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Multiscale modeling of three-dimensional cell cultures for type 2 diabetes studies
Full text linkThe pressing needs related to the processes of drug and therapy development for diseases such as type 2 diabetes, have increased the demand for new technologies and protocols. In vitro models are extremely helpful for physiological and drug screening studies; however they are time consuming and expensive.
A theoretical approach through mathematical modeling could help in the understanding of the biology of the system and to rationalize these experiments.
The aim of this thesis has been the development of a multiscale and multidisciplinary approach to model three-dimensional cell and tissue culture.
The final application was the rationalization of an ex vivo model of human adipose tissue to characterize the pathophysiological conditions of type 2 diabetes mellitus.
Mathematical models were developed for describing cell culture in dynamic systems, aiming to analyze the effect of macroscopic variables on cell microenvironment properties and therefore on their fate.
We investigated the role of experimental conditions like the flow rate and the culture chamber configuration on cell proliferation, and we tested the efficiency of a discontinuous management of cell cultures in a microfluidic chip.
In addition, in a three-dimensional perfusion bioreactor, we studied the effect of heterogeneous culture conditions (within porous scaffolds) in the cell microenvironment on the cell growth. The model linked the macroscopic variables to the cell microenvironment properties predicting cell growth as a function of the experimental conditions and the scaffold pore size distribution.
The effect of endogenous and exogenous factors on the intracellular processes was also investigated. In particular, we coupled the mass transport model to the insulin signaling pathways model, studying the influence of this hormone on the cell glucose metabolism.
We finally proposed a mathematical model for the ex vivo human adipose tissue culture in a microfluidic platform, supporting the design and realization of the device itself. This system has been used for studying the tissue response to an insulin stimulation and the pathophysiological conditions of type 2 diabetes.
These results show interesting applications for the experimental design and optimization of culture conditions, and they mark a step towards an efficient development of drug tests or therapies.Le forti necessità legate allo sviluppo di farmaci e terapie per malattie quali il diabete di tipo 2, hanno portato alla crescente domanda di nuove tecnologie e protocolli. Modelli in vitro sono estremamente utili per studi fisiologici e screening farmacologici; tuttavia questi sono dispendiosi e richiedono molto tempo.
Un approccio teorico attraverso la modellazione matematica può facilitare lo studio della biologia del sistema e aiutare nella razionalizzazione degli esperimenti.
Lo scopo di questa tesi è stato lo sviluppo di un approccio multi-scala e multi-disciplinare per modellare colture tridimensionali cellulari e di tessuto.
L’applicazione finale ha previsto la razionalizzazione di un modello ex vivo di tessuto adiposo umano con lo scopo di caratterizzare le condizioni fisiopatologiche del diabete di tipo 2.
Modelli matematici sono stati sviluppati per descrivere colture cellulari in sistemi dinamici, al fine di analizzare l’effetto di variabili macroscopiche sulle proprietà del micro-ambiente e quindi sull’evoluzione cellulare.
Abbiamo investigato il ruolo delle condizioni sperimentali, come ad esempio la portata e la configurazione della camera di coltura, e testato l’efficienza di una gestione discontinua delle colture cellulari in piattaforme microfluidiche.
Inoltre, nel caso di un bioreattore a perfusione per colture cellulari tridimensionali, abbiamo studiato l’effetto di condizioni di coltura eterogenee nel micro-ambiente cellulare (a causa dello scaffold poroso) sulla crescita cellulare. Il modello relaziona le variabili macroscopiche alle proprietà del micro-ambiente cellulare predicendo la crescita in funzione delle condizioni sperimentali e della distribuzione della dimensione dei pori dello scaffold.
In aggiunta è stato analizzato l’effetto di fattori endogeni ed esogeni sui processi intra-cellulari. In particolare, abbiamo integrato il modello di trasporto di materia con il modello per il signaling dell’insulina, studiando l’influenza di questo ormone sul consumo cellulare di glucosio.
Infine, è stato proposto un modello matematico per descrivere la coltura ex vivo di tessuto adiposo umano in una piattaforma microfluidica per assistere nella fase di progettazione e realizzazione della piattaforma stessa. Questo sistema è stato utilizzato per studiare la risposta del tessuto sottoposto ad uno stimolo di insulina e le condizioni fisiopatologiche del diabete di tipo 2.
Questi risultati hanno interessanti risvolti applicativi per la progettazione di esperimenti e l’ottimizzazione delle condizioni di coltura, segnando un passo in avanti verso lo sviluppo di terapie e tests farmacologici
Application of a spatio-temporal mathematical model of cell growth in 3D scaffold with a pore size distribution
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Spatio-temporal mathematical model of multiple substrate dependent growth of mammalian cell within 3D scaffold
No full textSpatio-temporal mathematical model of cell growth in 3D scaffold with a pore size distribution
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Theoretical analysis of insulin-dependent glucose uptake heterogeneity in 3D bioreactor cell culture
No full textThree-dimensional (3D) cell cultures in bioreactors are becoming relevant as models for biological and physiological in vitro studies. In such systems, mathematical models can assist the experiment design that links the macroscopic properties to single-cell responses. We investigated the relationship between biochemical stimuli and cell response within a 3D cell culture in scaffold with heterogeneous porosity. Specifically, we studied the effect of insulin on the local glucose metabolism as a function of 3D pore size distribution. The multiscale mathematical model combines the mass transport within a 3D scaffold and a signaling pathways model. It considers the scaffold heterogeneity, and it describes spatiotemporal concentration of metabolites, biochemical stimuli, and cell density. The signaling model was integrated into this model, linking the local insulin concentration at cell membrane to the glucose uptake rate through glucose transporter type 4 (GLUT4) translocation from the cytosol to the cell membrane. The integrated model determines the cell response heterogeneities in a single channel, hence the biological response distribution in a 3D system. It also provides macroscopic outcomes to evaluate the feasibility of an experimental measurement of the system response. From our analysis, it became apparent that the flow rate is the most important operative variable, and that an optimum value ensures a fast and detectable cell response. This model on insulin-dependent glucose consumption rate offers insight into the cell metabolism physiology, which is a fundamental requirement for the study metabolic disorder such as Type 2 diabetes mellitus, in which the physiological insulin-dependent glucose metabolism is impaired
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
Cell culture distribution in a three-dimensional porous scaffold in perfusion bioreactor
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