1,721,032 research outputs found
Development of microfluidic cell culture technology for the study of type 2 diabetes
Full text linkType 2 Diabetes Mellitus (T2DM) is a multi-factorial disease due to metabolic disorder with injuries in glucose homeostasis and body’s glucose uptake. The complexity of this disease led to the use of different classes of drugs acting with different mechanisms and targets and with effects that often change between patients.
The number of people in the world with diagnosed T2DM is constantly increasing and consequently the cost for healthcare. Nowadays, a defined cure for T2DM patients has been not clearly identified.
In the study of diabetes, animal models are one of the most popular systems used to underline its pathogenesis and to screen new drugs before clinical trials on humans. Even though their undeniable utility, they showed many limitations. Moreover, studies in vivo in humans are possible but tremendously expensive and require a huge effort in terms of ethical approval and safety issues. Therefore in vivo studies often do not permit an evaluation at specific tissue level: their interplay complexity allow a very difficult outcome interpretation. For all these reasons there is a great interest in developing alternative in vitro models that facilitate pharmaceutical and pathology studies.
Thus, the aim of this thesis is the development of an in vitro model that closely resemble the human physiology and mimic the pathophysiological conditions of type 2 diabetes. In particular, this work concerns the design and development of microfluidic technology for the study of insulin resistance and glucose uptake in cell and tissue culture from Type 2 Diabetes patients. High temporal resolution glucose uptake measurements were achieved by coupling microfluidic technologies and glucose detection measurements with a non invasive manner. The technology was applied to skeletal muscle and ex vivo adipose tissue, with the obtainment of high sensitive and reproducible experiments.
During this PhD, a microfluidic platform was developed and fabricated with multilayer soft lithography techniques. The platform was able to integrate 2D (cells) and 3D (ex vivo tissue) culture allowing long term viability and metabolic activity. High experiment feasibility was achieved by the long term culture capability.
Micro components were included into the device allowing automation and liquid handling control. Integrated microvalves and micropumps allowed the development of injection systems for high spatio temporal control of biochemical stimulus delivery, such as insulin and other anti-diabetic drugs.
Glucose uptake was investigated measuring high temporal resolution glucose concentration in the downstream culture chamber medium by high sensitive analytical measurements on nanoliter sampling, providing glucose dynamic with temporal resolution of minutes.
The measurement of intracellular glucose concentration was evaluated by encoded FRET nanosensor. The coupling between intracellular and extracellular glucose detection allowed the determination of novel glucose uptake and glycolytic rate evaluation technique within the cell.
These results show a good potential in future pharmaceutical and clinical experimentation, in which the use of a microfluidic ex vivo human patient assays could be useful in drug screening studies and patient specific therapies
Microfluidic technology for multi-parametric studies on patient-derived three-dimensional human adipose tissue model
No full textBackground 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
Determination of glucose metabolic fluxes in live myoblasts by microfluidic nanosensing and data analysis
No full textA Study about the Effects of Supercritical Carbon Dioxide Drying on Apple Pieces
Full text linkThe work explores the feasibility of supercritical carbon dioxide (SC-CO2) as alternative drying food process. Experiments carried out with apple pieces at 10 MPa and 35°C, aimed to investigate the effect of the drying time (5÷120 min) and the addition of a pre-dehydration step in graded ethanol (EtOH) solutions on the dehydration of the final product in terms of weight loss and structure. The treatment with pure EtOH leaded to a weight reduction of 50.0 ± 0.2 % after 120 min while SC-CO2 induced a reduction down to 18.53±1.57 % after the same drying time. Significant improvements in terms of weight loss were obtained when apples were first dehydrated in pure EtOH for 40 min, and subsequently in SC-CO2 for 10 min reaching a final reduction of 11.67±2.55%. Color measurements indicated that both SC-CO2 and EtOH-SC-CO2 drying treatments induced a significant increase of L*, while an increase of a* and b* parameters was detected only for SC-CO2 dried apples. The overall changes in color ΔΕ did not shown significant differences between the two dried samples. SEM images indicated that the EtOH-SC-CO2 dried apples presented more shrinking and a wider pore distribution compared to the SC-CO2 dried ones. EtOH-SC-CO2 drying confirmed the potential to apply the technology for drying apple slices in short time
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