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EN
Additive manufacturing methods give the opportunity to produce interesting, new structures with a more complicated topology than would be possible using traditional methods. Methods: Using the selective laser sintering method, a disk with a high roughness and porous structure was produced. Studies of material surface were performed on microscopic devices. An in vitro experiment was performed on the manufactured disk using mice fibroblastic cells. Results: The designed shape enabled the growth of the cell culture in the disc pores and ensured impermeability of the disc base. Based on average viability 79%, which is close to reference well (80%), preliminary results confirmed that the manufactured structures create sufficiently comfortable conditions for the cell cultures without the need to design its internal topography. Conclusions: Controlling the production parameters of SLS printing allows to obtain structures characterized by spatial and surface porosity without designing inner geometry of the structure. Polyamide 2200 (PA2200) powder with a laser beam, offers new possibilities for producing surfaces used in the tissue engineering, bioreactors, and microfluidics devices.
EN
Microspheres (MS) made of biostable polymer, namely polystyrene, have been used as substrates for cell culture enabling rapid cell expansion in dynamic conditions. However, due to non-resorbability, polystyrene (PS) MS when repopulated with cells cannot be directly used in tissue engineering. Our concept was to produce MS from resorbable polymer – poly(L-lactide- -co-glycolide) (PLGA) as a support for adherent cells, e.g. osteoblasts. We hypothesize that such MS can be applied to the injured site to act as cell carriers or as modules for modular tissue engineering (MTE). In this article, we present the results of optimizing the PLGA MS manufacturing conditions via oil-in-water emulsification. Due to such a technique, MS with the required size, size distribution and properties suitable for cell culturing can be obtained. Three parameters of the oil-in-water emulsification were examined: the stirring speed of a water phase during MS manufacturing, the surfactant concentration, i.e. poly(vinyl alcohol) (PVA) in a water phase and concentration of PLGA in dichloromethane (DCM) as an oil phase. The results proved that the 7.5% PLGA concentration in DCM solution as an oil phase, the 0.5-2% concentration of PVA solution as a water phase and the stirring speed of water phase of 1000 rpm provided MS with the 160 μm mean diameter, which is suitable for cell culture. Moreover, the developed sieving and cleaning procedures were efficient to collect MS with the mean diameter of 280 μm, the more coherent size distribution and the ability to sink in the cell culture medium. The presence on the bottom of cell culture wells is crucial for MTE.
EN
Multipotent mesenchymal stromal cells also known as mesenchymal stem cells according to the signal from the damaged tissue have the ability to differentiate into several types of specialized cells forming tissues of mesodermal origin such as bone, cartilage, tendon, skeletal muscle or adipose tissue. This ability of MSC is used in regenerative medicine. For the clinical and experimental purposes in order to increase the amount of MSC their ability to proliferate in vitro is used. The best-known source of mesenchymal stem cells is the adipose tissue and bone marrow which is the most common source material used for primary culture. This paper presents the next steps of culturing mesenchymal stem cells in vitro.
EN
One of the main scopes of modern cell engineering is development of cellular models that can replace animals in drug screening and toxicological tests, so called alternative methods. Construction of the alternative model is a very challenging task due to a richness of factors creating the in vivo environment. The monolayer cell culture — cultivation of adhesive cells on artificial surfaces such as glass or polymer — lack most of the in vivo-like interactions, but still is the only tool for the majority of applications. One of the most prospective approaches on mimicking in vivo environment is “Lab-on-a-chip” technology. Microfluidic devices offer lots of advantages over traditional in vitro culture, e.g. much higher cell volume-to-extracellular fluid volume ratio or possibility of regulation of hydrodynamic stress. This presentation aims to introduce latest advances of our team in microfluidic cell culture devices. Our novel approach is to cultivate three dimensional multicellular aggregates (spheroids) in microenvironments arranged in a microfluidic system. The geometry and materials of the system allow for cultivation, observation and analysis of multicellular spheroids. The results presented concern multicellular tumor spheroids (MCTS) rising from human cancer cells, which are considered to represent most of the conditionings of cancer tumor in vivo. The fully developed MCTS microdevice will be a reliable tool for anticancer drug screening, as the results most likely will be in a close accordance with the results obtained in vivo.
EN
A swine vesicular disease virus (SVDV) replication assay in IB-RS-2, SK-6, and PK-15 cell cultures was performed using the xCELLigence system. The cell status was monitored by impedance measurement, expressed as cell index (CI). Proliferation of particular cells was examined at the beginning of the study. The cells exhibited the ability to form a monolayer, and the CI values increased with the cell culture growth. After about 23 h and while still in the growth phase, the cells were infected with decimal virus dilutions (10⁻¹-10⁻⁶) containing from 100 000 to 1 median tissue culture infectious doses (TCID₅₀). SVDV replication in cell cultures induced a change in cell index; together with the occurrence of cytopathic effect (CPE), the CI values declined. A significant correlation between the concentration of the virus used and CPE occurrence was found. The results also enabled determination of cell sensitivity to SVDV infection. The highest sensitivity was exhibited by IB-RS-2, followed by SK-6. To conclude, the xCELLigence System was used effectively and evaluated as being an efficient tool for CPE detection and SVDV replication analysis in cell cultures. Compared to the standard method, it enabled a more precise assessment of viral replication based on the quantitative CI measurement, providing additional current information.
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PL
Celem badań było opracowanie biomimetycznej matrycy z nośnikiem tlenu do długoterminowej hodowli komórek. Przedstawiono wyniki wstępne obejmujące opracowanie warunków procesu produkcji dwufazowych matryc oraz przeprowadzenie hodowli ludzkich chondrocytów w uproszczonej matrycy. Przeprowadzone badania dowodzą, iż rozmiar produkowanych mikrokapsułek maleje wraz ze wzrostem częstotliwości drgań dyszy. Hodowla komórek CP5 wykazuje konieczność zwiększenia stopnia dotlenienia enkapsulowanych komórek.
EN
The purpose of this work was to develop biomimetic matrix with oxygen carrier for long-term cell culture. Preliminary research results including: microcapsules production conditions and results of human chondrocytes culture in simplified matrices are presented. The size of microcapsules decreases with the increase of nozzle vibration frequency. The chondrocytes culture showed that a greater oxygen supply to the cultured cells is needed.
EN
Investigation with time-lapse microscopy of the clonal growth of stem cells provides to the analysis of image sequences, which document development of a single clone in constant time increments. The cell doubling time (T2) determines the dynamics of the cell culture development as average time taken for a cell to complete the cell cycle and is typically estimated with cytometric measurements within several days. However our monitoring of the cell culture lasts shorter, so the dependence of T2 from measurements available from image sequences has been found and applied to the collected data. The results are compared with the cell doubling time estimated and published for the cell line by the lab, in which it has been established.
PL
Wiedza o wpływie pola elektromagnetycznego na organizmy żywe jest gromadzona w studium eksperymentalnym. Dotyczy ono oceny stanu zdrowia organizmu żywego poddanego wpływowi pola elektromagnetycznego w ściśle kontrolowany sposób. Przedmiotem takich badań mogą być ludzie, zwierzęta, rośliny, ale i izolowane z ich organizmów komórki żyjące w warunkach hodowli in vitro. W artykule opisano doświadczenia autorów dotyczące ustanawiania warunków ekspozycji hodowli komórkowych na pole elektromagnetyczne.
EN
The knowledge about the biological effects of electromagnetic fields comes from an experimental study. The experimental study refers to the assessment of health status of a living organism exposed to the electromagnetic field in a strictly controlled manner. The subject of such studies are people, animals, plants, but also isolated cells that are living in a in vitro culture. The article describes the authors' own experience in the establishment of exposure conditions to electromagnetic fields for cell cultures.
EN
n this work, morphology of Beta vulgaris L. cells permeabilized with 0.7 mM of Triton X-100® was evaluated using digital image processing and concepts of fractal dimension (perimeter- area relations). Important morphometric changes were found when the contact-time with chemical agent was increased.The size of cells decreased, the cells lost the roundness and their shape was more sinuous; this behaviour was a result of a probable shrinkage caused by the excess of exposure with the permeabili- zation agent. Morphology of B. vulgaris cells after permeabili- zation, exhibited a fractal nature since the slope of the ratio of the logarithm of the perimeter vs logarithm of the area was higher than unit. Fractal geometry of the cell morphology was affected as a re- sult of the exposure to Triton X-100®. Those changes can be attri- buted to the loss of turgor and structure of the cell wall.
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2010
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tom 54
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nr 2
223-228
EN
Cytotoxic potential of melamine was evaluated with the use of two in vitro models i.e. cell cultures of rat hepatoma (line FaO) and rat skeletal muscle (line L6). The cultures were exposed for 24, 48, and 72 h to melamine at eight concentrations, ranged from 0.01 to 10 mM. Four different assays were applied in which various biochemical endpoints were assessed: mitochondrial activity - MTT reduction assay, proliferation - Commassie Brilliant Blue (CBB) dye binding assay, lysosomal activity - neutral red uptake (NRU) assay, and membrane integrity - LDH release assay. Effective concentrations (EC₂₀, EC₅₀, EC₈₀) were calculated from concentration-response curves, and then they were averaged over three independently conducted experiments. It was found that MTT assay was the most sensitive to this compound. After 48 h exposure EC₅₀ values (mM, mean ± SD) for FaO and L6 cells were 6.4 ± 0.62, and 8.2 ± 1.51, respectively. The inhibition of lysosomal activity measured by NRU assay, and damage of plasma membrane measured by LDH assay were detected in L6 (but not in FaO) cells; however, the effects took place after longer (72 h) exposure. At that time EC₅₀ values were 5.2 mM and 9.2 mM for NRU and LDH, respectively. In spite of the low cytotoxicity of melamine, more studies are needed for hazard identification and characterization of the compound.
EN
Walnut (Juglans regia L., fam. Juglandaceae) fruit is found to be very rich in phenolic compounds and thus to show wide spectrum of biological activities like antioxidant, anti-inflammatory, or antitumour properties. Ethanol or methanol are preferentially used for preparation of walnut extracts for cosmetic applications. However, it is commonly known that alcohol causes dehydration and redness of the skin. The aim of this work was to prepare aqueous extract of undamaged walnut seeds rich in pellicles and to evaluate its antioxidant, anti-apoptotic, anti-inflammatory, and anti-aging properties in vitro. Conducted research clearly demonstrated that simple water extraction of undamaged kernels with pellicles allows to obtain rich in phenolic compounds (36-38 mg per g of lyophilisate) walnut extract, which protects fibroblasts and keratinocytes against oxidative stress induced by UVB dose of 35 mJ/cm2 (5-6-h exposure on a sunny day), protects keratinocytes against UVB-induced apoptotic death, limits the development of the inflammation in epidermis, and also possesses ability to inhibit collagenase and elastase. Thus, obtained aqueous walnut extract (at relatively low concentration of 5 µg/ml) is found to be a promising compound of sunscreen and anti-aging cosmetic formulations.
EN
Osteoblasts are cells of mesenchymal origin, which rebuild resorbed bone by synthesizing bone matrix proteins and by inducing bone matrix mineralization. Osteoblasts play a crucial role in creating and maintenance of healthy bone architecture, bone repair, and peri-implant bone healing (osseointegration). These bone-forming cells are also involved in regulation of osteoclasts function, and hence bone resorption in osteoclastogenesis process. We have presented our own studies on the subsequent stages of differentiation of Human Bone-Derived Cells (HBDCs) that could be a good candidate as an autogenous source for reconstruction and rebuilding of own patient's bone using tissue engineering methods. In this review we discussed the biology of osteoblasts, compared with the HBDCs cultures, under the influence of growth factors (FGF-2, TGF-ß, IGF, PDGF) and hormones (PTH, 1,25-dihydroxyvitamin D3, leptin). Our review is also focused on the participation of intercellular adhesion proteins (cadherins, claudins, connexin, 'OsteoMacs'), transcription factors (Cbfal, Msx-2, Osx, ATF4), and others molecules (RANKL, OPG, BMP2, lactofferin, PPARY) in modulating osteoblasts functions on the basis of current reports, throwing new light on the involvement of osteoblasts during osteogenesis and peri-implant bone healing.
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