RECELLULARIZATION: GROWING PANCREATIC BETA CELLS ON Leaf SCAFFOLDS

RECELLULARIZATION: GROWING PANCREATIC BETA CELLS ON Leaf SCAFFOLDS Umair Masood Awan Tissue engineering seeks to overcome the donor organ shortage for diabetes treatment. We present a novel approach using decellularized plant scaffolds as a biological matrix for cultivating pancreatic beta cells. Spinach leaves were decellularized to create 3D cellulose scaffolds featuring a natural, perfusable vascular network. Upon seeding with insulin producing beta cells, these plant-based structures supported robust cell growth and, crucially, restored glucose-stimulated insulin secretion. The plant's intrinsic vascular design enabled efficient nutrient delivery, addressing a key challenge in engineering thick tissues. This work demonstrates that recellularized plant scaffolds provide a pro-functionality niche for beta cells, offering a scalable, inexpensive, and ethically favorable platform for regenerative medicine strategies in diabetes. Fig:1.1 Culturing pancreatic beta cells: Culturing pancreatic beta cells on plant-derived scaffolds utilizes decellularized plant tissues, such as spinach leaves or apple hypanthium, to provide a natural 3D microstructure of cellulose that mimics the in vivo extracellular matrix. The process begins with decellularizing the plant material using detergents to remove all cellular content, leaving a porous, biocompatible cellulose scaffold. This scaffold is then sterilized and coated with adhesion-promoting proteins like laminin or collagen. Isolated pancreatic beta cells or islets are seeded onto the scaffold and cultured in a specialized medium, often supplemented with glucose and growth factors, within a bioreactor. The bioreactor provides dynamic perfusion of nutrients and oxygen through the scaffold's innate vascular channels, promoting cell viability, function, and the formation of 3D insulin secreting clusters. A suitable culture medium for promoting the growth of pancreatic beta cells on a plant leaf scaffold is RPMI-1640 (Roswell Park Memorial Institute medium). This medium is commonly supplemented with 10% Fetal Bovine Serum (FBS), 11.1 mM glucose, 1% penicillin-streptomycin, and sometimes additional growth factors. The RPMI-1640 formulation provides the essential nutrients, vitamins, and amino acids necessary for beta cell survival and proliferation. When used with a decellularized leaf scaffold, which offers a natural 3D structure, this medium helps maintain beta cell viability and function, creating a supportive microenvironment that mimics aspects of the native pancreatic niche. Results: Based on the successful culture of pancreatic beta cells using a leaf scaffold, the viability and growth of the cells were confirmed using Calcein-AM staining. This fluorescent dye binds to metabolically active cells, producing a characteristic green fluorescence. As clearly observed in Figures 1.2 and 1.3, the presence of widespread green fluorescence across the leaf scaffold demonstrates that the beta cells were not only present but were also growing and thriving in this natural, three-dimensional environment. This successful outcome validates the leaf scaffold as an effective and promising substrate for supporting beta cell culture. Fig: 1.2 Fig: 1.3 References:  Gershlak, J. R., Hernandez, S., Fontana, G., Perreault, L. R., Hansen, K. J., Larson, S. A., Binder, B. Y., Dolivo, D. M., Yang, T., Dominko, T., Rolle, M. W., Weathers, P. J., MedinaBolivar, F., Cramer, C. L., Murphy, W. L., & Gaudette, G. R. (2017). Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds. Biomaterials, *125*, 68–78.  Modulevsky, D. J., Lefebvre, C., Haase, K., Al-Rekabi, Z., & Pelling, A. E. (2014). Apple derived cellulose scaffolds for 3D mammalian cell culture. PLoS ONE, *9*(5), e97835.  Shapiro, A. M. J., Pokrywczynska, M., & Ricordi, C. (2017). Clinical pancreatic islet transplantation. Nature Reviews Endocrinology, *13*(5), 268–277.  Shapiro, A. M. J., Pokrywczynska, M., & Ricordi, C. (2017). Clinical pancreatic islet transplantation. Nature Reviews Endocrinology, *13*(5), 268–277.