells

Background context Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. Purpose To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). Study design/setting Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. Methods We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. Results As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. Conclusions These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states. © 2014 Elsevier Inc. All rights reserved.

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100mL stirred spinner flasks at a density of 3×10⁵cells.mL^-1 in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63±0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8±1.4% with a similar 3h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.

Human mesenchymal stem cells stimulate EaHy926 endothelial cell migration:combined proteomic and in vitro analysis of the influence of donor-donor variability

Mesenchymal stem cells (MSCs) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present. Here we report that human MSC-conditioned medium (n=3 donors) significantly increased EaHy-926 endothelial cell adhesion and cell migration, but that this stimulatory effect was markedly donor-dependent. MALDI-TOF/TOF mass spectrometry demonstrated that whilst collagen type I and fibronectin were secreted by all of the MSC cultures, the small leucine rich proteoglycan, decorin was secreted only by the MSC culture that was least effective upon EaHy-926 cells. These individual extracellular matrix components were then tested as culture substrata. EaHy-926 cell adherence was greatest on fibronectin-coated surfaces with least adherence on decorin-coated surfaces. Scratch wound assays were used to examine cell migration. EaHy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin. However, EaHy-926 cell migration was stimulated by the addition of MSC-conditioned medium irrespective of the types of culture substrates. These data suggest that whilst the MSC secretome may generally be considered angiogenic, the composition of the secretome is variable and this variation probably contributes to donor-donor differences in activity. Hence, screening and optimizing MSC secretomes will improve the clinical effectiveness of pro-angiogenic MSC-based therapies.

Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development

Cell-based therapies have the potential to contribute to global healthcare, whereby the use of living cells and tissues can be used as medicinal therapies. Despite this potential, many challenges remain before the full value of this emerging field can be realized. The characterization of input material for cell-based therapy bioprocesses from multiple donors is necessary to identify and understand the potential implications of input variation on process development. In this work, we have characterized bone marrow derived human mesenchymal stem cells (BM-hMSCs) from multiple donors and discussed the implications of the measurable input variation on the development of autologous and allogeneic cell-based therapy manufacturing processes. The range of cumulative population doublings across the five BM-hMSC lines over 30 days of culture was 5.93, with an 18.2% range in colony forming efficiency at the end of the culture process and a 55.1% difference in the production of interleukin-6 between these cell lines. It has been demonstrated that this variation results in a range in the process time between these donor hMSC lines for a hypothetical product of over 13 days, creating potential batch timing issues when manufacturing products from multiple patients. All BM-hMSC donor lines demonstrated conformity to the ISCT criteria but showed a difference in cell morphology. Metabolite analysis showed that hMSCs from the different donors have a range in glucose consumption of 26.98 pmol cell−1 day−1, Lactate production of 29.45 pmol cell−1 day−1 and ammonium production of 1.35 pmol cell−1 day−1, demonstrating the extent of donor variability throughout the expansion process. Measuring informative product attributes during process development will facilitate progress towards consistent manufacturing processes, a critical step in the translation cell-based therapies.

Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation.

Brain-derived neurotrophic factor as an indicator of chemical neurotoxicity:an animal-free CNS cell culture model

Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current 'whole animal' approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated-dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.

The development of functional astrocyte-neuron networks derived from stem cells

There is currently great scientific and medical interest in the potential of tissue grown from stem cells. These cells present opportunities for generating model systems for drug screening and toxicological testing which would be expected to be more relevant to human outcomes than animal based tissue preparations. Newly realised astrocytic roles in the brain have fundamental implications within the context of stem cell derived neuronal networks. If the aim of stem cell neuroscience is to generate functional neuronal networks that behave as networks do in the brain, then it becomes clear that we must include and understand all the cellular components that comprise that network, and which are important to support synaptic integrity and cell to cell signalling. We have shown that stem cell derived neurons exhibit spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling (1). Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, astrocytes exhibit morphology and functional properties consistent with this glial cell type. Astrocytes also respond to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. Astroctyes also generate propagating calcium waves that are gap junction and purinergic signalling dependent. Our results show that stem cell derived astrocytes exhibit appropriate functionality and that stem cell neuronal networks interact with astrocytic networks in co-culture. Using mixed cultures of stem cell derived neurons and astrocytes, we have also shown both cell types also modulate their glucose uptake, glycogen turnover and lactate production in response to glutamate as well as increased neuronal activity (2). This finding is consistent with their neuron-astrocyte metabolic coupling thus demonstrating a tractable human model, which will facilitate the study of the metabolic coupling between neurons and astrocytes and its relationship with CNS functional issues ranging from plasticity to neurodegeneration. Indeed, cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose (3). Both co-cultures of neurons and astrocytes and purified cultures of astrocytes showed a significant decrease in glucose uptake after treatment with 2 and 0.2 μmol/L Aβ at all time points investigated (p <0.01). In addition, a significant increase in the glycogen content of cells was also measured. Mixed neuron and astrocyte co-cultures as well as pure astrocyte cultures showed an initial decrease in glycogen levels at 6 hours compared with control at 0.2 μmol/L and 2 μmol/L P <0.01. These changes were accompanied by changes in NAD+/NADH (P<0.05), ATP (P<0.05), and glutathione levels (P<0.05), suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. As numerous cell types interact in the brain it is important that any in vitro model developed reflects this arrangement. Our findings indicate that stem cell derived neuron and astrocyte networks can communicate, and so have the potential to interact in a tripartite manner as is seen in vivo. This study therefore lays the foundation for further development of stem cell derived neurons and astrocytes into therapeutic cell replacement and human toxicology/disease models. More recently our data provides evidence for a detrimental effect of Aβ on carbohydrate metabolism in both neurons and astrocytes. As a purely in vitro system, human stem cell models can be readily manipulated and maintained in culture for a period of months without the use of animals. In our laboratory cultures can be maintained in culture for up to 12 months months thus providing the opportunity to study the consequences of these changes over extended periods of time relevant to aspects of the disease progression time frame in vivo. In addition, their human origin provides a more realistic in vitro model as well as informing other human in vitro models such as patient-derived iPSC.

Nicotine exposure reduces cell viability but induces anti-inflammatory effects in human cystic fibrosis bronchial epithelial cells

Background: Mouse models of cystic fibrosis (CF) fail to truly represent the respiratory pathology. We have consequently developed human airways cell culture models to address this. The impact of cigarette smoke within the CF population is well documented, with exposure being known to worsen lung function. As nicotine is often perceived to be a less harmful component of tobacco smoke, this research aimed to identify its effects upon viability and inflammatory responses of CF (IB3-1) and CF phenotype corrected (C38) bronchial epithelial cells. Methods: IB3-1 and C38 cell lines were exposed to increasing concentrations of nicotine (0.55-75μM) for 24 hours. Cell viability was assessed via Cell Titre Blue and the inflammatory response with IL-6 and IL-8 ELISA. Results: CF cells were more sensitive; nicotine significantly (P<0.05) reduced cell viability at all concentrations tested, but failed to have a marked effect on C38 viability. Whilst nicotine induced anti-inflammatory effects in CF cells with a significant reduction in IL-6 and IL-8 release, it had no effect on chemokine release by C38 cells. Conclusion: CF cells may be more vulnerable to inhaled toxicants than non-CF cells. As mice lack a number of human nicotinic receptor subunits and fail to mimic the characteristic pathology of CF, these data emphasise the importance of employing relevant human cell lines to study a human-specific disease.