IQUIMEFA   05518
INSTITUTO QUIMICA Y METABOLISMO DEL FARMACO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Immobilization of hybridoma cells in silicate matrices for monoclonal antibody production
Autor/es:
GISELA S. ALVAREZ; MAURICIO DE MARZI; EMILIO MALCHIODI; LUIS DIAZ; MARTIN DESIMONE
Lugar:
Rosario, Santa Fe
Reunión:
Taller; Primer taller de organos artificiales, biomateriales e ingenieria de tejidos; 2009
Resumen:
The encapsulation of biosystems including antibodies, enzymes, bacteria, and mammalian cells is becoming popular in several fields such as building of biosensors, bioreactors and cell encapsulation for bioartificial organs. The sol-gel process is an inorganic polymerization taking place, in some cases, in mild conditions allowing the association of mineral phases with biological systems. These gel matrices enable prolonged use of cells in an optimum environment and may provide the conditions for high productivity. In this work, we studied the effect of different parameters such as the concentration of different sol-gel precursors and immobilized cell density on the function of hybridoma cell viability and Mab production. When studying the effect of the concentration of sol-gel precursors (150-600 mM), a higher metabolic activity was observed in matrices containing the lowest silica concentration. However, when low silica concentrations are used the physical properties of the material change and they lack of mechanical strength. For that reason we decided to use 300 mM silica matrices. Cell morphology and membrane integrity were conserved as it was observed by optical and fluorescence microscopy. Cellular activity of immobilized cells was determined by the tetrazolium (MTT) assay and a LIVE/DEAD vitality fluorescent assay. Viability was 89.5% in tetrakis (2-hydroxyethyl) ortosilitate matrices and was similar to the values obtained in sodium silicate matrices. Hybridoma cells were not detected in the supernatant indicating matrices are efficient in retaining cells. Significant differences were observed in cellular activity when various cells densities were employed, being higher for lower cell densities when expressed on a per cell basis. Cellular activities observed for matrices containing 5x105, 1x106 and 1.5x106 cells were 44%, 75% and 82% lower than those observed for matrices containing 2.5x105 cells. The highest Mab concentration in the supernatant was found for 5x105 immobilized cells. Antibody diffusion out of silicate gels was studied showing a slow released during the first 24 hs and rising in the next days. MAb production from immobilized cells was detected by ELISA for a period of 15 days. The production of Mab by immobilized cells was 10-20% of the concentration obtained with the same initial number of free cells. However, as immobilized cells are not able to grow inside matrices in the same level as free cells, at the end of the experiment, there were 70-80% less cells in silicate matrices than in suspension. Once we have accomplished the first step of our investigation achieving good viability results and preserving cellular ability to produce monoclonal antibodies, the next step is to optimized Mab yields.