Download PDF (all abstracts) - BioMed Central

More documents

Recommendations

Info

BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 8 of 151 Results: A miniaturized circulation system has been established over a period of 14 days by fully covering all channels and surfaces of the MOC with human microvascular endothelial cells. By injecting 2 × 10 7 cells ml -1 into the channels, a homogeneous distribution of cells throughout all channels was achieved (Figure 1b). During the following static incubation, cells adhered well to the air plasma treated channel walls. A peristaltic micro-pump was used to create culture medium circulation. After adaption to shear stress, HDMEC showed an elongation and alignment parallel to the flow direction. Three-dimensional reconstitutions of image stacks indicate that cells formed confluent monolayers on all walls of the channels (Figure 1c). During the whole cultivation time they maintained adherence to the channel walls and were positive for Calcein AM viability staining (Figure 1b). After 14 days of culture HDMEC forming the microvascular circuit were positive for ac-LDL uptake (Figure 1d) and expressed the endothelialspecific marker CD31 and von Willebrand Factor (vWF) (Figure 1e). Conclusion: A robust procedure applying pulsatile shear stress has been established to cover all fluid contact surfaces of the system with a functional, tightly closed layer of HDMEC. Long-term cultivation of elongated and flow-aligned HDMEC inside the chipbased microcirculation was demonstrated over a period of 14 days. For such endothelialized microfluidic devices to be useful for substance testing, it is essential to show long-term viability and function in the presence of physiological flow rates as shown here. These artificial vessels are an important approach for systemic substance testing in Multi-Organ-Chips. The miniaturized circulation system creates the conditions for circulation of nutrients through the organoid culture chamber, allows for in vivo-like crosstalk between endothelial cells and tissues and prevents clumping inside the channels. Compared with conventional cell culture techniques, a microfluidic-based cell culture may mimic more accurate in vivo-like extracellular conditions, as the culture of cells and organ models in perfused microfluidic systems can improve their oxygen and nutrient supply. This makes it suitable for long-term cultivation and more efficient drug studies. In future, such endothelialized bioreactors might be used for testing vasoactive substances. Finally, the described system can now be used for the establishment of organ-specific capillary networks. Here, we will adhere to our recently published roadmap toward vascularized ‘’human-on-a-chip’’ models to generate systemic data fully replacing the animals or human beings currently used [2]. Acknowledgements: The work has been funded by the German Federal Ministry for Education and Research, GO-Bio Grant No. 0315569. References 1. Wu M-H, Huang S-B, Cui Z, Cui Z, Lee G-B: A high throughput perfusionbased microbioreactor platform integrated with pneumatic micropumps for three-dimensional cell culture. Biomedical microdevices 2008, 10:309-319. 2. Marx U, Walles H, Hoffmann S, Lindner G, Horland R, Sonntag F, Klotzbach U, Sakharov D, Tonevitsky A, Lauster R: “Human-on-a-chip” developments: a translational cutting-edge alternative to systemic safety assessment and efficiency evaluation of substances in laboratory animals and man? Alternatives to laboratory animals: ATLA 2012, 40:235-257. O7 Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering Kyoungho Lee 1 , Kohsuke Honda 1 , Hisao Ohtake 1 , Takeshi Omasa 1,2* 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; 2 Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjimacho, Tokushima 770-8506, Japan E-mail: omasa@bio.tokushima-u.ac.jp BMC Proceedings 2013, 7(Suppl 6):O7 Introduction: Dihydrofolate reductase (DHFR)-mediated gene amplification has been widely used to establish high-producing mammalian cell lines [1-3]. However, since gene amplification is an infrequent event, in that many rounds of methotrexate (MTX) selection to amplify the transgene and screening of over several hundred individual clones are required to obtain cells with high gene copy numbers [4]. Consequently, the process for DHFRmediated gene amplification is a time-consuming and laborious step for cell line construction. Here, we present a novel concept to accelerate gene amplification through cell cycle checkpoint engineering. In our knowledge, there is no previous report which focused on controlling cell cycle checkpoint to enhance the efficiency of DHFR gene amplification system. Materials and methods: A small interfering RNA (siRNA) expression vector against Ataxia-Telangiectasia and Rad3-Related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The effects of ATR down-regulation on gene amplification and productivity in CHO cells producing green fluorescent protein (GFP) and monoclonal antibody (mAb) were investigated. Results and discussion: Analysis of GFP expression level during gene amplification process: The ratio of GFP-expressing cells was evaluated by flow cytometry analysis during the gene amplification process at 100-, 250-, and 500-nM MTX concentrations. In the process of gene amplification at all MTX concentrations, the pools of ATR-downregulated cells showed a much higher percentage of GFP-positive cells as compared with the pools of mock cells. At 100-nM MTX concentration, the percentage of GFPpositive cells in the CHO-siATR cell pool was 18.7% of total cells, which was approximately twice of the 8.4% in the mock cells. At 250- and 500-nM MTX concentrations, CHO-siATR cell pools had 28.6 and 39.2% GFP-positive cells, respectively, which were up to six times higher than the 4.6 and 6.8% of the pools of mock cells. Comparison of IgG productivity: IgG-producing cell lines were generated to confirm the previous results obtained in GFP-producing cell lines. The ATR-downregulated cells showed a significant increase in specific production rate of an average of 0.08 pg cell −1 day −1 , which was approximately four times higher than the average of 0.02 pg cell −1 day −1 in the mock cells. The volumetric productivity of each cell line was also investigated to evaluate the influence of ATR downregulation. The volumetric productivity of ATR knockdown cells was an average of 0.035 mg L −1 day −1 , which was approximately three times higher than the average of 0.013 mg L −1 day −1 of the mock cells, suggesting that ATR knockdown generated the pool of higher-producing cells during the gene amplification process. Estimation of amplified transgene copy number: Quantitative real-time PCR was used to estimate the amplified transgene copy number of GFPproducing cell lines during the gene amplification process. The average copy number of ATR-downregulated cells was 15.4 ± 0.8, 27.6 ± 0.3, and 62.0 ± 2.9 at 100-, 250-, and 500-nM MTX concentrations, respectively. These numbers were up to 24 times higher than 3.98 ± 0.09, 2.20 ± 0.03, and 2.59 ± 0.07 of the mock cells. Interestingly, the amplified transgene copy numbers in the pools of ATR-downregulated cells were increased proportionally with the MTX concentration. The amplified transgene copy numbers in the IgG-producing cells were also investigated during the gene amplification process at 100-nM MTX concentration. The amplified light- and heavy-chain copy numbers of the pool of ATR knockdown cells were 13.2 ± 3.8 and 11.8 ± 1.8, respectively, which were up to seven times higher than 6.95 ± 0.07 and 1.68 ± 0.04 of the mock cells. The results from both the GFP- and IgG-producing cells showed that the pools of ATR-downregulated cells had much higher amplified transgene copy numbers as compared with the pools of mock cells during the gene amplification process. Conclusions: In conclusion, we have demonstrated that gene amplification can be accelerated by the downregulation of a cell cycle checkpoint kinase, ATR, and a pool of high-producing cells can be rapidly derived in a short time after MTX treatment. This novel method focuses on generating more high-producing cells in a heterogeneous pool as compared with the conventional method and would thus contribute to reducing the time and labor required for cell line establishment by increasing the possibility of selecting high-producing clones. Acknowledgements: This work is partially supported by grants from the Program for the Promotion of Fundamental Studies in Health Sciences of NIBIO and a Grant-in-Aid for Scientific Research of JSPS. We thank Prof. Yoshikazu Kurosawa at Fujita Health University for kindly providing heavyand light-chain genes of humanized IgG. References 1. Gandor C, Leist C, Fiechter A, Asselbergs FA: Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells. FEBS Lett 1995, 377:290-294. 2. Kim JY, Kim YG, Lee GM: CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 2012, 93:917-930. 3. Wurm FM: Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 2004, 22:1393-1398.
BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 9 of 151 4. Cacciatore JJ, Chasin LA, Leonard EF: Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system. Biotechnol Adv 2010, 28:673-681. O8 1 H-NMR spectroscopy for human 3D neural stem cell cultures metabolic profiling Daniel Simão 1,2 , Catarina Pinto 1,2 , Ana P Teixeira 1,2 , Paula M Alves 1,2 , Catarina Brito 1,2* 1 iBET, Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal; 2 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal E-mail: anabrito@itqb.unl.pt BMC Proceedings 2013, 7(Suppl 6):O8 Background: The current lack of predictable central nervous system (CNS) models in pharmaceutical industry early stage development strongly contributes for the high attrition rates registered for new therapeutics [1]. Thus, there is an increasing need for a paradigm shift towards more human relevant cell models, which can closely recapitulate the in vivo cell-cell interactions, presenting higher physiological relevance by bridging the gap between animal models and human clinical trials. In this context, human 3D in vitro models are promising tools with great potential for pre-clinical research, as they can mimic some of the main features of tissues, such as cell-cell and cell-extracellular matrix (ECM) interactions [2,3]. Moreover these complex cell models are suitable for high-throughput screening (HTS) platforms, essential in drug discovery pipelines by reducing both costs and time in clinical trials [2,4]. However, despite important advances in the last years and the increasing clinical and biological relevance, the full establishment of human 3D in vitro models in pre-clinical research requires a significant increase in the power of the available analytical methodologies towards more robust and comprehensive readouts [4]. With the emergence of systems biology field and several “-omics” technologies, such as metabolomics, it became possible to have a more mechanistic approach in the understanding of cellular programs. 1 H-nuclear magnetic resonance ( 1 H-NMR) spectroscopy is a powerful and widely accepted high resolution methodology for a number of applications, including metabolic profiling [5]. Despite the low sensitivity when compared with mass spectrometry (MS), 1 H-NMR profiling presents several advantages, enabling a non-invasive and non-destructive quantitative analysis requiring only minimal sample preparation [5]. In this work we present the development of a robust and optimized workflow for the exometabolome profiling of 3D in vitro cultures of human midbrain-derived neural progenitor cells (hmNPC). Materials and methods: Cell culture: hmNPC were isolated and routinely propagated in static conditions, on poly-L-ornithine-fibronectin (PLOF) coated plates, in serum-free expansion medium, containing basic fibroblast growth factor and epidermal growth factor, as previously reported [6]. hmNSC were cultured in stirred systems as neurospheres for 7 days, with a 50% media changes every at day 3 [7]. All experiments were performed in 500 mL shake flasks (80 mL working volume), with orbital shaking at 100 rpm. Cultures were maintained at 37°C, in 3% O 2 and 5% CO 2 . Sample Preparation: Neurospheres harvested at day 7 were plated on PLOF-coated plates. A washing step with PBS was performed before adding fresh medium (Neurobasal medium (Invitrogen) supplemented with 2% of B27, 2 mM of Glutamax (Invitrogen), 100 μM dibutyryl c-AMP (Sigma- Aldrich), and 10 μg/mL gentamycin (Invitrogen)) to the culture. Samples of supernatant were then collected at 6, 12, 24 and 48 hours after media exchange and stored at -20°C. Neurospheres were harvested and total protein was quantified with Micro BCA Protein Assay Kit (Pierce), according to manufacturer’s instructions. Prior to NMR analysis, samples were thawed and filtered using Vivaspin 500 columns (Sigma-Aldrich) at 14,000xg, in order to remove high molecular weight proteins and lipids that induce baseline distortions and peak broadening due to protein binding. To minimize variations in pH, 400 μL of filtered samples were mixed with 200 μL of phosphate buffer (50 mM, pH 7.4) with 5 mM DSS-d 6 [8]. 1 H-NMR spectra acquisition and profiling: For NMR analysis, 500 μL of the resulting supernatants were placed into 5 mm NMR tubes. All 1 H-NMR spectra were recorded at 25°C on a Bruker Avance II+ 500 MHz NMR spectrometer. One-dimensional (1D) spectra were recorded using a NOESYbased pulse sequence (4 s acquisition time, 1 s relaxation time and 100 ms mixing time). Typically, 256 scans were collected for each spectrum. All spectra were phase and baseline corrected automatically, with fine adjustments performed manually. Spectra analysis was performed using Chenomx NMR Suite 7.1, using DSS-d 6 as internal standard for quantification of metabolites. Results: The approach applied in this study for metabolic profiling of the hmNPC cultures using 1 H-NMR enables an accurate screening of a wide range of metabolites in the extracellular environment (Figure 1A), including amino acids, glucose, lactate, among other substrates and by-products. Metabolism plasticity has been widely described as closely related with cell pluri/multipotency and cell fate. Stemness programs and cell identity determination are driven mainly by genetic and epigenetic switches, which can modulate cell metabolism, among other cell fate pathways [9]. Thus, the transition from pluri/multipotency towards somatic cell lineages is accompanied by significant metabolic shifts, mainly at energy metabolism levels. In this context, the metabolic study of in vitro cultures of stem cells may contribute with valuable knowledge for the mechanistic understanding of stemness and differentiation pathways. Our results showed that the hmNPC in an undifferentiated state presented a highly glycolytic metabolism, with high glucose consumption and lactate production rates (Figure 1B), in agreement with previous reports for murine NPC [10]. The profiles observed for glucose consumption and lactate synthesis suggest an almost complete conversion of pyruvate, generated as the final product of glycolysis, to lactate. One key culture parameter that can greatly contribute for a low oxidative metabolism is the fact that neural stem/progenitor cells are typically cultured under physiological low oxygen tension environments. Hypoxic conditions have been widely described as critical for maintaining cell viability and selfrenewal, while promoting proliferation and influencing cell fate during differentiation [11]. Moreover, the consumption and depletion of pyruvate present in culture media may suggest not only its conversion to lactate, but may also contribute for the observed alanine synthesis. Interestingly, even though glutamate could not be detected at significant levels, an accumulation of pyroglutamate was observed, which can be found as N-terminal modification in many neuronal peptides, including pathological accumulating peptides as b-amyloid in Alzheimer’s disease. As a free metabolite pyroglutamate can derive both from degradation of proteins containing N-terminal residues or from glutamate/glutamine cyclization. Although it is still a matter of debate, pyroglutamate may act as a reservoir of neural glutamate, which is the main excitatory neurotransmitter in CNS and in high levels becomes a major neurotoxicant [12]. Concerning branched-chain amino acids (BCAA) metabolism it was possible to observe the extracellular accumulation of 2-oxoisocaproate and methylsuccinate as main by-products, although in low rates. In brain metabolism the balance between leucine and 2-oxisocaproate has particular relevance through the establishment of a nitrogen turnover cycle where astroglia cells catabolize leucine into 2-oxoisocaproate, which is then taken up by neurons and converted back into leucine [13,14]. Conclusions: The methodology presented in this work, enables a straightforward approach for an accurate and reproducible metabolic profiling of multipotent hmNPC 3D cultures. This methodology provides a robust alternative to an array of laborious analytical methods, by taking advantage of the fast and simple sample preparation for NMR spectroscopy and the ease of user-friendly software for spectra profiling, which is often a challenging and time-consuming process due to peak overlapping in complex mixtures such as the mammalian cell culture media. Moreover, this approach can be applied to other multi/pluripotent cell sources, not only for metabolic profiling of in vitro cultures but also to study the impact of new therapeutics or toxicants, contributing to generate invaluable data in drug development cascades. Acknowledgements: The authors acknowledge Dr J. Schwarz (Technical University of Munich, Germany) for the supply of hmNPC, within the scope of the EU project BrainCAV (FP7-222992); this work was supported by PTDC/EBB-BIO/112786/2009 and PTDC/EBB-BIO/119243/2010, FCT, Portugal; BrainCAV (FP7-222992), EU. The NMR spectrometers are part of The National NMR Facility, supported by Fundação para a Ciência e a Tecnologia (RECI/BBB-BQB/0230/2012). Daniel Simão acknowledges the PhD fellowship (SFRH/BD/78308/2011, FCT).
Page 1 and 2: BMC Proceedings 2013, Volume 7 Supp
Page 7: BMC Proceedings 2013, Volume 7 Supp
Page 59 and 60:
BMC Proceedings 2013, Volume 7 Supp
Page 61 and 62:
Page 63 and 64:
Page 65 and 66:
Page 67 and 68:
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
Page 75 and 76:
Page 77 and 78:
Page 79 and 80:
Page 81 and 82:
Page 83 and 84:
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151:
show all

Download PDF (all abstracts) - BioMed Central

Create successful ePaper yourself

Delete template?

Save as template?