Incubation Of Cell Cultures Research Articles

Fact, Fiction, and Momentum Investing

To compare the performances of conventional <i>in vitro</i> indicator cell culture, quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS) as detection methods for adventitious agents, a preliminary assessment was performed using human cytomegalovirus (HCMV) as a model virus. HCMV was spiked into a crude viral harvest at various concentrations and inoculated onto MRC-5 cell monolayers. The cultures were observed for cytopathic effects (CPEs) as per the compendial method requirements, and samples were taken at various time points for analysis by qPCR or NGS. When using NGS, the detection of HCMV is 10 fold more sensitive than observed using the conventional CPE endpoint method. It may be possible for qPCR to achieve the detection level demonstrated by NGS, but further optimization of the technique would be required. In addition, NGS was able to detect HCMV in the initial inoculum when it was spiked in at 10 CCID₅₀/mL, suggesting the potential to eliminate cell culture amplification with an NGS-based assay. This study highlights the advantage of NGS as a surrogate broad-spectrum technology for the detection of adventitious agents in biologics. <i>LAY ABSTRACT:</i> Human cytomeglovirus (HCMV) is highly prevalent in the general population and can lead to serious health issues in both immumocompromised individuals and/or newborns. The testing of HCMV in biological materials is stipulated by multiple regulatory agencies where HCMV is a potential risk. This test involves inoculating cell lines that are susceptible to HCMV infection, incubating the cultures for 28 days, and observing the cells for signs of viral infection. Next-generation sequencing and quantitative polymerase chain reaction (qPCR) are two technologies that can potentially shorten the extended 28 day cell culture incubation. In this study, we compared the sensitivity of the compendial cell culture assay with NGS and qPCR for the detection of HCMV. Our results show that NGS can potentially achieve a detection limit that is 10 times more sensitive than the cell culture assay. In addition, NGS was able to detect HCMV in the initial inoculum, potentially eliminating the need for cell culture amplification of the virus. Finally, sequence data generated by NGS directly demonstrate the presence of HCMV, and such information can serve as the foundation for any follow-up investigation.

Induction, Cloning and Functional Expression of a Sesquiterpene Biosynthetic Enzyme, δ-Guaiene Synthase, of Aquilaria microcarpa Cell Cultures

A homology-based cloning strategy yielded a cDNA clone presumably encoding δ-guaiene synthase, a sesquiterpene cyclase, from tissue cultures of Aquilaria microcarpa, which were treated with methyl jasmonate. Incubation of cell cultures of the plant with yeast extract also induced transcriptional activation of the sesquiterpene synthase gene. The translated protein of the gene obtained by heterologous expression in Escherichia coli catalyzed the cyclization of farnesyl diphosphate to liberate δ-guaiene with δ-guaiene and germacrene A as the minor products. The results obtained in the present study, together with the previously reported results, suggest that two classes of δ-guaiene synthase occur in Aquilaria; the enzyme proteins from A. microcarpa and A. sinensis liberate germacrene A as a minor product, while the protein from A. crassna generates α-humulene instead of germacrene A.

Embryos developing individually in the embryoscope system are of comparable quality to embryos grown in group culture in large cell culture incubators

The EmbryoScope has shown great promise as a tool for recording the development of human embryos and then allowing the retrospective examination of markers of embryo quality, ultimately enabling the embryologist to make a fully informed decision about which embryos to transfer and freeze. However, there is a school of thought that embryos grown individually, as in the Embryoscope slide cells, may not benefit from paracrine factors available from group culture. Therefore, we collected and analyzed developmental data from patients whose sibling embryos were cultured in either traditional incubators in group culture or embryos in the EmbryoScope in single culture.

Construction and characterization of a novel vocal fold bioreactor.

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

Hemoglobin regulates the migration of glioma cells along poly(ε-caprolactone)-aligned nanofibers.

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O2 )-carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε-caprolactone) (PCL)-aligned fibers was studied in cell culture media supplemented with the O2 storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O2 tensions ranging from 1 to 19% O2 . Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL-aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O2 , while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O2 levels. The control data suggests that decreasing the O2 tension in the incubator from 5 to 1% O2 decreased aggregate dispersion on the PCL-aligned fibers. However, the addition of bovine Hb at 5% O2 significantly improved aggregate dispersion. At 19% O2 , Hb did not impact aggregate dispersion. Also at 1% O2 , aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb-based O2 carriers can be utilized to improve O2 availability and the migration of glioma spheroids on nanofibers.

Inhibition of the Polyamine System Counteracts β-Amyloid Peptide-Induced Memory Impairment in Mice: Involvement of Extrasynaptic NMDA Receptors

In Alzheimer's disease (AD), the β-amyloid peptide (Aβ) has been causally linked to synaptic dysfunction and cognitive impairment. Several studies have shown that N-Methyl-D-Aspartate receptors (NMDAR) activation is involved in the detrimental actions of Aβ. Polyamines, like spermidine and spermine, are positive modulators of NMDAR function and it has been shown that their levels are regulated by Aβ. In this study we show here that interruption of NMDAR modulation by polyamines through blockade of its binding site at NMDAR by arcaine (0.02 nmol/site), or inhibition of polyamine synthesis by DFMO (2.7 nmol/site), reverses Aβ25–35-induced memory impairment in mice in a novel object recognition task. Incubation of hippocampal cell cultures with Aβ25–35 (10 µM) significantly increased the nuclear accumulation of Jacob, which is a hallmark of NMDAR activation. The Aβ-induced nuclear translocation of Jacob was blocked upon application of traxoprodil (4 nM), arcaine (4 µM) or DFMO (5 µM), suggesting that activation of the polyamine binding site at NMDAR located probably at extrasynaptic sites might underlie the cognitive deficits of Aβ25–35-treated mice. Extrasynaptic NMDAR activation in primary neurons results in a stripping of synaptic contacts and simplification of neuronal cytoarchitecture. Aβ25–35 application in hippocampal primary cell cultures reduced dendritic spine density and induced alterations on spine morphology. Application of traxoprodil (4 nM), arcaine (4 µM) or DFMO (5 µM) reversed these effects of Aβ25–35. Taken together these data provide evidence that polyamine modulation of extrasynaptic NMDAR signaling might be involved in Aβ pathology.

Reassessing laboratory results of low-frequency electromagnetic field exposure of cells in culture [telecommunications health and safety

A systematic measurement has shown that the background static and ELF magnetic fields in biological incubators can vary by orders of magnitude within a single incubator and between different incubators. Measured incubator fields varied from below levels that the IARC had classified as possibly carcinogenic to humans (0.3 μT to 0.4 μT) to as high as 240 μT, above the Earth or natural geomagnetic field range (23 μT to 65 μT). The significance is that studies of biological effects of ELF field exposure on cell cultures involved magnetic fields ranging from 0.1 μT to 50 μT. The inconsistency or variability of in vitro laboratory data prompted many to declare that there was no compelling evidence that biological effects are causally related to ELF magnetic-field exposure. Most in vitro investigations involving cell cultures in the laboratory were unaware of and therefore did not account for the large variations in background static and ELF magnetic fields in cell-culture incubators. Given the potential confounding of unspecified incubator fields, it seems only reasonable to reassess the data and reevaluate the validity of any conclusions to date, positive or negative. Are we back to square one? In some sense, it may be so: the reported background static and ELF magnetic-field variations in cell-culture incubators could be a game changer.

Effects of plasma-emulating light-emitting diode (LED) versus conventional LED on cytotoxic effects and polymerization capacity of orthodontic composites

The aim of this study was to evaluate, the cytotoxicity of orthodontic composites in vitro as a function of degree of conversion (DC) and the light curing units (LCU) employed on mouse fibroblast (L929). Cured samples of the composites Light bond (Reliance Orthodontic Products, Itasca, Illinois, USA), Ortho bracket paste (Bisco, Schaumburg, Illinois, USA), Opal bond MV (OPAL, South Jordan, Utah, USA), and Transbond XT (3M, Monrovia, California, USA) were prepared. Polymerization was performed with two LCUs: VALO Ortho (Ultradent, South Jordan, Utah, USA) is a third-generation LCU and Elipar S10 (3M, USA) is a second-generation LCU. Four samples were immersed in cell culture medium to obtain composite extracts. After incubation of L929 cell cultures with the extracts obtained, cytotoxicity was determined using the methyl tetrazolium test. Fourier transform infrared spectroscopy (FTIR) was used to evaluate DC for five samples. A multivariate analysis of variance (ANOVA), two-way ANOVA, and Tukey's honestly significant difference test were utilized for statistical analyses. Cytotoxicity and DC of all tested composites (p < 0.001) and the interaction between composites and LCUs (p < 0.01) were significantly different. LCUs had no significant influence on the cytotoxicity and DC of composite materials (p > 0.05). The correlations between cell viability and DC were positive for three composites but statistically insignificant. Composites and LCUs must be matched with one another to result in satisfactory maximal biocompatibility and DC. Opal Bond plasma light-emitting diode combination was a better choice for cell viability. Three composites showed a positive correlation between cytotoxicity and DC. Therefore high-intensity LCUs can be said to efficiently affect polymerization, and so, higher DC rates may achieve higher cell viability rates.

102 In-vitro characterization of myocardial cryothermal injury

Cardiac balloon and catheter cryoablations for the treatment of atrial fibrillation have been gaining attention as approaches for treating arrhythmias, in particular for isolation of the pulmonary veins (PV). Despite widespread and growing clinical use of cardiac cryoablation, there are still questions regarding dosing and treatment time, which may affect both efficacy and collateral injury. To date injury thresholds for therapy of cardiac and/or PV tissues are largely unreported in the literature. Slices of ventricular myocardium ( n = 11) and left atrial tissue ( n = 6) from female Yorkshire Cross swine hearts were dissected including the native endocardial surface. Samples were placed in an infrared imaging apparatus consisting of a plastic petri dish with central ablation probe (Galil Medical) and 2 mm of Sylgard polymer formed to the bottom. The tissue slices were impaled on the central cryoprobe and infrared thermography was captured using a Flir A20 looking from the top down. Subsequently, samples were cultured for 24 h in a cell culture incubator (myocardium) at 37 °C for optimal staining with 1% TTC in Trizma buffer for 1 h at 37 °C. Photographs of the resultant TTC staining of each sample were captured and a custom Matlab program was used to normalize staining intensity and correlate the average staining intensity with the average thermal profile at any radial distance from the cryoprobe. The relationship between the staining intensity and end temperature was fit with a sigmoidal curve. Samples with a fit of greater than r 2 > 0.99 were analyzed. The transition from dead to injured tissue was defined as the point on the curve fit where the staining ratio was 10% of the range between the lower and upper asymptotes of the sigmoid (i.e., 10% of the grayscale value change at the lesion margin closest to the lesion). The maximum cooling rate correlated to this ratio and end temperature was then calculated from the thermal profiles. Eight thermal profiles were averaged to obtain a characteristic thermal profile for each sample. This approach has led to the creation of a database of thermal profiles that results in either completely or partially necrotic tissues. Initial results suggest that cooling rates of −103 ± 49 and −72 ± 9 °C/min and end temperatures of approximately −21 ± 6 and −17.2 ± 3.2 °C are necessary for complete necrosis of ventricular and left atrial tissue, respectively. Using a two-tailed T -test assuming equal variance the difference in end temperature and cooling rate between ventricular and left atrial tissue were non-significant ( P = .21, P = .20 respectively). Experiments are ongoing to determine cryothermal injury thresholds of tissues associated with cardiac ablation, such as: lung and esophagus. The TTC staining protocol is in the process of being validated by comparison to H& E stain read by a blinded pathologist. Cellular viability assays are being performed with dye exclusion assays and results are being compared with tissue assays. Source of funding: GAANN Fellowship, Institute for Engineering in Medicine at the University of Minnesota, Medtronic, Inc. Source of funding: Medtrain, Inc. Conflict of interest: Ryan Goff has an Internship with Medtronic, Inc. goffx073@umn.edu

Osteogenesis of Mesenchymal Stem Cells by Nanoscale Mechanotransduction

It is likely that mesenchymal stem cells will find use in many autologous regenerative therapies. However, our ability to control cell stem growth and differentiation is presently limited, and this is a major hurdle to the clinical use of these multipotent cells especially when considering the desire not to use soluble factors or complex media formulations in culture. Also, the large number of cells required to be clinically useful is currently a hurdle to using materials-based (stiffness, chemistry, nanotopography, etc.) culture substrates. Here we give a first demonstration of using nanoscale sinusoidal mechanotransductive protocols (10-14 nm displacements at 1 kHz frequency), "nanokicking", to promote osteoblastogenesis in human mesenchymal stem cell cultures. On the basis of application of the reverse piezo effect, we use interferometry to develop the optimal stem cell stimulation conditions, allowing delivery of nanoscale cues across the entire surface of the Petri dishes used. A combination of immunofluorescence, PCR, and microarray has then been used to demonstrate osteoblastogenesis, and the arrays implicate RhoA as central to osteoblastic differentiation in agreement with materials-based strategies. We validate this with pharmacological inhibition of RhoA kinase. It is easy to envisage such stimulation protocols being up-scaled to form large-scale osteoblast bioreactors as standard cell culture plates and incubators are used in the protocol.

Impact of H 2 O 2 Decontamination Technology Over High Heat Sterilization: Increased Efficiency and Efficacy with Significantly Reduced Downtime

BioTechniquesVol. 54, No. 3 Application Forum - Sponsored PaperOpen AccessImpact of H2O2 Decontamination Technology Over High Heat Sterilization: Increased Efficiency and Efficacy with Significantly Reduced DowntimeDeepak M. Mistry & Nehal KothariDeepak M. MistryStrategic Development & MarketingSearch for more papers by this author & Nehal KothariStrategic Marketing & Application SpecialistSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114016AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail IntroductionContamination of cell cultures is easily the most common problem encountered in cell culture laboratories and can lead to very serious consequences. Of all the likely sources, the cell culture incubator has often been considered one of the most probable sources of biological contamination. Many incubator manufacturers have addressed this issue using different types of sterilization technologies.Determining the Most Efficient and Effective TechniqueA study was undertaken to determine the effectiveness and efficiency of three industry-leading incubators that utilize different sterilization techniques: Panasonic's MCO- 19AIC UVH with H2O2 decontamination technology that requires 2 hours for its cycle with around 15 minutes of prep time, Brand A with a dry heat sterilization method that takes 10-12 hours for the complete process, and Brand B with a moist heat sterilization method that requires up to 25 hours from start to finish. Five different microorganisms, generally considered the most common contaminants in a cell culture environment, were tested in this study: Candida albicans, Acholeplasma laidlawii, Mycoplasma orale, Bacillus subtilis, and Staphylococcus aureus.At a specific number of colony forming units (CFUs), each organism was placed on both upper and lower shelves of the incubators, which were then allowed to go through their individual sterilization cycle. A time control coupon was also run simultaneously for the same amount of time as the sterilization cycle.Each of the three cell culture incubators showed complete bioburden reduction for each organism. However, Panasonic's MCO-19AIC UVH offered complete organism elimination in 2 hours, as compared to 10-12 hours and 25 hours for Brand A and Brand B, respectively. In addition, a reduction in viability was observed for Candida albicans, Acholeplasma laidlawii and Mycoplasma orale when each time control was compared. The Panasonic MCO-19AIC UVH effectively showed a greater bioburden reduction than Brand A and Brand B when the 2 hour time control was compared to the 10-12 hour and 25 hour time controls. This validates the effectiveness with which Panasonic's MCO-19 AIC UVH incubator decontaminates the chamber. For Staphylococcus aureus and Bacillus subtilis, an increase in viability was observed over time when compared to time controls. However, Panasonic's MCO-19 AIC UVH incubator efficiently eliminated all of the bioburden in a considerably shorter span of time without significantly increasing the original concentration of the organisms.Figure 1. Comparison of sterilization effectiveness.Bioburden reduction was measured for three types of incubators: Panasonic's MCO-19 AIC UVH, Brand A, and Brand B.ConclusionThe 2 hour uptime advantage offered by the Panasonic MCO-19AIC UVH incubator with 100% kill rate and at least a 6-log reduction for all the organisms tested, leads to increased productivity in a research or GMP environment.Go towww.SterisonicH2O2.comto download a full version of the Whitepaper.FiguresReferencesRelatedDetails Vol. 54, No. 3 Follow us on social media for the latest updates Metrics Downloaded 152 times History Published online 3 April 2018 Published in print March 2013 Information© 2013 Future Science LtdPDF download

Evaluation of the Relationship Between Microenvironment and Biofilm and Coagulase Responses of Staphylococcus aureus Strains Under the Effect of Gentamicin

The frequency of infections caused by Staphylococcus aureus strains and the rate of antibiotic resistance among these strains are gradually increasing. Accordingly, serious problems emerge in the treatment of community or hospital acquired S.aureus infections. This study was aimed to determine the role of MIC and sub-MIC concentrations of gentamicin on biofilm and coagulase forming effects of S.aureus in in vitro test systems and cell cultures. A standard S.aureus ATCC 25923 strain and two clinical S.aureus strains isolated from blood cultures (C1 and C2) were included in the study. Gentamicin MIC values of the strains were determined with microdilution method at the cation-adjusted Mueller Hinton broth according to CLSI standards. For each strain, MIC, 50% MIC and 25% MIC values of gentamicin were determined separately. At the determined MIC values, biofilm formations of strains were determined with crystal violet method spectrophotometrically. Also, coagulase activities of the strains were evaluated in glass tubes. Human origin epithelial cell cultures namely HEp-2 cell lines, were infected with the standard and clinical S.aureus strains (Multiplicity of infection: 50/1) and left for incubation for two hours. After all, MIC, 50% MIC and 25% MIC values of gentamicin, were added to infected cell lines and incubated for 18 hours. Cells were blown up with distilled water and then bacteria were collected. Biofilm formation and coagulase production of these bacteria were evaluated. When S.aureus ATCC 25923 strain and C1 strains' biofilm formation was evaluated before (in vitro) and after incubation in cell culture, no difference was observed. However in C2 strain, under the effect of MIC level gentamicin, biofilm formation was occurred after interaction with the cell. In the same way, when coagulase responses were evaluated, after interaction with the cell, coagulase production of C2 strain was inhibited. These results indicated that, phenotypic characteristics such as biofilm formation and coagulase production might change during the process of bacterial adaptation to microenvironment. Further advanced experimental modelling designed with different combinations of antibiotics and different cell lines may provide data about the causes and timing of these phenotypic changes and shed light on the development of new treatment policies.

Assessment of RNA integrity in the postmortem pig colonic tissue ex vivo1

Surgical removal of porcine intestinal tissue followed by an ex vivo challenge is an alternative technique of testing the anti-inflammatory effect of bioactive compounds in the intestine of live pigs. We investigated the effects of ex vivo incubation of porcine colonic tissue on the quantity and quality of total RNA over a 12-h time period. Colonic tissue of pig (n = 6) was surgically removed immediately postslaughter and incubated for 0, 3, 6, and 12 h in a humidified cell culture incubator with 5% CO(2) at 37°C. Tissue samples were processed for RNA extraction. The quantity and quality of total RNA were assessed on a NanoDrop Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. Ex vivo incubation had an effect on both the quantity (P < 0.001) and quality (P < 0.001) of total RNA. Relative to the RNA yield at 0 h (505.0 ± 48.64 μg/mg), the yield was significantly reduced after 6 h (227.6 ± 25.52 μg/mg; P < 0.001) and 12 h (159.3 ± 24.19 μg/mg; P < 0.001) of incubation. The 28S and 18S rRNA bands were visibly intact after 0, 3, and 6 h of incubation. However, after 12 h of incubation, a degraded RNA profile was evident. The RNA integrity number (RIN) values for the 0, 3, 6, and 12 h of incubation were 9.4 ± 0.10, 9.0 ± 0.10, 6.7 ± 0.17 (P < 0.001), and 3.3 ± 0.24 (P < 0.001), respectively. The transcript abundances of 4 constitutively expressed genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), beta 2-microglobulin (B2M), and peptidylprolyl isomerase A (PPIA) were reduced at both 6 and 12 h of incubation. It is concluded that ex vivo incubation of porcine colonic tissue up to 3 h postmortem generates good quality total RNA suitable for gene expression studies.

An innovative stand-alone bioreactor for the highly reproducible transfer of cyclic mechanical stretch to stem cells cultured in a 3D scaffold

Much evidence in the literature demonstrates the effect of cyclic mechanical stretch in maintaining, or addressing, a muscle phenotype. Such results were obtained using several technical approaches, useful for the experimental collection of proofs of principle but probably unsuitable for application in clinical regenerative medicine. Here we aimed to design a reliable innovative bioreactor, acting as a stand-alone cell culture incubator, easy to operate and effective in addressing mesenchymal stem cells (MSCs) seeded onto a 3D bioreabsorbable scaffold, towards a muscle phenotype via the transfer of a controlled and highly-reproducible cyclic deformation. Electron microscopy, immunohistochemistry and biochemical analysis of the obtained pseudotissue constructs showed that cells 'trained' over 1 week: (a) displayed multilayer organization and invaded the 3D mesh of the scaffold; and (b) expressed typical markers of muscle cells. This effect was due only to physical stimulation of the cells, without the need of any other chemical or genetic manipulation. This device is thus proposed as a prototypal instrument to obtain pseudotissue constructs to test in cardiovascular regenerative medicine, using good manufacturing procedures.

Cytotoxic and antioxidant effects of grape seed oil on the treatment of leukemia with methotrexate

Methotrexate (MTX), a widely used cytotoxic chemotherapeutic agent, is often limited by its severe toxicity. Regarding the mechanisms of its adverse effects, several hypotheses have been put forward, among which oxidative stress is highly noticeable. Additive effect of oxidative damage caused by MTX to oxidative stress induced by cancer makes the situation dramatically bad. In order to reduce the damage, several approaches have been suggested. Grape seed is one of the most significant prophylactic agents due to its antioxidant and bioflavonoids composition. The aim of this study was to investigate the protective effect of grape seed oil against MTX-induced oxidative stress in K-562 human chronic myeloid leukemia cell lines. Cells were divided into groups as following control, GSOH (tumor cells treated with 200 mg/mL of grape seed oil), GSOL (tumor cells treated with 100 mg/mL of grape seed oil), MTX (tumor cells treated with 50 nM methotrexate) and MTX + GSOH ( tumor cells treated with 200 mg/mL of grape seed oil and methotrexate). For antioxidant statue; superoxide dismutase (SOD), catalase (CAT), paraoxonase (PON) and aryl esterase (ARE) activities, for lipid peroxidation; malondialdehyde (MDA) level and also for cytotoxicity; cell viability were detected in 24 th and 48 th hours of the cell culture incubation. Based on the data, 200 mg/mL of grape seed oil indicates synergic effects with MTX on K562 regarding cytotoxicity especially in 48 th hour. In case of GSOH + MTX combined treatment for 24 hours, antioxidant system take part preventing lipid peroxidation and a possible oxidative damage. Upon 48 hour-GSOH treatment, antioxidant parameters show significant increase and hence, prevent lipid peroxidation in cancer cells. In conclusion, GSOH complimentary treatment may be suggested for leukemia therapy with MTX to reduce side effects and enhance the cytotoxicity of MTX.

A new concept to measure cell proliferation using Fourier transform infrared spectroscopy

In our laboratory, we recently developed a new technique to measure cell proliferation that is based on infrared spectro-photometry and the density of cell culture medium. The fluid exchange between intra and extra cellular environments is the key to our method. Using this technique, we found that cell proliferation assessment may be performed much faster than presently existing methods, using two separate pieces of equipment (a cell culture incubator and an interferometer). We confirmed the viability of our method using five different cell lines, breast cancer cells (BT20), mouse normal embryonic fibroblast (NEF) as well as those expressing E6/E7 onco-proteins of human papillomavirus (HPV) type 16, and a human cervical cancer cell line (HeLa). Based on our findings, we propose the design of a mini-incubator allowing cell culture as well as infrared spectra collection of the culture medium, as an accessory to the FTIR work bench, which would become a promising arrangement for continuous cell proliferation monitoring. Moreover, the use of the collected culture medium will be highly efficient in terms of reducing both experimental time and cost.

Magnetoresistive-based real-time cell phagocytosis monitoring

The uptake of large particles by cells (phagocytosis) is an important factor in cell biology and also plays a major role in biomedical applications. So far, most methods for determining the phagocytic properties rely on cell-culture incubation and end-point detection schemes. Here, we present a lab-on-a-chip system for real-time monitoring of magnetic particle uptake by human fibroblast (NHDF) cells. It is based on recording the time evolution of the average position and distribution of magnetic particles during phagocytosis by giant-magnetoresistive (GMR) type sensors. We employ particles with a mean diameter of 1.2μm and characterize their phagocytosis-relevant properties. Our experiments at physiological conditions reveal a cellular uptake rate of 45 particles per hour and show that phagocytosis reaches saturation after an average uptake time of 27.7h. Moreover, reference phagocytosis experiments at 4°C are carried out to mimic environmental or disease related inhibition of the phagocytic behavior, and our measurements clearly show that we are able to distinguish between cell-membrane adherent and phagocytosed magnetic particles. Besides the demonstrated real-time monitoring of phagocytosis mechanisms, additional nano-biointerface studies can be realized, including on-chip cell adhesion/spreading as well as cell migration, attachment and detachment dynamics. This versatility shows the potential of our approach for providing a multifunctional platform for on-chip cell analysis.

Multifrequency impedance measurement technique for wireless characterization of microbiological cell cultures

An impedance measurement system with probe signal frequencies up to 50 kHz with AC-probe voltages below 30 mV rms was integrated for wireless and battery-free monitoring of microbiological cell cultures. The here presented modular design and the use of state-of-the-art components greatly eases adoptions to a wide range of biotechnological applications without the need of bulky LCR-meters or potentiostats. The device had a power consumption of less than 2.5 mA at a 3.3 V single power supply and worked trouble-free within the humid environment of a cell culture incubator. Measurements on lumped RC-elements showed an error of less than 1% for absolute values and less than 1° regarding the phase of the complex impedance. The performance of sensor devices with interdigitated electrode structures for the measurement of adherent cell cultures was tested in the presence of phosphate-buffered saline solution in the humid atmosphere of an incubator for biological cell cultures.

Effect of excess iodine on immune system; An in-vitro study

Abstract: Background & Aims: While iodine is an essential element for the synthesis of thyroid hormones, epidemiological studies have showed that excessive iodine intake leads to autoimmune thyroid diseases, with an unknown mechanism. Previous studies have showed disturbance in the circulating cytokines could lead to autoimmune diseases. To determine the role of iodine in cytokine production and development of thyroid autoimmune diseases, whole blood was stimulated with NaI (10 mm) and I 2 (0.5 mm). Methods: After evaluation of laboratory results of 25 healthy females (aged 40-45 years), 10 subjects with matched results were selected. Ten ml of sterile heparinized peripheral blood was taken from each subject and immediately were divided into 6 groups (control, NaI stimulated, I 2 stimulated and matching groups in presence of standard stimulators (LPS 1µg/ml & PHA 10µg/ml). Three identical sets were setup to investigate cytokine production at 24, 48, and 72 hours. All samples were incubated in cell culture incubator (95% O2 and 5% CO2) and after elapse of appropriate time, plasma was separated from each well and kept at -70 °C till the time of cytokines (IL-4, IL-10, INF-γ and TGF-β1) analysis. Results: NaI could significantly decrease the production of TGF-β1at all time points (P 2 significantly decreased the level of IL-4 and IL-10 (P 2 decreased the level of IL-4 as well as IL-10 (P<0.01). Conclusion: For the first time, the results of this study indicated that high levels of NaI and I 2 may reduce the level of protective cytokines in circulation. Finally, since neither thyroid hormones nor thyroid gland had role in this process, it may be concluded that thyroid autoimmunity is initiated from high consumption of iodine leading to the imbalance in cytokine production. Keywords: Autoimmune thyroid diseases, High iodine intake, Iodine, Sodium iodide, TGF-β1, INF-γ,

Na+ Channel Scn1b Gene Regulates Dorsal Root Ganglion Nociceptor Excitability in Vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).