Browsing by Author "Perestrelo, Ana Rubina"
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- Locust bean gum as an alternative polymeric coating for embryonic stem cell culturePublication . Perestrelo, Ana Rubina; Grenha, Ana; Costa, Ana M. Rosa da; Belo, José A.Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro differentiation assay revealed that mouse ESCs cultured in LBG preserve their tri-lineage differentiation capacity. In conclusion, our data indicate that LBG coating promotes mouse ESC growth in an undifferentiated state demonstrating to be a viable, non-animal derived alternative to gelatin to support pluripotent mouse ESCs in culture.
- Microfluidic Organ/Body-on-a-Chip Devices at the Convergence of Biology and MicroengineeringPublication . Perestrelo, Ana Rubina; Águas, Ana C. P.; Rainer, Alberto; Forte, GiancarloRecent advances in biomedical technologies are mostly related to the convergence of biology with microengineering. For instance, microfluidic devices are now commonly found in most research centers, clinics and hospitals, contributing to more accurate studies and therapies as powerful tools for drug delivery, monitoring of specific analytes, and medical diagnostics. Most remarkably, integration of cellularized constructs within microengineered platforms has enabled the recapitulation of the physiological and pathological conditions of complex tissues and organs. The so-called organ-on-a-chip technology, which represents a new avenue in the field of advanced in vitro models, with the potential to revolutionize current approaches to drug screening and toxicology studies. This review aims to highlight recent advances of microfluidic-based devices towards a body-on-a-chip concept, exploring their technology and broad applications in the biomedical field.
- Novel triblock co-polymer nanofibre system as an alternative support for embryonic stem cells growth and pluripotencyPublication . Perestrelo, Ana Rubina; Mouffouk, Fouzi; Costa, Ana M. Rosa da; Belo, José A.Conventionally, embryonic stem cells (ESCs) are cultured on gelatin or over a mitotically inactivated monolayer of mouse embryonic fibroblasts (MEFsi). Considering the lack of versatile, non-animal-derived and inexpensive materials for that purpose, we aimed to find a biomaterial able to support ESC growth in a pluripotent state that avoids the need for laborious and time-consuming MEFsi culture in parallel with mouse ESC (mESC) culture. Undifferentiated mESCs were cultured in a new nanofibre material designed for ESC culture, which is based on the self-assembly of a triblock co-polymer, poly(ethyleneglycol-β-trimethylsilyl methacrylate-β-methacrylic acid), conjugated with the peptide glycine-arginine-glycine-aspartate-serine, to evaluate its potential application in ESC research. The morphology, proliferation, viability, pluripotency and differentiation potential of mESCs were assessed. Compared to conventional stem cell culture methodologies, the nanofibres promoted a higher increase in mESCs number, enhanced pluripotency and were able to support differentiation after long-term culture. This newly developed synthetic system allows the elimination of animal-derived matrices and provides an economic method of ESC culture, made of a complex network of nanofibres in a scale similar to native extracellular matrices, where the functional properties of the cells can be observed and manipulated.
- Study and optimization of mouse embryonic stem cell culturePublication . Perestrelo, Ana Rubina; Belo, José AntónioStem cell research has grown from unexplored to an important field in biomedical sciences today. Indeed, a deeper understanding of the basic biology of stem cells holds the key to unlock new hopes to various incurable human diseases. Embryonic stem (ES) cells have the unique ability to proliferate indefinitely in an undifferentiated state and to give rise to any type of somatic cell lineage depending on specific signals. ES cells have been typically studied in two-dimension (2D) Petri dishes, 2D multi-well plates or 2D glass slides coated with different substrates which differ radically from the three-dimension (3D) microenvironment in the body. Consequently, cells isolated from higher organisms frequently modify their metabolism, morphology and gene expression profile. The substitution of the 2D systems by a 3D micro or nanomolecular network (scaffold) is a very promising approach in replicating the architecture of the in situ environment of a cell in a living organism. However, the existing 3D culture systems formed from animal-derived biomaterials pose problems for replacement therapies. The goal of this dissertation was to study the possibility of using a new artificial 3D nanowire scaffold formed from self-assembly of amphiphilic biodegradable peptidecopolymers, instead of conventional mitotically inactivated mouse embryonic fibroblasts, for culture of mouse ES cells (mES cells). Undifferentiated mouse ES cells were cultured using the bioconjugated polymeric nanowire scaffold and control conditions. The potential of the scaffold for applications in ES cell research was evaluated by assessing the morphology, proliferation, survival rate, self-renewal and pluripotency of mES cells. The results obtained suggest that, in low concentrations (50 and 25 Eg/ml) of Arginine– Glycine–Aspartate (RGD) bioconjugated polymeric nanowires scaffold the cells retain their viability and pluripotency. However, an improvement of the scaffold would be required in order to promote better survival rates and making this 3D system in an alternative substrate for mES cell culture, which saves time and is easy to use.
- Study of the application potential of synthetic and natural polymeric coatings in stem cell researchPublication . Perestrelo, Ana Rubina; Belo, JoséSelf-renewability and the ability to differentiate into various functional cells are characteristics of embryonic stem cells (ESCs) that make them attractive for applications in biomedical field, namely in restoring the function of damaged cells/tissues. In research, ESCs are usually cultured in gelatin or over a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard to maintain pluripotent ESCs in culture. A variety of alternative technologies have been suggested to control stem cell fate and function, but examples of versatile, non-animal derived and inexpensive materials able to support pluripotent ESCs are limited. To circumvent this, we aimed to find a biomaterial able to support pluripotent ESC cultures that would avoid the laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. There is an increasing interest in regulating stem cells under a specific microenvironment using biomaterials as artificial extracellular matrices (ECMs) to control their self-renewability and differentiation capacity. In the present work we developed and tested the applicability of two biomaterials, one natural and one synthetic polymer, to support mouse ESC (mESC) culture. Accordingly, undifferentiated mESCs were cultured in coatings of Locust Bean Gum (LGB) and of a new synthetic nanofiber (nf) material based on the self-assembly of a triblock copolymer, poly (ethyleneglycol-β-trimethylsilyl methacrylate-β-methacrylic acid), conjugated with the peptide Glycine-Arginine-Glycine-Aspartate-Serine. According to our data, compared to conventional stem cell culture methodologies, ESCs grown in LBG and nanofiber coatings maintained their self-renewability and trilineage differentiation capacity even after long term culture. In parallel, this work also comprises the functional study of collagen and calciumbinding EGF domains 1 (Ccbe1) using primary fibroblasts as a tool. It has been shown that Ccbe1 is involved in lymphangiogenesis, cardiogenesis and carcinogenesis, however, its function and molecular action remains unknown. The data presented here suggests that Ccbe1 coordinates cell adhesion, migration and proliferation, thus playing a key role in the ECM.