## 3 Result  
### Conceptual Design and Assembly of the Microfluidic Perifusion System  
The first goal of this study was to design an MPS entirely based on commercially available components (Figure 1A-C). Thus, components from different providers were evaluated in terms of versatility, simplicity, and costs before selecting the French company Fluigent for pressure and flow regulator systems and the Dutch company Micronit Microfluidics for the cell culture chambers, microreactors, and clamping apparatus (see Materials and Methods for technical specifications). Unlike classic peristaltic pumps, the combination of both pressure and flow regulator platforms coupled  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Flow Rate (LG)** & **Flow Rate (HG)** & **Expected Concentration (mM)** \\ \hline
30.0 & 0 & 3 \\
27.7 & 2.3 & 5 \\
21.8 & 8.2 & 10 \\
15.8 & 14.2 & 15 \\
9.8 & 20.2 & 20 \\
3.8 & 26.2 & 25 \\
0 & 30 & 28 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Flow-rate ratios (\(\upmu\)L/min) of the stimulatory solutions (LG and HG) and expected glucose concentration in the ascending ramp of glucose (ARG) protocol.  
with flow-rate sensors (Figure 1A,C) allowed the control (either manually or automatically) of flow rates of stimulatory solutions in a highly accurate and stable manner. The perfusion components (pressure and flow regulator, and flow-rate sensors) of our MPS were specifically calibrated to be used with (but not limited to) a Micronit cell culture chamber (Figure 1B) (see Materials and Methods for technical specifications) for the following reasons: (1) its three-layer design allows easy seeding and retrieval of PIs, (2) it has a small internal volume corresponding to 300 \(\upmu\)L, and (3) it retains the possibility of using the cell culture membranes (middle layer) up to three times before disposal. However, the standard Micronit cell culture chambers had an important limitation for the purpose of dynamic perifusion of PIs--the lack of a second inlet to allow the influx of two different stimulatory solutions. To overcome this limitation and to avoid the need for culture-chamber customization, we incorporated a microreactor with two inlets and one outlet connected to the standard cell culture chamber (Figure 1A,B).  
By using this approach, we could stimulate PIs not only with two different concentrations of glucose or potassium (Figure 2A-D), but also create an ascending ramp of glucose concentration by fine-tuning the flow ratio of low-glucose (LG) and high-glucose (HG) solutions (Figure 2C). Moreover, to limit shear stress exerted on the PIs and to avoid dilution of the collected samples, we set the flow rate at 30 \(\upmu\)L/min, which was the lowest achievable using our system configuration. Additionally, over-dilution in the collected samples was not detected, as demonstrated by the reduced number of human PIs (10 IEQ) required to assess the dynamic glucose-stimulated C-peptide secretion profiles (DGSCS). Furthermore, the flow rate of 30 \(\upmu\)L/min was highly stable through the different perifusion protocols due to the flow-control software (FRCM, Fluigent, Okabe, France), which is based on an innovative algorithm that automatically controls the pressures to maintain the flow rate with intra-experimental variations lower than 0.1 \(\upmu\)L/min.  
Figure 2: Validation of glucose concentration in four perifusion protocols. (**A**) Single pulse of glucose (filled line; HG = 28 mM) and potassium protocol (dotted line; KCl = 50 mM) SPGP. (**B**) Double pulse of high glucose (HG = 17 mM) protocol (DPG). (**C**) Ascending ramp of glucose concentration from 3–30 mM in 130 min (ARG). (**D**) Constant high-glucose concentration (HG = 17 mM) protocol (CHG). Data are expressed as mean \(\pm\) SD of \(n\) = 4 independent experiments. Arrows indicate the direction of transition between stimuli.