Synthesis Of PLGA Porous Microspheres And Their Application In The Construction Of Artificial Pancreatic Islet

Diabetes is a chronic disorder that threatens the overall health and well-being of people worldwide due to its high incidence,challenging curability,and inclination for triggering severe chronic complications.Clinical islet transplantation has been considered the most promising antidiabetic approaches offering the possibility for complete diabetes recovery,confronts significant limitations in its clinical application such as the shortage of islet donors and low islet isolation efficiency.In recent years,as one of excellent polymer microcarriers,porous microspheres(PMs)have garnered extensive attention in tissue engineering either as injectable modularized units or PMsconstituted scaffold-supported microtissues owing to PMs’ excellent cell loading capacity.Various cell loading approaches using PMs have been developed for tissue regeneration,however,construction efficiency remains limited regarding organoids,especially vascularized organoids/microtissues,that require input of multiple cell types.Thus,it remains a need to enhance both the cell seeding efficiency and the clinical feasibility of the PMs-based approach.Furthermore,despite it exists structural similarity between PMs and islets,PMs-based multicellular bioartificial pancreatic islets construction has been scarcely reported.Overall,the development of the technology platform based on centrifugation-based cell seeding technique and PMs holds immense importance.The research in this dissertation was partitioned into two parts as delineated below:1.Poly(lactic-co-glycolic acid)(PLGA)PMs were prepared using double emulsionsolvent evaporation method.The surface morphology,pore structure,and mechanical properties of the as-prepared PMs were characterized systematically.The results showed that PMs exhibit regular spherical shape and uniform pores,characterized by interconnected interior and exterior pore structure.The size of PMs was in accordance with the optimal injectable microcarriers and islet models,allowing for efficient diffusion of oxygen and nutrients.Simultaneously,the PMs possess the interior nido-like pore structure that is conducive to the retention of seeding cells in cell perfusion technology.The in vitro degradation results showed that the mass,molecular weight,and glass transition temperature(Tg)of the PLGA PMs decrease along with the degradation time.Additionally,the surface skeleton of the PMs exhibited cracking and dissolution gradually,and the PMs’ weight loss rate reached 30%after 10 weeks of degradation,demonstrating favorable in vitro biodegradability.Investigation of in vivo implantation demonstrated that PLGA PMs exhibited prosperous mechanical properties and biocompatibility.2.After verifying the feasibility of centrifugal perfusion technology for seeding pancreatic islet cells on PMs using MS1(a mouse pancreatic islet endothelial cell line)and MIN6(a mouse insulinoma cell line)cells respectively in vitro,in this study,MIN6 and MS1 cells were co-seeded into PLGA PMs via an upgraded centrifugation-based cell perfusion seeding technique for construction of PLGA PM-based islets,which demonstrated the desirable cell distribution,viability,and proliferation in the PMs.Otherwise,addition of the MS1 cells enhance the glucose-responsiveness of MIN6 cells.Compared to the MS1+MIN6 group(2D-culturing),PMs’ 3D-culturing environment enhanced MIN6’s glucose-stimulated index which represents glucose-stimulated insulin secretion capability in the presence of MS1.The RNA-seq results showed that compared to the 2D-cultured MS1+MIN6 group and primary pancreatic islets,PM-loaded MS 1+MIN6 cells upregulated vascular-related genes,suggesting that the 3D-cultured environment of PMs may enhance MS1 vascularization thus improving insulin-secreting function of MIN6 cells.Further exploration had been made applying centrifugation perfusion on primary islet cells,and the islet cells were evenly distributed in the PMs.Despite PM-loaded islet cells had poor insulin-secreting function.PLGA PM-based artificial pancreatic islets might provide an alternative strategy for potential treatment of diabetes in the future.