2.3 DSP ESI-MS Application to 2D Cell Culture
The MC3T3 cells were cultured in a lab separate from the lab where the mass spectrometer used for online analysis was located, requiring a transport and sterile sampling method that preserved the cell growth processes throughout the entire culturing period. When ready for sampling, the cells were removed from the incubator and placed into a sterile fume hood. The 6 well plate cover was removed and replaced by autoclaved aluminum foil, which was taped down in a manner that allowed for visual inspection through the side of the culture plate. During sampling, a digital microscope was positioned orthogonally to the cell culture plate, allowing for visual confirmation of sampling mode (i.e., local or bulk) as shown in the Figure 2 inset image. After the cells were ready for transport, they were placed in a Styrofoam cooler and brought to the mass spectrometer lab, where they were immediately placed on a hotplate to maintain the media at ~37 °C throughout the entire sampling process.
 Direct from culture DSP-ESI-MS analysis was carried out every 3 days immediately before media changes. The MS (BrukerTM MicrOTOF) was tuned and calibrated using BrukerTM tune mix at the start of each experiment to facilitate accurate mass identification. To remove all air bubbles, which interfere with continuous ESI, the entire fluidic system was primed with sterilized DI water. A 22 gauge sterilized hypodermic needle was used to puncture the foil and the sampling inlet was inserted through the hole for sampling. Directly before each sampling event, 2 μL of the sterilized water was pumped out of the sampling inlet at 100 μL/hr to purge the sample inlet line, reducing sample carry over effects. Samples of 1 μL volume were drawn into the sampling interface at 50 μL/hr and subsequently infused through the DSP for direct ESI-MS analysis at 30 μL/hr. Conditioning flow (1% acetic acid, 1% m-NBA) was run continuously at 50 mL/hr throughout the entire experiment. The conditioning flow channel also served as the electrical connection for a picoammeter, which was connected to a stainless steel wire submerged in the conditioning liquid reservoir. The electrospray current during all experiments was maintained at 10-20 nA so that the ESI characteristics remained consistent between experiments. Between each sample, DI water was infused through the DSP and into the MS via ESI to remove residual signal before starting another sampling event. 6 samples were captured from each cell group (i.e., differentiated/undifferentiated) at each time point, 3 local and 3 bulk (Fig. 2, inset), with each sample taken at a spatially disparate location to probe heterogeneities throughout the volume.