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In the last few years, quantum dots (QDs) have attracted research interest in different fields of science and technology. Despite their applications, it is essential to understand how nanomaterials (with different crystal sizes) are metabolized inside organisms. Thus, the focus of this study was on an evaluation of how crystal sizes of CdSTe QDs affect the viability and response of the cytochrome P450 system in CHO-K1 and HEP-G2 cells. CdSTe QDs were synthesized using a microwave-assisted system at different reaction temperatures (60, 120, 150, and 180 °C) to obtain different crystal sizes. The optical and structural characterization confirmed four crystal sizes from 3 to 8 nm. Fluorescence microscopy confirmed that CdSTe QDs are incorporated into both cell lines. Viability studies suggested that CHO-K1 cells are more sensitive than HEP-G2 cells to CdSTe QDs and Cd+2 ions. The responsible mechanisms for the toxicity of QDs and Cd+2 are apoptosis followed by necrosis. The activity of CYP 1A1, 1A2, and 3A4 isoenzymes suggests that the smallest CdSTe crystals are recognized in a manner similar to that of Cd+2. Furthermore, the largest CdSTe crystals can have different metabolic routes than Cd+2.

Antibodies (Abs) are among the most important class of biologicals, showcasing a high therapeutic and diagnostic value. In the global therapeutic Ab market, fully-human monoclonal Abs (FH-mAbs) are flourishing thanks to their low immunogenicity and high specificity. The rapidly emerging field of single-cell technologies has paved the way to efficiently discover mAbs by facilitating a fast screening of the antigen (Ag)-specificity and functionality of Abs expressed by B cells. This review summarizes the principles and challenges of the four key concepts to discover mAbs using these technologies, being confinement of single cells using either droplet microfluidics or microstructure arrays, identification of the cells of interest, retrieval of those cells and single-cell sequence determination required for mAb production. This review reveals the enormous potential for mix-and-matching of the above-mentioned strategies, which is illustrated by the plethora of established, highly integrated devices. Lastly, an outlook is given on the many opportunities and challenges that still lie ahead to fully exploit miniaturized single-cell technologies for mAb discovery. ispartof: LAB ON A CHIP vol:21 issue:19 pages:3627-3654 ispartof: location:England status: published

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

Article history:Received 21 January 2015Accepted 2 May 2015Available online 28 May 2015Keywords:CHO-K1 cellsGrowth-arrestedHuman melanoma cellsPolycosanols Background:Polycosanolsderivedfromplantspecieshavetraditionallybeenusedinmedicineasantiproliferativeagentsfortreatingvariousviruses(primarilytheherpessimplexvirus).However,fewstudieshavestudiedtheireffects on hyperproliferative cell lines. In this work, the antiproliferative capacity of polycosanols from tall-oilpitch, obtained from black liquor soaps in the kraft pulping process of cellulose (specifically from Pinus radiata,Pinus taede,andEucalyptus globulus), was evaluated on CHO-K1 and CRL-1974 human melanoma cell lines.Results: The proliferative capacities and cell viabilities were measured for 72 and 140 h, respectively. TreatmentwithdocosanolproduceddifferentialeffectsontheCHO-K1andhumanmelanomacellsandsignificantlyaffectedtheir proliferation rates, but not their cell viabilities. Tetracosanol produced a significant negative effect on theproliferation of human melanoma cells, and this effect was less than that caused by docosanol. However, it hadno effect on the proliferation of CHO-K1 cells and did not induce any significant effect on the viability of thestudied cell lines.Conclusion: Docosanol and tetracosanol induced antiproliferative effects on the studied cell lines and exhibitedsignificantly greater effects on the oncogenic cell lines. Prior to this study, the capacity of these polycosanolshas never been investigated. Future studies will be necessary to determine their mechanisms of action onthese cell systems.©2015Pontificia UniversidadCatolicadeValparaiso. Production and hosting byElsevierB.V. Allrights reserved.

Chinese hamster ovary (CHO) derived cell lines are the preferred host system for the production of therapeutic proteins. The aim of this work was to explore the regulation of suspension-adapted CHO-K1 host cell line bioprocesses, especially under a temperature gradient from 37 °C to 31 °C. We analyzed cell cycle behavior through flow cytometry of propidium iodide stained cells and high throughput transcriptome dynamics by RNA sequencing. We found a cell culture state characterized by G0/G1 synchronization, mainly during the late exponential growth phase and towards the last days of the stationary phase. We successfully identified key genes and pathways connected with the particular culture states, such as response to low temperature, modulation of the cell cycle, regulation of DNA replication and repair, apoptosis, among others. The most important gene expression changes occurred throughout the stationary phase when gene up-regulation markedly prevailed. Our RNA-seq data analysis enabled the identification of target genes for mechanism-based cell line engineering and bioprocess modification, an essential step to translate gene expression data from CHO-K1 host cells into bioprocess-related knowledge. Further efforts aim at increasing desirable phenotypes of CHO cells, and promoting efficient production of high quality therapeutic proteins can highly benefit from this type of studies. Fil: Tossolini, Ileana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina Fil: López Díaz, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Intellectics Inc.; Estados Unidos. Salk Institute for Biological Studies; Estados Unidos Fil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina Fil: Prieto, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Cellargen Biotech S.R.L.; Argentina

Yingxiao Li,1,2 Kai Chun Cheng,1 Chiang-Shan Niu,3 Shih-Hsiang Lo,3,4 Juei-Tang Cheng,2,5 Ho-Shan Niu31Department of Psychosomatic Internal Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan; 2Department of Medical Research, Chi Mei Medical Center, Yong Kang, Tainan City, 3Department of Nursing, Tzu Chi College of Technology, Hualien City, 4Division of Cardiology, Department of Internal Medicine, Chung Hsing Branch of Taipei City Hospital, 5Institute of Medical Sciences, College of Health Science, Chang-Jung Christian University, Guei-Ren, Tainan City, TaiwanBackground: G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) has been shown to participate in glucose homeostasis. In animal models, a TGR5 agonist increases incretin secretion to reduce hyperglycemia. Many agonists have been developed for clinical use. However, the effects of TGR5 blockade have not been studied extensively, with the exception of studies using TGR5 knockout mice. Therefore, we investigated the potential effect of triamterene on TGR5.Methods: We transfected the TGR5 gene into cultured Chinese hamster ovary cells (CHO-K1 cells) to express TGR5. Then, we applied a fluorescent indicator to examine the glucose uptake of these transfected cells. In addition, NCI-H716 cells that secrete incretin were also evaluated. Fura-2, a fluorescence indicator, was applied to determine the changes in calcium concentrations. The levels of cyclic adenosine monophosphate (cAMP) and glucagon-like peptide (GLP-1) were estimated using enzyme-linked immunosorbent assay kits. Moreover, rats with streptozotocin (STZ)-induced type 1-like diabetes were used to investigate the effects in vivo.Results: Triamterene dose dependently inhibits the increase in glucose uptake induced by TGR5 agonists in CHO-K1 cells expressing the TGR5 gene. In cultured NCI-H716 cells, TGR5 activation also increases GLP-1 secretion by increasing calcium levels. Triamterene inhibits the increased calcium levels by TGR5 activation through competitive antagonism. Moreover, the GLP-1 secretion and increased cAMP levels induced by TGR5 activation are both dose dependently reduced by triamterene. However, treatment with KB-R7943 at a dose sufficient to block the Na+/Ca2+ exchanger (NCX) failed to modify the responses to TGR5 activation in NCI-H716 cells or CHO-K1 cells expressing TGR5. Therefore, the inhibitory effects of triamterene on TGR5 activation do not appear to be related to NCX inhibition. Blockade of TGR5 activation by triamterene was further characterized in vivo using the STZ-induced diabetic rats.Conclusion: Based on the obtained data, we identified triamterene as a reliable inhibitor of TGR5. Therefore, triamterene can be developed as a clinical inhibitor of TGR5 activation in future studies.Keywords: triamterene, CHO-K1 cells, TGR5, transfection, sitagliptin&nbsp

Aim Investigation of the bystander effect in Chinese Hamster Ovary cells (CHO-K1) co-cultured with cells irradiated in the dose range of 0.1–4 Gy of high LET 12C ions and X-rays. Background The radiobiological effects of charged heavy particles on a cellular or molecular level are of fundamental importance in the field of biomedical applications, especially in hadron therapy and space radiation biology. Materials and methods A heavy ion 12C beam from the Heavy Ion Laboratory of the University of Warsaw (HIL) was used to irradiate CHO-K1 cells. Cells were seeded in Petri dishes specially designed for irradiation purposes. Immediately after irradiation, cells were transferred into transwell culture insert dishes to enable co-culture of irradiated and non-irradiated cells. Cells from the membrane and well shared the medium but could not touch each other. To study bystander effects, a clonogenic survival assay was performed. Results The survival fraction of cells co-cultured with cells irradiated with 12C ions and X-rays was not reduced. Conclusions The bystander effect was not observed in these studies.

Many short peptides selectively permeabilize the bacteria plasma membrane, leading to their lyses and death: they are therefore a source of antibacterial molecules and inspiration for novel and more selective drugs, which may have wider application in many other fields, as selective anticancer drugs. In this chapter, it is presented a new method to investigate the permeabilization properties of antimicrobial peptides under strict physiological conditions, employing the patch-clamp technique coupled to a fast perfusion system.

A CHO cell clone (CHO-PoIFN-β) with stable porcine IFN-β expression under control of CMV promoter was selected under G418 pressure. In a 25cm(2) cell culture flask (5 ml culture medium), the cumulative protein yield of recombinant PoIFN-β reached 2.3×10(6) IU/ml. This cells clone maintained stable expression for at least 20 generations even in the absence of G418 selection pressure. The expressed recombinant PoIFN-β could induce the expression of porcine Mx protein in PK15 cells, and activate the chicken Mx promoter-controlled luciferase reporter gene expression, confirming that the recombinant PoIFN-β has the biological activity of natural porcine type-I interferon. In addition, the recombinant PoIFN-β fully protected PK15 cells against 1000 TCID(50) of porcine transmissible gastroenteritis virus and pseudo-rabies virus infection, demonstrating its high potential in therapeutic applications. This is the first report of establishing a mammalian cell line with stable expression of porcine IFN-β.

The aim of this study was to determine the toxic effect of atrazine at the ovarian cellular level. Chinese Hamster Ovary (CHO-K1) cell line was used to evaluate the degree of in vitro atrazine cytotoxicity and the morphological changes were followed during the cell death. Application of four bioassays confirmed that atrazine decreases ovarian cell proliferation and IC(50) were determined with each assay after 72 h of exposure. The level of apoptosis in atrazine treated cells was low.

In vitro effects of the carbamates pirimicarb and zineb and their formulations Aficida® (50% pirimicarb) and Azzurro® (70% zineb), respectively, were evaluated in Chinese hamster ovary (CHO-K1) cells. Whereas the cytokinesis-blocked micronucleus cytome assay was employed to test for genotoxicity, MTT, neutral red (NR), and apoptosis evaluation were used as tests for estimating cell viability and succinic dehydrogenase activity, respectively. Concentrations tested were 10–300 μg/ml for pirimicarb and Aficida®, and 1–50 μg/ml for zineb and Azzurro®. All compounds were able to increase the frequency of micronuclei. A marked reduction in the nuclear division index was observed after treatment with 5 μg/ml of zineb and Azzurro® and 10 μg/ml of Azzurro®. Alterations in the cellular morphology not allowing the recognition of binucleated cells exposed to 300 μg/ml pirimicarb and Aficida® as well as 10–50 μg/ml zineb and Azzurro®. All four compounds induced inhibition of both cell viability and succinic dehydrogenase activity and trigger apoptosis in CHO-K1 cells, at least when exposed for 24 h. The data herein demonstrate the genotoxic and cytotoxic effects exerted by these carbamates and reveal the potential risk factor of these pesticides, still extensively used worldwide, for both human health and the environment. Fil: Soloneski, Sonia Maria Elsa. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Kujawski, Maciej. City of Hope Comprehensive Cancer Center; Estados Unidos Fil: Scuto, Anna. City of Hope Comprehensive Cancer Center; Estados Unidos Fil: Larramendy, Marcelo Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Electroporation is an efficient, low-toxic physical method which is used to deliver impermeant macromolecules such as genes and drugs into cells. Genetic modification of the cell is critical for many cell and gene therapy techniques. Common electroporation protocols can only handle small volumes of cell samples. Also, most of the conventional electroporation methods require expensive and sophisticated electro-pulsation equipment. In our lab, we have developed new electroporation methods conducted in microfluidic devices. In microfluidic-base electroporation, exogenous macromolecules can be delivered into cells continuously. Flow-through electroporation systems can overcome the issue of low sample volume limitation. In addition, in our method, electro-pulsation can be done by using a simple dc power supply, without the need for any extra equipment. Furthermore, our microfluidic chips are completely disposable and cheap to produce. We show that electroporation and electroporation-based gene delivery can be conducted employing tapered asymmetric curving channels. The size variation in the channel's cross-sectional area makes it possible to produce electric pulses of various parameters by using a dc power supply. We successfully delivered Enhanced Green Fluorescent Protein, EGFP, plasmid DNA into Chinese Hamster Ovary, CHO-K1, cells in our microfluidic chips. We show that the particles/cells undergo Dean flow in our asymmetric curving channels. We demonstrate that there are three main regimes for particle motion in our channels. At low flow rates (from 0 to ~75μl/min) cells do not focus and they randomly follow stream lines. However, as flow rate increases (~75 to 500μl/min), cells begin to focus into one line and they follow a single path throughout the micro-channel. When flow rate exceeds ~500μl/min, cells do not follow a single line and demonstrate more complex pattern. We show that the electric parameters affect the transfection efficiency and cell viability. Higher electric field intensity results in higher transfection efficiency. This is also true in the cases with longer electroporation duration time. In our experimental work, we executed flow-through electroporation for various duration times (t = 2 ms, 5 ms, and 7 ms), and at various electric field intensities (from 300 to 2200 V/cm) while we utilized different flow rates as well, i. e. 150 μl/min (focused flow) and 600 μl/min (complex flow). To explore the impact of individual electric pulse length and electric pulse number on electroporation results, we designed control channels with straight narrow sections. Cells experience different hydrodynamic forces in straight channels compared to curving channels. Flow pattern and cell focusing were also studied in control channels as well. Also, electroporation on CHO-K1 cells was successfully conducted in control channels. The hydrodynamic forces under the conditions we used do not appear to show substantial impact on transfection efficiency. Master of Science

This article has been made available through the Brunel Open Access Publishing Fund. This article is distributed under the terms of the creative commons Attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s)and the source are credited. Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive. UK Food Standards Agency

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes. The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide. This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

The effects of synthetic estrogens 17α-ethynylestradiol (EE2) and diethylstilbestrol (DES) were compared on cell proliferation and morphology in Channel Catfish Ovary (CCO) and Chinese Hamster Ovary (CHO-K1) cells. EE2 exposure (0.1 and 0.5 μg/mL) induced stimulatory effect on CCO and CHO-K1 cell proliferation, while higher concentrations (1-10 μg/mL) showed cytotoxic effects. Increase in DES concentrations mainly resulted in dose-depended increase in cytotoxicity in both cell lines. Morphological changes induced by EE2 and DES exposure (5 μg/mL) showed disrupted cell monolayer and increased number and size of lysosomes. Comparison of IC(50) values showed almost equal sensitivity towards cytotoxicity of tested compounds in CCO and CHO-K1 cells.

Lindane, a toxic insecticide from the persistent organic pollutants (POP's) group, may act as an endocrine disrupter affecting crucial tissues of reproductive system. In this study a Chinese Hamster Ovary cell line (CHO-K1) was applied to assess the potential of lindane cytotoxicity at the cellular level. The methods of Trypan blue exclusion, MTT and Kenacid blue assays were used to assess cytotoxicity and confirmed a decrease in the number of viable CHO-K1 cells at 34.4-344 microM lindane during 24, 48 and 72 hours of exposure. The cell proliferation tests showed significant inhibition (p < 0.025-0.001 vs control) and a progressive increase in toxicity with increasing lindane concentrations. Corresponding IC(50) values were determined with each applied method. After 72 h of lindane exposure, IC(50) values were 184 microM according to the Trypan blue method and 272 and 256 microM with the Kenacid blue and MTT assays, respectively. Morphological changes induced by the cytotoxicity of lindane were followed by the fluorescence microscopy and only necrotic cells were detected. Vitamin E (25 and 50 microg/mL) was used for protection of ovarian cells against lidane-induced oxidative stress damage, and lipid peroxidation was postulated as a possible mechanism of lindane toxicity. The viability of cells pre-incubated with vitamin E was significantly enhanced (up to p < 0.025) compared to the results observed in cells exposed to lindane only, but vitamin E treatment could not prevent complete lindane-induced cytotoxicity. Results suggest that vitamin E may exert a slightly protective role in cell defense against lipophilic pro-oxidant xenobiotics such as lindane.

PD 128907 [4a R, 10 b R-(+)-trans-3, 4, 4a, 10 b-tetrahydro-4-n-propyl2 H,5H-[1]benzop-yrano[4,3-b] 1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (K-i, 1 nM) versus human D-2 receptors (K-i, 1183 nM) and a 10000-fold selectivity versus human D-4 receptors (K-i, 7000 nM) using [H-3]spiperone as the radioligand in CHO-K1-cells. Studies with [H-3]PD 128907, showed saturable, high affinity binding to human D-4 receptors expressed in CHO-K1 cells (CHO-K1-D-3) with an equilibrium dissociation constant (K-d) of 0.99 nM and a binding density (B-max) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D-2 receptors (CHO-K1-D-2). The rank order of potency for inhibition of [H-3]PD 128907 binding with reference DA agents was consistent with reported values for D-3 receptors. These results indicate that [H-3]PD 128907 is a new, highly selective D-3 receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DAD(3) receptors.

There is considerable concern about the substances present in the environment and their potential to interfere with the endocrine system of vertebrates. Among these, the so-called endocrine-disrupting compounds, which can modulate or disrupt developmental and reproductive processes, substances with estrogenic activity have attracted most attention. Concerns about the presence of these compounds in the environment have led to the development of screening and testing assays that are able to detect such substances and evaluate their potential to induce adverse effects. In vitro systems such as mammalian and fish cell lines have become of growing importance in toxicity testing of such compounds. The cytotoxic and apoptotic effects induced by 17α-ethynylestradiol and diethylstilbestrol were studied on CHO-K1 cell line. Trypan blue exclusion method was used to determine the cell viability. Cytotoxicity of 17α-ethynylestradiol (0.34–34 μM) and diethylstilbestrol (0.37–37 μM) was found to be concentration-dependent with IC50 values of 12.8 and 10.4 μM after 72 h of exposure, respectively. In treated CHO-K1 culture cell death was assessed by determining morphological changes by haematoxilyn and eosin staining, nuclear morphology by fluorescein diacetate/propidium iodide staining and fluorescence microscopy, DNA fragmentation by TUNEL method and translocation of phosphatidyl serine by flow cytometry. The obtained results showed that 17α-ethynylestradiol induced apoptosis, while diethylstilbestrol induced necrosis in the treated CHO-K1 cells.

Metabolomische Analysen bilden einen entscheidenden Schritt zum Verständnis zellulärer Funktionen auf genomischer Ebene. Verlässliche metabolomische Analysen unter physiologischen oder in vivo Bedingungen werden insbesondere für systembiologische Untersuchungen zur Beschreibung der Interaktionen von Stoffwechselreaktionen und ihrer Dynamik benötigt. Während genomische, proteomische und transkriptomische Daten heutzutage mit hoher Präzision generiert werden können, gestaltet sich die Bestimmung von Metabolitenprofilen nach wie vor schwierig. Dies liegt einerseits an den schnellen Umsatzraten, den Wechselwirkungen zwischen verschiedenen Stoffwechselwegen und der chemischen Diversität von Metaboliten. Auf der anderen Seite bildet die Kompartimentierung des Stoffwechsels tierischer Zellen eine große Herausforderung, da hierdurch eine dynamische Spannbreite von Metabolitkonzentrationen innerhalb des Zytosols und der Mitochondrien vorliegt. Zur Bewältigung der Schwierigkeiten der Zellkompartimentierung wurde in der vorliegenden Arbeit der Versuch unternommen, die schnelle und effiziente Separation zytosolischer und mitochondrialer Kompartimente von Säugetierzellen durch neue technologische Ansätze sowohl im Makromaßstab als auch im Mikromaßstab, auf einem „Lab-on-a-Chip“ (LoaC), zu erzielen. Wegen ihrer beachtlichen industriellen Bedeutung wurde innerhalb dieser Forschungsarbeit die Suspensionszelllinie CHO-K1 als Modell-organismus verwendet. Für die Mitochondrienisolation im Makromaßstab wurden verschiedene Zellaufschlussverfahren modifiziert und getestet. Die Effizienz und Sensitivität einer jeden Methode wurden durch die Messung der Mitochondrienausbeute und der Integrität isolierter Mitochondrien bestimmt. Hierbei konnten die höchsten Ausbeuten intakter Mitochondrien für einen Zellaufschluss mittels Ultraschall und anschließender differentieller Zentrifugation erzielt werden. Parallel zu den makroskopischen Methoden wurde als Kooperationsprojekt ein Mikrochipsystem entwickelt, das innerhalb weniger Sekunden eine vollständige Probenverarbeitung (einschließlich Zellaufschluss) für metabolische Analysen ermöglichen sollte. Der Prototyp des LoaC wurde innerhalb dieser Arbeit erfolgreich in einen Bioreaktor integriert und für eine Gesamtdauer von 18 h betrieben. Eine kontinuierliche Probenahme konnte hierbei mittels Überdruck realisiert werden. Neben dynamischen Pulsexperimenten wurde durch dieses Mikrochip-Bioreaktor-System eine effiziente Separation der Zellen vom extrazellulären Medium erzielt. Durch eine vorangestellte Zelllyse mittels Digitonin und die anschließende Abtrennung des freigesetzten Zytosols von den verbleibenden zellulären und mitochondrialen Kompartimenten, erwies das integrierte Mikrochipsystem großes Potential, um die Limitierungen zur Analyse des kompartimentierten Metabolismus zu überwinden. Im letzten Teil der Arbeit wurden die CHO-K1 Zellen für systembiologische Untersuchungen in einem geregelten Kleinmaßstab-bioreaktor kultiviert. Hierfür wurden neben Batch-Verfahren ebenfalls kontinuierliche Kultivierungen von Mischpopulationen durchgeführt und detaillierte extrazelluläre Metabolitanalysen mittels LC-MS vorgenommen. Darüber hinaus wurden durch konfokale und hochauflösende STED-Mikroskopie die Mitochondrienstrukturen und die Verteilung mitochondrialer Proteine in Abhängigkeit von den Kultivierungsbedingungen analysiert. Zudem konnte der relative Gehalt stoffwechselrelavanter Enzyme intrazellulär durch Einsatz der ELISA-Technologie bestimmt werden. Zur Untersuchung der Abhängigkeit meta-bolischer Reaktionen vom Zellzyklus wurden Kultivierungen synchronisierter Zellen erfolgreich durchgeführt. Hierzu wurden die Zellen im Vorfeld aus heterogenen Populationen mittels Elutriationszentrifugation in der G1- bzw. der S-Phase angereichert. Die Synchronität über den Zeitraum einer Batch-Kultivierung konnte mittels durchflusszytometrischer Analysen zur Bestimmung des DNA-Gehalts und des Zelldurchmessers sowie durch die erfolgreiche Applikation eines at-line Mikroskops zur kontinuierlichen Bestimmung der Zellzahl verfolgt werden. Metabolische und mikroskopische Untersuchungen synchronisierten Zellen wurden parallel dazu durchgeführt und mit den Daten der Kultivierungen von Mischpopulation verglichen. Unter anderem durch deutlich erhöhte Verbrauchsraten der Hauptsubstrate Glukose und L-Glutamin in der S-Phase und eine versetzt eintretende erhöhte Bildung von Laktat während der G1-Phase, konnte eine Zellzyklusabhängigkeit des Metabolismus gezeigt werden., Metabolomic analysis constitutes a crucial step for understanding cellular functions at a genomic level. In particular for systems biology studies, e.g. to describe the interplay of metabolic reactions and their dynamics, a reliable metabolomic analysis under physiological or in vivo conditions is required. While genomic, proteomic, and transcriptomic data can be obtained with high accuracy nowadays, the determination of metabolic profiles and fluxes still proves to be complicated. On the one hand, this is due to the fast turnover rates, the interaction between various metabolic pathways, and the chemical diversity of metabolites. On the other hand, the compartmentalization of metabolism in mammalian cells poses a huge challenge because of the dynamic range of metabolite concentration within the cytosol and the mitochondria. To overcome these limitations of metabolomic analysis, attempts have been made in this work to achieve a fast and efficient separation of the cytosolic and mitochondrial compartments by using new technological approaches in both macro and microscales. Due to its significant value, the well-known industrial cell line CHO-K1 was used as a model organism within this research project. To investigate the isolation of mitochondria in macroscale, different cell disruption methods were modified and tested. The efficiency and sensitivity of each were determined by the measure of mitochondrial yield and the integrity of isolated mitochondria. Here, the highest yields of intact mitochondria were obtained using ultrasound for cell disruption and subsequent differential centrifugation. In addition to the large-scale procedures, a microfluidic device for metabolic analysis, providing a complete sample preparation (including cell disruption) within seconds, was developed in project cooperation. In this work, a prototype of the Lab-on-a-Chip (LoaC) was successfully integrated into a bioreactor and operated for a period of 18 h. Continuous sampling was carried out by applying overpressure onto the bioreactor. Besides dynamic pulse experiments, this microchip-bioreactor system implemented an efficient separation of cells and extracellular media. Moreover, a selective cell lysis using digitonin and a subsequent separation of released cytosol from the remaining cellular and mitochondrial compartments was realised on-chip. Thus, this integrated microfluidic system provides high potential to overcome the limitations of analyses concerning compartmentalized metabolism. In the last part of this work, CHO-K1 cells were cultivated in a controlled small-scale bioreactor in order to perform systems biology studies. Therefore, detailed analyses of extracellular metabolites using LC-MS were performed under batch and continuous cultivation conditions. In addition, the mitochondrial structure and distribution of mitochondrial proteins were studied using confocal and high resolution STED microscopy. Furthermore, the relative level of enzymes involved in metabolic reactions was measured intracellularly by using the in-cell ELISA technology. To investigate the dependence of metabolic reactions on different cell cycle phases, the cultivation of synchronized cells was performed. For this purpose, cells from heterogeneous populations were enriched in the G1- and S-phase, respectively, using the centrifugal elutriation method. The synchrony during batch cultivation was determined flow cytometrically by analysing the DNA content and the cell size distribution. In addition, the cell count was observed continuously due to the effective integration of an at-line microscope. Metabolomic and microscopic studies of synchronized cells were performed during cultivation and the resultant data were compared to that obtained during cultivation of heterogeneous cell populations. The cell cycle dependency of metabolism was shown by the increased consumption rates of glucose and glutamine within the S-phase and the shifted increased lactate production during G1-phase, amongst others.