Effects of Ca++ and Light Exposure of 3T3 Cells in Tissue Culture

The presence of high Ca++ concentrations in culture medium has been shown to induce DNA replication and to preserve the integrity of cell culture morphology in a variety of systems [1]. Furthermore, the exposure of cells to light also promote increased cell division, and stimulate cell secretion leading to faster wound healing and bone regeneration, possibly related to an increase of the influx of extracellular Ca++ upon light irradiation [2]. To understand the ultrastructural modifications of the cells underlying these effects, we have performed a TEM study of Balb/c 3T3 cells submitted to the irradiation with low intensity red light and treated with variable concentrations of Ca++, in the presence of vitamin D3. Sub-confluent cultures of Balb/c 3T3 cells were kept at 37ºC in Dulbecco's MEM supplemented with 10% bovine calf serum. 10-8 M of vitamin D3 and 40 mM CaCl2 were added to the cells. Cultures were irradiated with a laser Arctic in the visible red region at 650 nm with 5mW of power for 1 minute at a distance of 100 mm and processed for transmission electron microscopy 48h after treatment. Cell cultures were fixed in 3% glutaraldehyde in 0.1M cacodylate buffer pH 7.3. Fixed cells were further fixed in 1% osmium tetroxide in the same buffer and in 0.5% uranyl acetate in acetate-acetic acid buffer pH 5 with appropriate washings in between. Dehydration was performed in increasing concentrations of ethanol. After passage through propylene oxide, cell pellets were embedded in a mixture of epon and araldite, and sectioned with glass knives. One micrometer sections were stained with toluidine blue for light microscope examination and ultrathin sections were stained with uranyl acetate and lead citrate and observed in a JEOL 1200EX electron microscope.