Journal of Cellular Biotechnology - Volume 2, issue 2

Authors: Nowak, E. | Martin, F. | George, S. | Röber, N. | Conrad, K. | Küpper, J.-H.

Article Type: Research Article

Abstract: Paraneoplastic neurological syndromes (PNS) are caused by an immune response against neuronal proteins upon their ectopical expression in tumor cells. Ma2 belongs to the protein family of paraneoplastic Ma antigens (PNMA). Detection of Ma2-specific autoantibodies is relevant for diagnostics of anti-Ma2 PNS and an underlying tumor such as germ testicular cancer, small cell lung cancer or breast cancer. Thus, early tumor treatment should improve the outcome for PNS therapy either. Dot blot immunoassay based on recombinantly expressed and purified autoantigens could offer a sensitive method for identification of paraneoplastic autoantibodies from sera of PNS patients. Here we present purification …with IMAC and FPLC of human Ma2 autoantigen upon its recombinant expression in E.coli . Furthermore, we provide evidence that dot blot immunoassays with purified Ma2 autoantigen can be used for detection of Ma2-specific autoantibodies from sera of PNS patients. Show more

Keywords: Autoimmune disease, autoantibodies, dot blot immunoassay, FPLC, IMAC, Ma2, PNMA2, PNS, T7 expression system

DOI: 10.3233/JCB-15029

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 85-91, 2017

Authors: Ponce, Leslie | Heintz, Friedel | Schäfer, Ina | Klusch, Andreas | Holloschi, Andreas | Schmelz, Martin | Petersen, Marlen | Hafner, Mathias

Article Type: Research Article

Abstract: Keratinocytes are the main cell population in the epidermis, where they coexist with a variety of other cell types. Their successful isolation and cultivation have afforded opportunities to study epidermal functions. Human keratinocytes have been studied most extensively, but their source is limited by the skin supply. In a previous work, we developed an in vitro co-culture model of porcine keratinocytes with porcine sensory neurites to investigate functional interaction. However, a detailed description of the isolation of porcine keratinocytes and their culture conditions has not been given in detail. Here, we present the isolation procedure and a characterization of …keratinocytes derived from new-born piglets, using simple assays based on conventional and fluorescence microscopy. Media, coating substrates and plating densities were tested with respect to cell viability, proliferation and morphology. Growing keratinocytes in EpiLife keratinocyte growth medium (EKGM) on human collagen type I substrate was best to support proliferation. The minimum plated density was 500 viable cells/cm2 for primary and 1000 viable cells/cm2 for subcultured cells. Population doubling (PD) and generation time (tg) depended on the plating densities. Keratinocytes seeded at a density of 5000 viable cells/cm2 had a PD of 4.36±0.60 per passage and tg of 1.69±0.24 days. Our results show that the optimal isolation and culture conditions for keratinocytes from piglets differ from those for keratinocytes from adult pigs and humans. Thus, the information obtained from the characterization allowed the performance and optimization of a co-culture and contributes to further investigations in epidermal homeostasis and cutaneous sensation. Show more

Keywords: Keratinocytes, dorsal root ganglion neuron, piglet, porcine, primary cell culture, co-culture

DOI: 10.3233/JCB-15030

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 93-115, 2017

Authors: Muravyov, Alexie V. | Skorkina, Marina Yu. | Shamray, Elena A. | Tikunova, Tatyana S.

Article Type: Research Article

Abstract: The aim of this paper was to study the elastic and electrical properties of lymphoid cells in patients with acute lymphoblastic leucosis (ALL, n  = 15). The mechanical properties of the membrane of lymphoid cells was recorded by atomic force microscopy (AFM). The electrical properties of the lymphoid cell membranes were detected by the Kelvin method. The elastic and electrical membrane properties of lymphoid cells were recorded by incubation with doxorubicin, one of chemotherapy drugs. We used different concentrations and incubation time. It was shown that in the acute phase of the disease and the stage of stable remission the lymphoid …cell clones with a reduced stiffness and increased cell surface charge were found. In the experiments in vitro was demonstrated that the increased cell membrane rigidity may be one of the factors determining the tumor cell resistance to the chemotherapy. It was found that if to use the highest concentration of drug in the incubation medium (0.5–1.0 mg/ml) and its longest time, then the surviving cells had more elastic membrane (0.25–1.0 μPa) and the positive potential of the membrane surface (15–29 mV). These obtained data may have a significant prognostic importance for the evaluation of drug resistance of tumor cells. Show more

Keywords: Elastic and electrical properties of cells, acute lymphoblastic leucosis, doxorubicin

DOI: 10.3233/JCB-15031

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 117-123, 2017

Authors: Vahldiek, Janis L. | Thieme, Stefan F. | Gemeinhardt, Ole | Poch, Franz | Hiebl, Bernhard | Lehmann, Kai S. | Hamm, B. | Niehues, Stefan M.

Article Type: Research Article

Abstract: PURPOSE: Thermal ablation is an important interventional option in the management of liver tumors. Immediate postablational imaging regularly shows periablational enhancement. This peripheral hyperperfusion may induce heat-sink effects which could contribute to incomplete tumor ablation. To reduce the effect of hyperperfusion the feeding vessels source must be known. The aim of this study was to dynamically characterize the type of blood supply of the periablational enhancement zone immediately after hepatic radiofrequency ablation (RFA) using perfusion CT. METHODS: We used an in-vivo porcine liver model. Multipolar RFA was performed in healthy pig livers. Immediate post-ablational perfusion CT …was acquired. The contrast enhancement over time of the peripheral ablation zone, the aorta and the portal vein were recorded. Time differences of the peak periablational enhancement to the peak arterial perfusion and to the peak portalvenous perfusion were calculated and analyzed. RESULTS: The perfusion peak of the periablational enhancement zone always occurred in mean 8.1 s after the arterial peak in the aorta and in mean 16.9 s before the peak in the portal vein. CONCLUSIONS: Benign periablational enhancement is a result of primary arterial and not portalvenous hyperperfusion. In order to reduce heat sink effects, peri-ablational arterial balloon occlusion or transarterial chemoembolization may be beneficial during RFA. Show more

Keywords: Ablation techniques, CT-perfusion, liver, animal investigations

DOI: 10.3233/JCB-15032

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 125-130, 2017

Authors: Mhuka, Christine | Chatiza, Fungayi P. | Chidzwondo, Farisai | Sithole-Niang, Idah | Makuza, Stanley | Mlambo, Sibonani S.

Article Type: Research Article

Abstract: Lack of resources for characterization and the indiscriminate crossbreeding of cattle in Africa have led to the loss of some breeds. This study was aimed at screening for polymorphic primers for cattle breed identification in Zimbabwe. The Nguni and other local breeds have suffered genetic losses due to indiscriminate crossbreeding. Genetic characterization and semen cryopreservation are important in conservation and breeding programs. Genomic DNA was extracted from whole blood using a DNA extraction kit and was amplified using RAPD-PCR. A total of 8 primers was screened and five (OPX-15, OPB-04, 0PG-07, OPG-12 and OPD-02) were polymorphic and three (OPD-01, OPB-05 …and OPB-09) were monomorphic, as revealed by agarose gel electrophoresis. RAPD-PCR was found to be effective in detecting the polymorphisms within the bovine species. The polymorphic primers can be used to determine genotype variations in crossbred animals to determine genotype variations in different breeds. Show more

Keywords: Bovine, polymorphic, characterization, RAPD-PCR

DOI: 10.3233/JCB-15033

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 131-137, 2017

Authors: Peters, S. | Gemeinhardt, O. | Gemeinhardt, I. | Dietze, K. | Jung, F. | Hiebl, B.

Article Type: Research Article

Abstract: According to the DIN EN ISO 10993-1, assessing the potential of a substance to cause skin irritation represents an essential part of the biocompatibility test portfolio for all medical devices. Traditionally, the in vivo Draize skin test is used for skin irritation hazard assessment. However, in order to improve animal welfare, considerable efforts have been undertaken to develop and validate alternative in vitro test methods for the replacement of in vivo testing. In these assays, fully differentiated three-dimensional reconstructed epidermal tissue (RhE) grown from physiological human keratinocytes in a chemically defined medium at the air liquid interface is exposed to …potential irritants for defined periods of time to assess certain changes caused by inflammatory processes (e.g., viability or interleukin 1α release). Unfortunately, time periods of exposure of RhE to irritants are not defined clearly. Thus, objective of the presented study was to examine RhE viability and IL-1α release after 15 min, 24 hrs, and 48 hrs of exposure to a certain irritant. After RhE exposure to the negative control and an extract of an exemplary biomaterial (polypropylene) for 15 min and 24 hrs, Il-1α release did not exceed the threshold value and RhE viability remained unchanged. However, after 48 hrs RhE viability was reduced by 54.2% and IL-1α concentration in the cell culture medium reached 10.1 ± 1.9 pg/ml. In contrast, growth of RhE exposed to the positive control medium for 15 min caused a reduction in RhE viability of about 27.1% and a high IL-1α release (23.8 ± 13.0 pg/ml). During the following 24 hrs of exposure to the positive control medium, RhE viability was reduced by 99%. IL-1α concentration in the cell culture medium remained unchanged for the whole testing interval (48 hrs), which was probably due to the significantly lowered cellular capacity for IL-1α internalization and metabolization of RhE cells. Based on these results we suggest that a time period of 24 hrs for irritation testing of extracts of medical devices using RhE might be advantageous. Show more

Keywords: epidermis, skin irritation, biocompatibility, IL-1α, MTT

DOI: 10.3233/JCB-15034

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 139-144, 2017

Authors: Yerer, Mukerrem Betul | Tiryaki, Mehmet Kaan | Demirpolat, Eren

Article Type: Research Article

Abstract: BACKGROUND: GSK-3β, has been shown to regulate APP cleavage resulting in the increased production of Aβ and the inhibition of the GSK-3β reduced the amyloid beta induced neurotoxicity in several studies. OBJECTIVE: This study was designed to investigate the GSK-3β inhibitory effects of 6-gingerol and 6-shogaol the major components of Zingiber officinale could be able to recover the SHSY-5Y cells from Amyloid beta 1–42 oligomer and aggregate toxicity. METHODS: SHSY-5Y (ATCC; CRL-2266) cells were maintained in Dulbecco’s modified eagle medium (DMEM, Gibco) containing 10% fetal calf serum, 100 U/mL penicillin, 50 μg/mL streptomycin, at 37°C …and 5% CO2 in a humidified incubator. Cells were transferred to sterile 96-well e-plates at 5×104 cells per well and followed real time for 78 hours via Xcelligence system. Ferulic acid was used as a positive control as a GSK-3β inhibitor at 4 μM dose and 6-gingerol and 6-shogaol were applied to the cells at 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM doses/50 μL with or without β-Amyloid 1–42 oligomers or aggregates. GSK-3β activity was determined by Kinase-Glo assay. RESULTS: Both the amyloid beta aggregates and the oligomers had cytotoxic effect on SHSY-5Y cells followed by the real time cell analyzer system. 6-gingerol and 6 shogaol inhibited the GSK-3β enzyme up to % 20 levels in a dose dependent manner until 1 μM. However, parallel with the reduction of the inhibition for 6-shogaol over 1 μM, showed a toxicity itself on SHSY-5Y cells, whereas 6-gingerol did not show any cytotoxic effect at any doses applied. CONCLUSIONS: 6-gingerol and 6-shogaol increased the cell viability followed in a real time manner after around 24 hours than the amyloid beta aggregate or oligomer toxicity as much as the ferulic acid could. Furthermore these effects supported the GSK-3β inhibitory effects of both compounds at low doses up to 1 μM. These results reveal that 6-gingerol and 6-shogaol the major components of Zingiber officinale help to recovery from amyloid beta toxicity which might further be investigated by clinical trials. Show more

Keywords: GSK-3β, Amyloid β1–42 oligomer, Amyloid β1–42 aggregate, 6-gingerol, 6-shogaol, ferulic acid

DOI: 10.3233/JCB-15035

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 145-157, 2017

Authors: Degenhardt, Svenja | Janz, Olga | Slijepcevic, Mirjana | Wenzel, F.

Article Type: Research Article

Abstract: Apheresis products are regularly stored in bags containing phthalates. This study was initialized to find a possible relation between storage period, storage temperature, and diverse organic and non-organic substances with the migration of phthalates out of PVC-containing medical products as bags. Data were obtained by the observation of 20 autogenic peripheral stem cell donors, 21 allogenic peripheral stem cell donors as well as 10 bone marrow donors. Identification and quantification of DEHP and MEHP (primary metabolite of DEHP) was realized due to high performance liquid chromatography-mass spectrometry (LCMS). The results revealed that DEHP diffused out of the tube system (as …part of apheresis device) into the blood and was then also detectable in apheresis products. Further experiments focused on storage conditions could demonstrate that migration of DEHP out of PVC-containing storage bags directly correlated with storage temperature. In addition, concentrations of DEHP and MEHP were also influenced by storage period. All concentrations of DEHP remained below the recommended “tolerable daily intake” (TDI). Show more

Keywords: Di(2-ethylhexyl) phthalate, DEHP, MEHP, stem cell apheresis, storage conditions, PVC

DOI: 10.3233/JCB-15036

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 159-169, 2017

Article Type: Other

Citation: Journal of Cellular Biotechnology, vol. 2, no. 2, pp. 171-173, 2017