Engineering pseudoislets for type 1 diabetes: GLP-1 gene modulation, gelma nanogel immunoisolation, and endothelial support

Type 1 diabetes arises from autoimmune destruction of pancreatic 𝛽-cells. This leads to absolute insulin deficiency and persistent hyperglycaemia. Exogenous insulin remains the standard of care, non-curative, and cannot replicate the dynamic physiology of endogenous 𝛽 cells. Islet transplantation offers an alternative. However, its broader adoption is constrained by scarce high quality donors and the need for lifelong systemic immunosuppression with associated risks. These constraints motivate continued pursuit of cell replacement strategies. In this thesis, an injectable pseudoislet is a three dimensional, self-assembled spheroid of insulin producing cells by using hanging drop, providing a scaffold free architecture. As the transplanted spheroids face immune rejection, they must be shielded from immune cell attack without compromising mass transport. Macroencapsulation commonly suffers from diffusion limits, and alginate microencapsulation adds bulk. In contrast, an ultrathin conformal nanogel coating could confer immunoisolation without compromising glucose, oxygen, or insulin diffusion. This nanobarrier also facilitates direct contact and integration to the host vasculature, which can enhance engraftment and reduce foreign-body responses. In this thesis, bovine gelatin was methacrylated to obtain gelatin methacryloyl (GelMA). GelMA is a well established biomaterial that preserves gelatin's RGD adhesion motifs and MMP-degradable sites, supporting cell attachment and remodeling. It is highly cytocompatible, readily functionalized via methacrylate groups, and tunable in stiffness. Unlike bulk or macroencapsulation, GelMA nanogels can assemble via electrostatic interactions and RGD mediated adhesions as an ultrathin, conformal barrier around individual spheroids. This configuration minimizes diffusion barriers for glucose and oxygen while providing an immunoisolating shield that helps protect the spheroid from immune attack. To improve spheroid viability and functional performance with emphasis on insulin secretion we implemented a glucagon-like peptide-1 (GLP-1) based genetic strategy in 𝛽TC-6 cells. Specifically, we engineered 𝛽TC-6 cells to express glucagon-like peptide-1 (GLP-1) via lentiviral transduction. This enables sustained, local (auto/paracrine) delivery of an incretin that potentiates glucose-stimulated insulin secretion (GSIS) while supporting 𝛽-cell survival. We co-cultured GLP-1 transduced 𝛽TC-6 cells and endothelial cells to make self-assembled spheroids using the hanging-drop method. We need to preserve endothelial–𝛽-cell crosstalk, which stabilizes the 𝛽-cell phenotype and primes constructs for vascular integration after implantation. The total active GLP-1 protein from transduced 𝛽-TC-6 cells was confirmed and showed increase in stimulation index. The overall design aim was that (i) engineering 𝛽-TC-6 cells with GLP-1 would improve insulin secretion, (ii) co-culture with endothelial cells would provide revascularization, and (iii) coating the spheroid with GelMA nanogels would support immunoisolation. As a result, functional enhancement and immunoprotection are brought together in one graft-ready spheroid.This thesis study contributes to the research field of type-1 diabetes treatment by engineering and improving insulin secreting co-cultured pseudoislets with immunoisolation functions.