Advancing a MEMS-Based 3D Cell Culture System for in vitro Neuro-Electrophysiological Recordings

In this work we present advances in three dimensional (3D) neuronal cell culture systems based on a reversible assembly of a microbioreactor with a microelectrode array (MEA) to create a MEMS-based 3D cell culture system for in vitro neuro-electrophysiological recordings. A batch of six molds were milled in poly (methyl methacrylate). The molds were used for soft lithography of polydimethylsiloxane (PDMS). In the center of the PDMS shape, a porous polyethersulfone (PES) cylindrical tube was press-fitted to form a growth barrier between the culture chamber inside the PES tube and the microfluidic channel surrounding the PES tube. A thin layer of partially cured PDMS was used to seal the bottom of the microbioreactor and provide reversible adhesion with the glass surface of a MEA. SH-SY5Y cells were successfully differentiated inside the microbioreactors in Matrigel and demonstrated extended neuronal networks over a height of at least 184 micrometers within the system. In previous microbioreactor designs visibility was limited due to the closed top with the dispensing holes. The new open top design allows for a better evaluation of the cell culture by optical detection methods during the experiment. Electrophysiological activity was recorded within the microbioreactor using human induced pluripotent stem cell-derived cortical neurons cultured in Matrigel, in 3D, up until 21 days in vitro. In summary, we present advances made in the design, the fabrication process and integration of microbioreactors with MEAs. Optical imaging capabilities improved significantly with an open top and the culture time was further extended from 7 to 21 DIV without leakage or degradation thanks to introducing PES as a barrier material and an enhanced assembly procedure. The latter facilitated a sufficient long-term culture for neurons to mature in an environment free from flow-induced stress and provided a proof of principle for the recording of electrophysiological activity of cortical neurons cultured in 3D

Long-term exposure to ultrafine particles and incidence of cardiovascular and cerebrovascular disease in the EPIC-NL cohort

Background: There is a small but growing evidence base that exposure to ultrafine particles (UFP – particles smaller than 100nm) may play an important role in the etiology of several illnesses, including cardiovascular disease (CVD). However, this has been under-explored in population-level studies. Methods: Using Cox proportional hazard models we studied the association between long-term exposure to UFP (predicted via recently developed land use regression models) and incident cardiovascular disease in the Dutch arm of the European Prospective Investigation into Cancer cohort (EPIC-NL), which contains 33,831 Dutch residents. Hazard ratios (HR) for UFP were compared to HRs for more routinely monitored air pollutants, including PM$_{10}$, PM$_{coarse}$, PM$_{2.5}$, PM$_{2.5}$ absorbance, NO$_{x}$, and NO$_{2}$. Joint-pollutant effects were also evaluated in two-pollutant models. Results: Long-term exposure to UFP was associated with increased HRs for all incident cardiovascular disease (HR = 1.18 per 10,000 particles/cm$_{3}$, 95% CI: 1.03, 1.34), myocardial infarction (HR = 1.34, 95% CI: 1.00, 1.79), and heart failure (HR = 1.76, 95% CI: 1.17, 2.66). Positive associations were also observed for NO2 (HR for heart failure = 1.22, 95% CI: 1.01, 1.48 per 20 μg/m$^{3}$) and coarse PM (HR for all CVD = 1.21, 95% CI: 1.01, 1.45 per 10 μg/m$^{3}$). CVD was not positively associated with PM$_{2.5}$ (HR for all CVD = 0.95, 95% CI: 0.75, 1.28 per 5 μg/m$^{3}$). HRs for UFP and cerebrovascular diseases were positive, but not significant. In two-pollutant models (UFP + NO$_{2}$ and UFP + PM$_{coarse}$), positive associations tended to remain for UFP, while HRs for PM$_{coarse}$ and NO$_{2}$ generally attenuated towards the null. Conclusions: These findings strengthen the overall evidence that UFP exposure plays an important role in cardiovascular health and that risks of ambient air pollution, based on conventional air pollution metrics, may underestimate the true population risk.ment data and biological responses as viability (AlamarBlue assay), cytotoxicity (LDH release), and release of cytokines during long-term exposure are reported

RENAL REPLACEMENT THERAPY IN A POLYTRAUMATIZED PATIENT WITH HEMOPHILIA

Zatajenje bubrežne funkcije je rijetka pojava u bolesnika s nasljednim koagulacijskim poremećajima. Međutim, kada nastupi veoma brzo napreduje do završnog stadija bubrežne bolesti i potrebe za nadomještanjem bubrežne funkcije. Javljaju se problemi vezani uz odabir metode dijalize, periproceduralne nadoknade nedostatnog faktora koagulacije te heparinizacije dijaliznog sustava. Kod hemoiličara uvijek treba biti oprezan tijekom samog postupka dijalize zbog mogućeg razvoja komplikacija koje ih mogu vitalno ugroziti. Ovo je prikaz slučaja teško politraumatiziranog bolesnika koji boluje od hemofilije A. Tijekom intenzivnog liječenja razvio je akutno bubrežno zatajenje te tešku sepsu. S obzirom na okolnosti najbolja metoda izbora za njega je bila kontinuirana veno-venska hemodijaliza. Unato uspješno provedenoj dijalizi bez komplikacija bolesnik umire od protrahirane sepseRenal failure is a rare complication of hereditary coagulopathies. However, when it occurs, it rapidly progresses to a stage that requires replacement of renal function. Major problems include the choice of dialysis method, prevention of complications and supplementation of deicient factor. In hemodialysis, it is challenging to prevent system clotting and avoid bleeding. We present a case of polytraumatized male patient with hemophilia A, who developed compartment syndrome with acute renal failure. Continuous venovenous hemodialysis (CVVHD) improved his condition and he recovered his kidney function. However, over the next few days he developed severe sepsis with deterioration of renal function. CVVHDF (hemodiailtration) was restarted. Several large hematomas were found in the abdominal cavity and in the inguinal region, one of them inducing compartment syndrome with leg necrosis. The patient died from cardiorespiratory arrest

Variability of the Human Serum Metabolome over 3 Months in the EXPOsOMICS Personal Exposure Monitoring Study

Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and untargeted metabolomics are increasingly used in exposome studies to study the interactions between nongenetic factors and the blood metabolome. To reliably and efficiently link detected compounds to exposures and health phenotypes in such studies, it is important to understand the variability in metabolome measures. We assessed the within- and between-subject variability of untargeted LC-HRMS measurements in 298 nonfasting human serum samples collected on two occasions from 157 subjects. Samples were collected ca. 107 (IQR: 34) days apart as part of the multicenter EXPOsOMICS Personal Exposure Monitoring study. In total, 4294 metabolic features were detected, and 184 unique compounds could be identified with high confidence. The median intraclass correlation coefficient (ICC) across all metabolic features was 0.51 (IQR: 0.29) and 0.64 (IQR: 0.25) for the 184 uniquely identified compounds. For this group, the median ICC marginally changed (0.63) when we included common confounders (age, sex, and body mass index) in the regression model. When grouping compounds by compound class, the ICC was largest among glycerophospholipids (median ICC 0.70) and steroids (0.67), and lowest for amino acids (0.61) and the O-acylcarnitine class (0.44). ICCs varied substantially within chemical classes. Our results suggest that the metabolome as measured with untargeted LC-HRMS is fairly stable (ICC > 0.5) over 100 days for more than half of the features monitored in our study, to reflect average levels across this time period. Variance across the metabolome will result in differential measurement error across the metabolome, which needs to be considered in the interpretation of metabolome results

Land Use Regression Models for Ultrafine Particles in Six European Areas

Long-term ultrafine particle (UFP) exposure estimates at a fine spatial scale are needed for epidemiological studies. Land use regression (LUR) models were developed and evaluated for six European areas based on repeated 30 min monitoring following standardized protocols. In each area; Basel (Switzerland), Heraklion (Greece), Amsterdam, Maastricht, and Utrecht ("The Netherlands"), Norwich (United Kingdom), Sabadell (Spain), and Turin (Italy), 160-240 sites were monitored to develop LUR models by supervised stepwise selection of GIS predictors. For each area and all areas combined, 10 models were developed in stratified random selections of 90% of sites. UFP prediction robustness was evaluated with the intraclass correlation coefficient (ICC) at 31-50 external sites per area. Models from Basel and The Netherlands were validated against repeated 24 h outdoor measurements. Structure and model R2 of local models were similar within, but varied between areas (e.g., 38-43% Turin; 25-31% Sabadell). Robustness of predictions within areas was high (ICC 0.73-0.98). External validation R2 was 53% in Basel and 50% in The Netherlands. Combined area models were robust (ICC 0.93-1.00) and explained UFP variation almost equally well as local models. In conclusion, robust UFP LUR models could be developed on short-term monitoring, explaining around 50% of spatial variance in longer-term measurements

Advancing a MEMS-Based 3D cell culture system for in vitro neuro-electrophysiological recordings

In this work we present advances in three dimensional (3D) neuronal cell culture systems based on a reversible assembly of a microbioreactor with a microelectrode array (MEA) to create a MEMS-based 3D cell culture system for in vitro neuro-electrophysiological recordings. A batch of six molds were milled in poly (methyl methacrylate). The molds were used for soft lithography of polydimethylsiloxane (PDMS). In the center of the PDMS shape, a porous polyethersulfone (PES) cylindrical tube was press-fitted to form a growth barrier between the culture chamber inside the PES tube and the microfluidic channel surrounding the PES tube. A thin layer of partially cured PDMS was used to seal the bottom of the microbioreactor and provide reversible adhesion with the glass surface of a MEA. SH-SY5Y cells were successfully differentiated inside the microbioreactors in Matrigel and demonstrated extended neuronal networks over a height of at least 184 micrometers within the system. In previous microbioreactor designs visibility was limited due to the closed top with the dispensing holes. The new open top design allows for a better evaluation of the cell culture by optical detection methods during the experiment. Electrophysiological activity was recorded within the microbioreactor using human induced pluripotent stem cell-derived cortical neurons cultured in Matrigel, in 3D, up until 21 days in vitro. In summary, we present advances made in the design, the fabrication process and integration of microbioreactors with MEAs. Optical imaging capabilities improved significantly with an open top and the culture time was further extended from 7 to 21 DIV without leakage or degradation thanks to introducing PES as a barrier material and an enhanced assembly procedure. The latter facilitated a sufficient long-term culture for neurons to mature in an environment free from flow-induced stress and provided a proof of principle for the recording of electrophysiological activity of cortical neurons cultured in 3D

Spatial Variation of Endotoxin Concentrations Measured in Ambient PM10 in a Livestock-Dense Area: Implementation of a Land-Use Regression Approach

BACKGROUND: Results from studies on residential health effects of livestock farming are inconsistent, potentially due to simple exposure proxies used (e.g., livestock density). Accuracy of these proxies compared with measured exposure concentrations is unknown. OBJECTIVES: We aimed to assess spatial variation of endotoxin in PM10 (particulate matter ≤10μm) at residential level in a livestock-dense area, compare simple livestock exposure proxies to measured endotoxin concentrations, and evaluate whether land-use regression (LUR) can be used to explain spatial variation of endotoxin. METHODS: The study area (3,000 km2) was located in Netherlands. Ambient PM10 was collected at 61 residential sites representing a variety of surrounding livestock-related characteristics. Three to four 2-wk averaged samples were collected at each site. A local reference site was used for temporal variation adjustment. Samples were analyzed for PM10 mass by weighing and for endotoxin by using the limulus amebocyte lysate assay. Three LUR models were developed, first a model based on general livestock-related GIS predictors only, followed by models that also considered species-specific predictors and farm type-specific predictors. RESULTS: Variation in concentrations measured between sites was substantial for endotoxin and more limited for PM10 (coefficient of variation: 43%, 8%, respectively); spatial patterns differed considerably. Simple exposure proxies were associated with endotoxin concentrations although spatial variation explained was modest (R2<26%). LUR models using a combination of animal-specific livestock-related characteristics performed markedly better, with up to 64% explained spatial variation. CONCLUSION: The considerable spatial variation of ambient endotoxin concentrations measured in a livestock-dense area can largely be explained by LUR modeling based on livestock-related characteristics. Application of endotoxin LUR models seems promising for residential exposure estimation within health studies. https://doi.org/10.1289/EHP2252

Long-Term Exposure to Ultrafine Particles and Incidence of Cardiovascular and Cerebrovascular Disease in a Prospective Study of a Dutch Cohort

BACKGROUND: There is growing evidence that exposure to ultrafine particles (UFP; particles smaller than [Formula: see text]) may play an underexplored role in the etiology of several illnesses, including cardiovascular disease (CVD). OBJECTIVES: We aimed o investigate the relationship between long-term exposure to ambient UFP and incident cardiovascular and cerebrovascular disease (CVA). As a secondary objective, we sought to compare effect estimates for UFP with those derived for other air pollutants, including estimates from two-pollutant models. METHODS: Using a prospective cohort of 33,831 Dutch residents, we studied the association between long-term exposure to UFP (predicted via land use regression) and incident disease using Cox proportional hazard models. Hazard ratios (HR) for UFP were compared to HRs for more routinely monitored air pollutants, including particulate matter with aerodynamic diameter [Formula: see text] ([Formula: see text]), PM with aerodynamic diameter [Formula: see text] ([Formula: see text]), and [Formula: see text]. RESULTS: Long-term UFP exposure was associated with an increased risk for all incident CVD [[Formula: see text] per [Formula: see text]; 95% confidence interval (CI): 1.03, 1.34], myocardial infarction (MI) ([Formula: see text]; 95% CI: 1.00, 1.79), and heart failure ([Formula: see text]; 95% CI: 1.17, 2.66). Positive associations were also estimated for [Formula: see text] ([Formula: see text]; 95% CI: 1.01, 1.48 per [Formula: see text]) and coarse PM ([Formula: see text]; HR for all [Formula: see text]; 95% CI: 1.01, 1.45 per [Formula: see text]). CVD was not positively associated with [Formula: see text] (HR for all [Formula: see text]; 95% CI: 0.75, 1.28 per [Formula: see text]). HRs for UFP and CVAs were positive, but not significant. In two-pollutant models ([Formula: see text] and [Formula: see text]), positive associations tended to remain for UFP, while HRs for [Formula: see text] and [Formula: see text] generally attenuated towards the null. CONCLUSIONS: These findings strengthen the evidence that UFP exposure plays an important role in cardiovascular health and that risks of ambient air pollution may have been underestimated based on conventional air pollution metrics. https://doi.org/10.1289/EHP3047

Long-Term Exposure to Ultrafine Particles and Incidence of Cardiovascular and Cerebrovascular Disease in a Prospective Study of a Dutch Cohort.

There is growing evidence that exposure to ultrafine particles (UFP; particles smaller than [Formula: see text]) may play an underexplored role in the etiology of several illnesses, including cardiovascular disease (CVD). We aimed o investigate the relationship between long-term exposure to ambient UFP and incident cardiovascular and cerebrovascular disease (CVA). As a secondary objective, we sought to compare effect estimates for UFP with those derived for other air pollutants, including estimates from two-pollutant models. Using a prospective cohort of 33,831 Dutch residents, we studied the association between long-term exposure to UFP (predicted via land use regression) and incident disease using Cox proportional hazard models. Hazard ratios (HR) for UFP were compared to HRs for more routinely monitored air pollutants, including particulate matter with aerodynamic diameter [Formula: see text] ([Formula: see text]), PM with aerodynamic diameter [Formula: see text] ([Formula: see text]), and [Formula: see text]. Long-term UFP exposure was associated with an increased risk for all incident CVD [[Formula: see text] per [Formula: see text]; 95% confidence interval (CI): 1.03, 1.34], myocardial infarction (MI) ([Formula: see text]; 95% CI: 1.00, 1.79), and heart failure ([Formula: see text]; 95% CI: 1.17, 2.66). Positive associations were also estimated for [Formula: see text] ([Formula: see text]; 95% CI: 1.01, 1.48 per [Formula: see text]) and coarse PM ([Formula: see text]; HR for all [Formula: see text]; 95% CI: 1.01, 1.45 per [Formula: see text]). CVD was not positively associated with [Formula: see text] (HR for all [Formula: see text]; 95% CI: 0.75, 1.28 per [Formula: see text]). HRs for UFP and CVAs were positive, but not significant. In two-pollutant models ([Formula: see text] and [Formula: see text]), positive associations tended to remain for UFP, while HRs for [Formula: see text] and [Formula: see text] generally attenuated towards the null

Short-term personal and outdoor exposure to ultrafine and fine particulate air pollution in association with blood pressure and lung function in healthy adults

Studies reporting on associations between short-term exposure to outdoor fine (PM2.5), and ultrafine particles (UFP) and blood pressure and lung function have been inconsistent. Few studies have characterized exposure by personal monitoring, which especially for UFP may have resulted in substantial exposure measurement error. We investigated the association between 24-h average personal UFP, PM2.5, and soot exposure and dose and the health parameters blood pressure and lung function. We further assessed the short-term associations between outdoor concentrations measured at a central monitoring site and near the residences and these health outcomes. We performed three 24-h personal exposure measurements for UFP, PM2.5, and soot in 132 healthy adults from Basel (Switzerland), Amsterdam and Utrecht (the Netherlands), and Turin (Italy). Monitoring of each subject was conducted in different seasons in a one-year study period. Subject's activity levels and associated ventilation rates were measured using actigraphy to calculate the inhaled dose. After each 24-h monitoring session, blood pressure and lung function were measured. Contemporaneously with personal measurements, UFP, PM2.5 and soot were measured outdoor at the subject's residential address and at a central site in the research area. Associations between short-term personal and outdoor exposure and dose to UFP, PM2.5, and soot and health outcomes were tested using linear mixed effect models. The 24-h mean personal, residential and central site outdoor UFP exposures were not associated with blood pressure or lung function. UFP mean exposures in the 2-h prior to the health test was also not associated with blood pressure and lung function. Personal, central site and residential PM2.5 exposure were positively associated with systolic blood pressure (about 1.4 mmHg increase per Interquartile range). Personal soot exposure and dose were positively associated with diastolic blood pressure (1.2 and 0.9 mmHg increase per Interquartile range). No consistent associations between PM2.5 or soot exposure and lung function were observed. Short-term personal, residential outdoor or central site exposure to UFP was not associated with blood pressure or lung function. Short-term personal PM2.5 and soot exposures were associated with blood pressure, but not lung function