3D Bioprinting of Insulin-Producing Cell Aggregates-Derived from Human Pluripotent Stem Cells with Pancreatic Tissue-Derived Bioink

Abstract

Native pancreatic islets are clustered with diverse endocrine cells as functional units and surrounded by microvasculature networks communicating with neighboring cells for releasing hormones in response to glucose stimulation. 3D bioprinting technology has emerged as an promising strategy for the fabrication of engineered pancreatic tissue constructs because it enables implementation of complex tissue microarchitecture by precise positioning of multiple cells within functional bioink. In this study, we fabricated a pancreatic tissue construct through 3D aggregate bioprinting method using tissue-derived bioink with stem cell-derived insulin producing cells (IPC) to replicate the structural and functional features of native human pancreatic tissue. Selection of suitable bioink is a first step towards building functional 3D pancreatic tissue constructs. In previous study, we suggested pancreatic tissue derived-decellularized extracellular matrix (pdECM) as a attractive printable material to recapitulate native pancreatic cell niche. Here, we investigated representative constituents of pdECM bioink through proteomic analysis to evaluate that pdECM bioink can provide sufficient microenviromental cues. Additionally, functional gene classification regarding matrix-mediated characteristics were observed via gene ontology (GO) analysis. Collagen type VI was most abundant protein in pdECM bioink and other crucial ECM proteins for cell-matrix interactions were also enriched compared to the collagen bioink. Furthermore, we differentiated human embryonic stem cells (hESC) into IPC via four-stage protocol to generate functional human pancreatic cells. The differentiated cells we obtained at each stage were characterized by gene expression profile and flow cytometry analysis. Key markers of beta cells including PDX1, NKX6.1, and insulin were highly expressed after stage 3. After generation of IPC, printing conditions regarding the size of IPC aggregates were optimized for mimicking native pancreatic tissue geometry. In addition, we demonstrated interaction capacity between IPC aggregates with co-culture condition using human umbilical vein endothelial cells (HUVEC) and human mesenchymal stem cells (hMSC). Immunofluorescent staining results of printed constructs revealed that rapid induction of IPC aggregates networks can occur in tri-culture condition. The developed 3D human pancreatic tissue constructs will be able to broaden the application of in vitro disease models of diabetes and transplantable constructs for in vivo study.