Microfluidic technology for multi-parametric studies on patient-derived three-dimensional human adipose tissue model

Background and aims: Type 2 Diabetes Mellitus is a complex disease affecting many pathways in different tissues. The complexity of this disease led to the use of several classes of drugs acting with different mechanisms and targets and with effects which often change between patients. The screening of all these anti-diabetic drugs with animal models is not economically and timing sustainable and often not giving reliable results for human. On the other hand, specific study on human patients are possible but are tremendously expensive and require a huge effort in term of ethical approval and safety issues. Within this scenario we aim at developing a microfluidic platform allowing to perform in vitro highthroughput patient-specific tests of anti-diabetic drugs on patient-derived three-dimensional human adipose tissue. In particular, the first step is the realization of a microfluidic system for culturing human adipose tissue able to control the temporal evolution of culture conditions in terms of concentration of oxygen, metabolites, and insulin and able to perform multi-parametric analyses of the adipose tissue behaviour. Materials and methods: Biopsies of subcutaneous and visceral adipose tissues were obtained from both patients affected by Type 2 Diabetes and insulin-sensitive individuals. 1cm3 biopsy was minced right after surgery into 10-20mg tissues. Each piece was placed in a 24well plate with 1ml medium for 24h. Then the tissue was either cultured for additional time in the 24well plate with fresh medium or placed into the microfluidic system. A microfluidic platform including micro-valves, injectors, pumps, mixers was realized by soft-lithographic technique and its design, development, and application was assisted by mathematical modeling. In line measurements of tissue metabolic activity were performed using micro-biosensors placed downstream the culture chambers and able to detect glucose, lactate and oxygen concentration. The tissue responses to insulin were investigated also through analyses of free fatty acids and glycerol. Viability and histological analyses were performed at the end of the cultures. Results: Microscale adipose tissues were cultured within the microfluidic platform for up to 4 days. MTT assay at the end of the culture showed high tissue viability and no significant differences with controls in 24well plates. On the other end, the microfluidic system allowed a two times higher glucose uptake then the controls by reducing the glucose diffusive resistance. We then investigated the effect of different insulin concentrations (20, 40 and 100nM). Preliminary results obtained with tissues of insulin-sensitive individuals showed an high variability between biopsies and between cultures from the same biopsy. However, we observed an enhancement of glucose uptake for increasing insulin concentration when using 25mM glucose medium. We also investigated the difference on glucose uptake between insulin-sensitive individuals and patients affected by Type 2 Diabetes. Conclusion: We developed a microfluidic platform for culturing small-scale human adipose tissue and allowing to accurately control the temporal evolution of the culture conditions in terms of concentration of metabolites, oxygen, and insulin concentration. This system with in line biosensors open important perspectives towards the realization of high-throughput dynamic screening of anti-diabetic drugs on human adipose tissue