Process development for increased MSC production in single use stirred tank bioreactors

Industry trends in regenerative medicine highlight a critical need for closed, single use cell culture systems that support scalable manufacturing of adherent cell therapies. Due to the limited downstream processing steps and shelf-life requirements for cell therapies, single use technologies are essential for cell therapy production. However, typical single-use static in vitro culture methods, are often too cumbersome and inefficient to support commercial scale production of mesenchymal stem/stromal cells (MSCs). Single-use stirred tank bioreactor systems are a platform that can address this scaling limitation by decreasing labor, footprint, and overall cost. When developing a stirred tank bioreactor process, bioreactor seeding and process control strategies, such as agitation, must be optimized to enable the process to scale for commercial manufacturing. Herein, case studies are presented illustrating solutions to this need. The first case study demonstrates the application of Zwietering’s equation for suspension of solids to overcome scaling challenges often associated with microcarrier culture in stirred tanks. The second case study reviews strategies to further close the bioreactor seeding process. Identifying optimal seeding and process control strategies for microcarrier-based bioreactor expansion of adherent cells is paramount for the development of robust cell therapy manufacturing platforms

Student Nurses Who Witness Critical Events in the Clinical Setting: A Grounded Theory Qualitative Study

Hood, Tiffany Lee. Student Nurses Who Witness Critical Events in the Clinical Setting: A Grounded Theory Qualitative Study. Published Doctor of Philosophy dissertation, University of Northern Colorado, 2020. Background: Nursing students often experience critical events in the clinical setting, and too often, the clinical instructor does not have the training to help students through these situations. The literature shows that students often feel alone and abandoned, requiring them to endure these experiences without proper psychological recovery. Clinical nurse educators and staff nurses may not fully understand their role in emotional support, pre-briefing, and debriefing, not knowing what to do to help students through such difficult situations. Procedure: A grounded theory qualitative study was conducted to better understand the experiences of student nurses who have witnessed critical events in the clinical setting, and to better understand the types of support provided and the effectiveness of the support. Fourteen undergraduate student nurses from three four-year universities in Utah, United States, participated in this study. Results: Using a four-stage coding procedure, 50 initial categories were categorized into one core category, nine primary categories, and nine secondary categories. Relationships between categories were identified, and a theory of student nurse support and recovery through critical events in the clinical setting emerged. Conclusion: Student nurses need active faculty and/or staff support during critical events, and pre-briefing whenever possible. Students should be taught coping skills and have risk and support systems assessed prior to entering the clinical setting. Nursing knowledge, life experiences, values, beliefs, coping skills, current mental health state, and prior history of trauma affect student responses to critical events. Immediate debrief positively affects post-event stress response and coping by providing the opportunity for students to gain closure, decrease anxiety, increase understanding, time to mentally process the event, and emotional support. Lack of debrief increases post-event psychological distress and decrease coping and resilience. Support after critical events should continue in the days, weeks, and months following the event. Students should be monitored for signs of increased psychological distress and psychological trauma and be provided resources for help in coping. Students who do not receive adequate support prior to, during, or after a critical event are at risk for psychological trauma

Moral and Cultural Awareness in Emerging Adulthood: Preparing for Multi-Faith Workplaces

The study evaluates a pilot course designed to respond to findings from the National Study of Youth and Religion (NSYR) and similar findings reporting changes in U.S. life course development and religious participation through an intervention based on sociological theories of morality. The purpose of the study is to investigate the impacts of a business course in a public university designed to prepare emerging adults for culturally and religiously diverse workplaces. The intended outcomes are for students to better identify their personal moral values, while also gaining cultural awareness of the moral values in six different value systems: five major world religions and secular humanism. The study response rate was 97 percent (n = 109). Pre- and post-test survey data analyze changes in the reports of students enrolled in the course (primary group) compared to students in similar courses but without an emphasis on morality (controls). Qualitative data include survey short answer questions, personal mission statements, and student essays describing course impacts. Quantitative and qualitative results indicate reported increases in identification of personal moral values and cultural awareness of other moral values, providing initial evidence that the course helps prepare emerging adults for multi-faith workplaces

Process development for improved Car-t production utilizing an automated single use perfusion stirred-tank bioreactor

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T-cells (CAR-T), have generated significant clinical and commercial outcomes due to their unparalleled response rates against refractory/relapsed blood cancers. However, the development and manufacture of these advanced therapies face a number of translational bottlenecks that must be addressed to ensure long-term commercial viability. The cost and variability associated with these personalized advanced therapies presents a critical manufacturing and translational challenge. Due to the nature of cell therapies, single-use technologies are typically used for their production. This work demonstrates the importance of determining critical manufacturing process parameters using single-use technologies and incorporating automation into the process. The work builds off of previous proof of concept work completed in the group for the production of CAR-T cells in automated, stirred tank, single-use ambr250® bioreactors. These follow-on experiments utilize concepts of quality by design and design of experiments (DoE) to systematically determine the impact of process parameters on CAR-T production. Please click Additional Files below to see the full abstract

Process development for improved Car-t production utilizing an automated perfusion stirred-tank bioreactor

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T-cells (CAR-T), have generated significant clinical and commercial outcomes due to their unparalleled response rates against refractory/relapsed blood cancers. However, the development and manufacture of these advanced therapies face a number of translational bottlenecks that must be addressed to ensure long-term commercial viability. Please click Additional Files below to see the full abstract

Process development approaches for expansion of adherent stem cells in microcarrier-based bioreactor culture

Industry trends in regenerative medicine show an increased need for scalable and closed manufacturing of cell therapies. Single-use bioreactor systems have proven suitable as a platform to meet the industry’s needs. Key process parameters for cell culture performance in these systems include pH, dissolved oxygen (DO) and agitation rates. Especially important is the understanding and application of appropriate solid-liquid mixing, which is essential for microcarrier-based cultures used for adherent stem cells. Agitation rates that are too high in microcarrier-based cultures can be correlated with smaller eddy lengths that impact cellular shear. Conversely, agitation rates that are low do not support the consistent microcarrier turnover required for cell access to nutrients, DO, and maintenance of pH. Moreover, suboptimal agitation rates may impact cell-to-microcarrier attachment and transfer. Here, we summarize a stepwise approach to optimizing pH, DO and agitation set-points in the Mobius® 3L single-use bioreactor for mesenchymal stem/stromal cells (MSCs). The theoretical agitation operating range best suited for microcarrier cultures was calculated based on the Zwietering equation for suspension of solids in stirred tanks, and verified experimentally with human bone marrow-derived MSCs. Upper agitation limits were defined by Kolmogorov\u27s theory of turbulent eddy lengths, and were substantially higher than the agitation rates required to keep microcarriers suspended. Identifying optimal pH, DO and agitation rates for microcarrier-based bioreactor expansion of adherent cells is paramount to developing a robust platform for use in a controlled manufacturing environment

Thermally drawn fibers as nerve guidance scaffolds

a b s t r a c t Synthetic neural scaffolds hold promise to eventually replace nerve autografts for tissue repair following peripheral nerve injury. Despite substantial evidence for the influence of scaffold geometry and dimensions on the rate of axonal growth, systematic evaluation of these parameters remains a challenge due to limitations in materials processing. We have employed fiber drawing to engineer a wide spectrum of polymer-based neural scaffolds with varied geometries and core sizes. Using isolated whole dorsal root ganglia as an in vitro model system we have identified key features enhancing nerve growth within these fiber scaffolds. Our approach enabled straightforward integration of microscopic topography at the scale of nerve fascicles within the scaffold cores, which led to accelerated Schwann cell migration, as well as neurite growth and alignment. Our findings indicate that fiber drawing provides a scalable and versatile strategy for producing nerve guidance channels capable of controlling direction and accelerating the rate of axonal growth

Varicella zoster virus infection of highly pure terminally differentiated human neurons

In vitro analyses of varicella zoster virus (VZV) reactivation from latency in human ganglia have been hampered by the inability to isolate virus by explantation or cocultivation techniques. Furthermore, attempts to study interaction of VZV with neurons in experimentally infected ganglion cells in vitro have been impaired by the presence of nonneuronal cells, which become productively infected and destroy the cultures. We have developed an in vitro model of VZV infection in which highly pure (>95 %) terminally differentiated human neurons derived from pluripotent stem cells were infected with VZV. At 2 weeks post-infection, infected neurons appeared healthy compared to VZV-infected human fetal lung fibroblasts (HFLs), which developed a cytopathic effect (CPE) within 1 week. Tissue culture medium from VZV-infected neurons did not produce a CPE in uninfected HFLs and did not contain PCR-amplifiable VZV DNA, but cocultivation of infected neurons with uninfected HFLs did produce a CPE. The nonproductively infected neurons contained multiple regions of the VZV genome, as well as transcripts and proteins corresponding to VZV immediate-early, early, and late genes. No markers of the apoptotic caspase cascade were detected in healthy-appearing VZV-infected neurons. VZV infection of highly pure terminally differentiated human neurons provides a unique in vitro system to study the VZV-neuronal relationship and the potential to investigate mechanisms of VZV reactivation

The Habitable Exoplanet Observatory (HabEx) Mission Concept Study Final Report

The Habitable Exoplanet Observatory, or HabEx, has been designed to be the Great Observatory of the 2030s. For the first time in human history, technologies have matured sufficiently to enable an affordable space-based telescope mission capable of discovering and characterizing Earthlike planets orbiting nearby bright sunlike stars in order to search for signs of habitability and biosignatures. Such a mission can also be equipped with instrumentation that will enable broad and exciting general astrophysics and planetary science not possible from current or planned facilities. HabEx is a space telescope with unique imaging and multi-object spectroscopic capabilities at wavelengths ranging from ultraviolet (UV) to near-IR. These capabilities allow for a broad suite of compelling science that cuts across the entire NASA astrophysics portfolio. HabEx has three primary science goals: (1) Seek out nearby worlds and explore their habitability; (2) Map out nearby planetary systems and understand the diversity of the worlds they contain; (3) Enable new explorations of astrophysical systems from our own solar system to external galaxies by extending our reach in the UV through near-IR. This Great Observatory science will be selected through a competed GO program, and will account for about 50% of the HabEx primary mission. The preferred HabEx architecture is a 4m, monolithic, off-axis telescope that is diffraction-limited at 0.4 microns and is in an L2 orbit. HabEx employs two starlight suppression systems: a coronagraph and a starshade, each with their own dedicated instrument

The Habitable Exoplanet Observatory (HabEx) Mission Concept Study Final Report

The Habitable Exoplanet Observatory, or HabEx, has been designed to be the Great Observatory of the 2030s. For the first time in human history, technologies have matured sufficiently to enable an affordable space-based telescope mission capable of discovering and characterizing Earthlike planets orbiting nearby bright sunlike stars in order to search for signs of habitability and biosignatures. Such a mission can also be equipped with instrumentation that will enable broad and exciting general astrophysics and planetary science not possible from current or planned facilities. HabEx is a space telescope with unique imaging and multi-object spectroscopic capabilities at wavelengths ranging from ultraviolet (UV) to near-IR. These capabilities allow for a broad suite of compelling science that cuts across the entire NASA astrophysics portfolio. HabEx has three primary science goals: (1) Seek out nearby worlds and explore their habitability; (2) Map out nearby planetary systems and understand the diversity of the worlds they contain; (3) Enable new explorations of astrophysical systems from our own solar system to external galaxies by extending our reach in the UV through near-IR. This Great Observatory science will be selected through a competed GO program, and will account for about 50% of the HabEx primary mission. The preferred HabEx architecture is a 4m, monolithic, off-axis telescope that is diffraction-limited at 0.4 microns and is in an L2 orbit. HabEx employs two starlight suppression systems: a coronagraph and a starshade, each with their own dedicated instrument.Comment: Full report: 498 pages. Executive Summary: 14 pages. More information about HabEx can be found here: https://www.jpl.nasa.gov/habex