174 research outputs found
Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells
The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. This work describes the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions.
Since initial cell density and aggregate size have an important impact in the expansion and commitment of these cells into a particular lineage, a combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 ”m) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was then performed in 50 mL spinner flasks, and process optimization using a factorial design approach was developed involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. Furthermore, after scalable differentiation, hiPSC-derived neural progenitors still retained their multipotent potential, being able to give rise to neuronal and glial cells.
The results presented in this work should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using totally controlled, automated and reproducible large-scale bioreactor culture systems
hiPSC-based model of prenatal exposure to cannabinoids: effect on neuronal differentiation
Copyright © 2020 Miranda, Barata, Vaz, Ferreira, Quintas and Bekman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Phytocannabinoids are psychotropic substances ofcannabis with the ability to bind endocannabinoid (eCB) receptors that regulate synaptic activity in the central nervous system (CNS). Synthetic cannabinoids (SCs) are synthetic analogs of Î9-tetrahydrocannabinol (Î9-THC), the psychotropic compound of cannabis, acting as agonists of eCB receptor CB1. SC is an easily available and popular alternative to cannabis, and their molecular structure is always changing, increasing the hazard for the general population. The popularity of cannabis and its derivatives may lead, and often does, to a child's exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with an altered rate of mental development and significant changes in nervous system functioning. However, the understanding of mechanisms of its action on developing the human CNS is still lacking. We investigated the effect of continuous exposure to cannabinoids on developing human neurons, mimicking the prenatal exposure by drug-consuming mother. Two human induced pluripotent stem cells (hiPSC) lines were induced to differentiate into neuronal cells and exposed for 37 days to cannabidiol (CBD), Î9-THC, and two SCs, THJ-018 and EG-018. Both Î9-THC and SC, at 10 ÎŒM, promote precocious neuronal and glial differentiation, while CBD at the same concentration is neurotoxic. Neurons exposed to Î9-THC and SC show abnormal functioning of voltage-gated calcium channels when stimulated by extracellular potassium. In sum, all studied substances have a profound impact on the developing neurons, highlighting the importance of thorough research on the impact of prenatal exposure to natural and SC.This work was supported by the Fundação para a CiĂȘncia e a Tecnologia (FCT), Portugal (SFRH/BPD/81627/2011 to SV), by iBB â Institute for Bioengineering and Biosciences â project UIDB/04565/2020, and by Egas Moniz Higher Institute of Health Science (Egas Moniz, CRL). Funding was also received from the European Unionâs Horizon 2020 Research and Innovation programme, under the Grant Agreement number 739572âThe Discoveries Centre for Regenerative and Precision Medicine H2020-WIDESPREAD-01-2016-2017 to EB.info:eu-repo/semantics/publishedVersio
Human pluripotent stem cell expansion in vertical-wheel bioreactors
Human induced pluripotent stem cells (hiPSC) have been regarded as an enormous breakthrough for medicine, since they can be derived from patients and be used to generate virtually all types of cells in the human body. One of the great bottlenecks in the usage of these cells for regenerative medicine or drug discovery applications is their expansion to relevant quantities. The Vertical-Wheel Bioreactors (PBS Biotech) present a novel scalable bioreactor configuration, whose agitation mechanism allows for homogeneous mixing conditions inside the single-use vessel, while conveying less shear stress to the cells when compared to traditional alternatives. These characteristics are advantageous for hiPSC expansion and thus, in this work, hiPSC were expanded in the Vertical-Wheel Bioreactor using different strategies, namely culturing the cells 1) on microcarriers and 2) as floating aggregates.
In the first approach, cells were cultured under xeno-free conditions, using the Essential 8 medium together with microcarriers and coatings devoid of any animal-derived products [1]. The culture conditions were optimized in terms of initial cell/microcarrier ratio, inoculation method and agitation rate, in the PBS 0.1 vessel (working volume: 80 mL). The cells were successfully expanded, maintaining a normal karyotype, up to a 6.7-fold increase in cell number, after 6 days. These optimized culture conditions were successfully repeated in a larger vessel, the PBS 0.5 (300 mL working volume) demonstrating the scalability of the Vertical-Wheel system.
In the second approach, hiPSC were expanded as floating aggregates, a methodology which does not require a separation step at the end of culture, to remove microcarriers, facilitating the downstream processing and Good Manufacturing Practice-compliance of the process. Cells were cultured in the PBS 0.1 (working volume: 60 mL), using mTeSR1, a serum-free medium and were monitored throughout culture regarding growth kinetics, aggregate size distribution and expression of pluripotency markers. The Vertical-Wheel Bioreactors were shown to efficiently keep the cell aggregates in suspension, under lower linear agitation speeds than an equivalent volume spinner flask (7 cm/s vs. 13 cm/s). Following 7 days of culture, cells were expanded up to a 5.2 ± 0.6-fold increase in cell number. The hiPSC aggregates increased in size over time, from an average diameter of 135 ± 61 ”m to 397 ± 119 ”m after 7 days. Pluripotency was maintained throughout time, as assessed by sustained high (\u3e 80%) expression of pluripotency markers OCT4, SOX2 and TRA-1-60, and low (\u3c 10%) expression of early differentiation marker SSEA-1. The results were validated using a second hiPSC line.
This study revealed that the Vertical-Wheel Bioreactor allows hiPSC growth either on microcarriers and as aggregates and suggested it to have advantages versus other configurations. These results make the Vertical-Wheel Bioreactor a promising platform for hiPSC expansion and, prospectively, differentiation approaches, contributing for the generation of bona fide cells for various biomedical applications, namely drug screening, disease modelling, and, ultimately, for Regenerative Medicine.
[1] Rodrigues CAV, Silva TP, Nogueira DES, Fernandes TG, Hashimura Y, Wesselschmidt R, Diogo MM, Lee B, Cabral JMS (2018), âScalable Culture Of Human Induced Pluripotent Cells On Microcarriers Under XenoâFree Conditions Using SingleâUse VerticalâWheelâą Bioreactorsâ, Journal of Chemical Technology and Biotechnology, DOI: 10.1002/jctb.573
Utilização de placas protrusivas em tempo parcial no tratamento da dor e disfunção da ATM: estudo controlado de um ano
This study aimed at evaluating the effectiveness of partial use of anterior repositioning appliances in the management of TMJ pain and dysfunction when compared to stabilization splints and a control group in a one-year follow-up. Sample was initially constituted by 60 patients, randomly divided into three groups: I- stabilization splints, II- repositioning splints and III- no treatment. The whole sample was evaluated by means of TMJ and muscle palpation, mandibular AROM, analysis of occlusal contacts, joint sounds inspection and Visual Analogue Scale (VAS) for one year; 52 patients composed the final sample. A significant (after 15 days) improvement in pain report (VAS) and palpation index was found for group II (pO objetivo desse estudo foi avaliar a efetividade das placas oclusais reposicionadoras no controle de patologias intra-articulares da ATM, quando comparadas a um tratamento convencional (placa estabilizadora) e um grupo sem tratamento (controle). A amostra final constou de 52 pacientes portadores de sinais e sintomas de desordens intra-articulares da ATM, divididos, aleatoriamente em trĂȘs grupos, de acordo com o procedimento empregado: grupo I (n=20), utilizou placa estabilizadora, grupo II (n=18) utilizou placa reposicionadora e grupo III (n=14), sem nenhum tratamento.Toda a amostra foi acompanhada durante um ano, sendo avaliada atravĂ©s de questionĂĄrios anamnĂ©sicos, palpação muscular e da ATM, movimentação mandibular e detecção de sons articulares.Uma avaliação da condição oclusal tambĂ©m foi realizada nos diferentes perĂodos de exame. Os resultados demonstraram uma maior efetividade das placas reposicionadoras na redução inicial da dor relatada pelo paciente, assim como uma diminuição na sensibilidade a palpação na ATM.Todos os grupos mostraram-se semelhantes apĂłs seis meses de controle em relação aos sintomas, assim como em relação aos ruĂdos articulares. Os Ăndices de palpação muscular mostraram-se melhor para todos os grupos, sendo que a oclusĂŁo tambĂ©m nĂŁo se alterou significantemente. Baseado no acima exposto, conclui-se que a terapia de uso parcial das placas reposicionadoras constitui-se num meio efetivo de controle das patologias intra-articulares da ATM
Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides
Peer reviewedPublisher PD
A releitura de portfólios para a construção do trabalho de conclusão de curso de Pedagogia
Low-intensity resistance exercise does not affect cardiac autonomic modulation in patients with peripheral artery disease
OBJECTIVE: To analyze the effect of a single bout of resistance exercise on cardiac autonomic modulation in patients with peripheral artery disease. METHODS: Fifteen patients with peripheral artery disease (age: 58.3±4.0 years) underwent the following sessions in a random order: resistance exercise (three sets of 10 repetitions of the six resistance exercises with a workload of 5-7 in the OMNI-RES scale) and control (similar to the resistance session; however, the resistance exercises were performed with no load). The frequency domain (low frequency, high frequency and sympathovagal balance) and symbolic analysis (0V, 1V and 2V patterns) of heart rate variability were obtained before and until one hour after the interventions. RESULTS: After the resistance exercise and control sessions, similar increases were observed in the consecutive heartbeat intervals (control: 720.8±28.6 vs. 790.9±34.4 ms; resistance exercise: 712.9±30.1 vs. 756.8±37.9 ms;
Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages
After the injection of saliva into the host's skin by sand flies, a transient erythematous reaction is observed, which is related to an influx of inflammatory cells and the release of various molecules that actively facilitate the blood meal. It is important to understand the specific mechanisms by which sand fly saliva manipulates the host's inflammatory responses. Herein, we report that saliva from Lutzomyia (L.) longipalpis, a widespread Leishmania vector, induces early production of eicosanoids. Intense formation of intracellular organelles called lipid bodies (LBs) was noted within those cells that migrated to the site of saliva injection. In vitro and ex vivo, sand fly saliva was able to induce LB formation and PGE2 release by macrophages. Interestingly, PGE2 production induced by L. longipalpis saliva was dependent on intracellular mechanisms involving phosphorylation of signaling proteins such as PKC-α and ERK-1/2 and subsequent activation of cyclooxygenase-2. Thus, this study provides new insights into the pharmacological properties of sand fly saliva and opens new opportunities for intervening with the induction of the host's inflammatory pathways by L. longipalpis bites
Restless legs syndrome in patients on chronic hemodialysis in a Brazilian city: frequency, biochemical findings and comorbidities
Caracterização farmacognóstica das folhas e sementes de Albizia lebbeck (L.) Benth. (Fabaceae)
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