Browsing by Author "Costa, Emanuel Fonseca Dinis"
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- Development of a new microcarrier culture based in matrix of natural polymerPublication . Costa, Emanuel Fonseca Dinis; Passarinha, Luís António Paulino; Sousa, Ângela Maria Almeida deGellan gum is a low cost polysaccharide manufactured by microbial fermentation of the Sphingomonas paucimobilis microorganism. This polymer is commonly used in food and pharmaceutical industry. It can be dissolved in water, and when heated and mixed with mono or divalent cations, forms a gel upon lowering the temperature under mild conditions. Gellan gum can be found in two forms, one with high acetylate content, called high acyl gellan gum and other form with low acetylate content, called low acyl gellan gum. Each form of gellan gum presents different gel characteristics. Microcarrier cell culture seems to be a promising method for the growth of large amounts of cells. This technique has been used not only for the production of pharmaceuticals but also in biomedical engineering and in drug screening using 3D cell culture. However microcarriers available on the market have some disadvantages such as opacity, low density or low porosity. Also the large diameter of the macroporous microcarriers makes them disadvantageous for some functions such as viewing adhered cells. But the main disadvantage lies in the high cost of processing. Bringing together these two topics, the aim of this work was create a new microcarrier made by the conjugation of the two forms of gellan gum. To achieve this aim, the work was divided in two main topics, the production and recovery of high acyl gellan gum and the development and characterization of the microcarrier structure. For the production and recovery of high acyl gellan gum, different procedures were tested and results revealed that medium with low nitrogen source and salt solution containing 10 g/L of Na2HPO4, 3 g/L of KH2PO4, 1 g/L of K2SO4, 1 g/L of NaCL, 0.2 g/L of MgSO4.7H2O, 0.01 g/L of CaCl2, 0.001 g/L of FeSO4.7H2O is more suitable for the growth of bacteria. The procedure of two centrifugation steps showed the best results for the removal of bacteria content. In addition, acetonitrile was the organic solvent that showed the best results for the precipitation of high acyl gellan gum compared to the other organic solvent tested (isopropanol, ethanol and acetone). In the second phase of the project was used the combination of gellan gum of high and low acetylation to develop a new microcarrier for cell culture. The combination of these two forms is related to the fact of trying to decrease the density of negative charges of low acyl gellan gum by using gellan gum high acetylation. Furthermore it was also desired to analyse the effect of the two forms in the swelling capacity and the degree of porosity of the final microcarrier. For this aim was then developed an experimental design in order to get the possible combinations between the concentrations of gellan gum high (0%, 1%, 1.5%, 2%, 2.5%, 3%) and low acetylation (0%, 0,16% , 12:33%, 0.5%). The water-in-oil emulsion technique was applied to the construction of microcarrier structures. The microcarriers developed were then evaluated for porosity degree by mercury microporosimetry and for swelling ability by the difference between the hydrated and lyophilized form of microcarrier. Through the results obtained, two initial models for the prediction of swelling behaviour and porosity degree were developed. Scanning electron microscopy was also performed to visualize the topographic surface of the microcarrier. Microcarriers with high (1.672 m2/g) and low level of porosity (0.209m2/g) were chosen for a cell culture assay and demonstrated similar behaviour regarding cell adhesion. The ability to adsorb compounds with positive charge was proved with a bovine serum albumin adsorption study by the surface of the microcarrier. In overall, these results suggest that the gellan microcarriers show advantageous characteristics that can be used for microcarrier cell culture. However more studies are needed in order to improve the microcarrier characteristics.