Diabetes is a metabolic disease characterized by hyperglycemia caused by lack of insulin secretion from the pancreatic β-cells or impaired sensitivity to insulin. The goal of conventional therapies for patients with type 1 diabetes is to maintain glucose homeostasis and prevent long term complications associated with the disease. This is possible through the administration of exogenous insulin and monitoring the glycemia. Instead, transplantation therapies have several drawbacks. Scarce supplies of pancreases and the need for chronic immunosuppressive treatments limit the pancreatic islet transplantation to more liable diabetic patients with the incapacity to stabilize the disease with intensive insulin treatment, pumps and/or glucose monitoring therapies. Transplantation of immunoisolated pancreatic islets would spread the safe and minimally invasive procedure to a larger group of type 1 patients. Conformal coating allows the encapsulation of single pancreatic islets in a polymeric membrane that conforms to the shape and size of the islets, resulting in a small coating uniform in thickness. The membrane allows small molecules like glucose, oxygen, and nutrients to enter, and insulin and metabolic waste to exit. However, it does not allow the passage of immune cells and antibodies, preventing autoimmune rejection (immunoisolation). The present work shows how a soft lithographic microfluidic device can be exploited to obtain conformally coated islets-like cell clusters in a PEG-MAL membrane. The use of such devices allows for a high control over the capsule properties, still maintaining lower costs, related to reagents volumes and production process, compared to the macrodevices’. The in vitro assays assess that the obtained conformal coating does not affect the viability of the cells whilst permits the physiological insulin release in response to glucose stimulation. Overall, the results show how it is possible to coat cell clusters in a conformal way that is able to adapt to the non-homogeneous surface and completely immunoisolate the cells, which remain viable and functional by keeping a proper insulin-glucose kinetics.
Il diabete è una malattia cronica caratterizzata da un alterato metabolismo dei carboidrati, grassi e proteine causata dalla mancanza di secrezione di insulina dalle cellule pancreatiche o da una diminuzione della sensitività dei tessuti all’insulina. Il principale obiettivo delle terapie convenzionali per il diabete di tipo 1 è quello di mantenere l’omeostasi e prevenire complicanze legate alla patologia. Questo avviene grazie alla somministrazione di insulina esogena e il continuo controllo della glicemia, concentrazione di glucosio presente nel sangue. Sono presenti delle terapie che si basano sul trapianto dell’intero pancreas o delle sole isole pancreatiche, ma presentano diverse problematiche. La scarsa disponibilità di organi e la necessità di somministrare farmaci immunosoppressivi limita l’utilizzo di queste terapie a pazienti per cui non è possibile tenere sotto controllo la malattia solamente tramite l’utilizzo di microinfusori e/o sensori continui per la glicemia. La possibilità di trapianto di isole pancratiche immunoisolate potrebbe portare alla diffusione della procedura ad un numero maggiore di pazienti diabetici. La tecnica di conformal coating (CC) permette l’incapsulamento di singole isole pancreatiche in una membrana polimerica completamente adattata alla forma e dimensione delle isole stesse; il risultato è una membrana con spessore uniforme e costante intorno ai 20-30 μm. Le capsule permettono la diffusione di molecole quali glucosio, ossigeno ed insulina, ma non permettono il passaggio alle cellule immunitarie e anticorpi del paziente, evitando il rigetto del materiale trapiantato (imunoisolamento). Il seguente lavoro di tesi volge a dimostrate come un dispositivo microfluidico ottenuto tramite soft litografia possa essere utilizzato per ottenere dei cluster cellulari incapsulati in una membrana conformale realizzata in PEG-MAL. L’utilizzo di questo dispositivo permette di avere il controllo sulle proprietà delle capsule, mantenendo i costi relativi a volumi di reagenti e processo di produzione, minori rispetto ai dispositivi ad oggi utilizzati. I test in vitro dimostrano come il processo di incapsulamento non ha effetti negativi sulla vitalità cellulare e al contempo permette un rilascio fisiologico di insulina in risposta alla stimolazione tramite glucosio. In conclusione, i risultati mostrano come sia possibile ottenere una membrana polimerica che si adatta completamente alla superficie non omogenea di cluster cellulari, permettendone l’immunoioslamento e il mantenimento di vitalità e funzionalità.
Design and development of a flow focusing microfluidic device for the encapsulation of pancreatic islets
Valente Perrone, Annelisa
2020/2021
Abstract
Diabetes is a metabolic disease characterized by hyperglycemia caused by lack of insulin secretion from the pancreatic β-cells or impaired sensitivity to insulin. The goal of conventional therapies for patients with type 1 diabetes is to maintain glucose homeostasis and prevent long term complications associated with the disease. This is possible through the administration of exogenous insulin and monitoring the glycemia. Instead, transplantation therapies have several drawbacks. Scarce supplies of pancreases and the need for chronic immunosuppressive treatments limit the pancreatic islet transplantation to more liable diabetic patients with the incapacity to stabilize the disease with intensive insulin treatment, pumps and/or glucose monitoring therapies. Transplantation of immunoisolated pancreatic islets would spread the safe and minimally invasive procedure to a larger group of type 1 patients. Conformal coating allows the encapsulation of single pancreatic islets in a polymeric membrane that conforms to the shape and size of the islets, resulting in a small coating uniform in thickness. The membrane allows small molecules like glucose, oxygen, and nutrients to enter, and insulin and metabolic waste to exit. However, it does not allow the passage of immune cells and antibodies, preventing autoimmune rejection (immunoisolation). The present work shows how a soft lithographic microfluidic device can be exploited to obtain conformally coated islets-like cell clusters in a PEG-MAL membrane. The use of such devices allows for a high control over the capsule properties, still maintaining lower costs, related to reagents volumes and production process, compared to the macrodevices’. The in vitro assays assess that the obtained conformal coating does not affect the viability of the cells whilst permits the physiological insulin release in response to glucose stimulation. Overall, the results show how it is possible to coat cell clusters in a conformal way that is able to adapt to the non-homogeneous surface and completely immunoisolate the cells, which remain viable and functional by keeping a proper insulin-glucose kinetics.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/188471