We demonstrate the use of a microfluidic cell culture chip with Braille pin-driven pumping, capable of on-chip CO2 incubation that does not require an external chamber or gas supply. The proposed chip consists of a poly(dimethylsiloxane)(PDMS)-made microfluidic chip, flip-mounted on a glass slide, that contains a nested pair of cell culture media reservoirs and water-jacket, insulated by a permeable PDMS wall. By using 0.8 M sodium bicarbonate with 65 mM sodium carbonate as the water-jacket and placing on a 37 °C surface, the chip maintained osmolality shift and the pCO2 in the media reservoir stabilized within < 3 mmol/kg and 5.0% ± 0.2% over at least 24 hours. The incubation capabilities were demonstrated through microfluidic culture of CV-1 epithelial cells under an inverted microscope for at least 12 days.