Development of microfluidic cell culture technology for the study of type 2 diabetes

Autor: Zambon, Alessandro
Rok vydání: 2013
Předmět:
Popis: Type 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.
Databáze: OpenAIRE
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