Analytical Chemistry the device at a ﬂow rate of 0.5−1 μL/min. When ASW was ﬂowed, the microvalves were closed to prohibit chemical stimulation solution from entering the channels. When chemical stimulations ﬂowed through the device, ASW ﬂow was stopped and the microvalves opened. Small pieces of silicone tubing connected the pressure channels to an in-house built solenoid valve system. Prior to cell loading, each pressure channel was ﬁlled with water to prevent bubble formation in the channels. If bubbles entered the channels, a small amount of negative pressure was applied to the channel to remove them. During each stimulation experiment, cells were visualized to monitor cell viability with an inverted ﬂuorescence microscope (Carl Zeiss, Inc. Peabody, MA). Sample Preparation and Mass Spectrometric Detec- tion. Sample solutions exited the device through polytetra- ﬂuoroethylene tubing (Cole Palmer Instrument Company, Vernon Hills, IL) and between 10 and 20 μL of sample were collected and then desalted and concentrated using ZipTip C18 pipet tips (Millipore, Billerica, MA) according to manufacturer instructions. Brieﬂy, the ZipTip was wetted with 100% acetonitrile (ACN), equilibrated with 0.1% triﬂuoroacetic acid (TFA) in deionized water, loaded with sample containing 0.1% TFA, washed with 5% methanol/0.1% TFA in deionized water, and eluted onto a ground steel MALDI target (Bruker Daltonics Inc., Billerica, MA) ﬁrst with 50% ACN and then with 75% ACN solutions. Samples were then combined with 50 mg/mL 2,5-dihydroxybenzoic acid matrix in 1:1 acetone/water for analysis. An ultraﬂeXtreme TOF mass spectrometer (Bruker Daltonics) in the reﬂectron mode was used to analyze peptide releasates. Mass calibration was performed with peptide calibration standard II (Bruker Daltonics). Between 500 and 1500 shots were collected per spot. Individual mass spectra were analyzed with ﬂexAnalysis (version 3.3, Bruker Daltonics). ■ RESULTS AND DISCUSSION Device Design and Fabrication. The goal of this work was to create a small-volume cell culturing region that allows neuronal network formation and access to cells. Fluidics were added to enable the extracellular media to be exchanged with various solutions (media or stimulation solutions) and a collection port/channel to enable oﬀ-line sample character- ization, in this case via MS. In order to enable temporal control of the media surrounding the cells, we used microvalve- controlled stimulation channels (Figure 1A). The main and outlet reservoirs are 1 mm in diameter; this reduces dilution within the device while allowing for eﬀective ﬂuid ﬂow. The main reservoir serves as the cell culture chamber and is open for cell loading (and physiological recordings, if necessary). Flow channels are 200 μm wide by 50 μm high and rounded; pressure channels are 200 μm wide by 100 μm high. Pressure channels can be selectively opened (or closed) to allow for chemical stimulation additions to the cultured neurons within the device. Studies were performed with colored ink solutions (as shown in Figure 1B) to verify that this arrangement allows ﬂuidic isolation between the stimulation channels and the main channel. PDMS channels are OEGylated to reduce peptide losses into the PDMS.29 The culture chamber is where the neurons are cultured and form the network. Cell Culture. A signiﬁcant amount of chemical hetero- geneity exists among diﬀerent cell types and, perhaps surprisingly, even among cell populations that are considered to be homogeneous. One of the challenges in studying high- Article Figure 1. (A) Schematic of the device, 8 mm wide, 8 mm long, and 2 mm high. Bottom layer (blue) denotes the ﬂow channels whereas the top layer (red) denotes the pressure channels. (B) Image of an ink solution in the stimulation channels with both valves closed. Ink is not observed in the main channel, indicating that valves ﬂuidically isolate stimulation solutions from the main channel. Scale bar = 500 μm. (C) Representative bag cell culture after 1 DIV. Neurons cultured in the device appear viable and form networks. Scale bar = 100 μm. density or larger cultures is due to the small chemical diﬀerences that can be lost when measurement approaches report average values from within a high-density cell population. There are a number of reports documenting high-density cultures in microﬂuidic devices,34−37 but signiﬁ- cantly fewer protocols exist for low-density cultures. While individual cells can be assayed for their peptide contents using MS,24,25,38,39 chemical signaling requires multiple cells and so sparse neuronal networks appear to be of the appropriate scale for investigation of signaling networks using microﬂuidics. For these reasons, we fabricated a device that is capable of maintaining low-density cultures. Each device reported here contained 10−20 bag cell neurons that were removed from the animal and cultured for 24 h prior to stimulation experiments. A representative image of a bag cell culture is shown in Figure 1C; this image shows the cells forming a network with putative chemical or electrical connections between the neurons. Even after 1 day in vitro (DIV), neurons appeared healthy and viable with long process growth and network formation. Cells can be maintained for at least 12 DIV (data not shown) with media changes, allowing for multiple-day stimulation experiments on the same cell cluster in the device. Elevated K+ Stimulation. Following cell culture, neurons within a device were exposed to media that was known or expected to cause the release of peptides. Elevated K+ acts as a secretagogue, which causes the exocytosis of neurotransmitters from a neuron through the depolarization of the cell membrane.40 The addition of K+ to bag cell neurons and the detection of peptide release have been extensively studied, and their subsequent peptide proﬁles have been widely docu- mented.29,41−44 Therefore, A. californica provides an excellent model for validating this system; by observing known released peptides, we validate device function. To that end, nine diﬀerent devices, with each containing a diﬀerent network of bag cell neurons, were subjected to increasing time periods of K+ exposure (Figure 2). A pre- and postcontrol was performed on each group of neurons with ASW before and after chemical stimulation, respectively. ASW is a minimal culture media (as it contains the required inorganic ions) and should not elicit peptide release. We did not observe peptide release with the addition of ASW, which suggests that we did not induce release 9448 dx.doi.org/10.1021/ac302283u | Anal. Chem. 2012, 84, 9446−9452