RESUMO
Helicobacter pylori is a gram-negative, spiral-shaped, flagellated bacterium that colonizes the stomach of half the world's population. Helicobacter pylori infection causes pathologies of varying severity. Standard oral therapy fails in 15-20% since the barriers of the oral route decrease the bioavailability of antibiotics and the intrinsic factors of bacteria increase the rates of resistance. Nanoparticles and microparticles are promising strategies for drug delivery into the gastric mucosa and targeting H. pylori. The variety of building blocks creates systems with distinct colloidal, surface, and biological properties. These features improve drug-pathogen interactions, eliminate drug depletion and overuse, and enable the association of multiple actives combating H. pylori on several fronts. Nanoparticles and microparticles are successfully used to overcome the barriers of the oral route, physicochemical inconveniences, and lack of selectivity of current therapy. They have proven efficient in employing promising anti-H. pylori compounds whose limitation is oral route instability, such as some antibiotics and natural products. However, the current challenge is the applicability of these strategies in clinical practice. For this reason, strategies employing a rational design are necessary, including in the development of nano- and microsystems for the oral route.
RESUMO
Immobilizing enzymes into microcarriers is a strategy to improve their long-term stability and reusability, hindered by (UV) light irradiation. However, in such approaches, enzyme-substrate interaction is mediated by diffusion, often at slow kinetics. In contrast, enzyme-linked self-propelled motors can accelerate this interaction, frequently mediated by the convection mechanism. This work reports on a new photosensitive polymeric Janus micromotor (JM) for UV-light protection of enzymatic activity and efficient degradation of substrates accelerated by the JMs. The JMs were assembled with UV-photosensitive modified chitosan, co-encapsulating fluorescent-labeled proteins and enzymes as models and magnetite and platinum nanoparticles for magnetic and catalytic motion. The JMs absorbed UV light, protecting the enzymatic activity and accelerating the enzyme-substrate degradation by magnetic/catalytic motion. Immobilizing proteins in photosensitive JMs is a promising strategy to improve the enzyme's stability and hasten the kinetics of substrate degradation, thereby enhancing the enzymatic process's efficiency.
Assuntos
Quitosana/química , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Movimento (Física) , Armoracia/enzimologia , Compostos Azo/química , Compostos Azo/efeitos da radiação , Catalase/química , Quitosana/efeitos da radiação , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Lacase/química , Fenômenos Magnéticos , Nanopartículas de Magnetita/efeitos da radiação , Platina/química , Platina/efeitos da radiação , Raios UltravioletaRESUMO
Vero cells are used to develop viral vaccines approved for human use, thus including a vaccine against the SARSCoV2 virus. Our objective was to establish a production protocol for this cultivation condition. Cells grown in spinner flask were tested at different concentrations. The μmax values allow us to observe that the best performance was with the assay whose value was 0.0334 h . Cell duplication occurred at 1=37.47 h, 2=21.15 h and 3=22.29 h. The concentration and quality of the inoculum are crucial for the performance of cells when cultivated in a pseudostirred system with microcarriers
Células Vero são utilizadas para o desenvolvimento de vacinas virais, aprovadas para uso humano, incluindo assim uma vacina contra o vírus SARSCoV2. Nosso objetivo foi estabelecer um protocolo de produção para esta condição de cultivo. As células cultivadas em frasco spinner foram testadas em diferentes concentrações. Os valores de μmax permitem observar que o melhor desempenho foi com o ensaio cujo valor ficou em 0,0334 h1. A duplicação celular ocorreu em 1= 37,47 h, 2= 21,15 h e 3= 22,29 h. A concentração e a qualidade do inóculo são determinantes para o desempenho das células quando cultivadas em sistema pseudo agitado com microcarregadores.
RESUMO
Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Reatores Biológicos , Contagem de Células , Diferenciação Celular , Proliferação de Células , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/imunologia , Cordão Umbilical/imunologiaRESUMO
Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.