Journal of Cellular Biotechnology - Volume 5, issue 2

Authors: Keller, Florian | Rudolf, Rüdiger | Hafner, Mathias

Article Type: Research Article

Abstract: BACKGROUND: Reproducibility and physiological relevance are essential features for test systems used in preclinical cancer drug development. In this context, 3D cell culture models like spheroids or organoids have recently become attractive due to their potential of mimicking native biology. Further, to increase screening throughput and effectiveness, full automation of sample preparation and data analysis is important. OBJECTIVE: This work addressed methodological factors of spheroid cell culturing that are of particular relevance for reproducibility and physiological significance in studies investigating metabolic effects of drug treatment, including media composition, extracellular matrix, addition of stromal cells, and the quality of …contrast-based readouts. METHODS: Using standardized and enriched media as well as additional basal membrane extract, spheroids were made from MCF10A human breast epithelial cells and MDA-MB-231 human breast cancer cells. In addition, co-culture spheroids of MDA-MB-231 with CCD-1137Sk human fibroblast cells were prepared. Samples were compared in terms of metabolic behaviour. Spheroid analysis employed SpheroidSizer software, confocal microscopy, and Western blotting. RESULTS: Media composition, supportive additives and the co-culture situation can massively alter the growth and metabolic behaviour of spheroids. In particular, spheroid integrity was affected by the presence of extracellular matrix components and upon long-term culturing. Due to drug- or culture-induced spheroid disintegration, automated spheroid size analysis data needed careful evaluation. CONCLUSION: Media composition, extracellular matrix, addition of stromal cells, and automated readouts are important parameters for standardized spheroid-based drug screening approaches. Contrast-based spheroid size analyses in automated high-throughput screenings need special attention, since spheroid-disaggregation or proliferation of surrounding cells may sophisticate the readouts. Show more

Keywords: High-throughput screening methods, cell culture, co-culture, breast cancer, multicellular spheroids, automation, tumor cell line

DOI: 10.3233/JCB-199001

Citation: Journal of Cellular Biotechnology, vol. 5, no. 2, pp. 89-101, 2019

Authors: Keil, Karolin | Dierenbach, Jonas | Tillich, Ulrich M. | Steuer, Ralf | Frohme, Marcus

Article Type: Research Article

Abstract: High-throughput screening and cultivation methods offer significant potential to accelerate the development of phototrophic microorganisms into microbial cell factories. To date, however, only few suitable platforms have been reported, often lacking adequate methods for automated data handling and analysis. Here we report an improved high-throughput cultivation and screening platform that incorporates a newly developed open-source laboratory information and management system implemented using the R package Shiny. The PhotoScreener Viewer allows the user to automatically backup the collected data, provides methods for interactive graphical representation of data, including a set of statistical tests. Furthermore, the platform enables authorized users to …create, view and edit their experiments online. As an advanced application study for the improved platform, we consider the response of an ethanol-producing cyanobacteria to varying salinity of the culture medium. Based on the conjecture that high-salt conditions impact the productivity of a ethanol-producing modified strain of Synechocystis sp. PCC 6803, we monitor growth, vitality and ethanol production using cultivation in 96-deep-well plates within the robotic platform. We could not observe an increased ethanol production, but rather the opposite. Show more

Keywords: Synechocystis sp. PCC 6803, ethanol, salt tolerance, biofuel, high-throughout screening (HTS), laboratory information and management systems (LIMS), salt acclimation

DOI: 10.3233/JCB-199002

Citation: Journal of Cellular Biotechnology, vol. 5, no. 2, pp. 103-114, 2019

Authors: German, Jasmin | Ruchlak, Ralf | Witt, Thomas | Wenzel, Folker

Article Type: Research Article

Abstract: INTRODUCTION: Nowadays obesity is a widespread health challenge, be it for the person, the society or the health system. Obesity is divided into different classifications using the BMI. In this study, the cutaneous microcirculation of obese patients was measured during a rehabilitation stay. MATERIALS AND METHODS: The study involved 25 patients, who were treated because of their obesity in rehabilitation (mean BMI of 44.75 kg/m2 (7 females and 18 males)). The 11 controls had an average BMI of 24.93 kg/m2 (3 females and 8 males). The PeriFlux 5000 by Perimed Instruments GmbH was used in order to …measure the microcirculation in a skin area of about 1 mm3 on the left middle finger. RESULTS: The baseline LDF-value of the obese patients was at the beginning of the rehabilitation 256.73±11.64 PU and at the end at 312.46±15.51 PU. Immediately after exercise, the microcirculation decreased to a mean value of 254.57±11.67 PU at beginning of the rehabilitation. At the end, the microcirculation decreased to a mean value of 306.94±11.75 PU. The control group’s microcirculation at the baseline was at 205.2±20.91 PU and under heat provocation at 394.36±25.03 PU. CONCLUSION: In obese patients, it could be observed that during a rehabilitation interval of three weeks not only the body weight showed a reduction but also in parallel an improvement in cutaneous microcirculation could be seen. So stimulating und monitoring of cutaneous microcirculation may be a supportive parameter in therapeutic strategies for obese patients. Show more

Keywords: Cutaneous microcirculation, obese patients, rehabilitation

DOI: 10.3233/JCB-199003

Citation: Journal of Cellular Biotechnology, vol. 5, no. 2, pp. 115-123, 2019

Authors: Jung, Friedrich | Jung, Conrad G.H. | Krüger-Genge, Anne | Waldeck, Peter | Küpper, Jan-Heiner

Article Type: Research Article

Abstract: Since there is growing interest throughout the world in photosynthetic microbes as a potential source of food or food supplements, an assessment of factors which influence the biomass obtained in bioreactors, protein contents and constituents is important. This work reviews the autotrophic cultivation conditions of Spirulina platensis especially the dependency on the strain, the composition of the nutrient solution, pH, temperature of the medium, light intensity and color as well as exposure rhythm, the flow rate and composition of the aerating gas mixture and the bubble size, the content of oxygen, CO2 and HCO3 in the medium and …last but not least from the optical density of the spirulina suspension during growth. Show more

Keywords: Spirulina platensis, influencing factors, temperature, light intensity, wave length

DOI: 10.3233/JCB-199004

Citation: Journal of Cellular Biotechnology, vol. 5, no. 2, pp. 125-134, 2019

Authors: Seifert, Verena | Liers, Josephine | Kniess, Torsten | Richter, Susan | Bechmann, Nicole | Feldmann, Anja | Bachmann, Michael | Eisenhofer, Graeme | Pietzsch, Jens | Ullrich, Martin

Article Type: Research Article

Abstract: BACKGROUND: Pheochromocytomas and paragangliomas (PPGLs) are rare catecholamine-producing tumors arising from chromaffin tissue. In a PPGL subgroup, dysregulation of hypoxia signaling pathways, in particular mediated through stabilization of hypoxia-inducible factor 2 alpha (HIF2α), have been suggested to drive tumorigenesis through altering downstream transcriptional activity. OBJECTIVE: This study evaluated the use of mCherry-transgenic mouse pheochromocytoma (MPCmCherry ) spheroids as in vitro models for investigating consequences of HIF2 α expression on aggregation behavior, morphology, growth, glucose consumption, amino acid uptake, and somatostatin type 2 receptors under stable hypoxic conditions. METHODS: MPCmCherry spheroids were monitored using confocal …laser scanning microscopy. Hypoxic regions were detected using pimonidazole. Radiotracer incubation was performed using 2- [18 F]fluoro-2 -deoxyglucose ([18 F]FDG), O -3 -(2 [18 F]fluoroethoxy)-4 -hydroxyphenylalanine ([18 F]OFED), and [68 Ga]Ga-(Tyr3 )octreotate ([68 Ga]Ga-DOTA-TATE). RESULTS: Both HIF2 α-expressing and empty vector (EV) control spheroids showed regions of stable cellular hypoxia. Expression of HIF2 α in MPCmCherry spheroids was associated with less symmetric morphology, faster growth, and decreased uptake of [68 Ga]Ga-DOTA-TATE (somatostatin type 2 receptors) compared to controls, whereas, uptake of [18 F]FDG (glucose transporter 1 and hexokinases) and [18 F]OFED (system L amino acid transporter 1) remained unaffected. CONCLUSIONS: The recent study proved MPCmCherry spheroids to be complex three-dimensional tumor cell models for investigating morphologic and metabolic consequences of dysregulated hypoxia pathways under hypoxic conditions. Show more

Keywords: Amino acid transporters, confocal laser scanning microscopy, neuroendocrine tumors, paraganglioma, pimonidazole, positron emitters, radiotracer uptake, somatostatin receptors

DOI: 10.3233/JCB-199005

Citation: Journal of Cellular Biotechnology, vol. 5, no. 2, pp. 135-151, 2019