The use of Transwells™ improves the rates of differentiation and growth of cultured 3T3L1 cells

The use of Transwells (TM) for routine cultures of 3T3L1 cells results in a much improved rate of differentiation of fibroblasts to adipocytes (100 % in 9 of 10 tests) compared with bottom-well layer cultures. Mean size of cells was not different, but the cell number and overall cell mass was 3x larger in transwell in spite of a smaller surface area. The difference between both models was the accessibility in transwells of both sides of the cells to the medium (and oxygen). Cells were counted, and their size estimated using a handheld cell counter, Scepter (TM), designed for blood cells, but adjusted to the larger size of adipocytes. Finally, the effect of nitric oxide was tested using spermineNONOate, a nitric oxide (NO center dot) donor. The product was released to cultures at a constant 1 mu l/h rate for up to 3 days using osmotic Alzet (TM) minipumps held in wells with water and discharging their contents to the cultured cell-laden wells through a short capillary tube. A rate of 0.3 pmol/min/ml of medium did not affect the cells' size, but 0.4 pmol/min/ml significantly increased cell mass. The methodological improvements presented here allow for more uniform cultured cell yields and a more flexible environment for control of cell size and administration of signaling agents.