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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.
- Development of scalable processes to produce therapeutic mesenchymal stromal cell-derived extracellular vesicles and their characterizationPublication . Cunha, Raquel M. S.; Vargas, Elga; Pires, Filipa; Calado, Cecília; Cabral, Joaquim M.S.; Silva, Cláudia; Fernandes, AnaIn summary, this study contributes to the establishment of a scalable process for MSC-EV production.
- Impact of the human mesenchymal stem cells donor on conditional medium compositionPublication . Pereira, Maria João Canha; Ramalhete, Luís; Aleixo, Sandra; da Silva, Cláudia L.; Cabral, Joaquim M.S.; Calado, Cecília; Fernandes, AnaExosomes produced by Mesenchymal Stem Cells (MSCs) can represent a very appealing strategy for cell-free therapies. However, to achieve this reality it is necessary to further understand the process associated to the MSC culture when conditioned to produce exosomes. In the present work, it was evaluated how different MSC obtained from different donors may affect the conditioned media composition and how this can be influenced by the conditioned media type (DMEM versus Xeno-Free medium, XF). The molecular fingerprint of the conditioned media composition was obtained by mid-infrared (MIR) spectroscopy, as this technique reflects fundamental vibrations of a high diversity of functional chemical groups present in biological samples. It was observed by principal component analysis of the second derivative spectra of conditioned media that the media chemical composition depends more from the MSCs donor than the conditioning days. Diverse spectral regions, characteristic of defined chemical groups, enabled to discriminate the chemical composition of the media according to the MSC donor. All of this was observed in both types of media (DMEM and XF). This work is a step forward to understand how different MSC donors and conditioned media may affect the exosomes characteristics.
- Scaling-up the manufacturing of well-characterized mesenchymal stromal cell-derived extracellular vesicles for biomedical applicationsPublication . Fernandes, Ana; Rosa, Sara; Silva, Ricardo; Cunha, Raquel M. S.; Fuzeta, Miguel de Almeida; Calado, Cecília; Carvalho, Carla; Cabral, Joaquim M.S.; Azevedo, Ana; Silva, CláudiaThe platform established herein could be applied to the production of well-characterized SPC-EVs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as EVs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer.
- Design and operation of a fully controlled platform for the production and purification of well-defined mesenchymal stromal cell (MSC)-derived extracellular vesiclesPublication . Fernandes, Ana; Rosa, Sara; Silva, Ricardo; Cunha, Raquel; Fuzeta, Miguel Almeida; Calado, Cecília; Carvalho, Carla; Cabral, Joaquim M.S.; Azevedo, AnaCurrently, the production of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC) is performed in laborious and time-consuming static culture systems using serum-containing media, and their purification is through ultracentrifugation based methods. The whole process lacks on resolution, selectivity and capacity. Additionally, several differences were observed, in terms of the cargo of EVs, between EVs isolated from culture supernatants of MSC expanded under different culture conditions, stressing the importance of controlling all culture process parameters. A robust and indepth process to manufacture MSC-EVs may provide a new therapeutic paradigm for cell-free MSC-based therapies.
- Evaluating the impact of culture conditions on human mesenchymal stromal cell-derived extracellular vesicles molecular fingerprint through FTIR spectroscopyPublication . Fernandes, Ana; Pereira, Maria; Ramalhete, Luís; Aleixo, Sandra; Silva, Cláudia; Cabral, Joaquim M.S.; Calado, CecíliaThis work is a step forward into understanding how different culture conditions affect MSC-derived EVs characteristics.