Advances in high throughput cell culture technologies for therapeutic screening and biological discovery applications.

Cellular phenotypes and functional responses are modulated by the signals present in their microenvironment, including extracellular matrix (ECM) proteins, tissue mechanical properties, soluble signals and nutrients, and cell-cell interactions. To better recapitulate and analyze these complex signals within the framework of more physiologically relevant culture models, high throughput culture platforms can be transformative. High throughput methodologies enable scientists to extract increasingly robust and broad datasets from individual experiments, screen large numbers of conditions for potential hits, better qualify and predict responses for preclinical applications, and reduce reliance on animal studies. High throughput cell culture systems require uniformity, assay miniaturization, specific target identification, and process simplification. In this review, we detail the various techniques that researchers have used to face these challenges and explore cellular responses in a high throughput manner. We highlight several common approaches including two-dimensional multiwell microplates, microarrays, and microfluidic cell culture systems as well as unencapsulated and encapsulated three-dimensional high throughput cell culture systems, featuring multiwell microplates, micromolds, microwells, microarrays, granular hydrogels, and cell-encapsulated microgels. We also discuss current applications of these high throughput technologies, namely stem cell sourcing, drug discovery and predictive toxicology, and personalized medicine, along with emerging opportunities and future impact areas.

Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells.

Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness-variable, extracellular matrix (ECM) protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6 kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple-component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.

Regulatory role of cholesterol in modulating actin dynamics and cell adhesive interactions in the trabecular meshwork.

The trabecular meshwork (TM) tissue plays a crucial role in maintaining intraocular pressure (IOP) homeostasis. Increased TM contractility and stiffness are directly correlated with elevated IOP. Although cholesterol is known to be a determinant of glaucoma occurrence and elevated IOP, the underlying mechanisms remain elusive. In this study, we used human TM (HTM) cells to unravel the effects of cholesterol on TM stiffness. We achieved this by performing acute cholesterol depletion with Methyl-ß-cyclodextrin (MßCD) and cholesterol enrichment/replenishment with MßCD cholesterol complex (CHOL). Interestingly, cholesterol depletion triggered notable actin depolymerization and decreased focal adhesion formation, while enrichment/replenishment promoted actin polymerization, requiring the presence of actin monomers. Using a specific reporter of phosphatidylinositol 4,5-bisphosphate (PIP2), we demonstrated that cholesterol depletion decreases PIP2 levels on the cell membrane, whereas enrichment increases them. Given the critical role of PIP2 in actin remodeling and focal adhesion formation, we postulate that cholesterol regulates actin dynamics by modulating PIP2 levels on the membrane. Furthermore, we showed that cholesterol levels regulate integrin α5ß1 and αVß3 distribution and activation, subsequently altering cell-extracellular matrix (ECM) interactions. Notably, the depletion of cholesterol, as a major lipid constituent of the cell membrane, led to a decrease in HTM cell membrane tension, which was reversed upon cholesterol replenishment. Overall, our systematic exploration of cholesterol modulation on TM stiffness highlights the critical importance of maintaining appropriate membrane and cellular cholesterol levels for achieving IOP homeostasis.

Physical job demands in pregnancy and associated musculoskeletal health and employment outcomes: a systematic review.

OBJECTIVE: A decline in musculoskeletal health during pregnancy is an underappreciated adverse outcome of pregnancy that can have immediate and long-term health consequences. High physical job demands are known risk factors for nontraumatic musculoskeletal disorders in the general working population. Evidence from meta-analyses suggest that occupational lifting and prolonged standing during pregnancy may increase risk of adverse pregnancy outcomes. This systematic review examined associations between occupational lifting or postural load in pregnancy and associated musculoskeletal disorders and related sequalae. DATA SOURCES: Five electronic databases (Medline, Embase, CINAHL, NIOSHTIC-2, and Ergonomic Abstracts) were searched from 1990 to July 2022 for studies in any language. A Web of Science snowball search was performed in December 2022. Reference lists were manually reviewed. STUDY ELIGIBILITY CRITERIA: Eligible studies reported associations between occupational lifting or postural load and musculoskeletal health or sequelae (eg, employment outcomes) among pregnant and postpartum workers. METHODS: Data were extracted using a customized form to document study and sample characteristics; and details of exposures, outcomes, covariates, and analyses. Investigators independently assessed study quality for 7 risk-of-bias domains and overall utility, with discrepant ratings resolved through discussion. A narrative synthesis was conducted due to heterogeneity. RESULTS: Sixteen studies (11 cohort studies, 2 nested case-control studies, and 3 cross-sectional studies) from 8 countries were included (N=142,320 pregnant and N=1744 postpartum workers). Limited but consistent evidence with variable quality ratings, ranging from critical concern to high, suggests that pregnant workers exposed to heavy lifting (usually defined as ≥22 lbs or ≥10 kg) may be at increased risk of functionally limiting pelvic girdle pain and antenatal leave. Moreover, reports of dose-response relationships suggest graded risk levels according to lifting frequency, ranging from 21% to 45% for pelvic girdle pain and 58% to 202% for antenatal leave. Limited but consistent evidence also suggests that postural load increases the risk of employment cessation. CONCLUSION: Limited but consistent evidence suggests that pregnant workers exposed to heavy lifting and postural load are at increased risk of pelvic girdle pain and employment cessation. Job accommodations to reduce exposure levels may promote safe sustainable employment for pregnant workers.

Engineered matrix microenvironments reveal the heterogeneity of liver sinusoidal endothelial cell phenotypic responses.

Fibrosis is one of the hallmarks of chronic liver disease and is associated with aberrant wound healing. Changes in the composition of the liver microenvironment during fibrosis result in a complex crosstalk of extracellular cues that promote altered behaviors in the cell types that comprise the liver sinusoid, particularly liver sinusoidal endothelial cells (LSECs). Recently, it has been observed that LSECs may sustain injury before other fibrogenesis-associated cells of the sinusoid, implicating LSECs as key actors in the fibrotic cascade. A high-throughput cellular microarray platform was used to deconstruct the collective influences of defined combinations of extracellular matrix (ECM) proteins, substrate stiffness, and soluble factors on primary human LSEC phenotype in vitro. We observed remarkable heterogeneity in LSEC phenotype as a function of stiffness, ECM, and soluble factor context. LYVE-1 and CD-31 expressions were highest on 1 kPa substrates, and the VE-cadherin junction localization was highest on 25 kPa substrates. Also, LSECs formed distinct spatial patterns of LYVE-1 expression, with LYVE-1+ cells observed in the center of multicellular domains, and pattern size regulated by microenvironmental context. ECM composition also influenced a substantial dynamic range of expression levels for all markers, and the collagen type IV was observed to promote elevated expressions of LYVE-1, VE-cadherin, and CD-31. These studies highlight key microenvironmental regulators of LSEC phenotype and reveal unique spatial patterning of the sinusoidal marker LYVE-1. Furthermore, these data provide insight into understanding more precisely how LSECs respond to fibrotic microenvironments, which will aid drug development and identification of targets to treat liver fibrosis.

High throughput interrogation of human liver stellate cells reveals microenvironmental regulation of phenotype.

Liver fibrosis is a common feature of progressive liver disease and is manifested as a dynamic series of alterations in both the biochemical and biophysical properties of the liver. Hepatic stellate cells (HSCs) reside within the perisinusoidal space of the liver sinusoid and are one of the main drivers of liver fibrosis, yet it remains unclear how changes to the sinusoidal microenvironment impact HSC phenotype in the context of liver fibrosis. Cellular microarrays were used to examine and deconstruct the impacts of bio-chemo-mechanical changes on activated HSCs in vitro. Extracellular matrix (ECM) composition and stiffness were found to act individually and in combination to regulate HSC fibrogenic phenotype and proliferation. Hyaluronic acid and collagen III promoted elevated collagen I expression while collagen IV mediated a decrease. Previously activated HSCs exhibited reduced lysyl oxidase (Lox) expression as array substrate stiffness increased, with less dependence on ECM composition. Collagens III and IV increased HSC proliferation, whereas hyaluronic acid had the opposite effect. Meta-analysis performed on these data revealed distinct phenotypic clusters (e.g. low fibrogenesis/high proliferation) as a direct function of their microenvironmental composition. Notably, soft microenvironments mimicking healthy tissue (1 kPa), promoted higher levels of intracellular collagen I and Lox expression in activated HSCs, compared to stiff microenvironments mimicking fibrotic tissue (25 kPa). Collectively, these data suggest potential HSC functional adaptations in response to specific bio-chemo-mechanical changes relevant towards the development of therapeutic interventions. These findings also underscore the importance of the microenvironment when interrogating HSC behavior in healthy, disease, and treatment settings. STATEMENT OF SIGNIFICANCE: In this work we utilized high-throughput cellular microarray technology to systematically interrogate the complex interactions between HSCs and their microenvironment in the context of liver fibrosis. We observed that HSC phenotype is regulated by ECM composition and stiffness, and that these phenotypes can be classified into distinct clusters based on their microenvironmental context. Moreover, the range of these phenotypic responses to microenvironmental stimuli is substantial and a direct consequence of the combinatorial pairing of ECM protein and stiffness signals. We also observed a novel role for microenvironmental context in affecting HSC responses to potential fibrosis therapeutics.

A Mixed-Methods Study of the Impact of Mild Cognitive Impairment Diagnosis on Patient and Care Partner Perception of Health Risks.

BACKGROUND: Older patients (≥65 years) with mild cognitive impairment (MCI) are undertreated for cardiovascular disease (CVD). One reason for this disparity could be that patients with MCI might underestimate the chances of CVD and overestimate dementia. OBJECTIVE: To compare conceptions of health risk between older patients with MCI and normal cognition (NC) and their care partners. METHODS: We conducted a multi-center mixed-methods study of patient-care partner dyads completing written quantitative surveys (73% response rate; 127 dyads: 66 MCI and 61 NC) or semi-structured interviews (20 dyads: 11 MCI, and 9 NC). Surveys assessed two-year patient risks of dementia, heart attack, stroke, and fall. Interviews assessed similar health risks and reasons for risk perceptions. RESULTS: On surveys, a similarly low proportion of MCI and NC patients felt they were at risk of stroke (5% versus 2%; p = 0.62) and heart attack (2% versus 0%; p = 0.99). More MCI than NC patients perceived dementia risk (26% versus 2%; p < 0.001). Care partners' survey findings were similar. Interviews generally confirmed these patterns and also identified reasons for future health concerns. For both MCI and NC dyads, personal experience with cognitive decline or CVD (personal or family history) increased concerns about each disease. Additionally, perceptions of irreversibility and lack of treatment for cognitive decline increased concern about dementia. CONCLUSION: Less use of CVD treatments in MCI seems unlikely to be driven by differential perceptions of CVD risk. Future work to improve awareness of CVD risks in older patients and dementia risk in patients with MCI are warranted.

From Self-Esteem to Selflessness: An Evidence (Gap) Map of Self-Related Processes as Mechanisms of Mindfulness-Based Interventions.

Self-related processes (SRPs) have been theorized as key mechanisms of mindfulness-based interventions (MBIs), but the evidence supporting these theories is currently unclear. This evidence map introduces a comprehensive framework for different types of SRPs, and how they are theorized to function as mechanisms of MBIs (target identification). The evidence map then assesses SRP target engagement by mindfulness training and the relationship between target engagement and outcomes (target validation). Discussion of the measurement of SRPs is also included. The most common SRPs measured and engaged by standard MBIs represented valenced evaluations of self-concept, including rumination, self-compassion, self-efficacy, and self-esteem. Rumination showed the strongest evidence as a mechanism for depression, with other physical and mental health outcomes also supported. Self-compassion showed consistent target engagement but was inconsistently related to improved outcomes. Decentering and interoception are emerging potential mechanisms, but their construct validity and different subcomponents are still in development. While some embodied self-specifying processes are being measured in cross-sectional and meditation induction studies, very few have been assessed in MBIs. The SRPs with the strongest mechanistic support represent positive and negative evaluations of self-concept. In sum, few SRPs have been measured in MBIs, and additional research using well-validated measures is needed to clarify their role as mechanisms.

Emotion-related constructs engaged by mindfulness-based interventions: A systematic review and meta-analysis.

OBJECTIVES: Mindfulness-based interventions (MBIs) have been widely implemented to improve self-regulation behaviors, often by targeting emotion-related constructs to facilitate change. Yet the degree to which MBIs engage specific measures of emotion-related constructs has not been systematically examined. METHODS: Using advanced meta-analytic techniques, this review examines construct and measurement engagement in trials of adults that used standardized applications of the two most established MBIs: Mindfulness-Based Stress Reduction (MBSR) and Mindfulness-Based Cognitive Therapy (MBCT), or modified variations of these interventions that met defined criteria. RESULTS: Seventy-two studies (N=7,378) were included (MBSR k=47, MBCT k = 21, Modified k=4). MBIs led to significant improvement in emotion-related processing overall, compared to inactive controls (d=0.58; k =36), and in all constructs assessed: depression (d=0.66; k=26), anxiety (d =0.63; k=19), combined mental health (d =0.75; k=7 ) and stress (d =0.44; k=11). Reactions to pain, mood states, emotion regulation, and biological measures lacked sufficient data for analysis. MBIs did not outperform active controls in any analyses. Measurement tool and population-type did not moderate results, but MBI-type did, in that MBCT showed stronger effects than MBSR, although these effects were driven by a small number of studies. CONCLUSIONS: This review is the first to examine the full scope of emotion-related measures relevant to self-regulation, to determine which measures are most influenced by MBCT/MBSR. Compared to extant reviews, which typically focused on MBI outcomes, this work examined mechanistic processes based on measurement domains and tools. While effect sizes were similar among measurement tools, this review also includes a descriptive evaluation of measures and points of caution, providing guidance to MBI researchers and clinicians for selection of emotion-related measurement tools.

The Use of Music in the Treatment and Management of Serious Mental Illness: A Global Scoping Review of the Literature.

Mental and substance use disorders have been identified as the leading cause of global disability, and the global burden of mental illness is concentrated among those experiencing disability due to serious mental illness (SMI). Music has been studied as a support for SMIs for decades, with promising results; however, a lack of synthesized evidence has precluded increased uptake of and access to music-based approaches. The purpose of this scoping review was to identify the types and quantity of research at intersections of music and SMIs, document evidentiary gaps and opportunities, and generate recommendations for improving research and practice. Studies were included if they reported on music's utilization in treating or mitigating symptoms related to five SMIs: schizophrenia, bipolar disorder, generalized anxiety disorder, major depressive disorder, or post-traumatic stress disorder. Eight databases were searched; screening resulted in 349 included studies for data extraction. Schizophrenia was the most studied SMI, with bipolar disorder studied the least. Demographics, settings, and activity details were found to be inconsistently and insufficiently reported; however, listening to recorded music emerged as the most common musical activity, and activity details appeared to have been affected by the conditions under study. RCTs were the predominant study design, and 271 unique measures were utilized across 289 primary studies. Over two-thirds of primary studies (68.5%) reported positive results, with 2.8% reporting worse results than the comparator, and 12% producing indeterminate results. A key finding is that evidence synthesis is precluded by insufficient reporting, widely varied outcomes and measures, and intervention complexity; as a result, widespread changes are necessary to reduce heterogeneity (as feasible), increase replicability and transferability, and improve understandings of mechanisms and causal pathways. To that end, five detailed recommendations are offered to support the sharing and development of information across disciplines.

Outcome choice and definition in systematic reviews leads to few eligible studies included in meta-analyses: a case study.

BACKGROUND: There is broad recognition of the importance of evidence in informing clinical decisions. When information from all studies included in a systematic review ("review") does not contribute to a meta-analysis, decision-makers can be frustrated. Our objectives were to use the field of eyes and vision as a case study and examine the extent to which authors of Cochrane reviews conducted meta-analyses for their review's pre-specified main outcome domain and the reasons that some otherwise eligible studies were not incorporated into meta-analyses. METHODS: We examined all completed systematic reviews published by Cochrane Eyes and Vision, as of August 11, 2017. We extracted information about each review's outcomes and, using an algorithm, categorized one outcome as its "main" outcome. We calculated the percentage of included studies incorporated into meta-analyses for any outcome and for the main outcome. We examined reasons for non-inclusion of studies into the meta-analysis for the main outcome. RESULTS: We identified 175 completed reviews, of which 125 reviews included two or more studies. Across these 125 reviews, the median proportions of studies incorporated into at least one meta-analysis for any outcome and for the main outcome were 74% (interquartile range [IQR] 0-100%) and 28% (IQR 0-71%), respectively. Fifty-one reviews (41%) could not conduct a meta-analysis for the main outcome, mostly because fewer than two included studies measured the outcome (21/51 reviews) or the specific measurements for the outcome were inconsistent (16/51 reviews). CONCLUSIONS: Outcome choice during systematic reviews can lead to few eligible studies included in meta-analyses. Core outcome sets and improved reporting of outcomes can help solve some of these problems.

Psychometric Properties of Functional, Ambulatory, and Quality of Life Instruments in Lower Limb Amputees: A Systematic Review.

OBJECTIVE: Summarize the psychometric properties of functional, ambulatory, and quality of life instruments among adult lower limb amputees, highlighting evidence deemed generalizable to the United States Medicare population. DATA SOURCES: Six databases and existing systematic reviews through October 30, 2017. Searches included terms for lower limb amputation or prostheses and outcome measures in humans, without language restriction. STUDY SELECTION: We included peer-reviewed studies of at least 20 adults (≥18 years) with lower limb amputation. Eligible studies reported on psychometric properties of functional, ambulatory, or quality of life instruments. Fifty-three of 425 retrieved articles (12%) met criteria. DATA EXTRACTION: Study characteristics and psychometric property data (validity, reliability, responsiveness, minimum detectable change, minimal important difference, or floor or ceiling effect) were extracted into a customized form based on standardized criteria. All extracted data were confirmed by 2 experts in systematic review and rehabilitation outcome measurement. Instruments were categorized regarding having been validated and found reliable. Other reported psychometric properties were recorded. Studies were also assessed for applicability to the Medicare population based on age and amputation etiology (dysvascular). DATA SYNTHESIS: Fifty-six studies (in 53 articles) reported psychometric properties of 50 instruments. There is evidence for both validity and reliability for 30 instruments, 17 of which have evidence that was deemed generalizable to the Medicare population. Most of the remaining instruments have evidence of either validity or reliability, but not both. Twelve instruments have been assessed specifically among lower limb amputees prior to prosthesis prescription. Thirteen instruments have been assessed regarding their predictive properties for future outcomes. CONCLUSIONS: Numerous instruments assessing ambulation, function, quality of life, and other patient-centered outcomes have evidence of validity and reliability for adults with lower limb amputations. Researchers and clinicians should use validated, reliable instruments when feasible. Many existing and new instruments require validation for use with lower limb amputees.

Pharmacologic and Nonpharmacologic Treatments for Urinary Incontinence in Women: A Systematic Review and Network Meta-analysis of Clinical Outcomes.

Background: Urinary incontinence (UI), a common malady in women, most often is classified as stress, urgency, or mixed. Purpose: To compare the effectiveness of pharmacologic and nonpharmacologic interventions to improve or cure stress, urgency, or mixed UI in nonpregnant women. Data Sources: MEDLINE, Cochrane Central Register of Controlled Trials (Wiley), Cochrane Database of Systematic Reviews (Wiley), EMBASE (Elsevier), CINAHL (EBSCO), and PsycINFO (American Psychological Association) from inception through 10 August 2018. Study Selection: 84 randomized trials that evaluated 14 categories of interventions and reported categorical cure or improvement outcomes. Data Extraction: 1 researcher extracted study characteristics, results, and study-level risk of bias, with verification by another independent researcher. The research team collaborated to assess strength of evidence (SoE) across studies. Data Synthesis: 84 studies reported cure or improvement outcomes (32 in stress UI, 16 in urgency UI, 4 in mixed UI, and 32 in any or unspecified UI type). The most commonly evaluated active intervention types included behavioral therapies, anticholinergics, and neuromodulation. Network meta-analysis showed that all interventions, except hormones and periurethral bulking agents (variable SoE), were more effective than no treatment in achieving at least 1 favorable UI outcome. Among treatments used specifically for stress UI, behavioral therapy was more effective than either α-agonists or hormones in achieving cure or improvement (moderate SoE); α-agonists were more effective than hormones in achieving improvement (moderate SoE); and neuromodulation was more effective than no treatment for cure, improvement, and satisfaction (high SoE). Among treatments used specifically for urgency UI, behavioral therapy was statistically significantly more effective than anticholinergics in achieving cure or improvement (high SoE), both neuromodulation and onabotulinum toxin A (BTX) were more effective than no treatment (high SoE), and BTX may have been more effective than neuromodulation in achieving cure (low SoE). Limitation: Scarce direct (head-to-head trial) evidence and population heterogeneity based on UI type, UI severity, and history of prior treatment. Conclusion: Most nonpharmacologic and pharmacologic interventions are more likely than no treatment to improve UI outcomes. Behavioral therapy, alone or in combination with other interventions, is generally more effective than pharmacologic therapies alone in treating both stress and urgency UI. Primary Funding Source: Patient-Centered Outcomes Research Institute. (PROSPERO: CRD42017069903).

Real-Time Emergency Department Electronic Notifications Regarding High-Risk Patients: A Systematic Review.

Background: To systematically review evidence on the feasibility and efficacy of real-time electronic notifications about patients at high risk of emergency department (ED) recidivism. Methods: Eight electronic databases were searched for empirical studies of real-time ED-based electronic tools, identifying adult patients at high risk of frequent utilization. Study selection and data extraction were performed independently by two reviewers. Qualitative data synthesis and assessment of strength of evidence were conducted through consensus discussion. Results: Of 2,256 records found through the search, 210 were duplicates, 2,004 were excluded based on abstract review, and 31 were excluded after full text review. The final sample consisted of 10 studies described in 11 articles describing the effect of real-time ED-based electronic notifications for high-risk patients. Three were randomized controlled trials (RCTs). All notifications were based on prespecified markers of risk. Seven studies integrated complex care plans into the electronic health record. Effect on ED use and length of stay (LOS) was mixed: nine studies reported decreased ED use, although results were statistically significant in only three studies; for LOS, one study reported a statistically significant reduction. Impact on cost and financial metrics was promising, with three (of three studies reporting this metric) showing improved organizational financial metrics. Three RCTs reported a reduction in opioid prescriptions. Conclusions: Real-time electronic notifications of ED providers regarding patients at high risk of ED recidivism are feasible. They may help reduce resource utilization and costs. Large knowledge gaps remain regarding patient- and provider-centered outcomes.