Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation.

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

Background aims: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. Methods: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R² = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost-effective manufacturing processes. Microcarriers enable the culture of anchorage-dependent cells in stirred-tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow-derived MSC (hBM-MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM-MSC1 underwent more cumulative population doublings over three passages in comparison to hBM-MSC2 and hBM-MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM-MSC expansion. HBM-MSCs were successfully harvested and characterised, demonstrating hBM-MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier-based allogeneic cell therapy manufacture. Large-scale production of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) requires expansion on microcarriers in agitated systems. This study demonstrates the importance of microcarrier selection and presents a systematic methodology for selection of an optimal microcarrier. The study also highlights the impact of an agitated culture environment in comparison to a static system, resulting in a significantly higher hBM-MSC yield under agitated conditions.

Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate

Background aims: The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. Methods: In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. Results: The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. Conclusions: This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.

Charles E. Perry Having Fun

Charles Perry, Alvin Dark, Lee Danielson playing golf. Charles Edward Perry (Chuck), 1937-1999, was the founding president of Florida International University in Miami, Florida. He grew up in Logan County, West Virginia and received his bachelor's and masters's degrees from Bowling Green State University. He married Betty Laird in 1960. In 1969, at the age of 32, Perry was the youngest president of any university in the nation. The name of the university reflects Perry’s desire for a title that would not limit the scope of the institution and would support his vision of having close ties to Latin America. Perry and a founding corps opened FIU to 5,667 students in 1972 with only one large building housing six different schools. Perry left the office of President of FIU in 1976 when the student body had grown to 10,000 students and the university had six buildings, offered 134 different degrees and was fully accredited. Charles Perry died on August 30, 1999 at his home in Rockwall, Texas. He is buried on the FIU campus in front of the Graham Center entrance. Alvin Ralph Dark (1972-), AKA “Blackie” and “The Swamp Fox”, played for the National Football League and was a shortstop and manager in Major League Baseball.

Global compilation of benthic data sets I

Approaches to quantify the organic carbon accumulation on a global scale generally do not consider the small-scale variability of sedimentary and oceanographic boundary conditions along continental margins. In this study, we present a new approach to regionalize the total organic carbon (TOC) content in surface sediments (<5 cm sediment depth). It is based on a compilation of more than 5500 single measurements from various sources. Global TOC distribution was determined by the application of a combined qualitative and quantitative-geostatistical method. Overall, 33 benthic TOC-based provinces were defined and used to process the global distribution pattern of the TOC content in surface sediments in a 1°x1° grid resolution. Regional dependencies of data points within each single province are expressed by modeled semi-variograms. Measured and estimated TOC values show good correlation, emphasizing the reasonable applicability of the method. The accumulation of organic carbon in marine surface sediments is a key parameter in the control of mineralization processes and the material exchange between the sediment and the ocean water. Our approach will help to improve global budgets of nutrient and carbon cycles.

Resonance Energy Transfer-Based Molecular Switch Designed Using a Systematic Design Process Based on Monte Carlo Methods and Markov Chains

A RET network consists of a network of photo-active molecules called chromophores that can participate in inter-molecular energy transfer called resonance energy transfer (RET). RET networks are used in a variety of applications including cryptographic devices, storage systems, light harvesting complexes, biological sensors, and molecular rulers. In this dissertation, we focus on creating a RET device called closed-diffusive exciton valve (C-DEV) in which the input to output transfer function is controlled by an external energy source, similar to a semiconductor transistor like the MOSFET. Due to their biocompatibility, molecular devices like the C-DEVs can be used to introduce computing power in biological, organic, and aqueous environments such as living cells. Furthermore, the underlying physics in RET devices are stochastic in nature, making them suitable for stochastic computing in which true random distribution generation is critical.

In order to determine a valid configuration of chromophores for the C-DEV, we developed a systematic process based on user-guided design space pruning techniques and built-in simulation tools. We show that our C-DEV is 15x better than C-DEVs designed using ad hoc methods that rely on limited data from prior experiments. We also show ways in which the C-DEV can be improved further and how different varieties of C-DEVs can be combined to form more complex logic circuits. Moreover, the systematic design process can be used to search for valid chromophore network configurations for a variety of RET applications.

We also describe a feasibility study for a technique used to control the orientation of chromophores attached to DNA. Being able to control the orientation can expand the design space for RET networks because it provides another parameter to tune their collective behavior. While results showed limited control over orientation, the analysis required the development of a mathematical model that can be used to determine the distribution of dipoles in a given sample of chromophore constructs. The model can be used to evaluate the feasibility of other potential orientation control techniques.

A Molecular-scale Programmable Stochastic Process Based On Resonance Energy Transfer Networks: Modeling And Applications

While molecular and cellular processes are often modeled as stochastic processes, such as Brownian motion, chemical reaction networks and gene regulatory networks, there are few attempts to program a molecular-scale process to physically implement stochastic processes. DNA has been used as a substrate for programming molecular interactions, but its applications are restricted to deterministic functions and unfavorable properties such as slow processing, thermal annealing, aqueous solvents and difficult readout limit them to proof-of-concept purposes. To date, whether there exists a molecular process that can be programmed to implement stochastic processes for practical applications remains unknown.

In this dissertation, a fully specified Resonance Energy Transfer (RET) network between chromophores is accurately fabricated via DNA self-assembly, and the exciton dynamics in the RET network physically implement a stochastic process, specifically a continuous-time Markov chain (CTMC), which has a direct mapping to the physical geometry of the chromophore network. Excited by a light source, a RET network generates random samples in the temporal domain in the form of fluorescence photons which can be detected by a photon detector. The intrinsic sampling distribution of a RET network is derived as a phase-type distribution configured by its CTMC model. The conclusion is that the exciton dynamics in a RET network implement a general and important class of stochastic processes that can be directly and accurately programmed and used for practical applications of photonics and optoelectronics. Different approaches to using RET networks exist with vast potential applications. As an entropy source that can directly generate samples from virtually arbitrary distributions, RET networks can benefit applications that rely on generating random samples such as 1) fluorescent taggants and 2) stochastic computing.

By using RET networks between chromophores to implement fluorescent taggants with temporally coded signatures, the taggant design is not constrained by resolvable dyes and has a significantly larger coding capacity than spectrally or lifetime coded fluorescent taggants. Meanwhile, the taggant detection process becomes highly efficient, and the Maximum Likelihood Estimation (MLE) based taggant identification guarantees high accuracy even with only a few hundred detected photons.

Meanwhile, RET-based sampling units (RSU) can be constructed to accelerate probabilistic algorithms for wide applications in machine learning and data analytics. Because probabilistic algorithms often rely on iteratively sampling from parameterized distributions, they can be inefficient in practice on the deterministic hardware traditional computers use, especially for high-dimensional and complex problems. As an efficient universal sampling unit, the proposed RSU can be integrated into a processor / GPU as specialized functional units or organized as a discrete accelerator to bring substantial speedups and power savings.

A new genus Abyssogena from deep-water vents and seeps

A new genus Abyssogena is established for A. phaseoliformis (Métivier, Okutani & Ohta, 1986) and A. kaikoi (Okutani & Métivier, 1986), which were previously assigned to the genus Calyptogena Dall, 1891, and also for two new species, A. southwardae and A. novacula. The most characteristic features of Abyssogena are an elongate shell up to about 280 mm in length; a pallial line starting from the ventral margin of the anterior adductor scar; secondary pallial attachment scars developed dorsal to the pallial line; radially arranged hinge teeth with a reduced anterior cardinal tooth in the right valve; and presence of an inner ctenidial demibranch only. Abyssogena occurs in deep water from 2,985 to 6,400 m and is distributed in the Pacific and Atlantic Oceans at cold seeps along continental margins and hydrothermal vents at mid-oceanic ridges. Some species have a remarkably wide geographic distribution; A. southwardae is present throughout the Atlantic and A. phaseoliformis is present in Japan, Kuril-Kamchatka, as well as Aleutian Trenches. No fossils of Abyssogena are known.

Collecting information and identification of New England Seamount Geodia species

This dataset gives the collecting information of New England Seamount Geodia species from the Yale Peabody Museum. Museum numbers, fixation processing and Genbank accession numbers are also given.

La Musicoterapia, una vía de expresión global

La Musicoterapia se ha revelado últimamente como una herramienta de intervención efectiva en la prevención y rehabilitación de patologías muy diversas, ayudando a mantener o mejorar las funciones físicas, psico-emocionales y sociales de las personas. En este articulo explicamos cómo se articula una intervención en Musicoterapia partiendo del conocimiento de los efectos de la música en el ser humano y cómo estos llegan de una manera global a todas las áreas de la persona y utilizando adecuadamente las diferentes técnicas de musicoterapia basadas en la escucha y la improvisación musical, podemos influir en el bien estar de nuestros pacientes o clientes

Audiovisual Storytelling and Ideological Horizons : Audiences, Cultural Contexts and Extra-textual Meaning Making

Abstract: Audiovisual Storytelling and Ideological Horizons: Audiences, Cultural Contexts and Extra-textual Meaning Making In a society characterized by mediatization people are to an increasing degree dependent on mediated narratives as a primary means by which we make sense of our experience through time and our place in society (Hoover 2006, Lynch 2007, Hjarvard 2008, Hjarvard & Lövheim 2012). American media scholar Stewart Hoover points to symbols and scripts available in the media environment, what he call the “symbolic inventory” out of which individuals make religious or spiritual meaning (Hoover 2006: 55). Vernacular meaning-making embedded in everyday life among viewers’ dealing with fiction narratives in films and tv-series highlight a need for a more nuanced understanding of complex audiovisual storytelling. Moving images provide individuals with stories by which reality is maintained and by which humans construct ordered micro-universes for themselves using film as a resource for moral assessment and ideological judgments about life (Plantinga 2009, Johnston 2010, Axelson 2015). Important in this theoretical context are perspectives on viewers’ moral frameworks (Zillman 2005, Andersson & Andersson 2005, Frampton 2006, Avila 2007).This paper presentation will focus on ideological contested meaning making where audiences of different cultural background engage emotionally with filmic narratives, possibly eliciting ideological and spiritual meaning-making related to viewers’ personal world views. Through the example of the Homeland tv-series I want to discuss how spectators’ cultural, religious, political and ideological identities could be understood playing a role in the interpretative process of decoding content. Is it possible to trace patterns of different receptions of the multilayered and ambiguous story depicted in Homeland by religiously engaged Christians and Moslems as well as non-believers, in America, Europe and Middle East? How is the fiction narrative dealt with by spectators in the audience in different cultural contexts and how is it interpreted through the process of extra-text evaluation and real world2understanding in a global era preoccupied with war on terror? The presentation will also discuss methodological considerations about how to reach out to audiences anchored in different cultural context.

Movies and the Enchanted Mind : Emotional Comprehension and Spiritual Meaning Making Among Young Adults in Contemporary Sweden

The outcome of an empirical audience study in Sweden including questionnaires, focus groups and ten in-depth individual interviews discussing favorite films supports claims about viewers as active and playful (cf. Frampton 2006, Hoover 2006, Plantinga 2009). The soft side of mediatization processes is illustrated showing young adults experiencing enchantment through films (Jerslev 2006, Partridge 2008, Klinger 2008, Oliver & Hartmann 2010). The outcome is in line with a growing number of empirical case studies which support conclusions that both thinking and behavior are affected by film watching (Marsh 2007, Suckfüll 2010, Oliver & Hartmann 2010, Axelson 2014). The results of the interviews exploring specific scenes of idiosyncratic relevance support theories about fiction films as important sources for moral and spiritual reflection (Partridge 2004, Zillman 2005, Lynch 2007, Plantinga 2009).  The concept thick viewing is proposed for capturing these moments of film experience when profound and enchanted emotional interpretations take place.