RESUMEN
Enhancement of endogenous neurogenesis after ischemic stroke may improve functional recovery. We previously demonstrated that medium B, which is a combination with epidermal growth factor (EGF) and fibronectin, can promote neural stem/progenitor cell (NSPC) proliferation and migration. Here, we showed that medium B promoted proliferation and migration of cultured NSPCs onto various 3-dimentional structures. When rat cortical neurons with oxygen glucose deprivation (OGD) were co-cultured with NSPCs, medium B treatment increased neuronal viability and reduced cell apoptosis. In a rat model with transient middle cerebral artery occlusion (MCAO), post-insult intraventricular medium B treatment enhanced proliferation, migration, and neuronal differentiation of NSPCs and diminished cell apoptosis in the infarct brain. In cultured post-OGD neuronal cells and the infarct brain from MCAO rats, medium B treatment increased protein levels of Bcl-xL, Bcl-2, phospho-Akt, phospho-GSK-3ß, and ß-catenin and decreased the cleaved caspase-3 level, which may be associated with the effects of anti-apoptosis. Notably, intraventricular medium B treatment increased neuronal density, improved motor function and reduced infarct size in MCAO rats. In summary, medium B treatment results in less neuronal death and better functional outcome in both cellular and rodent models of ischemic stroke, probably via promotion of neurogenesis and reduction of apoptosis.
Asunto(s)
Apoptosis , Isquemia Encefálica/tratamiento farmacológico , Ventrículos Cerebrales/patología , Factor de Crecimiento Epidérmico/uso terapéutico , Fibronectinas/uso terapéutico , Neurogénesis , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Isquemia Encefálica/complicaciones , Isquemia Encefálica/fisiopatología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ventrículos Cerebrales/fisiopatología , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/farmacología , Fibronectinas/farmacología , Glucosa/deficiencia , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Ventrículos Laterales/patología , Ventrículos Laterales/fisiopatología , Masculino , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/ultraestructura , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Oxígeno , Ratas Wistar , Recuperación de la Función/efectos de los fármacos , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/fisiopatologíaRESUMEN
To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay.
Asunto(s)
Reactores Biológicos , Técnicas de Cultivo de Célula/instrumentación , Ensayos de Selección de Medicamentos Antitumorales/métodos , Modelos Biológicos , Perfusión , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Humanos , Boca/metabolismo , Boca/patología , Neoplasias de la Boca/patología , Células Tumorales CultivadasRESUMEN
A perfusion three-dimensional (3D) cell culture microfluidic chip has been developed for real-time and non-invasive impedimetric monitoring of cell proliferation and chemosensitivity. In this study, human oral cancer cells (OEC-M1) were encapsulated in 3D agarose scaffold and cultured in a miniaturized chamber under perfusion of tested substance. This setting provides a more in vitro physiologically relevant microenvironment to better mimic the complex in vivo microenvironment. A pair of vertical electrodes was embedded at the opposite sidewalls of the culture chamber for the on-site impedance measurement. Cell density in the 3D construct was shown to be proportional to the impedance magnitude of the entire construct. Therefore, perfusion 3D cell culture was performed for up to 5 days and cell proliferation can be monitored by the impedimetric analysis. Moreover, real-time impedimetric monitoring of cell viability under the perfusion of anti-cancer drug in different concentrations was conducted and the impedance magnitude was directly correlated with the cell viability. From the confirmation of the endpoint cell viability assays, a concentration-dependent effect was shown; however, the response of cell viability during the drug treatment was able to be traced by the impedance measurement. The experimental results showed that cell proliferation and chemosensitivity in 3D cell culture format can be monitored by impedance measurement. This microfluidic chip has a high potential to develop a powerful analytical platform for cancer research.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Antineoplásicos/farmacología , Técnicas Biosensibles/instrumentación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Impedancia Eléctrica , Diseño de Equipo , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Sefarosa/química , Andamios del Tejido/químicaRESUMEN
In this work, a non-invasive measurement technique for the quantitative determination of cell viability in a three-dimensional (3D) cell culture construct is proposed. This technique is based on on-site electrical impedance measurement. A microfluidic chip with a 3D culture chamber is fabricated to demonstrate this technique. In vitro 3D cell culture has been interpreted for faithfully representation of the in vivo cellular responses in 3D cell culture construct is normally time-consuming and labor-intensive. In this study, the microfluidic chip consists of a culture chamber, in which a pair of vertical electrodes at its opposite sidewalls was embedded, and a fluidic channel for drug perfusion. Cancer cells encapsulated in agarose gel were loaded into the culture chamber to perform 3D cell culture under the perfusion of culture medium and anti-cancer drug in different concentrations (6, 12, 18, and 24 µg/ml) for 2 days. Since higher drug concentration led to more cell damage or death, the total impedance magnitude of the culture construct was shown to be reasonably proportional to the anti-cancer drug concentration. Moreover, cell proliferation can be also monitored using this technique. The proposed measurement method can determine cell viability without affecting the cellular behaviors during culture. It has a high potential to develop a fast and easy measurement compared with the conventional cellular analysis techniques.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Técnicas Analíticas Microfluídicas/métodos , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Impedancia Eléctrica , Humanos , Microtecnología , SefarosaRESUMEN
This study reports the utilisation of an optically switched dielectrophoretic (ODEP) force for the manipulation and assembly of cell-encapsulating alginate microbeads in a microfluidic perfusion cell culture system for bottom-up tissue engineering. One of the key features of this system is the ODEP force-based mechanism, which allows a commercial projector to be coupled with a computer to manipulate and assemble cell-encapsulating microbeads in an efficient, manageable, and user-friendly manner. Another distinctive feature is the design of the microfluidic cell culture chip, which allows the patterned cell-encapsulating microbeads to be cultivated on site under culture medium perfusion conditions. For demonstrating its application in bottom-up cartilage tissue engineering, chondrocyte-encapsulating alginate microbeads varying in encapsulated cell densities were generated. The manipulation forces associated with operating the alginate microbeads were experimentally evaluated. The results revealed that the measured manipulation forces increased with increases in both the applied electric voltage and the number of cells in the alginate microbeads. Nevertheless, the observed manipulation force was found to be independent of the size of the cell-free alginate microbeads. It can be speculated that the friction force may influence the estimation of the ODEP force within the experimental conditions investigated. In this study, chondrocyte-encapsulating alginate microbeads with three different cell densities were manipulated and assembled in the proposed microfluidic system to form a compact sheet-like cell culture construct that imitates the cell distribution in the cross-section of native articular cartilage. Moreover, the demonstration case also showed that the cell viability of the cultured cells in the microfluidic system remained as high as 96 ± 2%. In this study, four sheet-like cell culture constructs were stacked to create a larger assembled cell culture construct. The cell distribution inside the cell culture construct was further confirmed by a confocal microscopy observation, which showed that the distribution was similar to that in native articular cartilage. As a whole, the proposed system holds great promise as a platform for engineering tissue constructs with easily tunable inner cell distributions.