Long-term cell culture in microfluidic devices is an essential prerequisite for "on a chip" biological and physiological based studies. We investigated how medium delivery, from continuous to periodic perfusion, affects long-term cell cultures in a microfluidic platform. Computational simulations suggested that different delivery strategies result in different temporal profiles of accumulation and washing out of endogenous (EnF) and exogenous (ExF) factors, respectively. Thus, cultures exposed to the same overall amount of medium with different temporal profiles were analysed in terms of homogeneity, cell morphology and phenotype. Murine and human cell lines (C2C12 and HFF) and mouse embryonic stem cells (mESC) were cultured in microfluidic channels. An ad hoc experimental setup was developed to perform continuous and periodic medium delivery into the chip, tuning the flow rate, the perfusion time, and the interval of perfusion while using the same amount of medium volume. Periodic medium delivery with a short perfusion pulse ensured cell homogeneity compared to standard cell culture. Conversely, a continuous flow resulted in cell heterogeneity, with abnormal morphology and vesiculation. Only dramatic and unfeasible increasing of perfused medium volume in the continuous configuration could rescue normal cell behaviour. Consistent results were obtained for C2C12 and HFF. In order to extend these results to highly sensitive cells, mESC were cultured for 6 days in the microfluidic channels. Our analysis demonstrates that a periodic medium delivery with fast pulses (with a frequency of 4 times per day) resulted in a homogeneous cell culture in terms of cell viability, colony morphology and maintenance of pluripotency markers. According to experimental observations, the computational model provided a rational description of the perfusion strategies and of how they deeply shape the cell microenvironment in microfluidic cell cultures. These results provide new insight to define optimal strategies for homogeneous and robust long-term cell culture in microfluidic systems, an essential prerequisite for lab on chip cell-based applications.

Although obesity represents a risk factor for the development of type 2 diabetes mellitus (T2DM), the link between these pathological conditions is not so clear. The manner in which the different elements of adipose tissue (AT) interplay in order to grow has been suggested to have a role in the genesis of metabolic complications, but this has not yet been fully addressed in humans. Through IHC, transmission electron microscopy, cytometry, and in vitro cultures, we described the morphological and functional changes of subcutaneous and visceral AT (SAT and VAT) in normoglycemic, prediabetic and T2DM patients with obesity compared to lean subjects. In both SAT and VAT we measured a hypertrophic and hyperplastic expansion, causing similar vascular rarefaction in obese patients with different degrees of metabolic complications. Capillaries display dysfunctional basement membrane thickening only in T2DM patients evidencing VAT as a new target of T2DM microangiopathy. The largest increase in adipocyte size and decrease in adipose stem cell number and adipogenic potential occur both in T2DM and in prediabetes. We showed that SAT and VAT remodeling with stemness deficit is associated with early glucose metabolism impairment suggesting the benefit of an AT-target therapy controlling hypertrophy and hyperplasia already in prediabetic obese patients.

In mice and humans, reduced p66Shc levels protect from obesity, but not from ectopic fat accumulation, glucose intolerance and insulin resistance.

No correlation was found between the estimated volume of the remaining gastric fundus and weight loss (%EBL) after LSG. Patients showing a rapid gastroduodenal transit of the CM achieved a better weight loss than patients with a slow voiding rate.

A definitive relationship between adiposity and MP production is yet to be demonstrated. The aim of our study was to prospectively evaluate the levels of microparticles (MP) in a group of 20 III degree obese patients before and after weight loss. Plasma levels of annexin V-MP, endothelial-derived MP, platelet-derived MP (CD61+ and P-Selectin+), leukocyte-derived MP, tissue factor-bearing (TF+) and CD36+MP were prospectively measured in 20 patients with III degree obesity (BMI ≥ 40 kg/m(2)) before (T0) and 3 (T3) and 12 (T12) months after sleeve gastrectomy (SLG). Obese patients had lost 18 % of their body weight at T3 and 41 % at T12. We find that considering all MP, except for endothelial-derived MP, which had significantly decreased at T3, all MP subtypes had significantly decreased at T12. At T12, subjects showed a higher median level of all types of MP, except endothelial-derived MP, compared to T3, but without a statistically significant difference. The percentages of reduction of all the MP were significantly correlated with the percentage of reduction of BMI. The reductions of leukocyte-derived, TF+ and CD36+MP were significantly correlated with the reduction of leptin. Moreover, the reductions of leukocyte-derived and CD36+MP were significantly correlated with hs-CRP decrease. The decrease of BMI post-SLG in morbid obesity was matched with a decrease of circulating MP of endothelial, platelet, leukocyte origin, TF+ and CD36+. A trend of slight increase in all MP subtypes, except endothelial-derived, was detected 12 months after gastrectomy, indicating a possible underlying slow low-grade inflammatory/hypercoagulability state from adipose tissue before the potential overt weight gain.

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