Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single Cell Level

Biopharmaceutical manufacturing using Chinese hamster ovary (CHO) cells requires the generation of high-producing clonal cell lines. During cell line development, cell cloning using fluorescence activated cell sorting (FACS) has the potential to combine isolation of single cells with sorting based on specific cellular attributes that correlate with productivity and/or growth, identifying cell lines with desirable phenotypes for manufacturing. This study describes the application of imaging flow cytometry (IFC) to characterize recombinant cell lines at the single cell level to identify cell attributes predictive of productivity. IFC assays to quantify organelle content, and recombinant heavy (HC) and light (LC) chain polypeptide and mRNA amounts in single cells were developed. The assays were then validated against orthogonal standard flow cytometry, western blot and qRT-PCR methods. We describe how these IFC assays may be used in cell line development and show how cellular properties can be correlated with productivity at the single cell level, allowing the isolation of such cells during the cloning process. Our analysis found HC polypeptide and mRNA to be predictive of productivity early in the culture, however specific organelle content did not show any correlation with productivity

Extraordinarily rapid proliferation of cultured muscle satellite cells from migratory birds

Migratory birds experience bouts of muscle growth and depletion as they prepare for, and undertake prolonged flight. Our studies of migratory bird muscle physiology in vitro led to the discovery that sanderling (Calidris alba) muscle satellite cells proliferate more rapidly than other normal cell lines. Here we determined the proliferation rate of muscle satellite cells isolated from five migratory species (sanderling; ruff, Calidris pugnax; western sandpiper, Calidris mauri; yellow-rumped warbler, Setophaga coronata; Swainson\u27s thrush, Catharus ustulatus) from two families (shorebirds and songbirds) and with different migratory strategies. Ruff and sanderling satellite cells exhibited rapid proliferation, with population doubling times of 9.3 ± 1.3 and 11.4 ± 2 h, whereas the remaining species\u27 cell doubling times were greater than or equal to 24 h. The results indicate that the rapid proliferation of satellite cells is not associated with total migration distance but may be related to flight bout duration and interact with lifespan

Metabolic profile of long-distance migratory flight and stopover in a shorebird

Migrating birds often complete long non-stop flights during which body energy stores exclusively support energetic demands. The metabolic correlates of such long-distance travel in free-living migrants are as yet poorly studied. Bar-tailed godwits, Limosa lapponica taymyrensis, undertake a 4500 km flight to their single spring stopover site and thus provide an excellent model in which to determine the energy fuels associated with endurance travel. To this end, we evaluated plasma concentrations of six key metabolites in arriving godwits caught immediately upon landing near their stopover site. Initial metabolite levels were compared with levels after 5 h of inactive rest to determine how flight per se affects energy metabolism. Birds refuelling on the stopover site were also examined. Arriving godwits displayed elevated plasma free fatty acids, glycerol and butyrate, confirming the importance of lipid fuel in the support of extended migratory activity. Furthermore, elevated plasma triglycerides in these birds suggest that fatty acid provisioning is facilitated through hepatic synthesis and release of neutral lipids, as previously hypothesized for small migrants with high mass-specific metabolic rates. Finally, elevations in plasma uric acid suggest that protein breakdown contributes to the support of long-distance movement, to possibly maintain citric acid cycle intermediates, gluconeogenesis and/or water balance

Characterisation of Chinese Hamster Ovary (CHO) cells at the single cell level

Biopharmaceuticals are a class of biological macromolecules that include antibodies and antibody derivatives, generally produced from cultured mammalian cells line via secretion directly into the media. Manufacturing requires the generation of Chinese hamster ovary (CHO) clonal cell lines capable of expressing the biopharmaceutical product at commercially relevant quantities with desirable product quality. The isolation of cell clones based on random single cell deposition via fluorescence activated cell sorting (FACS) provides a heterogeneous panel of expressers. We hypothesize that the application of FACS to provide an additional sorting step based on cell characteristics that correlate with productivity, product quality or cell growth attributes could lead to the isolation of higher producing cell lines with enhanced product quality attributes. A panel of 20 cell lines expressing a model recombinant monoclonal antibody were characterised in terms of growth, productivity, and intracellular recombinant protein and mRNA amounts. Assays were also developed to investigate cell attributes and organelle content using the ImageStream instrument, an imaging flow cytometer, which enables the investigation of cellular characteristics that correlate with cell productivity at the single cell level. Characterisation revealed the cell lines exhibited a range of values for productivity, growth, and intracellular (IC) antibody mRNA and protein expression, ideal for further ImageStream characterisation. Western blot and qRT-PCR analysis demonstrated that final titre correlated with both IC heavy chain (HC) protein and mRNA amounts (Pearson Correlation Coefficient (R) = 0.70 and R = 0.80, respectively). To assess productivity at the single cell level, assays multiplexing IC HC protein and mRNA with organelles, such as mitochondria, endoplasmic reticulum and Golgi apparatus, were therefore developed. ImageStream quantification of HC mRNA and protein amounts also showed correlations between titre and IC HC protein and mRNA (R = 0.84 and R = 0.79, respectively), confirming results from western blots and qRT-PCR analysis. A cell attribute that correlates with specific productivity has been found, and current work is investigating whether this cell attribute could be used during cell sorting for the isolation of more productive clones. Future experiments will also look at cell attributes that could lead to improved product quality. The developed assays are expected to allow a greater understanding of the intracellular mechanisms underlying productivity and product quality in CHO cells. Moreover, outcomes from this study have the potential to not only integrate into the cell line development clonal selection process, shortening timelines and reducing cost and resource requirements, but also inform host cell engineering projects with the potential for the development of an improved CHO host

Effect of dietary fatty acid composition on depot fat and exercise performance in a migrating songbird, the red-eyed vireo

Most migrating birds accumulate lipid stores as their primary source of energy for fueling long distance flights. Lipid stores of birds during migration are composed of mostly unsaturated fatty acids; whether such a fatty acid composition enhances exercise performance of birds is unknown. We tested this hypothesis by measuring metabolic rate at rest and during intense exercise in two groups of red-eyed vireos, a long-distance migratory passerine, fed either a diet containing 82% unsaturated fat (82%U), or one containing 58% unsaturated fat (58%U). Vireos fed the 82%U diet had fat stores containing (77%) unsaturated fatty acids, whereas vireos fed the 58% U diet had fat stores containing less (66%) unsaturated fatty acids. Blood metabolites measured prior to and immediately following exercise confirmed that vireos were metabolizing endogenous fat during intense exercise. Mass-specific resting metabolic rate (RMR) was similar for vireos fed the 58%U diet (2.75±0.32 ml O2 g–1 h–1) and for vireos fed the 82%U diet (2.30±0.30 ml O2 g–1 h–1). However, mass-specific peak metabolic rate (MRpeak) was 25% higher in vireos fed the 58%U diet (28.55±1.47 ml O2 g–1 h–1) than in vireos fed the 82%U diet (21.50±1.76 ml O2 g–1 h–1). Such whole-animal energetic effects of fatty acid composition of birds suggest that the energetic cost of migration in birds may be affected by the fatty acid composition of the diet

Remove Debris Mission, From Concept to Orbit

The RemoveDebris mission will be the first European Active Debris Removal (ADR) missions to give an in orbit demonstration of the viability of a series of cost effective technologies that can be used to observe, capture and destroy space debris. RemoveDebris is a low cost mission performing key active debris removal (ADR) technology demonstrations including the use of a net, a harpoon, vision-based navigation (VBN) and a dragsail in a realistic space operational environment. For the purposes of the mission two CubeSats will be ejected and used as targets for experiments instead of real space debris, which is an important step towards a fully operational ADR mission. The craft has launched to the ISS on the 2nd of April 2018, on board a Dragon capsule (SpaceX CRS-14 ISS re-supply mission). From here the satellite is to be deployed via the NanoRacks Kaber system into an orbit of around 400 km. Aglietti 2 32nd Annual AIAA/USU Conference on Small Satellites This paper examines the design of the mission from initial concepts through to manufacture, AIT, testing and up to launch, and apart from a general consideration of the mission, will focus on the elements of design & testing that differ from a conventional mission

The Pacific as the world’s greatest theater of bird migration:Extreme flights spark questions about physiological capabilities, behavior, and the evolution of migratory pathways

The Pacific Basin, by virtue of its vastness and its complex aeroscape, provides unique opportunities to address questions about the behavioral and physiological capabilities and mechanisms through which birds can complete spectacular flights. No longer is the Pacific seen just as a formidable barrier between terrestrial habitats in the north and the south, but rather as a gateway for specialized species, such as shorebirds, to make a living on hemispherically distributed seasonal resources. This recent change in perspective is dramatic, and the research that underpins it has presented new opportunities to learn about phenomena that often challenge a sense of normal. Ancient Polynesians were aware of the seasonal passage of shorebirds and other landbirds over the Pacific Ocean, incorporating these observations into their navigational “tool kit” as they explored and colonized the Pacific. Some ten centuries later, systematic visual observations and tracking technology have revealed much about movement of these shorebirds, especially the enormity of their individual nonstop flights. This invites a broad suite of questions, often requiring comparative studies with bird migration across other ocean basins, or across continents. For example, how do birds manage many days of nonstop exercise apparently without sleep? What mechanisms explain birds acting as if they possess a Global Positioning System? How do such extreme migrations evolve? Through advances in both theory and tracking technology, biologists are poised to greatly expand the horizons of movement ecology as we know it. In this integrative review, we present a series of intriguing questions about trans-Pacific migrant shorebirds and summarize recent advances in knowledge about migratory behavior operating at temporal scales ranging from immediate decisions during a single flight, to adaptive learning throughout a lifetime, to evolutionary development of migratory pathways. Recent advances in this realm should stimulate future research across the globe and across a broad array of disciplines

Medical and Dental Students' Perception of Interdisciplinary Knowledge, Teaching Content, and Interprofessional Status at a German University: A Cross-Sectional Study.

Although oral health is considered a key indicator of overall health, dentistry is still neglected in medical education at the university level. Interprofessional education (IPE) is an important tool to promote collaboration among health care providers and to reduce barriers to access in health care. In this cross-sectional study, medical and dental students at Mainz University, Germany, were surveyed regarding their perception of interdisciplinary knowledge, teaching content, interprofessional standing, and attitudes toward IPE. Spearman's rank correlation was used to identify associated statements. Structural equation modeling (SEM) was performed to understand how sex, study progress, and prior education might influence student attitudes. In total, 426 medical students and 211 dental students were included in the study. Dental students rated their interdisciplinary knowledge higher than medical students. The relevance of IPE as assessed by the students correlated significantly with their motivation to continue IPE after graduation. Both groups of students valued the other discipline but rejected a combined graduate program. Students with prior professional training valued the synergy of medicine and dentistry more the students without prior training. Interprofessional knowledge and interest in IPE was higher among dental students. Understanding students' attitudes toward IPE is an important prerequisite for adapting university curricula to strengthen students' attitudes and motivation

Constitutive immune function in European starlings, \u3cem\u3eSturnus vulgaris\u3c/em\u3e, is decreased immediately after an endurance flight in a wind tunnel

Life-history theory predicts that animals face a trade-off in energy allocation between performing strenuous exercise, such as migratory flight, and mounting an immune response. We experimentally tested this prediction by studying immune function in European starlings, Sturnus vulgaris, flown in a wind tunnel. Specifically, we predicted that constitutive immune function decreases in response to training and, additionally, in response to immediate exercise. We compared constitutive immune function among three groups: (1) ‘untrained’ birds that were kept in cages and were not flown; (2) ‘trained’ birds that received flight training over a 15 day period and performed a 1-4 h continuous flight, after which they rested for 48 h before being sampled; and (3) ‘post-flight’ birds that differed from the ‘trained’ group only in being sampled immediately after the final flight. A bird in our trained group represents an individual during migration that has been resting between migratory flights for at least 2 days. A bird in our post-flight group represents an individual that has just completed a migratory flight and has not yet had time to recover. Three of our four indicators (haptoglobin, agglutination and lysis) showed the predicted decrease in immune function in the post-flight group, and two indicators (haptoglobin, agglutination) showed the predicted decreasing trend from the untrained to trained to post-flight group. Haptoglobin levels were negatively correlated with flight duration. No effect of training or flight was detected on leukocyte profiles. Our results suggest that in European starlings, constitutive immune function is decreased more as a result of immediate exercise than of exercise training. Because of the recent emergence of avian-borne diseases, understanding the trade-offs and challenges faced by long-distance migrants has gained a new level of relevance and urgency