Intensified cell culture using a linked bioreactor system

As manufacturing capacity becomes limited and demand and competition increases in the biologics space, the need for more flexible, cost effective, and productive biomanufacturing processes grows. We will describe a novel, intensified cell culture process that uses the cell-bleed of an N-1 (or seed) perfusion bioreactor to continuously feed a production-stage continuous-flow stirred-tank bioreactor. This continuously-linked bioreactor system effectively separates cells into two cultures, one a high growth phase, and the second a highly productive stationary phase. The design of the system allows it to operate indefinitely under steady-state conditions with volumetric productivities exceeding 1.0 gram/liter/day, regardless of any cell-specific productivity loss due to cell line instability. Multiple modes of system start-up will be discussed as part of the optimization work performed to date. Due to the unique design of the linked bioreactor system it may be effectively operated at very large scales typically deemed impractical for more conventional perfusion processes, potentially enabling more efficient use of installed production capacity. The linked bioreactor system attained productivities up to 2.4-fold those achieved in the respective commercial-ready, fed-batch processes, while drastically reducing overall process media consumption. Finally, since the production bioreactor operates as a continuous-flow stirred-tank bioreactor the system creates additional flexibility for downstream operations. Cell culture could be harvested continuously, or intermittently on a potentially variable cycle, depending upon facility fit and maximum efficiency using existing large-volume disc-stacked centrifuges. The resulting integrated continuous biomanufacturing system could operate under very nearly true steady-state conditions uninterrupted for months at a time

A Systematic Observational Study of a Juvenile Drug Court Judge

The shift of the juvenile justice system from its initial rehabilitative ideal toward a more punitive orientation highlights the need to systematically document key elements of the juvenile drug court model. In particular, it is important to clearly document the role of the juvenile court judge because he or she is considered vital to this program model. The current study used participant observation as well as confidential questionnaires on which youth shared their perceptions of the judge. Findings show the judge‐participant interactions typically were brief, varied by the participants\u27 level of compliance with the program, and that sanctions were given twice as often as rewards. Youth perceived the judge to be fair, respectful, and concerned about their lives. Discussion focuses on the significant opportunity that juvenile drug court judges have for positively influencing the lives of drug‐involved youth

Innovations in bioreactor operational modes – Hybrid semi-continuous processes to push beyond the limits of conventional fed-batch cultures

Due to the limits of feed volume addition and the problem of amino acid counter ion, miscellaneous osmolyte, and cell growth inhibitor accumulation, the fed-batch mode of bioreactor operation for CHO cell production of protein therapeutics is inherently limited with respect to the cell densities and productivities that are achievable. Continuous or perfusion culture with cell retention can overcome some of these limitations, but suffers from the disadvantages of large volume media consumption, long times to reach peak cell densities, and complications with cell retention devices. We will describe hybrid versions of continuous culture that overcome many of these limitations and utilize a unique and simple technology which allows cells to control their own rate of perfusion with continuous feedback. Volumetric productivities of greater than 1 gram/L/day (more than double the optimized fed-batch culture) for several moderate specific productivity cell lines have been achieved with very modest medium volumes, comparatively simple bioreactor operations, and a batch length that fits in a standard fed-batch window. Methods of operation and experimental results obtained at the pilot scale when coordinating these hybrid continuous cultures with a continuous downstream process will also be discussed

Cell-controlled high-intensity perfusion and hybrid fed-batch systems that drastically reduce perfusion rates and harmonize with continuous downstream processing

Peak cell densities in fed-batch cultures of mammalian cells can be limited by the depletion of nutrients or accumulation of inhibitors. Since addition of nutrients is relatively straight forward, inevitably it is growth inhibitor buildup, combined with the practical limits of feed volume addition and the problem of amino acid counter ion and miscellaneous osmolyte accumulation, that serve to limit productivity in the industry standard fed-batch bioreactor. To break these barriers we have begun to investigate mixed modes of bioreactor operation, some using novel methods for cell-controlled perfusion, which can substantially reduce the volumes of perfusion medium required, suppress lactic acid formation, shorten the time to reach peak cell densities, and reduce the complications associated with cell retention devices. Our methods of high-intensity perfusion and mixed mode bioreactor operation are also more compatible with the move to continuous downstream processing as the stream delivered is more concentrated and provides a more continuous daily mass of product to allow for reduced affinity chromatography scale. The techniques we will describe significantly increase viable cell densities and have achieved volumetric productivities of 1.0-1.5 grams/L/day (nearly 4X the optimized fed-batch culture in one case) for several moderate specific productivity cell lines, while using very modest medium volumes, highly concentrated perfusion media, comparatively simple bioreactor operations, and a batch length that fits in a standard fed-batch window. Methods of operation and experimental results obtained at the pilot (100 liter) scale when coordinating these hybrid continuous cultures with a continuous downstream process will also be discussed

The National Clinical Assessment Tool for Medical Students in the Emergency Department (NCAT-EM)

Introduction: Clinical assessment of medical students in emergency medicine (EM) clerkships is a highly variable process that presents unique challenges and opportunities. Currently, clerkship directors use institution-specific tools with unproven validity and reliability that may or may not address competencies valued most highly in the EM setting. Standardization of assessment practices and development of a common, valid, specialty-specific tool would benefit EM educators and students. Methods: A two-day national consensus conference was held in March 2016 in the Clerkship Directors in Emergency Medicine (CDEM) track at the Council of Residency Directors in Emergency Medicine (CORD) Academic Assembly in Nashville, TN. The goal of this conference was to standardize assessment practices and to create a national clinical assessment tool for use in EM clerkships across the country. Conference leaders synthesized the literature, articulated major themes and questions pertinent to clinical assessment of students in EM, clarified the issues, and outlined the consensus- building process prior to consensus-building activities. Results: The first day of the conference was dedicated to developing consensus on these key themes in clinical assessment. The second day of the conference was dedicated to discussing and voting on proposed domains to be included in the national clinical assessment tool. A modified Delphi process was initiated after the conference to reconcile questions and items that did not reach an a priori level of consensus. Conclusion: The final tool, the National Clinical Assessment Tool for Medical Students in Emergency Medicine (NCAT-EM) is presented here. [West J Emerg Med. 2018;19(1)66-74.

Dermal and muscle fibroblasts and skeletal myofibers survive chikungunya virus infection and harbor persistent RNA

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after the initial infection, yet we still have a poor understanding of what promotes chronic disease. While replicating virus has not been detected in joint-associated tissues of patients with persistent arthritis nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to pathogenesis during this chronic phase, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3'-Cre). CHIKV-3'-Cre replicated in myoblasts and fibroblasts, and it induced arthritis during the acute phase in mice. Importantly, it also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type virus. CHIKV-3'-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling revealed that these tdTomato+ cells predominantly were myofibers and dermal and muscle fibroblasts. Treatment with an antibody against Mxra8, a recently defined host receptor for CHIKV, reduced the number of tdTomato+ cells in the chronic phase and diminished the levels of chronic viral RNA, implicating these tdTomato+ cells as the reservoir of chronic viral RNA. Finally, isolation and flow cytometry-based sorting of the tdTomato+ fibroblasts from the skin and ankle and analysis for viral RNA revealed that the tdTomato+ cells harbor most of the persistent CHIKV RNA at chronic time points. Therefore, this CHIKV-3'-Cre and tdTomato reporter mouse system identifies the cells that survive CHIKV infection in vivo and are enriched for persistent CHIKV RNA. This model represents a useful tool for studying CHIKV pathogenesis in the acute and chronic stages of disease

An Adaptable, Portable Microarray Reader for Biodetection

We have developed an inexpensive portable microarray reader that can be applied to standard microscope slide-based arrays and other array formats printed on chemically modified surfaces. Measuring only 19 cm in length, the imaging device is portable and may be applicable to both triage and clinical settings. For multiplexing and adaptability purposes, it can be modified to work with multiple excitation/emission wavelengths. Our device is shown to be comparable to a commercial laser scanner when detecting both streptavidin-biotin and antibody interactions. This paper presents the development and characterization of a handheld microarray imager and directly compares it with a commercial scanner

A Model for Rigorously Applying the Exploration, Preparation, Implementation, Sustainment (EPIS) Framework in the Design and Measurement of a Large Scale Collaborative Multi-Site Study

Background This paper describes the means by which a United States National Institute on Drug Abuse (NIDA)-funded cooperative, Juvenile Justice-Translational Research on Interventions for Adolescents in the Legal System (JJ-TRIALS), utilized an established implementation science framework in conducting a multi-site, multi-research center implementation intervention initiative. The initiative aimed to bolster the ability of juvenile justice agencies to address unmet client needs related to substance use while enhancing inter-organizational relationships between juvenile justice and local behavioral health partners. Methods The EPIS (Exploration, Preparation, Implementation, Sustainment) framework was selected and utilized as the guiding model from inception through project completion; including the mapping of implementation strategies to EPIS stages, articulation of research questions, and selection, content, and timing of measurement protocols. Among other key developments, the project led to a reconceptualization of its governing implementation science framework into cyclical form as the EPIS Wheel. The EPIS Wheel is more consistent with rapid-cycle testing principles and permits researchers to track both progressive and recursive movement through EPIS. Moreover, because this randomized controlled trial was predicated on a bundled strategy method, JJ-TRIALS was designed to rigorously test progress through the EPIS stages as promoted by facilitation of data-driven decision making principles. The project extended EPIS by (1) elucidating the role and nature of recursive activity in promoting change (yielding the circular EPIS Wheel), (2) by expanding the applicability of the EPIS framework beyond a single evidence-based practice (EBP) to address varying process improvement efforts (representing varying EBPs), and (3) by disentangling outcome measures of progression through EPIS stages from the a priori established study timeline. Discussion The utilization of EPIS in JJ-TRIALS provides a model for practical and applied use of implementation frameworks in real-world settings that span outer service system and inner organizational contexts in improving care for vulnerable populations. Trial registration NCT02672150. Retrospectively registered on 22 January 2016

Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates

Abstract Background Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates. Results We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons. Conclusions These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.http://deepblue.lib.umich.edu/bitstream/2027.42/112375/1/12864_2012_Article_5193.pd