Clinical trials investigating extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) have rapidly increased in recent years. Scalable GMP-compatible production of MSC-EVs requires ...bioreactor expansion and downstream processing (DSP) for purification. We have published MSC-EV bioreactor processes from 3L to 50L with DSP operations including clarification, tangential flow filtration (TFF), chromatography, and formulation/fill. Cumulative post-DSP yields of MSC-EVs are often <10%, representing a major challenge in total EVs per lot and increased cost of goods. Recently, we discovered a novel reagent (Agent V, patent pending) that streamlines DSP and greatly increases total EV yield while maintaining MSC-EV critical quality attributes (CQAs). The goal of this study was to optimize the concentration of Agent V in a bioreactor feedstream of harvested conditioned medium (CM) that maximizes DSP yield while maintaining EV CQAs.
Human bone marrow MSC-EVs were produced in a 3L bioreactor (Eppendorf) using RoosterCollect™-EV for a 5-day collection as described previously. Agent V (RoosterBio) was added to harvested CM at 1, 0.1, and 0.01 U/mL for at least 15 minutes and MSC-EV yield though clarification filters (Sartorius) was quantified by NTA (Particle Metrix). The optimal Agent V dose (0.1 U/mL) was chosen to treat CM. MSC-EV yield and purity (particles/mg protein) for each additional unit operation (TFF, Repligen; chromatography, Cytiva) were measured +/- Agent V treatment. Purified MSC-EVs were tested for MSC-EV identity (CD63/CD9/CD81/ALIX/TSG101 via ProteinSimple Jess) and function (CD73 activity).
MSC-EV yield following DSP is often <10%, with ∼50-60% loss at the initial clarification step. Treating CM with Agent V led to a dose-dependent yield improvement through a 5µm clarification filter. Treated and untreated CM were processed sequentially through each DSP unit operation, demonstrating that treatment leads to significant cumulative yield improvements (∼5% vs. ∼50%, a 10-fold increase). Furthermore, MSC-EV markers were maintained (ProteinSimple Jess), and purified MSC-EVs from the Agent V treated group showed ∼3X greater CD73 activity per particle.
Agent V is a novel, GMP compatible reagent that simplifies and streamlines EV purification by increasing MSC-EV DSP yield 10-fold while maintaining CQAs and possibly enhancing functionality.
Background. Values clarification is a recommended element of patient decision aids. Many different values clarification methods exist, but there is little evidence synthesis available to guide design ...decisions. Purpose. To describe practices in the field of explicit values clarification methods according to a taxonomy of design features. Data Sources. MEDLINE, all EBM Reviews, CINAHL, EMBASE, Google Scholar, manual search of reference lists, and expert contacts. Study Selection. Articles were included if they described 1 or more explicit values clarification methods. Data Extraction. We extracted data about decisions addressed; use of theories, frameworks, and guidelines; and 12 design features. Data Synthesis. We identified 110 articles describing 98 explicit values clarification methods. Most of these addressed decisions in cancer or reproductive health, and half addressed a decision between just 2 options. Most used neither theory nor guidelines to structure their design. “Pros and cons” was the most common type of values clarification method. Most methods did not allow users to add their own concerns. Few methods explicitly presented tradeoffs inherent in the decision, supported an iterative process of values exploration, or showed how different options aligned with users’ values. Limitations. Study selection criteria and choice of elements for the taxonomy may have excluded values clarification methods or design features. Conclusions. Explicit values clarification methods have diverse designs but can be systematically cataloged within the structure of a taxonomy. Developers of values clarification methods should carefully consider each of the design features in this taxonomy and publish adequate descriptions of their designs. More research is needed to study the effects of different design features.
Background. Diverse values clarification methods exist. It is important to understand which, if any, of their design features help people clarify values relevant to a health decision. Purpose. To ...explore the effects of design features of explicit values clarification methods on outcomes including decisional conflict, values congruence, and decisional regret. Data Sources. MEDLINE, all EBM Reviews, CINAHL, EMBASE, Google Scholar, manual search of reference lists, and expert contacts. Study Selection. Articles were included if they described the evaluation of 1 or more explicit values clarification methods. Data Extraction. We extracted details about the evaluation, whether it was conducted in the context of actual or hypothetical decisions, and the results of the evaluation. We combined these data with data from a previous review about each values clarification method’s design features. Data Synthesis. We identified 20 evaluations of values clarification methods within 19 articles. Reported outcomes were heterogeneous. Few studies reported values congruence or postdecision outcomes. The most promising design feature identified was explicitly showing people the implications of their values, for example, by displaying the extent to which each of their decision options aligns with what matters to them. Limitations. Because of the heterogeneity of outcomes, we were unable to perform a meta-analysis. Results should be interpreted with caution. Conclusions. Few values clarification methods have been evaluated experimentally. More research is needed to determine effects of different design features of values clarification methods and to establish best practices in values clarification. When feasible, evaluations should assess values congruence and postdecision measures of longer-term outcomes.
The purpose of this article is to review the views of supralapsarianism and infralapsarianism which are two views within the Reformed theology camp. These two views are often misunderstood, ...supralapsarianism is often seen as making God a sin-maker and God acting unjustly. On the other hand, infralapsarianism is accused of falling on Arminianism. This article is an attempt to clarify the misunderstanding of these two views. The method used in this article is a literature review in the presentation of argumentative main ideas. The results of this study indicate that both supralapsarianism and infralapsarianism cannot be said to be absolute antitheses, but there are also differences that supralapsarianism focuses on the ideal and theological, while infralapsarianism focuses on the historical structure of Bible teachings.
When children are questioned, it is crucial they request clarification to resolve potential misunderstandings. The current research tested a method for increasing children's appropriate clarification ...requests during an interview, and examined the impact of age and question characteristics. Children (n = 81), ages 6‐ to 11‐years‐old, responded to scripted questions, some of which were designed to be “tricky” and required clarification. Half of the children received “I don't understand” (IDU) rule reminders during the interview. Older children and children who received IDU rule reminders requested clarification to a significantly greater proportion of tricky questions than younger children and children who did not receive reminders. Results indicate that children can recognize when they need clarification, and reminding them of the IDU rule increases the frequency with which they request clarification. Children's ability to request clarification provides insight into children's metacognitive abilities and has implications for those who question children across contexts (e.g., forensic, research).
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