Microfluidic platform for 3D cell culture with live imaging and clone retrieval.

Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes. Here, we present a method to encapsulate live cells in a 3D hydrogel matrix, via hydrogel bead compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we can long-term culture and subsequently extract individual cells-in-beads by media flow only, obviating the need for enzymatic cell removal from the platform. Specific beads may be extracted from the platform in isolation, without disrupting the adjacent beads. We show that we can differentiate mouse embryonic stem cells, monitor reporter expression by live imaging, and retrieve individual beads for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays

Come back when you’re infected: pharmacy access to sterile syringes in an Arizona Secret Shopper Study, 2023

Background: Pharmacies are critical healthcare partners in community efforts to eliminate bloodborne illnesses. Pharmacy sale of sterile syringes is central to this effort. Methods: A mixed methods “secret shopper” syringe purchase study was conducted in the fall of 2022 with 38 community pharmacies in Maricopa and Pima Counties, Arizona. Pharmacies were geomapped to within 2 miles of areas identified as having a potentially high volume of illicit drug commerce. Daytime venue sampling was used whereby separate investigators with lived/living drug use experience attempted to purchase syringes without a prescription. Investigator response when prompted for purchase rationale was “to protect myself from HIV and hepatitis C.” A 24-item instrument measured sales outcome, pharmacy staff interaction (hostile/neutral/friendly), and the buyer’s subjective experience. Results: Only 24.6% (n = 28) of 114 purchase attempts across the 38 pharmacies resulted in syringe sale. Less than one quarter (21.1%) of pharmacies always sold, while 44.7% never sold. Independent and food store pharmacies tended not to sell syringes. There emerged distinct pharmacy staff interactions characterized by body language, customer query, normalization or othering response, response to purchase request and closure. Pharmacy discretion and pharmacy policy not to sell syringes without a prescription limited sterile syringe access. Investigators reported frequent and adverse emotional impact due to pharmacy staff negative and stigmatizing interactions. Conclusions: Pharmacies miss opportunities to advance efforts to eliminate bloodborne infections by stringent no-sale policy and discretion about syringe sale. State regulatory policy facilitating pharmacy syringe sales, limiting pharmacist discretion for syringe sales, and targeting pharmacy-staff level education may help advance the achievement of public health goals to eliminate bloodborne infections in Arizona

Active GSK3β and an intact β-catenin TCF complex are essential for the differentiation of human myogenic progenitor cells

CA was in receipt of a KCL studentship. The work was part funded by the BBSRC ref BB/L009943/1

StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells.

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification

Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.Wellcome Trust WT108438/C/15/

Niche stiffness underlies the ageing of central nervous system progenitor cells.

Ageing causes a decline in tissue regeneration owing to a loss of function of adult stem cell and progenitor cell populations1. One example is the deterioration of the regenerative capacity of the widespread and abundant population of central nervous system (CNS) multipotent stem cells known as oligodendrocyte progenitor cells (OPCs)2. A relatively overlooked potential source of this loss of function is the stem cell 'niche'-a set of cell-extrinsic cues that include chemical and mechanical signals3,4. Here we show that the OPC microenvironment stiffens with age, and that this mechanical change is sufficient to cause age-related loss of function of OPCs. Using biological and synthetic scaffolds to mimic the stiffness of young brains, we find that isolated aged OPCs cultured on these scaffolds are molecularly and functionally rejuvenated. When we disrupt mechanical signalling, the proliferation and differentiation rates of OPCs are increased. We identify the mechanoresponsive ion channel PIEZO1 as a key mediator of OPC mechanical signalling. Inhibiting PIEZO1 overrides mechanical signals in vivo and allows OPCs to maintain activity in the ageing CNS. We also show that PIEZO1 is important in regulating cell number during CNS development. Thus we show that tissue stiffness is a crucial regulator of ageing in OPCs, and provide insights into how the function of adult stem and progenitor cells changes with age. Our findings could be important not only for the development of regenerative therapies, but also for understanding the ageing process itself.The work was supported by European Research Council (ERC) grant 772798 (to K.J.C.) and 772426 (to K.F.); the UK Multiple Sclerosis Society (to R.J.M.F.); Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/M008827/1 (to K.J.C and R.J.M.F.) and BB/N006402/1 (to K.F.); the Adelson Medical Research Foundation (R.J.M.F. and D.H.R.); an EMBO Long-Term Fellowship ALTF 1263-2015 and European Commission FP7 actions LTFCOFUND2013, GA-2013-609409 (to I.P.W.); and a core support grant from the Wellcome Trust and Medical Research Council (MRC) to the Wellcome Trust–MRC Cambridge Stem Cell Institute

Opioid use, motivation to quit, and treatment status related to COVID-19: a cross-sectional study

Persons who use opioids may be at elevated risk of harm from the coronavirus disease 2019 (COVID-19) pandemic, yet few data currently exist that can be used to examine this risk. As part of a rapid response survey, this study measured opioid users’ perceptions of risk or harm from COVID-19, as well as potential changes in motivation to quit, frequency of use, and engagement with treatment. Data collected from Amazon’s Mechanical Turk (n = 562) were analyzed

An Image Analysis Method for the Precise Selection and Quantitation of Fluorescently Labeled Cellular Constituents

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored

Perceived enablers and barriers of community engagement for vaccination in India: Using socioecological analysis

Background There is high level policy consensus in India that community engagement (CE) improves vaccination uptake and reduces burden of vaccine preventable diseases. However, to date, vaccination studies in the country have not explicitly focused on CE as an outcome in and of itself. Therefore, this study sought to examine the barriers and enablers of community engagement for vaccination in India. Methods Employing qualitative methods, twenty-five semi-structured elite interviews among vaccine decisionmakers were triangulated with twenty-four national-level vaccine policy documents and researcher field notes (December 2017 to February 2018). Data collected for this study included perceptions and examples of enablers of and barriers to CE for vaccination uptake. Concepts, such as the absence of formal procedures or data collection approaches related to CE, were confirmed during document review, and a final convening to review study results was conducted with study respondents in December 2018 and January 2019 to affirm the general set of findings from this study. The Social Ecological Model (SEM) was used to organize and interpret the study findings. Results Although decisionmakers and policy documents generally supported CE, there were more CE barriers than facilitators in the context of vaccination, which were identified at all socialecological levels. Interviews with vaccine decisionmakers in India revealed complex systemic and structural factors which affect CE for vaccination and are present across each of the SEM levels, from individual to policy. Policy-level enablers included decisionmakers political will for CE and policy documents and interviews highlighted social mobilization, whereas barriers were lack of a CE strategy document and a broad understanding of CE by decisionmakers. At the community level, dissemination of Social-behavioral Change Communication (SBCC) materials from the national-level to the states was considered a CE facilitator, while class, and caste-based power relations in the community, lack of family-centric CE strategies, and paternalistic attitude of decisionmakers toward communities (the latter reported by some NGO heads) were considered CE barriers. At the organizational level, partnerships with local organizations were considered CE enablers, while lack of institutionalized support to formalize and incentivize these partnerships highlighted by several decisionmakers, were barriers. At the interpersonal level, SBCC training for healthcare workers, sensitive messaging to communities with low vaccine confidence, and social media messaging were considered CE facilitators. The lack of strategies to manage vaccine related rumors or replicate successful CE interventions during the during the introduction and rollout of new vaccines were perceived as CE barriers by several decisionmakers. Conclusion Data obtained for this study highlighted national-level perceptions of the complexities and challenges of CE across the entire SEM, from individual to systemic levels. Future studies should attempt to associate these enablers and barriers with actual CE outcomes, such as participation or community support in vaccine policy-making, CE implementation for specific vaccines and situations (such as disease outbreaks), or frequency of sub-population-based incidents of community resistance and community facilitation to vaccination uptake. There would likely be value in developing a population-based operational definition of CE, with a step-by-step manual on how to do CE. The data from this study also indicate the importance of including CE indicators in national datasets and developing a compendium documenting CE best-practices. Doing so would allow more rigorous analysis of the evidencebase for CE for vaccination in India and other countries with similar immunization programs. © 2021 Public Library of Science. All rights reserved.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]