Browsing by Author "Carmelo, Joana G."
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- Mid-infrared spectroscopy: a groundbreaking tool for monitoring mammalian cells processesPublication . Rosa, Filipa O.P.; Ribeiro Da Cunha, Bernardo; Carmelo, Joana G.; Fernandes-Platzgummer, Ana; da Silva, Claudia; Calado, CecíliaMammalian cells are extensively used in cell biology studies, e.g. as a model system of human pathologies, or as a major source of very high-value biopharmaceuticals (that can be the cells itself or their products e.g. heterologous proteins). As such, it is highly pertinent to develop monitoring methods for mammalian cultivations capable of delivering detailed bioprocess information in a rapid and economic way. It is relevant to acquire information concerning the conventional critical variables (as cell growth, consumption of nutrients, production and consumption of by-products and the bioproduct production), and the cell metabolism towards a better understanding of the culture process and consequently for more efficient optimization and control procedures. In the present work, Mid-infrared (MIR) spectroscopy was evaluated as a monitoring technique enabling the acquisition of said bioprocess information in a simple (single step of dehydration), rapid (minutes), economic (without reagent consumption), label-free and high-throughput mode (using 96-wells microplates). The new method was evaluated across a highly diverse set of mammalian culture processes: The monitoring of ex vivo expansion of human mesenchymal stem/stromal cells (MSC) conducted under diverse culture strategies, where it was possible to accurately predict glucose, lactate and ammonia concentrations. The monitoring of recombinant human embryonic kidney cells producing green fluorescent protein, which enabled the estimation of transfection efficiency and the metabolic impact of protein production on the host cell metabolism. Finally, the monitoring of infected gastric cell lines with Helicobacter pylori, which enabled to identify spectral biomarkers for defining the status of infection (infected vs non infected) and to characterize the infection conducted by virulent strains, usually associated to severe gastric diseases as peptide ulcer and gastric cancer. In resume, high-throughput MIR spectroscopy enabled to adequately monitor diverse mammalian cell cultures, thus allowing to attain meaningful information concerning said bioprocesses, from traditional critical variables of the process, to the metabolic status of mammalian host cells and even to define disease biomarkers in a groundbreaking way.
- Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopyPublication . Rosa, Filipa; Sales, Kevin C.; Carmelo, Joana G.; Fernandes-Platzgummer, Ana; Silva, Cláudia L. da; Lopes, Marta B.; Calado, CecíliaHuman mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes.