Cryopreservation critical process parameters: Impact on post-thaw recovery of cellular product

Technological advances have transformed cells from mere drug targets into potent ‘living drugs’ with the potential to cure formerly incurable diseases such as cancer. Such Regenerative Medicine Advanced Therapies (RMATs) require stringent and complex vein-to-vein support to deliver their intended function. Cold Chain pertains to strategies designed to ensure the product potency and efficacy during ex-vivo transition, and includes various components starting from the source material collection, to culture and expansion process, formulation, fill-finish and packaging, storage, transportation, chain of custody, and delivery. Biopreservation is the overarching theme of the cold chain and refers to strategies to slow down or fully suspend the biological clock to allow logistical considerations. The two main modes of biopreservation are hypothermic storage and cryopreservation. This presentation aims to map the connection between a specific biopreservation strategy, namely cryopreservation, and formulation and fill-finish, and how implementation of Biopreservation Best Practices can improve the outcome of Cold Chain. There is more to Biopreservation than storage on ice or freezing at a rate of -1°C/min in 10% DMSO. To comprehend the rationale behind Biopreservation Best Practices, a basic understanding of cellular response to cold and freezing is essential. In this study, we highlight critical process parameters (CPPs) of cryopreservation, such as freezing and thawing rates, storage and post-thaw stability, and container type, among others. Using a Jurkat T-cell model, we will discuss the impact of these CPPs on critical quality attributes (CQAs) such as viability, yield, proliferation rate, and return to function. We will also discuss the connection between variability in CPPs and characterization assay results. In general, implementation of best practices in formulation can directly address multiple process bottlenecks, including GMP compliance, minimizing freezing damage, support stability during storage and against transient warming events, support post-thaw stability, and excipient use. The CQAs may also be significantly improved by adjusting a few parameters in the freezing profile. For example, a missed or improper nucleation step during freezing may result in decreased recovery and increased variability in post-thaw proliferation rates. We have also found that the feeding timeline prior to freezing can have a profound impact on post-thaw viability and recovery in Jurkat T cells. While discussing these results, we will also review the underlying biophysics of such phenomena. The basic knowledge of designing a freezing profile may introduce degrees of freedom to process engineers to minimize the DMSO concentration in the formulation, and improve the CQAs of “hard-to-freeze” cells such as Natural Killer (NK) cells. We will also discuss the interplay between the cryopreservation CPPs and the choice of container format and how it may impact the CQAs. Please click Additional Files below to see the full abstract

Tracheostomy timing and the duration of weaning in patients with acute respiratory failure

INTRODUCTION: The effect of various airway management strategies, such as the timing of tracheostomy, on liberation from mechanical ventilation (MV) is uncertain. We tested the hypothesis that tracheostomy, when performed prior to active weaning, does not influence the duration of weaning or of MV in comparison with a more selective use of tracheostomy. PATIENTS AND METHODS: In this observational prospective cohort study, surgical patients requiring ≥ 72 hours of MV were followed prospectively. Patients undergoing tracheostomy prior to any active weaning attempts (early tracheostomy [ET]) were compared with patients in whom initial weaning attempts were made with the endotracheal tube in place (selective tracheostomy [ST]). RESULTS: We compared the duration of weaning, the total duration of MV and the frequency of fatigue and pneumonia. Seventy-four patients met inclusion criteria. Twenty-one patients in the ET group were compared with 53 patients in the ST group (47% of whom ultimately underwent tracheostomy). The median duration of weaning was shorter (3 days versus 6 days, P = 0.05) in patients in the ET group than in the ST group, but the duration of MV was not (median [interquartile range], 11 days [9–26 days] in the ET group versus 13 days [8–21 days] in the ST group). The frequencies of fatigue and pneumonia were lower in the ET group patients. DISCUSSION: Determining the ideal timing of tracheostomy in critically ill patients has been difficult and often subjective. To standardize this process, it is important to identify objective criteria to identify patients most likely to benefit from the procedure. Our data suggest that in surgical patients with resolving respiratory failure, a patient who meets typical criteria for a trial of spontaneous breathing but is not successfully extubated within 24 hours may benefit from a tracheostomy. Our data provide a framework for the conduct of a clinical trial in which tracheostomy timing can be assessed for its impact on the duration of weaning. CONCLUSION: Tracheostomy prior to active weaning may hasten liberation from ventilation and reduce complications. However, this does not reduce the overall duration of MV

Stakeholder priorities for multi-functional coastal defence developments and steps to effective implementation

To fulfil international conservation commitments, governments have begun to recognise the need for more proactive marine planning policies, advocating sensitive engineering design that can deliver secondary benefits above and beyond the primary purpose of developments. In response, there is growing scientific interest in novel multi-functional coastal defence structures with built-in secondary ecological and/or socio-economic benefits. To ensure research efforts are invested effectively, it is first necessary to determine what secondary benefits can potentially be built-in to engineered coastal defence structures, and further, which of these benefits would be most desirable. It is unlikely that secondary benefits are perceived in the same way across different stakeholder groups. Further, their order of priority when evaluating different options is unlikely to be consistent, since each option will present a suite of compromises and trade-offs. The aim of this study was to investigate stakeholder attitudes towards multi-functional coastal defence developments across different sector groups. A preliminary questionnaire indicated unanimous support for implementing multi-functional structures in place of traditional single-purpose ones. This preliminary survey informed the design of a Delphi-like study, which revealed a more nuanced and caveated level of support from a panel of experts and practitioners. The study also elicited a degree of consensus that the most desirable secondary benefits that could be built-in to developments would be ecological ones – prioritised over social, economic and technical benefits. This paper synthesises these findings, discusses the perceived barriers that remain, and proposes a stepwise approach to effective implementation of multi-functional coastal defence developments

Improving energy efficiency of electrochemical blackwater disinfection through sequential reduction of suspended solids and chemical oxygen demand [version 2; referees: 2 approved]

Onsite reuse of blackwater requires removal of considerable amounts of suspended solids and organic material in addition to inactivation of pathogens. Previously, we showed that electrochemical treatment could be used for effective pathogen inactivation in blackwater, but was inadequate to remove solids and organics to emerging industry standards. Further, we found that as solids and organics accumulate with repeated recycling, electrochemical treatment becomes less energetically sustainable. Here, we describe a pilot study in which concentrated blackwater is pretreated with ultrafiltration and granular activated carbon prior to electrochemical disinfection, and show that this combination of treatments removes 75-99% of chemical oxygen demand, 92-100% of total suspended solids, and improves the energy efficiency of electrochemical blackwater treatment by an order of magnitude

Improving energy efficiency of electrochemical blackwater disinfection through sequential reduction of suspended solids and chemical oxygen demand [version 1; referees: 2 approved]

Onsite reuse of blackwater requires removal of considerable amounts of suspended solids and organic material in addition to inactivation of pathogens. Previously, we showed that electrochemical treatment could be used for effective pathogen inactivation in blackwater, but was inadequate to remove solids and organics to emerging industry standards. Further, we found that as solids and organics accumulate with repeated recycling, electrochemical treatment becomes less energetically sustainable. Here, we describe a pilot study in which concentrated blackwater is pretreated with ultrafiltration and granular activated carbon prior to electrochemical disinfection, and show that this combination of treatments removes 75-99% of chemical oxygen demand, 92-100% of total suspended solids, and improves the energy efficiency of electrochemical blackwater treatment by an order of magnitude

Trade-offs in marine protection : Multi-species interactions within a community-led temperate marine reserve

This study investigated the effects of a community-led temperate marine reserve in Lamlash Bay, Firth of Clyde, Scotland, on commercially important populations of European lobster (Homarus gammarus), brown crab (Cancer pagurus), and velvet swimming crabs (Necora puber). Potting surveys conducted over 4 years revealed significantly higher catch per unit effort (cpue 109% greater), weight per unit effort (wpue 189% greater), and carapace length (10-15 mm greater) in lobsters within the reserve compared with control sites. However, likely due to low levels of recruitment and increased fishing effort outside the reserve, lobster catches decreased in all areas during the final 2 years. Nevertheless, catch rates remained higher within the reserve across all years, suggesting the reserve buffered these wider declines. Additionally, lobster cpue and wpue declined with increasing distance from the boundaries of the marine reserve, a trend which tag-recapture data suggested were due to spillover. Catches of berried lobster were also twice as high within the reserve than outside, and the mean potential reproductive output per female was 22.1% greater. It was originally thought that higher densities of lobster within the reserve might lead to greater levels of aggression and physical damage. However, damage levels were solely related to body size, as large lobsters >110 mm had sustained over 218% more damage than smaller individuals. Interestingly, catches of adult lobsters were inversely correlated with those of juvenile lobsters, brown crabs, and velvet crabs, which may be evidence of competitive displacement and/or predation. Our findings provide evidence that temperate marine reserves can deliver fisheries and conservation benefits, and highlight the importance of investigating multispecies interactions, as the recovery of some species can have knock-on effects on others

Policy Recommendations for Meeting the Grand Challenge to Ensure Healthy Development for All Youth

This brief was created forSocial Innovation for America’s Renewal, a policy conference organized by the Center for Social Development in collaboration with the American Academy of Social Work & Social Welfare, which is leading theGrand Challenges for Social Work initiative to champion social progress. The conference site includes links to speeches, presentations, and a full list of the policy briefs

Selective role for superoxide in InsP3 receptor–mediated mitochondrial dysfunction and endothelial apoptosis

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2.− that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2.−-triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2.− and could be prevented by [Ca2+]i buffering. This study provides evidence that O2.− facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation