## 4 Discussion
With the DPG protocol, we demonstrated that HLSC-ILS were also able to secrete the C-peptide in response to consecutive pulses of 17 mM of glucose and to return to the basal state detected during pre-stimulation in the presence of low glucose. Both the kinetics of secretion and the SIs were comparable to those of PIs derived from healthy donors (being the SIs \(\geq\) 2). For a more detailed characterization, two additional perfusion protocols were carried out. The ARG protocol revealed that the glucose threshold for C-peptide secretion in HLSC-ILS occurred earlier (4 mM) than in PIs from healthy donors (5-8 mM). Additional differences were observed between the secretion profiles of healthy PIs and HLSC-ILS by using the CHG protocol. We found that HLSC-ILS displayed a robust first phase of secretion (SI-5) followed by an oscillatory second phase, characterized by peaks of secretion every 15 min. We hypothesize that such oscillations in the second phase of secretion were likely causedby a lack of enough \"reserve pool\" of secretory granules [19]. These data confirm the fairly immature secretion profile observed before undergoing further maturation after transplantation in diabetic mice [3]. Future studies will be performed to investigate the possibility to induce in vitro maturation of HLSC-ILS by using different culture media or adding differentiation factors [24]. Furthermore, our data provides evidence that the MPS we propose is a valuable device to assess \(\beta\)-cell function, and a useful tool to detect features of the \(\beta\)-cell secretion profile, otherwise missing in static conditions. Finally, the novelty of our MPS could be summarized as follows: (a) it is a more versatile, standardized, and less expensive microfluidic perfusion system, which is entirely based on commercially available components; (b) it allows the evaluation of several dynamic aspects of \(\beta\)-cell functionality; (c) it requires a low number of PIs (10 IEQ); (d) it could generate low flow rates (30 uL/min), which avoids sample dilution and limits the detrimental effects of shear stress on PIs, thanks to the combination of a pressure- and flow-regulator platform. Supplementary materials can be found at [[http://www.mdpi.com/2227-9059/8/2/26/s1](http://www.mdpi.com/2227-9059/8/2/26/s1)]([http://www.mdpi.com/2227-9059/8/2/26/s1](http://www.mdpi.com/2227-9059/8/2/26/s1)). [PERSON], [PERSON], [PERSON], [PERSON] and [PERSON] designed the research; [PERSON], performed the research; [PERSON], collected the data; [PERSON], and [PERSON] analyzed and interpreted the data; [PERSON], performed the statistical analysis; All authors contributed to writing the manuscript. All authors have read and agreed to the published version of the manuscript. **Funding:** This work was supported by a grant Unicyte Islet project no. 2019 (Oberdorf NW, Switzerland). **Acknowledgments:** We thank [PERSON] (University of Turin) for his guidance and advice to set-up the MPS. **Conflicts of Interest:** [PERSON] and [PERSON] are employed by a commercial company (Unicyte AG) and contributed to the study as researcher. [PERSON], [PERSON], [PERSON], and [PERSON] are named inventors in related patents. [PERSON] is a component of the scientific advisory board of Unicyte AG. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.