IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Impact of encapsulated folic acid on Caco-2 cell line in-vitro systems
Autor/es:
FERNANDA ELÍAS; JIMENA H MARTÍNEZ; FLORENCIA TARSIA; OSCAR E PÉREZ
Lugar:
La Plata
Reunión:
Workshop; Imaging Techniques for Biotechnology and Biomedical Applications; 2016
Institución organizadora:
Universidad Nacional de La Plata y CONICET
Resumen:
Folate is a type of B9 vitamin that cannot be synthesized by the human being and is primarily involved in the synthesis of amino acids and purines and pyrimidines. It is well known that low levels of this vitamin during pregnancy lead to neural tube malformations and anencephaly in the fetus. In adults, such low levels generate anemia, depression and lack of memory. Folate´s synthetic form, folic acid (FA), contains one mono glutamate on its most oxidized form. Argentine´s law dictates that wheat flour must be fortified with FA, although the percentage of FA that is truly absorbed is only 10-20%. It is of great importance to find new ways of supplied FA, hence promoting its protection in the intestine and avoiding pH degradation or alteration in the stomach. Last but not least, the characterization of an in-vitro system that allows transport studies and resembles de physiological conditions of the vitamin transport through de human epithelium is also important. FA (2,5, 10 and 20 µM) was encapsulated in a carboxymethylcellulose (CMC) matrix (0,1 % p/p) and conserved at 4°C. The cell line Caco-2 (ATCC) was seeded in a polycarbonate membrane transwell system (pore size: 4 µm) at three different densities: 1x105, 5x105 and 1x106 cells/insert in DMEM with 10% FBS and maintained at 37°C with an atmosphere of 5% CO2 during 20 days to achieve cell differentiation to enterocytes . Afterwards, the cells were fixed and the nucleus and actin were labeled with DAPI and faloidin-rodamine, respectively. The filter was subtracted from the insert and visualized with a confocal laser microscope (CLM) Olympus FluoView FV1000. Transepithelial electrical resistance (TEER) was measured with a voltohmeter (EVOM2, Millipore) to test enterocyte phenotype. The effects of encapsulated FA was evaluated on Caco-2 cell line, to this end 2.5x105 cells were seeded on 12 mm glass with DMEM 10% FBS and growth for 48 h. Cells were fixed and nucleus and actin were labeled with the fluorophores. Samples were visualized under CLM and the images processed with the program FIJI®. Cell monolayer was checked upon time. Once cells are differentiated, the actin cytoskeleton was redirected to the periphery of the cell. In the images, the majority of cells have fibroblastic form accompanied with the actin citoskeleton; nevertheless, there are some cells which are spherical and have the cortical actin, being indicative of differentiation.