Concentration Of Fetal Bovine Serum Research Articles

Research Note: Adipogenic differentiation of embryonic fibroblasts of chicken, turkey, duck, and quail in vitro by medium containing chicken serum alone

The study of adipogenesis is one of the most important areas for not only regulating meat quality, but production efficiency associated with fat accretion in the poultry species. Current in vitro models for avian adipogenesis require adipogenic inducers including dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), fatty acids, or insulin. However, problems still remain in these models for testing/screening potential nutritional, hormonal, and pharmaceutical factors because of interfering/overriding effects of the inducing factors. Therefore, the purpose of this study was to develop a simple in vitro method for avian adipogenesis. In this study, chicken serum (CS) and fetal bovine serum (FBS) were compared for adipogenic potential using chicken embryonic fibroblasts (CEF). Oil-red O staining at 4 d in culture of CEF under CS revealed that lipid droplet formation was increased by CS in a dose-dependent manner (0 to 10%). On the contrary, all concentrations of FBS (0 to 10%) alone did not show lipid droplet formation. In accordance with the morphological data of CEF, mRNA expression of genes involved in adipocyte differentiation/determination, fatty acid uptake, and triacylglycerol (TAG) synthesis, were most significantly up-regulated by 10% CS at d 4 compared to 1 or 5% CS. In addition, embryonic cells isolated from quail (QEF) at E5, duck (DEF) at E6, and turkey (TEF) at E6, were tested for adipogenic differentiation by media containing the same concentrations of CS. Similar to the morphological data from CEF, quantitative data of the Oil-red O staining showed that lipid droplet formation in QEF, DEF, and TEF was increased by CS in a dose-dependent manner (0 to 10%). The current study demonstrates that CS alone can induce adipogenesis on embryonic fibroblasts of various poultry species. By providing a new simple in vitro method of avian adipogenesis, diverse nutritional, hormonal, and pharmaceutical factors can be broadly and easily tested for scientific and industrial purposes.

CHO/LY‐B cell growth under limiting sphingolipid supply: Correlation between lipid composition and biophysical properties of sphingolipid‐restricted cell membranes

Sphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO linein whichserine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In this study, LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, that is, 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72hours. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of growth rates analogous to those of control LY-B cells. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led only to a sixfold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Smaller, though significant, changes were also detected in glycerophospholipids under SL-restriction conditions.

Clusterin levels in undernourished SH-SY5Y cells

Food-related disorders are increasingly common in developed societies, and the psychological component of these disorders has been gaining increasing attention. Both overnourishment with high-fat diets and perinatal undernourishment in mice have been linked to a higher motivation toward food, resulting in an alteration in food intake. Clusterin (CLU), a multifaced protein, is overexpressed in the nucleus accumbens (NAc) of over-fed rats, as well as in those that suffered chronic undernutrition. Moreover, an increase of this protein was observed in the plasma of obese patients with food addiction, suggesting the implication of CLU in this eating disorder. To characterize CLU’s cellular mechanisms, in vitro experiments of undernutrition were performed using dopaminergic SH-SY5Y cells. To mimic in vivo dietary conditions, cells were treated with different fetal bovine serum (FBS) concentrations, resulting in control (C group) diet (10% FBS), undernourishment (U group) diet (0.5% FBS), and undernourishment diet followed by restoration of control diet (UC group) (0.5 + 10% FBS). Undernourishment compromised cell viability and proliferation, and concomitantly increased CLU secretion as well as the cytosolic pool of the protein, while decreasing the mitochondrial level. The restoration of normal conditions tended to recover cell physiology, and the normal levels and distribution of CLU. This research study is a step forward toward the characterization of clusterin as a potential marker for food addiction and nutritional status.

Relationship between serum concentration, functional parameters and cell bioenergetics in IPEC-J2 cell line.

The foetal bovine serum (FBS) concentration could influence functional parameters of IPEC-J2 cells. IPEC-J2 is a non-transformed continuous epithelial cell line that represents an established in vitro model to study porcine gut inflammation and alterations of intestinal integrity. This cell line also represents a good translational model thanks to the high similitudes between pig and human gastrointestinal tract. With the aim to assess if the FBS-dependent functional variations are linked to the bioenergetic aspects, the addition of 5% and 10% FBS in the IPEC-J2 culture medium were tested. Doubling time and TEER measurement indicated that cells cultured at higher FBS dose grow faster and as a more compact monolayer. 10% FBS increases ATP production and mitochondrial oxidative phosphorylation (OxPhos) and does not affect glycolysis. Both at 5% and 10% FBS ATP production mainly comes from OxPhos and FBS concentration does not affect the cell respiration bioenergetic parameters. Noteworthy, IPEC-J2 treated with 5% and 10% FBS have a metabolic potential since both OxPhos and glycolysis increase by > 100% and < 50%, respectively in comparison with baseline metabolism. Moreover, glucose, fatty acids and glutamine constitute the preferred metabolic fuel for mitochondrial respiration at both FBS conditions tested. Accordingly, the cells flexibility to oxidize these substrates shows that IPEC-J2 mitochondria cannot maintain the basal ATP production without oxidizing all the substrates available irrespective of FBS concentration. To sum up, in IPEC-J2 cells OxPhos increases with the FBS-stimulated functional physiological parameters to fulfil ATP requirements.

Can we Dispense with Sphingolipids? Correlation between Membrane Lipid Composition and Biophysical Properties in Sphingolipid-Restricted Mammalian Cells

LY-B cells derive from a CHO line in which serine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed. in the present study LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, i.e. 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 h.

Cerium oxide nanoparticles mitigate retinal pigment epithelium (RPE) death using APRE19 cell model

Objective(s): In this study, we present the potential of cerium oxide nanoparticle pretreatment on ARPE-19 cells, a cell line of the Retinal Pigment Epithelium (RPE), as a therapeutic modality to cellular stresses such as low serum starvation.Materials and Methods: ARPE-19 cells were pretreated with nano-cerium oxide at a concentration of 500 µg/mL before low serum stress was induced for 24, 48, 72, and 96 hours. Starvation stress was induced by using low concentrations of Fetal Bovine Serum (FBS) media at three increments: 10%, 1%, 0.1%.Results: Contrast images demonstrated higher cell confluence and cell integrity in cells pretreated with cerium oxide nanoparticles compared to untreated cells. Increased cell viability for cerium oxide pretreated cells was confirmed by MTS assay after 96 hours of serum starvation.Conclusion: By using nanoparticles to influence pathways of apoptosis, we hope to rescue ARPE-19 cells from a range of stressors, including oxidative stress, and re-establish homeostasis for the cell. Nanoparticles may represent a novel class of therapeutics for diseases of the eye, like AMD and blue-light induced oxidative stress.

Direct and Label-Free Monitoring of Albumin in 2D Fatty Liver Disease Model Using Plasmonic Nanogratings.

Non-alcoholic fatty liver (NAFLD) is a metabolic disorder related to a chronic lipid accumulation within the hepatocytes. This disease is the most common liver disorder worldwide, and it is estimated that it is present in up to 25% of the world’s population. However, the real prevalence of this disease and the associated disorders is unknown mainly because reliable and applicable diagnostic tools are lacking. It is known that the level of albumin, a pleiotropic protein synthesized by hepatocytes, is correlated with the correct function of the liver. The development of a complementary tool that allows direct, sensitive, and label-free monitoring of albumin secretion in hepatocyte cell culture can provide insight into NAFLD’s mechanism and drug action. With this aim, we have developed a simple integrated plasmonic biosensor based on gold nanogratings from periodic nanostructures present in commercial Blu-ray optical discs. This sensor allows the direct and label-free monitoring of albumin in a 2D fatty liver disease model under flow conditions using a highly-specific polyclonal antibody. This technology avoids both the amplification and blocking steps showing a limit of detection within pM range (≈0.26 ng/mL). Thanks to this technology, we identified the optimal fetal bovine serum (FBS) concentration to maximize the cells’ lipid accumulation. Moreover, we discovered that the hepatocytes increased the amount of albumin secreted on the third day from the lipids challenge. These data demonstrate the ability of hepatocytes to respond to the lipid stimulation releasing more albumin. Further investigation is needed to unveil the biological significance of that cell behavior.

Establishment and Characterization of Eye Muscle Cell Line from Snow Trout, Schizothorax richardsonii (Gray, 1832), a Vulnerable Coldwater Fish, for In Vitro Studies

Schizothorax richardsonii, commonly called snow trout is a vulnerable coldwater fish distributed in India and adjoining countries, is an important resource as food, ornamental and game fishery. A cell line, designated as SREM-1, has been established from the eye muscle of S. richardsonii using explant method and cultured in Leibovitz’s L-15 medium. The growth studies at 20-32°C temperatures and 5-20% of fetal bovine serum (FBS) concentrations revealed that SREM-1 cells exhibited optimal growth at 28°C in L-15 medium containing 20% FBS. Cell type was confirmed primarily as epithelial in nature by immuno-phenotyping assay and was sub-cultured for &gt;55 times since its development. The origin of the cell line was confirmed by sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. SREM-1 cell line is free from mycoplasma contamination and has been cryopreserved at different passage levels with revival efficiency of 70-80% after 6 months. The SREM-1 cell line was successfully transfected and also employed for evaluating in vitro cytotoxicity against a heavy metal, mercuric chloride. The newly established cell line of this vulnerable species would be useful as a model for aquatic toxicological and transfection studies.

Fetal bovine serum (FBS) enhances proliferation and colonization of caprine spermatogonial stem cells

Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. This study was undertaken to investigate the effect of serum concentration on colony formation and development of different types of SSC colonies with respect to passage number. Cells were isolated from pre-pubertal buck testes by two step enzymatic digestion method. The filtered cells were enriched by differential adherence selection method. Cells were then randomly divided into 8 groups, depending on concentration of FBS in culture medium ranging from 0% to 35%. In experiment 1, effect of different concentrations of FBS on total number pSSCs with reference to differential plating was observed while in experiment 2, effect of different concentrations of FBS on types of pSSC colonies with respect to passage number was observed. No colony formation was observed in control group (0% FBS) while significantly higher number of single, paired, cluster and rosette colonies observed were with 20% FBS group in differential 2 (D2) as compared to other groups. Alkaline phosphatase staining and immunocytochemistry staining (PGP9.5 and OCT4) were positive in SSCs colonies. The growth rate of the culture was significantly and consistently higher with 20% FBS.

Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor

Self-inactivating lentiviral vectors (LVVs) are used regularly for genetic modification of cells, including T cells and hematopoietic stem cells for cellular gene therapy. As vector demand grows, scalable and controllable methods are needed for production. LVVs are typically produced in HEK293T cells in suspension bioreactors using serum-free media or adherent cultures with serum. The iCELLis® is a packed-bed bioreactor for adherent or entrained cells with surface areas from 0.53 to 500 m2. Media are pumped through the fixed bed and overflows, creating a thin film that is replenished with oxygen and depleted of CO2 as media return to the reservoir. We describe the optimization and scale-up of the production of GPRTG-EF1α-hγc-OPT LVV using a stable packaging cell line in the iCELLis Nano 2-cm to the 10-cm bed height low compaction bioreactors (0.53 and 2.6 m2 surface area) and compare to the productivity and efficacy of GPRTG-EF1α-hγc-OPT LVV manufactured under current Good Manufacturing Practice (cGMP) using 10-layer cell factories for the treatment of X-linked severe combined immunodeficiency. By optimizing fetal bovine serum (FBS) concentration, pH post-induction, and day of induction, we attain viral yields of more than 2 × 107 transducing units/mL. We compared transduction efficiency between LVVs produced from the iCELLis Nano and cell factories on healthy, purified CD34+ cells and found similar results.

A simple method for isolation and culture of primary hepatocytes from Salvelinus leucomaenis (White-spotted Charr).

White-spotted charr (Salvelinus leucomaenis, S. I.) is an anadromous cold water-adapted fish, distributed in the Far East. We have previously reported the complete mitochondrial DNA sequences of white-spotted chars (S. l. imbrius and S. l. pluvius) in Japan. In general, fish hepatocytes are useful for cellular and biochemical studies of fish. In this study, we isolated hepatocytes from the liver of white-spotted charr and used basic methods, such as enzyme digestion and low centrifugation, to analyze the molecular mechanisms involved in specific cellular responses. The isolated hepatocytes could be cultured at 5-20°C but not 37°C. The morphology of hepatocytes was altered in a temperature-dependent manner. The properties of hepatocyte were similar to those of living fish. Moreover, the proliferation rate and damage of isolated hepatocytes depended on the concentration of fetal bovine serum in the culture medium. Taken together, this study demonstrates that this simple method for isolation and culture of hepatocytes from white-spotted charr may be useful for other biochemical and cellular studies.

Impact of serum concentration in cell culture media on tight junction proteins within a multiorgan microphysiological system

A key problem hindering the regulation and approval of organ-on-chip technology by international drug regulatory agencies is the lack of standardization. This study aims to evaluate the impact of different concentrations of fetal bovine serum (FBS) on tight junction formation between cells in a microphysiological system (MPS). Hence, a standardized FBS concentration in cell culture media used for MPSs can be proposed. Caco-2, HepG2, and HK-2 immortal cell lines representing gut, liver and, kidney organ models, respectively were cultured in microphysiological devices and monitored in real-time for transepithelial electrical resistance (TEER) and pH over a six-days period. Glass-based microfluidic chips with embedded TEER electrodes were utilized to measure the impedance of the cell monolayers. Considering albumin, kidney injury molecule-1 (KIM-1), and alkaline phosphatase biomarkers, the results obtained using cell culture media containing 5% FBS were more physiologically relevant, when compared to media containing 0% and 10% FBS. E-cadherin confocal microscopy and a live dead cell assay were performed and compared with TEER data to confirm this claim. This optimized FBS concentration should enable microphysiological platforms to perform at an optimum cell culture capacity, facilitating robust pathophysiology, drug toxicity as well as, absorption, distribution, metabolism, and excretion (ADME) studies.

A modified fabrication procedure of retinal explant and optimized formulation of culture medium in a three-dimensional retinal culture system

BackgroundThree-dimensional culture system of retinal explant is commonly used to study retinal ganglion cell (RGC) axon regeneration in vitro. The retinal explants fabricated by traditional procedure in culture system, however, are usually too small (merely 0.5 × 0.5 mm) to be easily detected or treated by current experimental techniques. Also, the constituents of culture medium have not been fully elucidated. New methodA fabrication procedure was developed to enlarge the retinal explants and explore the reasonable concentration of fetal bovine serum (FBS) for evaluating axonal regeneration. ResultsThere were no significant differences in the density or length of regenerative neurites in the retinal explants fabricated by traditional and modified procedures. Increased FBS concentrations promoted neurite regeneration, decreased RGCs apoptosis, and activated tyrosine kinase B (TrkB) receptors, all reaching a plateau at 1 % FBS. Comparison with existing methodsCompared with traditional procedure, the modified fabrication procedure facilitates application of experimental techniques to retinal explants, increases the efficiency of obtaining observation area of regenerating neurites, and reduces the wastage of retinal tissues. The recommended FBS concentration determined in this study is shown to be more suitable for studying neuronal regeneration. ConclusionThe retinal explants made by the modified fabrication procedure are successfully applied to the three-dimensional culture system, and presented several advantages over the traditional one. Furthermore, a preliminary experiment must be performed to determine the suitable concentration of FBS in each study to ensure accuracy and stability of the results obtained from the three-dimension retinal culture system.

Various factors affect lipopolysaccharide sensitization in cell cultures.

Commercially available lipopolysaccharide (LPS) is commonly used in research. Although protocols for its use are well established, we experienced a loss of LPS responsiveness in our cell cultures despite no obvious experimental changes. Our cell lines were stimulated with LPS and the media quantified for LPS responsiveness via an IL-8 ELISA. We discovered that the major cause of signal loss was differences in fetal bovine serum (FBS) formulation and concentration. One FBS formulation was notably better at eliciting an IL-8 signal than the second FBS, and 10% FBS in media was better at inducing LPS responsiveness than lower concentrations. We urge researchers to be aware of inherent variations in seemingly commonplace reagents as they may be unexpected sources of inconsistencies.

Dedifferentiation of MCF-7 Breast Cancer Continuous Cell Line, Development of Breast Cancer Stem Cells (BCSCs) Enriched Culture and Biomarker Analysis

BACKGROUND: Cancer stem cells (CSCs) eradication might serve as a robust approach for cancer eradication. MCF-7 as breast cancer continuous cell line is known to contain breast CSCs (BCSCs) for its capability to maintain its original tumor population. CSCs enriched culture is a fundamental tool for CSCs targeted therapy development. Effective and unsophisticated CSCs dedifferentiation protocol for producing CSCs enriched culture is needed.METHODS: MCF-7 cells were cultured initially in Dulbecco's Modified Eagle Medium (DMEM) low glucose medium then changed to DMEM:F12. Serum starvation was performed during each medium refreshment gradually with fetal bovine serum (FBS) concentration of 10%, 5%, 2.5% until reaching 1% FBS concentration. Stable MCF-7 culture was then adapted to serum free culture system, containing DMEM:F12, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and B27 supplement as dedifferentiation protocol for 18 days. Cluster of differentiation (CD)44 and CD24 double staining immunocytochemistry was performed to evaluate cell stemness.RESULTS: The population of cells expressing BCSCs markers (CD44+/CD24low) in non-adherent single cells subpopulation was significantly increased after the dedifferentiation procedure (70.39%) compared to control groups (0.71%) (p&lt;0.05). In contrast, the expression of BCSCs marker in adherent single cells subpopulation and for both adherent and non-adherent mammosphere the BCSCs markers showed a stable expression.CONCLUSION: BCSCs enrichment of breast cancer cell cultures from MCF-7 breast cancer cell line can be performed. Breast cancer cell plasticity is observed during the dedifferentiation protocol. Development of dedifferentiation inducing protocols can serve as an important foundation for breast cancer therapy development through BCSCs elimination.KEYWORDS: breast neoplasms, cell line, dedifferentiation, immunohistochemistry, neoplastic stem cells

Establishment and characterization of a new cell line from the muscle of humpback grouper (Cromileptes altivelis).

The humpback grouper (Cromileptes altivelis) is a commercially important species of the family Epinephelidae. With the development in aquaculture industry, C. altivelis breeding has gradually increased in volumetric production, leading to the occurrence of various diseases. In this study, we established a new cell line (CAM) derived from the muscle tissue of C. altivelis. Our results showed that the optimal growth temperature and working concentration of fetal bovine serum (FBS) of CAM cells were 28°C and 15%, respectively. DNA sequencing and comparative analysis of 18S rRNA gene sequence showed that CAM cell line was originated from C. altivelis. Chromosome analysis showed that the modal chromosome number of CAM cells was 48. After transfection using pEGFP-N3 plasmid, CAM cells exhibited high transfection efficiency, indicating that CAM cells could be used in foreign gene expression studies. Further, cytotoxicity analysis revealed that CAM cells were sensitive to Vibrio harveyi and Edwardsiella tarda. Moreover, the cytotoxicity of heavy metals (Hg, Cd, and Cu) to CAM cells was dose-dependent. This CAM cell line might be used as an ideal tool in vitro for analyzing and understanding the mechanisms of pathogenesis, host-pathogen interactions, and toxicity assay of heavy metals.

Beneficial influence of fetal bovine serum on in vitro cryosurvival of chicken spermatozoa

Chicken spermatozoa are highly susceptible to cryopreservation often requiring extenders containing additives to enhance their post-thaw quality. Although protective properties of fetal bovine serum (FBS) during freezing of tissue cultured cells are widely known, its potential as a cryoprotectant for sperm cells has not been largely explored. Thus, the aims of our study were to (i) investigate the protective effect of FBS at different concentrations (0, 5, 10, 15 and 20%) against cryodamages in chicken spermatozoa, and (ii) test the FBS concentration that yielded the best preservation versus 1 mg/mL of cholesterol-loaded cyclodextrins (CLCs). Samples were assessed before and after freezing for sperm motility parameters, plasma membrane and acrosomal integrities, mitochondrial membrane potential, oxidative stress and plasma membrane peroxidation. Our findings showed that, despite their beneficial effects on fresh spermatozoa, higher FBS concentrations (15 and 20%) obtained the worst results for most motility and functional parameters after thawing. In contrast, lower FBS concentrations (5 and 10%) improved all post-thaw variables when compared to control. Afterwards, based on regression analysis, the concentration of 7% FBS was chosen to be assessed against CLCs in an experiment composed by four groups: control, FBS, CLCs, and FBS + CLCs. FBS and FBS + CLCs groups exhibited higher progressive motility in fresh samples, whereas only FBS maintained higher post-thaw progressive motility. Additionally, the incorporation FBS into extenders increased the percentage of rapid cells and reduced free radicals production and plasma membrane peroxidation. Together, these outcomes indicated that FBS minimize some harmful effects of cryopreservation, providing an alternative for chicken semen extenders that in many aspects appears to be superior to CLCs at 1 mg/mL.

Mobility and Invasion Related Gene Expression Patterns in Equine Sarcoid.

Simple SummaryThe current studies profiled the expression of five equine sarcoid cell genes related to cell mobility and invasion (cell cycle control binding protein alpha, coronin 1b, metalloproteinase 2, tissue inhibitor of metalloproteinases 3 and vimentin) and compared the expression of these genes in healthy skin fibroblasts. Cells were collected from healthy and sarcoid-affected skin biopsies obtained by a qualified veterinarian. Gene expression patterns were investigated under two different conditions of cell culture, with high and low availability of nutritional components in the culture medium. The results showed significant differences in the expression of the two analyzed genes (coronin 1b and vimentin) depending on culture conditions. The obtained results emphasize the complexity of the genomic background of sarcoids and indicate the importance of further research on genes related to the physiological changes that occur in sarcoids.Sarcoids are the most common skin neoplasm in the Equidae family. Sarcoids are benign, but may cause severe damage in affected animals. Due to the high risk of post-treatment recurrence and the lack of an effective method of treatment, it is reasonable to perform studies on the molecular aspects of this neoplasm. Therefore, the present studies analyzed five genes (cell cycle control binding protein alpha, coronin 1b, metalloproteinase 2, tissue inhibitor of metalloproteinases 3 and vimentin) related to cell mobility and invasion traits. Primary healthy fibroblasts and sarcoid cells were obtained from skin biopsies. Cell lines were cultured in two different medium types with different concentrations of foetal bovine serum (10% and 0.5% FBS) to study its influence on the analyzed genes. Gene expression was measured using the real-time PCR method. The results showed significant differences in two genes (coronin and vimentin) depending on culture conditions. In conclusion, the results enabled finding two new genes, related to cell motility and invasion traits, in which gene expression is deregulated. Results of the study may put new knowledge into the complexity of the genetic background of this disease and show the importance of further analysis on this subject.

Aspects of a Methodological Approach to Determination of Interferon Alpha Specific Activity

Specific antiviral activity is one of the key indicators characterising pharmaceutical quality and pharmacological efficacy of interferon alpha products (IFN-α). Specific activity is determined using a bioassay measuring antiviral activity in cell culture. The aim of the study was to select the most appropriate conditions for in vitro determination of IFN-α product specific activity. Materials and methods: Vero, MDBK, Hep-2, and A-549 homologous and heterologous cell cultures, as well as vesicular stomatitis Indiana virus (VSV) and murine encephalomyocarditis (EMC) virus at a dose of 100 TCD50 /0.1 mL were used for determination of specific antiviral activity. The international reference standard of recombinant interferon alpha-2b activity (Interferon alpha 2b, human, rDNA, E. coli-derived, 2nd WHO International Standard, 1999, NIBSC Code No. 95/566) and human recombinant interferon alpha 2b in the form of solution (batch No. 040214, Pharmapark LLC, Russia) were used as IFN-α samples. Results: the analysis of the obtained data helped to determine: the combinations of cell lines and the indicator virus most sensitive to IFN-α; the optimal concentration of fetal serum in the medium, and the optimal time parameters; the preferred method of reporting test results. Conclusions: the following test conditions were found to be optimal: the MDBK/VSV and Hep-2/EMC combinations proved to be the most sensitive to IFN-α; the optimal period of interferon and cell culture incubation—24 hours; the optimal concentration of fetal bovine serum in the culture medium used for diluting interferon products—2–5%. The instrumental procedure is preferred for reporting the results of interferon antiviral activity determination, because it is up-to-date, reliable, accurate and time-efficient.

Yangambin and Epi-yangambin Isomers: New Purification Method from Ocotea fasciculata and First Cytotoxic Aspects Focusing on In Vivo Safety.

Ocotea fasciculata presents yangambin (YAN) and its isomer epi-yangambin (EPI-YAN) as major lignans, which are employed as the plant markers for quality control purposes and as potential pharmacological compounds. However, a gap between the pure isomers and safety and efficacy protocols is faced by the scientific community. In this context, this work aimed to report (i) a new and advantageous purifying process in a semi-preparative scale for YAN and EPI-YAN isolation from Ocotea fasciculata, and (ii) an in vitro cytotoxicity study to estimate, for the first time, the LD50 values of the isolated epimers, as well as the influence of albumin concentration in cell culture medium. The best condition for epimers isolation was achieved in normal-phase liquid chromatography. The lignan fraction (LF), previously obtained from the plant ethanolic extract, was purified yielding 17% and 29% of YAN and EPI-YAN, respectively. The in vitro study demonstrated that YAN and EPI-YAN were safe, and only at the highest concentration studied, a decrease on cell viability was observed. The estimated LD50 value was higher than 1612 mg/kg for both epimers. The LF, on the other hand, demonstrated an estimated LD50 of 422 mg/kg. Lignan cytotoxicity studies also evidenced that the higher cell viability was related to the higher concentration of fetal bovine serum as a source of albumin in medium. This is the first time the LD50 and safety of the isolated epimers were estimated, opening up great perspectives of success in in vivo studies.