Integrated Cell Culture Research Articles

Persistence of human Aichi virus infectivity from raw surface water to drinking water.

Human Aichi virus 1 (AiV-1) is a water- and food-borne infection-associated picornavirus that causes gastroenteritis in humans. Recent studies on environmental waters showed a high frequency and abundance of AiV-1, suggesting that it might be an appropriate indicator of fecal contamination. We screened 450 surface and drinking water samples from a Tunisian drinking water treatment plant (DWTP) and the Sidi Salem dam for AiV-1 by real time reverse transcriptase PCR (RT-qPCR). The persistence of infectious particles was evaluated using an integrated cell culture approach coupled with quantitative molecular detection (ICC-RT-qPCR). In all, 85 (18.9%) water samples were positive for AiV-1 with viral loads ranging from 0.47 to 11.62 log10 cp/L and a median of 4.97 log10 cp/L, including 30/100 raw, 18/50 decanted, 14/50 flocculated, 9/100 treated, 1/50 tap, and 13/100 surface water samples. Of these, 15 (17.6%) samples contained infectious AiV-1 genotype A particles, including five raw, four decanted, one surface, three flocculated, and two treated water samples. Our data suggest that the persistence of infectious AiV-1 particles in environmental waters might represent a potential threat to public health. This study also indicates that the ICC-RT-qPCR is a practical tool for monitoring human waterborne viral risk in aquatic environments.IMPORTANCEHuman Aichi virus 1 (AiV-1) is a water- and food-borne infection-associated picornavirus that causes gastroenteritis in humans. Its high frequency and abundance in environmental waters would suggest that it might be an appropriate indicator of fecal contamination. The analysis of surface and drinking water samples from a Tunisian drinking water treatment plant (DWTP) and the Sidi Salem dam using an integrated cell culture approach coupled with a quantitative molecular detection (ICC-RT-qPCR) confirmed the persistence of infectious AiV-1 particles in samples at all stages of the treatment process, except in tap water. This suggests that the persistence of AiV-1 infectivity in environmental waters might represent a potential threat to public health. This study also indicates that the ICC-RT-qPCR is a practical tool for monitoring human waterborne viral risk in aquatic environments.

Molecular genetic methods for quality control of inactivated vaccines using a Chikungunya virus model: vaccine strain identification and completeness of virus inactivation

INTRODUCTION. The completeness of virus inactivation and the identity of the vaccine strain are essential parameters for the safety and quality of inactivated virus vaccines, which should be controlled during vaccine development and production. Currently, the most promising quality control methods for inactivated virus vaccines are molecular genetic methods, which provide rapid results with high sensitivity and specificity.AIM. The aim of this study was the development of a real-time quantitative polymerase chain reaction (qPCR) method and an integrated cell culture real-time quantitative polymerase chain reaction (ICC-qPCR) method to assess the completeness of virus inactivation, as well as a reverse-transcription polymerase chain reaction assay coupled with restriction fragment length polymorphism analysis (RT-PCR-RFLP) to confirm the identity of the vaccine virus strain.MATERIALS AND METHODS. This study used RNA of CHIKV genotypes (three strains of each of the four CHIKV genotypes, including Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL), which were identified by sequencing prior to analysis). Additionally, the study used the Nika21 CHIKV strain (ECSA genotype), the Nika21 CHIKV strain inactivated with β-propiolactone, and the Nika21 CHIKV strain antigen adsorbed on aluminium hydroxide. The methods used included real-time qPCR, RT-PCR-RFLP, and virus neutralisation.RESULTS. The study identified a 218 bp fragment of the nsP1 gene (positions 789 to 1006) with restriction endonuclease recognition sites. These sites were present or absent in combinations specific to each of the four CHIKV genotypes. The authors selected primers for amplification of the specified gene region and tested the conditions for real-time qPCR and RT-PCR-RFLP. The study demonstrated the possibility of using the ICC-qPCR method to confirm the completeness of virus inactivation and the RT-PCR-RFLP method to identify the vaccine strain.CONCLUSIONS. The study showed the advantages of using the ICC-qPCR method to confirm the completeness of antigen inactivation and the RT-PCR-RFLP method to identify the vaccine strain. These methods are more sensitive and faster than traditional culture methods. ICC-qPCR and RT-PCR-RFLP can be used at any stage of the production process for inactivated vaccines.

A biomimetic skin microtissue biosensor for the detection of fish parvalbumin

In this paper, a biomimetic skin microtissue biosensor was developed based on three-dimensional (3D) bioprinting to precisely and accurately determine fish parvalbumin (FV). Based on the principle that allergens stimulate cells to produce ONOO− (peroxynitrite anion), a screen-printed electrode for the detection nanomolar level ONOO− was innovatively prepared to indirectly detect FV based on the level of ONOO− release. Gelatin methacryloyl (GelMA), RBL-2H3 cells, and MS1 cells were used as bio-ink for 3D bioprinting. The high-throughput and standardized preparation of skin microtissue was achieved using stereolithography 3D bioprinting technology. The printed skin microtissues were put into the self-designed 3D platform that integrated cell culture and electrochemical detection. The experimental results showed that the sensor could effectively detect FV when the optimized ratio of RBL-2H3 to MS1 cells and allergen stimulation time were 2:8 and 2 h, respectively. The linear detection range was 0.125–3.0 μg/mL, and the calculated lowest detection limit was 0.122 μg/mL. In addition, the sensor had excellent selectivity, specificity, stability, and reliability. Thus, this study successfully constructed a biomimetic skin microtissue electrochemical sensor for PV detection.

Relative abundance and the fate of human rotavirus in wastewater during treatment processes: identification of potential infectious rotavirus in the final effluents and receiving aquatic milieu in Durban area, South Africa

The occurrence and persistence of rotaviruses in raw and treated wastewater and their discharge into rivers represent a significant health risk for humans and animals, worldwide. In this study, samples were collected monthly from each of the four Durban wastewater treatment plants (DWWTPs) and receiving rivers for a period of 3 months. Rotavirus was quantified by real-time quantitative PCR (RT-qPCR), and viability was assessed using integrated cell culture (ICC)-qPCR. Rotavirus was detected consistently in 100% of influent wastewaters (mean concentration range, 4.36–4.46 log10 genome equivalent (GE) copies/L) and final effluent samples of three DWWTPs (range, 3.35–3.61 log10 GE copies/L). Overall, 94% (45/48) of the wastewater analyzed and 95% (20/21) of the associated river water samples were positive for rotavirus (range, 2.04–6.77 log10 GE copies/L). The activated sludge process with 0.10–0.43 log10 reduction values (LRV) only moderately reduced the viral loads. Similarly, one of the DWWTPs that operated the biofilter modality produced 0.20 LRV. Though the additional treatment with chlorine produced higher LRV (range, 0.31–0.53) than the corresponding activated sludge or biofilter process, the difference in viral removals was not significant (p > 0.05). The equivalent treatment efficiencies of the four DWWTPs varied from 19 to 43% decay in the population of rotavirus. Further, infectious rotavirus ranging from 66.67 to 100%, 50 to 100%, and 66.67 to 100% were detected in the post-activated sludge, final effluents, and river water samples, respectively. In conclusion, the findings of infectious rotavirus in both the final effluents and associated rivers represent an infection risk for humans or animals during contact. Thus, close monitoring for rotavirus and risk assessment studies under distinct exposure scenarios may further shed light on the health-related risks associated with water recovery and reuse in urban settings.

Electrofluidic control for textile-based cell culture: Identification of appropriate conditions required to integrate cell culture with electrofluidics.

Electric field-driven microfluidics, known as electrofluidics, is a novel attractive analytical tool when it is integrated with low-cost textile substrate. Textile-based electrofluidics, primarily explored on yarn substrates, is in its early stages, with few studies on 3D structures. Further, textile structures have rarely been used in cellular analysis as a low-cost alternative. Herein, we investigated novel 3D textile structures and develop optimal electrophoretic designs and conditions that are favourable for direct 3D cell culture integration, developing an integrated cell culture textile-based electrofluidic platform that was optimised to balance electrokinetic performance and cell viability requirements. Significantly, there were contrasting electrolyte compositional conditions that were required to satisfy cell viability and electrophoretic mobility requiring the development of and electrolyte that satisfied the minimum requirements of both these components within the one platform. Human dermal fibroblast cell cultures were successfully integrated with gelatine methacryloyl (GelMA) hydrogel-coated electrofluidic platform and studied under different electric fields using 5mM TRIS/HEPES/300mM glucose. Higher analyte mobility was observed on 2.5% GelMA-coated textile which also facilitated excellent cell attachment, viability and proliferation. Cell viability also increased by decreasing the magnitude and time duration of applied electric field with good cell viability at field of up to 20Vcm-1.

Deep Learning Powered Identification of Differentiated Early Mesoderm Cells from Pluripotent Stem Cells.

Pluripotent stem cells can be differentiated into all three germ-layers including ecto-, endo-, and mesoderm in vitro. However, the early identification and rapid characterization of each germ-layer in response to chemical and physical induction of differentiation is limited. This is a long-standing issue for rapid and high-throughput screening to determine lineage specification efficiency. Here, we present deep learning (DL) methodologies for predicting and classifying early mesoderm cells differentiated from embryoid bodies (EBs) based on cellular and nuclear morphologies. Using a transgenic murine embryonic stem cell (mESC) line, namely OGTR1, we validated the upregulation of mesodermal genes (Brachyury (T): DsRed) in cells derived from EBs for the deep learning model training. Cells were classified into mesodermal and non-mesodermal (representing endo- and ectoderm) classes using a convolutional neural network (CNN) model called InceptionV3 which achieved a very high classification accuracy of 97% for phase images and 90% for nuclei images. In addition, we also performed image segmentation using an Attention U-Net CNN and obtained a mean intersection over union of 61% and 69% for phase-contrast and nuclear images, respectively. This work highlights the potential of integrating cell culture, imaging technologies, and deep learning methodologies in identifying lineage specification, thus contributing to the advancements in regenerative medicine. Collectively, our trained deep learning models can predict the mesoderm cells with high accuracy based on cellular and nuclear morphologies.

Organ-on-Chip: The Advancing Microfluidic Technology For Theranostics

Organ-on-chip (OoC) technology represents a revolutionary advancement in in-vitro modeling by closely mimicking the physiological and structural characteristics of human organs. It offers a significant improvement over existing microfluidic technology by integrating cell culture within these systems. This integration allows OoCs to simulate organ-level functions on a miniaturized platform, where a microenvironment resembling that within tissue is created through the dynamic flow of fluids and interactions between different cell types. This has valuable applications in drug discovery, disease modeling, and personalized medicine. This review delves into the engineering principles behind OoC systems, with an emphasis on their operating principles, construction, key materials, and various theranostic applications, supplemented by case studies. Key examples, such as lung-on-chip, liver-on-chip, and multi-organ systems, are discussed to illustrate OoC’s potential in theranostics, particularly in drug testing and disease progression studies. While OoC models provide significant improvements over traditional in-vitro methods, their potential to replace preclinical animal testing is still under investigation. Despite its promise, OoC technology faces several challenges, including integration with sensors, reproducibility, scalability, long-term stability, and regulatory hurdles. This review also explores future trends and technological advancements needed for OoC technology to become a standard tool in biomedical research and clinical applications.

Ultrathin and Flexible Bioelectronic Arrays for Functional Measurement of iPSC-Cardiomyocytes under Cardiotropic Drug Administration and Controlled Microenvironments.

Emerging heart-on-a-chip technology is a promising tool to establish in vitro cardiac models for therapeutic testing and disease modeling. However, due to the technical complexity of integrating cell culture chambers, biosensors, and bioreactors into a single entity, a microphysiological system capable of reproducing controlled microenvironmental cues to regulate cell phenotypes, promote iPS-cardiomyocyte maturity, and simultaneously measure the dynamic changes of cardiomyocyte function in situ is not available. This paper reports an ultrathin and flexible bioelectronic array platform in 24-well format for higher-throughput contractility measurement under candidate drug administration or defined microenvironmental conditions. In the array, carbon black (CB)-PDMS flexible strain sensors were embedded for detecting iPSC-CM contractility signals. Carbon fiber electrodes and pneumatic air channels were integrated to provide electrical and mechanical stimulation to improve iPSC-CM maturation. Performed experiments validate that the bioelectronic array accurately reveals the effects of cardiotropic drugs and identifies mechanical/electrical stimulation strategies for promoting iPSC-CM maturation.

Efficacy of disinfectants for inactivation of Ebola virus in suspension by integrated cell culture coupled with real-time RT–PCR

Ebola virus can be transmitted by contact with environmental surfaces (fomites) contaminated with secretions and excretions from infected individuals. Due to their potential to cause a public health emergency and the absence of efficacious drugs and vaccines, a crucial intervention may involve the use of an effective virucidal agent for disinfecting contaminated surfaces. To evaluate the virucidal efficacy of three disinfectants against Ebola virus: Micro-Chem Plus detergent disinfectant cleaner (MCP), FWD, and ethanol; in suspension tests according to the Technical Standard for Disinfection of China. All products at different concentrations were tested with application times ranging from 15s to 8min by using a quantitative suspension test, and a comparative inactivation analysis was performed. A reduction in the virus titre of ≥4 log10 was regarded as evidence of virucidal activity. MCP and FWD, which contain dual quaternary ammonium compounds, are highly effective at inactivating the Ebola virus within 15s of contact time, despite a slight difference between them at lower concentrations. In agreement with the literature, our results confirmed the excellent virucidal activity of medical ethanol for Ebola virus, which can reduce viral titres to background levels within 15s at a concentration of 38% (v/v). These three disinfectants display sufficient inactivation efficacy for the Ebola virus at reasonably short contact times, which may be practically achieved in the field. The use of these disinfectants for decontamination in healthcare settings and laboratories could mitigate the risk of Ebola virus transmission.

Recent advances of integrated microfluidic suspension cell culture system.

Microfluidic devices with superior microscale fluid manipulation ability and large integration flexibility offer great advantages of high throughput, parallelisation and multifunctional automation. Such features have been extensively utilised to facilitate cell culture processes such as cell capturing and culturing under controllable and monitored conditions for cell-based assays. Incorporating functional components and microfabricated configurations offered different levels of fluid control and cell manipulation strategies to meet diverse culture demands. This review will discuss the advances of single-phase flow and droplet-based integrated microfluidic suspension cell culture systems and their applications for accelerated bioprocess development, high-throughput cell selection, drug screening and scientific research to insight cell biology. Challenges and future prospects for this dynamically developing field are also highlighted.

The prevalence and levels of enteric viruses in groundwater of private wells in rural Alberta, Canada

The prevalence and levels of enteric viruses in untreated groundwater of private wells used for drinking and/or agricultural practices in rural Alberta were studied using the qPCR panel assay, integrated cell culture with qPCR and cell culture in the volume of 500 liters per sample through serial sampling. Seven viruses were assessed including adenovirus, rotavirus, norovirus, astrovirus, sapovirus, reovirus and JC virus. Five viruses were detected with an overall positive detection rate of 6.33 % (45 of 711 samples). The most frequently detected virus was adenovirus (48.9%, 22/45) followed by rotavirus (44.4%, 20/45), reovirus (20%, 9/45), JC virus (6.7%, 3/45) and norovirus (6.7%, 3/45). There was no significant difference in the positive detection rates, ranging from 1.1% to 3.4% by various well settings used for broiler farms, cow/calf farms, feedlots and rural acreages. Effects of well characteristics (aquifer type, well depth, static level of water, well seal) and well completion lithology on potential viral contamination of groundwater of private wells were also analyzed upon available data. The findings demonstrate that occurrence of enteric viruses is low and viral contamination is sporadic in groundwater of private wells in rural Alberta. Conventional fecal bacterial indicators (coliform and/or E. coli) were not a representative marker for viral contamination in groundwater wells in rural Alberta.

An integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously quantify the infectious concentrations of eight environmentally relevant enterovirus serotypes

Enterovirus (EV) infectivity is typically measured as a bulk parameter, yet EV serotypes vary in their susceptibility to natural and engineered stressors. Here we developed an integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously and specifically quantify the infectious concentrations of eight EV serotypes commonly encountered in sewage (coxsackieviruses A9, B1, B2, B3, B4 and B5, and echoviruses 25 and 30). The method uses two cell lines for virus replication and serotype-specific qPCR primers for quantification. Primers were designed to target multiple environmental strains of a given serotype and displayed high specificity. The ICC-RTqPCR method exhibited a linear calibration range between 50 and 1000 (echoviruses) or 5000 (coxsackieviruses) infectious units per mL. Over this range, measurements were not influenced by the presence of non-target serotypes, and calibration slopes were reproducible for different virus batches and cell ages. The ICC-RTqPCR method was able to accurately quantify the infectious concentration of a virus after inactivation by heat, and the concentration of a virus within a wastewater matrix. This method will be valuable to assess the differing fates of EV serotypes in natural or engineered systems, and to portray the associated changes in EV population composition.

Microbial Source Tracking in Small Farms: Use of Different Methods for Adenovirus Detection

This study aims to expand the knowledge about fecal contamination by humans and animals using Adenovirus (AdV) as bioindicators in different water sources from rural areas, to evaluate the viral infectivity, and to compare the different techniques used to detect the Human mastadenovirus (HAdV). For that, 124 samples were collected (86 from groundwater and 38 from surface water) along the Rio dos Sinos Basin. Escherichia coli count was carried out, and the samples were submitted for the detection and characterization tests of AdV by different methods (qPCR, multiplex qPCR, and nested PCR). In addition, the viral infectivity was realized by integrated cell culture quantitative PCR (ICC-qPCR). E. coli was detected in 63% of groundwater samples (geometric mean of 16.7 MPN/100 mL) and 68% (geometric mean: 5.08×102 MPN/100 mL) in surface waters. Among the viral indicator in the groundwater, the HAdV was detected in 49% of the samples, followed by Canine mastadenovirus (CAV, 20%), Bovine mastadenovirus (BAdV, 17%), Aviadenovirus (AvAdV, 15%), and Porcine mastadenovirus (PAdV, 03%). In surface water, HAdV was detected in 45%, followed by CAV (42%), BAdV (29%), and PAdV and AvAdV (13%). The quantification of genomic copies per liter ranged from 9.40×104 to 5.54×1010 gc/L. In groundwater samples, it was possible to observe infectious adenovirus in 12% of the samples, as well as in surface water for 18%. The results showed an increase in the sensitivity of positive samples when a combined set of techniques were used for HAdV detection.

Demonstrating the reduction of enteric viruses by drinking water treatment during snowmelt episodes in urban areas

This study investigates short-term fluctuations in virus concentrations in source water and their removal by full-scale drinking water treatment processes under different source water conditions. Transient peaks in raw water faecal contamination were identified using in situ online β-d-glucuronidase activity monitoring at two urban drinking water treatment plants. During these peaks, sequential grab samples were collected at the source and throughout the treatment train to evaluate concentrations of rotavirus, adenovirus, norovirus, enterovirus, JC virus, reovirus, astrovirus and sapovirus by reverse transcription and real-time quantitative PCR. Virus infectivity was assessed through viral culture by measurement of cytopathic effect and integrated cell culture qPCR. Virus concentrations increased by approximately 0.5-log during two snowmelt/rainfall episodes and approximately 1.0-log following a planned wastewater discharge upstream of the drinking water intake and during a β-d-glucuronidase activity peak in dry weather conditions. Increases in the removal of adenovirus and rotavirus by coagulation/flocculation processes were observed during peak virus concentrations in source water, suggesting that these processes do not operate under steady-state conditions but dynamic conditions in response to source water conditions. Rotavirus and enterovirus detected in raw and treated water samples were predominantly negative in viral culture. At one site, infectious adenoviruses were detected in raw water and water treated by a combination of ballasted clarification, ozonation, GAC filtration, and UV disinfection operated at a dose of 40 mJ cm−2. The proposed sampling strategy can inform the understanding of the dynamics associated with virus concentrations at drinking water treatment plants susceptible to de facto wastewater reuse.

Viral Interference as a Factor of False-Negative in the Infectious Adenovirus Detection Using Integrated Cell Culture-PCR with a BGM Cell Line

This study investigated the influence of viral interference on the detection of enteric viruses using the integrated cell culture (ICC)-PCR with a BGM cell line. It was possible to detect 102 plaque-forming units (PFU)/flask of enterovirus 71 (EV71) in spite of the presence of 104 PFU/flask of adenovirus 40 (AdV40). Meanwhile, 104 PFU/flask of AdV40 was not detected in the presence of 102 PFU/flask of EV71. This inhibition of AdV40 detection using ICC-PCR was attributable to the growth of EV71, because the addition of a growth inhibitor of EV71 (rupintrivir) neutralized the detection inhibition of AdV40. The growth inhibition of AdV40 under co-infection with EV71 is probably caused by the immune responses of EV71-infected cells. AdV is frequently used as a fecal contamination indicator of environmental water, but this study demonstrated that false-negative detection of infectious AdV using ICC-PCR could be caused by the co-existence of infectious EV in a water sample. The addition of rupintrivir could prevent false-negative detection of AdV using ICC-PCR. This study, therefore, emphasizes the importance of confirming the presence of multiple enteric viruses in a sample derived from environmental water prior to the application of ICC-PCR because the viral interference phenomenon may lead to the false-negative detection of target viruses.

Optimizing a Suspension Culture Method with a Decreased Cost to Detect Enteroviruses in Water to Increase Surveillance Access

Enteroviruses are a public health threat due to the high incidence of infections and potential for serious illness or death. Some laboratories in high-income countries detect enteroviruses in water by integrating cell culture and PCR (ICC/PCR). This combined method carries a high financial burden, due in part to specialized cell culture equipment. Therefore, we expanded upon a pilot study to reduce the cost by using common laboratory polypropylene tubes to create a cell culture in suspension. We optimized the protocol by determining minimal incubation periods post-infection as a function of the initial virus concentration. Cells in suspension and traditional monolayers were inoculated with poliovirus and incubated in 8-hour intervals up to 48 hours prior to extraction. Quantitative PCR (qPCR) was used to detect viral nucleic acid targets. Treated and raw water samples were seeded with virus and the suspension ICC/qPCR protocol used to ascertain whether the protocol performed similar to directly seeding cells. No variation in virus detection occurred using the suspension ICC/qPCR or monolayer ICC/qPCR (p = 0.95). In surface water samples, viral nucleic acid was successfully detected, with no significant increase after 32 h (p > 0.05). Suspension ICC/qPCR is as effective as monolayer ICC/qPCR in detecting enteroviruses in surface waters. Materials used in the suspension ICC/qPCR have a lower monetary cost than traditional cell culture materials without loss of sensitivity. More accessible testing of waters for enterovirus contamination through cost reduction has the potential to reduce human exposure and disease.

Occurrence of four waterborne viruses at five typical raw water resources in the Republic of Korea during August 2013 to February 2019.

Waterborne diseases have critical public health issues and socioeconomic relevancy worldwide. Various viral pathogens are ordinarily associated with waterborne diseases. Six-year-surveillance (a total of 20 times) of norovirus, hepatitis A virus, group C rotavirus, and enterovirus was conducted at five raw water sampling sites including two lakes (Lakes Soyang and Juam), Hyundo region of Geum River in Daejeon City, and Guui region of Han River in Seoul Metropolitan City and Moolgeum region of Nakdong River in Gimhae City which are located near two water intake plants. In this study, we routinely investigated virus contamination in water samples through reverse transcription polymerase chain reaction (RT-PCR) and integrated cell culture RT-PCR with high sensitivity and specificity. A total 100 samples were tested. Most of the targeted viruses were found in 32% of the samples and at least one of the indicator bacteria was detected in 65% of these occurrences. Among all the detected viruses, enterovirus was the most prevalent with a detection frequency of 12% and 2.71 MPN/10 L on average, while hepatitis A virus was the least prevalent with a detection frequency of 4%. Nearly all of the analyzed viruses (except for group C rotavirus) were present in samples from Han River (the Guui region), Geum River (the Hyundo region), Lake Juam, and Nakdong River (the Moolgeum region), while group C rotavirus was detected in those from the Guui region. During the six-year sampling period, the targeted waterborne viruses in water samples exhibited seasonal patterns in their occurrence that were different from the indicator bacteria levels in the water samples. The fact that they were detected in the five representative Korean water environments makes it necessary to establish the chemical and biological analysis systems for waterborne viruses and sophisticated management systems.

Investigation of drug dissolution and uptake from low-density DPI formulations in an impactor–integrated cell culture model

Besides deposition, pulmonary bioavailability is determined by dissolution of particles in the scarce epithelial fluid and by cellular API uptake. In the present work, we have developed an experimental in vitro model, which is combining the state-of-the-art next generation impactor (NGI), used for aerodynamic performance assessment of inhalation products, with a culture of human alveolar A549 epithelial cells to study the fate of inhaled drugs following lung deposition. The goal was to investigate five previously developed nano-milled and spray-dried budesonide formulations and to examine the suitability of the in vitro test model. The NGI dissolution cups of stages 3, 4, and 5 were transformed to accommodate cell culture inserts while assuring minimal interference with the air flow. A549 cells were cultivated at the air–liquid interface on Corning® Matrigel® -coated inserts. After deposition of aerodynamically classified powders on the cell cultures, budesonide amount was determined on the cell surface, in the interior of the cell monolayer, and in the basal solution for four to eight hours. Significant differences in the total deposited drug amount and the amount remaining on the cell surface at the end of the experiment were found between different formulations and NGI stages. Roughly 50% of budesonide was taken up by the cells and converted to a large extent to its metabolic conjugate with oleic acid for all formulations and stages. Prolonged time required for complete drug dissolution and cell uptake in case of large deposited powder amounts suggested initial drug saturation of the surfactant layer of the cell surface. Discrimination between formulations with respect to time scale of dissolution and cell uptake was possible with the present test model providing useful insights into the biopharmaceutical performance of developed formulations that may be relevant for predicting local bioavailability. The absolute quantitative result of cell uptake and permeation into the systemic compartment is unreliable, though, because of partly compromised cell membrane integrity due to particle impaction and professed leakiness of A549 monolayer tight junctions, respectively.

A keratinocyte and integrated fibroblast culture model for studying particulate matter-induced skin lesions and therapeutic intervention of fucosterol

Increased levels of particulate matter (PM) air pollutants in East Asia have resulted in detrimental health impacts increasing morbidity and mortality. Epidemiological studies suggest a possible relation between the cutaneous exposure of PM and increased oxidative stress and inflammation which lead to skin lesions. The present study utilizes an integrated cell culture model of keratinocytes and fibroblasts to mimic viable skin layers and investigate the possible effects of PM exposure after penetration through corneocytes. The skin perfection is upheld by homeostatic functionality of epidermal cells and the integrity of connective tissues. Exposure to xenobiotics could alter the skin cell homeostasis aggravating premature skin aging. Stimulation of HaCaT keratinocytes by PM collected from Beijing, China (CPM) increased the intracellular ROS levels triggering a cascade of events aggravating inflammatory responses and connective tissue degradation. In HDF fibroblasts, treatment with preconditioned keratinocyte culture media augmented inflammatory responses, cellular differentiation, and connective tissue degradation. Above events were marked by the increased intracellular ROS, inflammatory mediators, pro-inflammatory cytokines, matrix metalloproteinases (MMP)-1 and -2 levels, collagenase, and elastase activity. Fucosterol treatment of keratinocytes dose-dependently attenuated the detrimental effects both in keratinocytes and fibroblasts restoring the conditions near to physiological levels. Further evaluations could be advanced on developing fucosterol, in forms such as rejuvenating cosmeceuticals which could attenuate detrimental responses of CPM exposure.

Artemia spp., a Susceptible Host and Vector for Lymphocystis Disease Virus.

Different developmental stages of Artemia spp. (metanauplii, juveniles and adults) were bath-challenged with two isolates of the Lymphocystis disease virus (LCDV), namely, LCDV SA25 (belonging to the species Lymphocystis disease virus 3) and ATCC VR-342 (an unclassified member of the genus Lymphocystivirus). Viral quantification and gene expression were analyzed by qPCR at different times post-inoculation (pi). In addition, infectious titres were determined at 8 dpi by integrated cell culture (ICC)-RT-PCR, an assay that detects viral mRNA in inoculated cell cultures. In LCDV-challenged Artemia, the viral load increased by 2–3 orders of magnitude (depending on developmental stage and viral isolate) during the first 8–12 dpi, with viral titres up to 2.3 × 102 Most Probable Number of Infectious Units (MPNIU)/mg. Viral transcripts were detected in the infected Artemia, relative expression values showed a similar temporal evolution in the different experimental groups. Moreover, gilthead seabream (Sparus aurata) fingerlings were challenged by feeding on LCDV-infected metanauplii. Although no Lymphocystis symptoms were observed in the fish, the number of viral DNA copies was significantly higher at the end of the experimental trial and major capsid protein (mcp) gene expression was consistently detected. The results obtained support that LCDV infects Artemia spp., establishing an asymptomatic productive infection at least under the experimental conditions tested, and that the infected metanauplii are a vector for LCDV transmission to gilthead seabream.