## 4 Discussion  
The assessment of human PI functionality before transplantation could improve the patient's clinical outcome. A reliable and highly reproducible perfusion system for the assessment of \(\beta\)-cell function would be highly appreciated by the islet research community. In this study, we reported the design and validation of an MPS built entirely on commercially available components. This MPS allows the automatization of the perfusion protocols, an easy and simple loading and retrieval of PIs due to culture chips, a reproducible and stable flow rate without the need for pre-calibration, and the employment of components that can easily be set-up inside a standard cell incubator. An additional value of the MPS described herein is the possibility to design several highly reproducible GPPs to investigate specific aspects of PI function. As a validation model to evaluate the feasibility of our MPS, C-peptide secretion profiles of PIs derived from different donors (healthy, obese, and T2 DM) were analyzed for their ability to respond to (1) glucose in a concentration-dependent manner, (2) consecutive HG pulses, (3) an ascending ramp of glucose concentration, and 4) a constant perfusion of glucose. As expected, the C-peptide secretion profiles of PIs derived from healthy donors were characterized by the ability response to (a) different glucose concentrations, (b) consecutive pulses of high-glucose concentrations, (c) a glucose threshold ranging from 5-8 mM, and (d) a constant high-glucose perfusion in a biphasic manner. Such a profile recapitulates some features that have been previously reported for dynamic GSIS assays [15; 16; 17; 18]. When PIs derived from obese donors were evaluated, we found a dysregulated profile characterized by hyper-responsiveness to 28 mM but not to 17 mM of glucose, the failure to go back to basal concentration after KCl-induced depolarization, the failure to maintain glucose-dependent secretion after increasing glucose concentration, and a reduced amplitude of the first secretion phase. Of note, we detected further functional deteriorations when PIs derived from T2 DM donors were exposed to the GPPs. In particular, consistent with \(\beta\)-cell dysfunction they showed a lower amplitude in C-peptide secretion, a delayed glucose-response, and a virtually absent first phase of secretion. Similar results have been reported in models of obesity and T2 DM both in vitro and in vivo [19; 20; 21]. Since HbA1C levels were significantly higher in T2 DM compared to obese and healthy donors, it can be postulated that such dysfunctional response may reflect the impaired PI function driven by their long-lasting exposure to HG [22; 23]. In summary, despite the limited number of human samples analyzed, our MPS could be considered a useful tool to detect differences in the secretion profiles in clinically different donors. Hence, the results on human PIs validate the feasibility of our MPS to study \(\beta\)-cell function. Moreover, this device would also allow to investigate different endocrine cells, such as \(\alpha\)-cells, by simply perfusing stimulatory solutions containing lower glucose concentration (1-3 mM) or secretagogues. Recently, our lab has described a simple charge-based single-step protocol to differentiate human liver stem cells (HLSC) into islet-like structures [3]. These islet-like structures, not only express several genetic markers of \(\beta\)-cell differentiation, but were also glucose-responsive and, most importantly, were able to restore euglycemia in STZ-induced SCID diabetic mice when implanted at day 14 post-differentiation [3]. By using our MPS we were able to characterize the dynamic secretion profile of HLSC-ILS at day seven post-differentiation. The SPGP protocol indicates that HLSC-ILS were able to secrete C-peptide in a concentration-dependent manner when stimulated with 28 mM of glucose and 50 mM of KCl.