RESUMO
Accurate epidemiological models require parameter estimates that account for mobility patterns and social network structure. We demonstrate the effectiveness of probabilistic programming for parameter inference in these models. We consider an agent-based simulation that represents mobility networks as degree-corrected stochastic block models, whose parameters we estimate from cell phone co-location data. We then use probabilistic program inference methods to approximate the distribution over disease transmission parameters conditioned on reported cases and deaths. Our experiments demonstrate that the resulting models improve the quality of fit in multiple geographies relative to baselines that do not model network topology.
Assuntos
Simulação por Computador , Modelos Epidemiológicos , HumanosRESUMO
The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting. Graphical abstract â¢The embryonic stem cell test to predict teratogenicity was made automation-compatible. â¢Several key improvements to the assay procedure have been introduced to increase performance. â¢The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.
Assuntos
Desenvolvimento Embrionário , Ensaios de Triagem em Larga Escala , Células-Tronco Pluripotentes/metabolismo , Reprodução , Bibliotecas de Moléculas Pequenas/farmacologia , Testes de Toxicidade , Trifosfato de Adenosina/farmacologia , Animais , Automação , Bioensaio , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Células NIH 3T3 , Células-Tronco Pluripotentes/efeitos dos fármacos , Reprodução/efeitos dos fármacosRESUMO
The gold standard in cryopreservation is still conventional slow freezing of single cells or small aggregates in suspension, although major cell loss and limitation to non-specialised cell types in stem cell technology are known drawbacks. The requirement for rapidly available therapeutic and diagnostic cell types is increasing constantly. In the case of human induced pluripotent stem cells (hiPSCs) or their derivates, more sophisticated cryopreservation protocols are needed to address this demand. These should allow a preservation in their physiological, adherent state, an efficient re-cultivation and upscaling upon thawing towards high-throughput applications in cell therapies or disease modelling in drug discovery. Here, we present a novel vitrification-based method for adherent hiPSCs, designed for automated handling by microfluidic approaches and with ready-to-use potential e.g. in suspension-based bioreactors after thawing. Modifiable alginate microcarriers serve as a growth surface for adherent hiPSCs that were cultured in a suspension-based bioreactor and subsequently cryopreserved via droplet-based vitrification in comparison to conventional slow freezing. Soft (0.35%) versus stiff (0.65%) alginate microcarriers in concert with adhesion time variation have been examined. Findings revealed specific optimal conditions leading to an adhesion time and growth surface (matrix) elasticity dependent hypothesis on cryo-induced damaging regimes for adherent cell types. Deviations from the found optimum parameters give rise to membrane ruptures assessed via SEM and major cell loss after adherent vitrification. Applying the optimal conditions, droplet-based vitrification was superior to conventional slow freezing. A decreased microcarrier stiffness was found to outperform stiffer material regarding cell recovery, whereas the stemness characteristics of rewarmed hiPSCs were preserved.
Assuntos
Células-Tronco Pluripotentes Induzidas , Vitrificação , Alginatos , Criopreservação/métodos , Elasticidade , Congelamento , HumanosRESUMO
BACKGROUND: Phosphorylated histone H2AX, also known as γH2AX, forms µm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected. RESULTS: To overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of γH2AX foci in z-image stacks acquired by confocal fluorescence microscopy. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and γH2AX foci, as well as evaluation of the total foci number per cell nucleus. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. We validated the FocAn algorithm using fluorescence-labeled γH2AX in two glioblastoma cell lines, irradiated with 2 Gy and given up to 24 h post-irradiation for repair. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to ~ 200 DSB/nucleus. CONCLUSIONS: FocAn is freely available an open-source 3D foci analyzer. The user-friendly algorithm FocAn requires little supervision and can automatically count the amount of DNA-DSBs, i.e. fluorescence-labeled γH2AX foci, in 3D image stacks acquired by laser-scanning microscopes without additional nuclei staining.
Assuntos
Algoritmos , Reparo do DNA , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Histonas/análise , Histonas/metabolismo , HumanosRESUMO
BACKGROUND: Unequivocal identification of patients is a precondition for a safe medical journey through different information systems (ISs) and software applications that are communicating and exchanging interoperable data. A master patient index (MPI) can facilitate this task. Being a repository of patient identity traits, a MPI allows an accurate surveillance of the patients' "medical identities". Up to 2014, the Grand Duchy of Luxembourg did not possess a MPI. Here, we describe our experience in the establishment of a national MPI for the Grand Duchy of Luxembourg. METHODS: The different steps that were used to establish the MPI system are described. Firstly, through the identification of the suitable application and, secondly, through the implementation of the MPI to the eHealth national platform and its connection to the national health care system. In parallel to the first two phases, the identity management policies were defined and implemented. RESULTS: Since 2014, when the MPI was integrated to the eHealth platform, we observed a continuous increase of identity profiles. At the latest update (31 December 2018), 2.418.336 identity profiles have been counted, including almost the totality of Luxembourgish residents (95.2%) as well as all the cross-border workers that are affiliated to the Luxembourgish social security system. An analysis of the identification domains connected to the platform highlighted a yearly increase in the usage rate of the identities by external applications (currently representing 70%). The evaluation of the quality of information contained in each identity profile showed low rejection rates (0.2%), indicating a high quality and a good level of completeness in regards to the required identity traits. CONCLUSIONS: This paper presents the current state of patient identity management in Luxembourg and discusses how this synergistically supports the functioning of the national electronic health record (EHR) known as DSP (from the French Dossier de Soins Partagé) and the Luxemburgish health care system. The here described national MPI has refined the identification of patients, leading to an improvement of their safety during their medical journey. Nevertheless, the application regularly undergoes updates to better meet the current requirements of the Luxembourgish health system.
Assuntos
Atenção à Saúde , Registros Eletrônicos de Saúde , Sistemas de Identificação de Pacientes , Feminino , Humanos , Luxemburgo , Masculino , Fatores de TempoRESUMO
BACKGROUND: Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells. METHODS: Using single-cell tracking and wound healing migration tests, colony-forming assay, Western blotting, flow cytometry and electrorotation we examined the effects of MK-2206 and PI-103 and/or irradiation on the migration, radiation sensitivity, expression of several marker proteins, DNA damage, cell cycle progression and the plasma membrane properties in two glioblastoma (DK-MG and SNB19) cell lines, previously shown to differ markedly in their migratory behavior and response to PI3K/mTOR inhibition. RESULTS: We found that MK-2206 strongly reduces the migration of DK-MG but only moderately reduces the migration of SNB19 cells. Surprisingly, MK-2206 did not cause radiosensitization, but even increased colony-forming ability after irradiation. Moreover, MK-2206 did not enhance the radiosensitizing effect of PI-103. The results appear to contradict the strong depletion of p-Akt in MK-2206-treated cells. Possible reasons for the radioresistance of MK-2206-treated cells could be unaltered or in case of SNB19 cells even increased levels of p-mTOR and p-S6, as compared to the reduced expression of these proteins in PI-103-treated samples. We also found that MK-2206 did not enhance IR-induced DNA damage, neither did it cause cell cycle distortion, nor apoptosis nor excessive autophagy. CONCLUSIONS: Our study provides proof that MK-2206 can effectively inhibit the expression of Akt in two glioblastoma cell lines. However, due to an aberrant activation of mTOR in response to Akt inhibition in PTEN mutated cells, the therapeutic window needs to be carefully defined, or a combination of Akt and mTOR inhibitors should be considered.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Radiossensibilizantes/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Dano ao DNA , Furanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Mutação , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Análise de Célula Única , Serina-Treonina Quinases TOR/metabolismoRESUMO
Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (â¼200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy ( dSTORM) with a lateral resolution of â¼20 nm to analyze the focal nanostructure of the DSB marker histone γH2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained γH2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that γH2AX foci consisted of distinct circular subunits ("nanofoci") with a diameter of â¼45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that γH2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual γH2AX-containing nucleosomes. dSTORM-based γH2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of γH2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between â¼0.6 and â¼1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.-Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.
RESUMO
When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me2SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4â¯wt% changed the devitrification time with a factor of 342 and 271 for Me2SO and EG, respectively. Concentration changes in EG/Me2SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.
Assuntos
Varredura Diferencial de Calorimetria/métodos , Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Animais , Bancos de Espécimes Biológicos , Temperatura Baixa , Cristalização , Humanos , Vitrificação/efeitos dos fármacosRESUMO
The surface charge of a biomaterial represents a promising tool to direct cellular behavior, which is crucial for therapeutic approaches in regenerative medicine. To expand the understanding of how the material surface charge affects protein adsorption and mesenchymal stem cell behavior, differently charged surfaces with zeta potentials spanning from -25 mV to +15 mV were fabricated by the conjugation of poly(amidoamine) to alginate-based hydrogels. We showed that the increase of the biomaterials surface charge resulted in enhanced quantities of biologically available, surface-attached proteins. Since different surface charges were equalized after protein adsorption, mesenchymal stem cells interacted rather with diverse protein compositions instead of different surface features. Besides an enhanced cell attachment to increasingly positively charged surfaces, the cell spreading area and the expression of adhesion-related genes integrin α5 and tensin 1 were found to be increased after adhesion. Moreover, first results indicate a potential impact of the surface charge on mesenchymal stem cell differentiation towards bone and fat cells. The improved understanding of surface charge-related cell behavior has significant impact on the design of biomedical devices and artificial organs.
Assuntos
Alginatos/química , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Poliaminas/química , Adsorção , Materiais Biocompatíveis/química , Adesão Celular , Técnicas de Cultura de Células , Diferenciação Celular , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Integrina alfa5/metabolismo , Microscopia Eletrônica de Varredura , Fenótipo , Análise Espectral Raman , Propriedades de Superfície , Tensinas/metabolismo , Engenharia TecidualRESUMO
Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , PTEN Fosfo-Hidrolase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Citoesqueleto de Actina , Actinas/biossíntese , Benzotiazóis/farmacologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Interferência de RNA , RNA Interferente Pequeno , Serina-Treonina Quinases TOR/biossíntese , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
Animal testing plays a vital role in biomedical research. Stress reduction is important for improving research results and increasing the welfare and the quality of life of laboratory animals. To estimate stress we believe it is of great importance to develop non-invasive techniques for monitoring physiological signals during the transport of laboratory animals, thereby allowing the gathering of information on the transport conditions, and, eventually, the improvement of these conditions. Here, we study the suitability of commercially available electric potential integrated circuit (EPIC) sensors, using both contact and contactless techniques, for monitoring the heart rate and breathing rate of non-restrained, non-sedated laboratory mice. The design has been tested under different scenarios with the aim of checking the plausibility of performing contactless capture of mouse heart activity (ideally with an electrocardiogram). First experimental results are shown.
Assuntos
Anestesia , Capacitância Elétrica , Frequência Cardíaca/fisiologia , Monitorização Fisiológica/métodos , Respiração , Animais , Animais de Laboratório , Pressão Sanguínea/fisiologia , Camundongos Endogâmicos C57BL , Processamento de Sinais Assistido por Computador , Fatores de TempoRESUMO
Tissue metabolomics requires high sample quality that crucially depends on the biobanking storage protocol. Hence, we systematically analyzed the influence of realistic storage scenarios on the liver metabolome with different storage temperatures and repeated transfer of samples between storage and retrieval environments, simulating the repeated temperature changes affecting unrelated samples stored in the same container as the sample that is to be retrieved. By cycling between storage (-80 °C freezer, liquid nitrogen, cold nitrogen gas) and retrieval (room temperature, -80 °C), assuming three cycles per day and sample, we simulated biobank storage between 3 months and 10 years. Liver tissue metabolome was analyzed by liquid chromatography/mass spectrometry. Most metabolite concentrations changed <5% for the first "year" of time-compressed biobanking simulation, predominantly due to hydrolysis of peptides and lipids. Interestingly, storage temperature affected metabolite concentrations only little, while there was a linear dependence on the number of temperature change cycles. Elevated sample temperature during (prolonged) retrieval time led to a distinctly different signature of metabolite changes that were induced by cycling. Our findings allow giving recommendations for optimized storage protocols and provide signatures that allow detection of deviations from protocol.
Assuntos
Criopreservação , Fígado/metabolismo , Metabolômica , Cromatografia Líquida , Humanos , Espectrometria de MassasRESUMO
BACKGROUND: To investigate the periodontal disease status in a multi-center cross-sectional study in Germany. Associations of dental, socio-economic, blood and biomedical variables with periodontal outcome parameters were evaluated. METHODS: From 4 different centers N = 311 persons were included, drawn randomly from the registration offices. Maximal pocket depth (PD) was used as primary indicator for periodontitis. It was classified as: no/mild ≤3 mm, moderate 4-5 mm, severe ≥6 mm. Associations between socioeconomic (household income, education), lifestyle, and biomedical factors and PD or bleeding on probing (BOP) per site ("Yes"/"No") was analyzed with logistic regression analysis. RESULTS: Mean age of subjects was 46.4 (range 20-77) years. A significantly higher risk of deeper pockets for smokers (OR = 2.4, current vs. never smoker) or persons with higher BMI (OR = 1.6, BMI increase by 5) was found. Severity of periodontitis was significantly associated with caries lesions (p = 0.01), bridges (p < .0001), crowns (p < .0001), leukocytes (p = 0.04), HbA1c (p < .0001) and MCV (p = 0.04). PD was positively correlated with BOP. No significant associations with BOP were found in regression analysis. CONCLUSIONS: Earlier findings for BMI and smoking with severity of PD were confirmed. Dental variables might be influenced by potential confounding factors e.g. dental hygiene. For blood parameters interactions with unknown systemic diseases may exist.
Assuntos
Estilo de Vida , Índice Periodontal , Bolsa Periodontal/classificação , Classe Social , Adulto , Idoso , Índice de Massa Corporal , Estudos de Coortes , Estudos Transversais , Coroas/estatística & dados numéricos , Cárie Dentária/classificação , Prótese Parcial/estatística & dados numéricos , Escolaridade , Índices de Eritrócitos , Estudos de Viabilidade , Feminino , Alemanha , Hemoglobinas Glicadas/análise , Humanos , Renda/estatística & dados numéricos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Bolsa Periodontal/sangue , Periodontite/sangue , Periodontite/classificação , Fumar , Adulto JovemRESUMO
Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated K(V)1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K(2P)) channels in the RVD of human CD4(+) T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K(2P)5.1 and K(2P)18.1 as the most important K(2P) channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K(2P)5.1 and K(2P)18.1 on the RVD was comparable to that of K(V)1.3. K(2P)9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K(2P) channels.
Assuntos
Canais de Potássio de Domínios Poros em Tandem/química , Linfócitos T/metabolismo , Biofísica/métodos , Linfócitos T CD4-Positivos/citologia , Eletrofisiologia/métodos , Homeostase , Humanos , Inflamação , Íons , Microscopia de Vídeo/métodos , Osmose , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de TempoRESUMO
Hydrohalite, a crystalline rock salt hydrate, (NaCl·2H2O), can form in cryopreservation samples under certain circumstances changing the local chemical environment of the preserved cells. Evidence of this crystalline phase was recently found by microspectroscopy measurements, and believed to form exclusively extracellular. We have studied the spatial distribution of hydrohalite in frozen mouse fibroblast cell samples by means of confocal Raman scanning microscopy (CRM). Hydrohalite has a unique Raman spectrum with several bands in the high frequency tail of the OH-stretching band which can be used for unambiguous identification. Hydrohalite can only form through eutectic crystallization in saline solutions without any cryoprotective agents and the spatial distribution thus gives a more detailed view on this crystallization process. This is important since eutectic crystallization has been empirically correlated to cell death, but the exact injury mechanism is unclear. By the means of colocalization of Raman bands we show that hydrohalite can indeed form intracellularly and is not a strictly extracellular phenomenon. We furthermore found that intracellular ice and intracellular hydrohalite very often coincide. Finally we show that the addition of 0.5 wt.% dimethyl sulfoxide (Me2SO) inhibits formation of hydrohalite. This study shows how Raman microscopy and successive analysis can be employed non-invasively within cryobiology to give additional chemical and structural information compared to conventional imaging techniques.
Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Cloreto de Sódio/química , Análise Espectral Raman/métodos , Animais , Linhagem Celular , Cristalização , Fibroblastos , Congelamento/efeitos adversos , Camundongos , Microscopia ConfocalRESUMO
BACKGROUND: In 2011, almost 20.0% of the population of Germany had a migration background. Studies on their health tend to have low participation rates. The aim of our study was to compare different sampling strategies and to test different approaches to recruit migrants for an epidemiological study. METHODS: Four recruitment centres of the German National Cohort recruited persons of Turkish origin and ethnic German immigrants from former Soviet Union countries. A register-based (random samples from residents' registration offices) and a community-orientated strategy were applied. Participants underwent a medical examination and self-completed a questionnaire. RESULTS: Used approaches: The community-orientated strategies comprised the acquisition of key persons from migrant networks to support the recruitment, invitation talks and distribution of study materials in migrant settings, etc. The identifying variables in the registry data were name, nationality or country of birth. All but one centres used bilingual study material and study staff. PARTICIPATION: When comparing the two strategies, the register-based participation rates ranged from 10.1 to 21.0% (n = 668 participants) and the community-oriented recruitment resulted in 722 participants. CONCLUSION: Register-based recruitment should use a combination of name, nationality and country of birth in order not to be limited to identifying persons with a foreign nationality. However, according to the study staff, the community-oriented approach involving key persons of the same cultural background leads to a better acceptance by the participants. Also, it covers a more heterogeneous group. Yet, it is time-consuming and needs considerably more staff. Further research should establish the effectiveness of a combination of both strategies.
Assuntos
Emigrantes e Imigrantes/estatística & dados numéricos , Estudos Epidemiológicos , Seleção de Pacientes , Sistema de Registros/estatística & dados numéricos , Características de Residência/estatística & dados numéricos , Adulto , Idoso , Etnicidade/estatística & dados numéricos , Estudos de Viabilidade , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos de Amostragem , Inquéritos e Questionários , Turquia/etnologia , U.R.S.S./etnologia , Adulto JovemRESUMO
Cultivation and proliferation of stem cells in three-dimensional (3-D) scaffolds is a promising strategy for regenerative medicine. Mesenchymal stem cells with their potential to differentiate in various cell types, cryopreserved adhesion-based in fabricated scaffolds of biocompatible materials can serve as ready-to-use transplantation units for tissue repair, where pores allow a direct contact of graft cells and recipient tissue without further preparation. A successful cryopreservation of adherent cells depends on attachment and spreading processes that start directly after cell seeding. Here, we analyzed different cultivation times (0.5, 2, 24 h) prior to adhesion-based cryopreservation of human mesenchymal stem cells within alginate-gelatin cryogel scaffolds and its influence on cell viability, recovery and functionality at recovery times (0, 24, 48 h) in comparison to non-frozen control. Analysis with confocal laser scanning microscopy and scanning electron microscopy indicated that 2 h cultivation time enhanced cryopreservation success: cell number, visual cell contacts, membrane integrity, motility, as well as spreading were comparable to control. In contrast, cell number by short cultivation time (0.5 h) reduced dramatically after thawing and expanded cultivation time (24 h) decreased cell viability. Our results provide necessary information to enhance the production and to store ready-to-use transplantation units for application in bone, cartilage or skin regenerative therapy.
Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Criopreservação/métodos , Regeneração Tecidual Guiada/instrumentação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Engenharia Tecidual/instrumentação , Alicerces Teciduais , Alginatos/química , Técnicas de Cultura Celular por Lotes/métodos , Adesão Celular/fisiologia , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Criogéis/química , Desenho de Equipamento , Análise de Falha de Equipamento , Gelatina/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Medicina Regenerativa/instrumentaçãoRESUMO
The penetration kinetics of small-molecule compounds like nutrients, drugs, and cryoprotective agents into artificial cell aggregates are of pivotal relevance in many applications, from stem cell differentiation and drug screening through to cryopreservation. Depending on compound and tissue properties as well as aggregate size and shape, the penetration behavior can differ vastly. Here, we introduce bioorthogonal Raman microspectroscopy as a contactless technique to investigate the penetration of various compounds into spheroids, organoids, and other tissue models in terms of diffusion coefficients and perfusion times. We showcase the potential of the method by applying it to the radial perfusion of neural stem cell spheroids with the prevalent cryopreservation additive dimethyl sulfoxide. Employing a diffusion model for spherical bodies, the spectroscopic data were quantitatively analyzed. Perfusion times were obtained for spheroids in the sub-mm region, and interesting findings about the spheroid-size dependence of the diffusion coefficient are reported.
RESUMO
For the investigation of diseases and other harmful environmental influences (e.g., chemicals) epidemiological studies rely on high quality human samples, among others. Collecting samples and data in the field can pose an enormous challenge to the study team with regard to health protection and occupational safety, especially in the context of a pandemic where there was great uncertainty about the biological risks associated with SARS-CoV-2. The German Environmental Specimen Bank (German ESB) is a key element of environmental and human biomonitoring in Germany with the aim to document and assess trends of human and environmental exposure to chemicals over time and to provide scientific data for policy decision makers. Starting with a pilot study in 1978 human samples are now collected at four sampling locations annually, while sampling is carried out with a highly standardized mobile laboratory since 2013. Due to the corona pandemic 3 of 4 ESB sampling campaigns had to be cancelled in 2020. However, a continuous sampling is crucial to generate current policy relevant data on chemical exposure. Hence, a protection and hygiene concept has been developed including COVID-19 testing with the goal to protect the health of participants and employees during sampling and to meet legal requirements, while sustaining the standardized procedures of sampling and sample preparation. The concept is based on a flexible approach to allow adjustments to changing government regulations and recommendations in the course of the pandemic. By implementing this concept, all samplings were successfully carried out in 2021 & 2022, with the pandemic still ongoing. This paper provides an example of good practice and valuable insights in how to collect human samples during a pandemic.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , COVID-19/prevenção & controle , Alemanha/epidemiologia , Exposição Ambiental/efeitos adversos , Manejo de Espécimes , Monitoramento Biológico/métodos , Monitoramento Ambiental/métodos , Bancos de Espécimes Biológicos , Pandemias , Exposição Ocupacional/prevenção & controleRESUMO
Islet cell transplantation is a promising option for the restoration of normal glucose homeostasis in patients with type 1 diabetes. Because graft volume is a crucial issue in islet transplantations for patients with diabetes, we evaluated a new method for increasing functional tissue yield in xenogeneic grafts of encapsulated islets. Islets were labeled with three different superparamagnetic iron oxide nano particles (SPIONs; dextran-coated SPION, siloxane-coated SPION, and heparin-coated SPION). Magnetic separation was performed to separate encapsulated islets from the empty capsules, and cell viability and function were tested. Islets labeled with 1000 µg Fe/ml dextran-coated SPIONs experienced a 69.9% reduction in graft volume, with a 33.2% loss of islet-containing capsules. Islets labeled with 100 µg Fe/ml heparin-coated SPIONs showed a 46.4% reduction in graft volume, with a 4.5% loss of capsules containing islets. No purification could be achieved using siloxane-coated SPIONs due to its toxicity to the primary islets. SPION labeling of islets is useful for transplant purification during islet separation as well as in vivo imaging after transplantation. Furthermore, purification of encapsulated islets can also reduce the volume of the encapsulated islets without impairing their function by removing empty capsules.