Development of scaffold-free vascularized pancreatic beta-islets in vitro models by the anchoring of cell lines to a bioligand-functionalized gelatine substrate

Abstract

Bioengineered pancreatic β-islets have been widely advocated for the research and treatment of diabetes by offering both suitable cell culture models for the study of the pathology and the testing of new drugs and a therapy in those patients no longer responding to insulin administration and as an alternative to the shortage of donors for organ and islet transplantation. Unlike most of the studies published so far where pancreatic islets of pancreatic β-cells are encapsulated in hydrogels, this study demonstrate the formation of bioengineered pancreatic islets through cell anchoring to a gelatine-based biomaterial, PhenoDrive-Y, able to mimic the basement membrane of tissues. Through simple culture conditions, PhenoDrive-Y led human pancreatic β-cell lines and human umbilical endothelial cell lines to form organized structures closely resembling the natural vascularized pancreatic islets. When compared to gelatine, the cultures in presence of PhenoDrive-Y show higher degree of organization in tissue-like structures, a more pronounced endothelial sprouting and higher expression of typical cell markers. Noticeably, when challenged by hyperglycaemic conditions, the cells embedded in the PhenoDrive-Y assembled spheroids responded with higher levels of insulin production. In conclusion, the present work demonstrates the potential of PhenoDrive-Y as substrate for the development of bioengineered vascularized pancreatic islets and to be particularly suitable as a model for in vitro studies and testing of new therapeutics. Graphical abstract.

Graphical abstract

Fig. 1

Morphological analysis of the forming human pancreatic β-1.1B4 cell lines and HUVEC constructs after 2 h incubation with gelatine (A, C) and gelatine-based PhenoDrive-Y (B–D). Arrows highlight forming endothelial sprouting. A, B = × 10 objective lens, C, D = ×20 objective lens

Fig. 2

Morphological and morphometric analysis of the forming cell human pancreatic β-1.1B4 cell lines and HUVEC constructs after 24 h incubation with gelatine (A), gelatine-based PhenoDrive-Y (B), morphometry of engineered islets (C). Arrows highlight forming β-islet-like structures. Microscopy images were taken at ×20

Fig. 3

Immunostaining of the human pancreatic β-1.1B4 cell lines and HUVEC constructs for PDX-1⁺ human β-cells in co-culture obtained in presence of gelatine (A, C) and gelatine-based PhenoDrive-Y (B, D) at normo-glycaemic (A, B) and hyperglycaemic (C, D) conditions. Microscopy images were taken at ×10

Fig. 4

Immunostaining of the human pancreatic β-1.1B4 cell lines and HUVEC constructs for intracellular insulin synthesis when in normo-glycaemic and hyperglycaemic conditions. A Quantification of the immunostaining fluorescence, C gelatine constructs, B, D gelatine-based PhenoDrive-Y constructs. Microscopy images were taken at ×20 objective lens

Fig. 5

Immunostaining of the human pancreatic β-1.1B4 cell lines and HUVEC constructs for Connexin-43 expression in normo-glycaemic (A, B) and hyperglycaemic conditions (C, D). Gelatine constructs (A, C), gelatine-based PhenoDrive-Y constructs (B, D). Microscopy images were taken at ×20 objective lens

Fig. 6

Confocal analysis of the human pancreatic β-1.1B4 cell lines and HUVEC constructs obtained by gelatine-based PhenoDrive-Y. Merged images of CD31⁺ endothelial cells and the PDX-1⁺ human β-cells (A) and single analysis of PDX-1⁺ (green-fluorescent staining, B) and CD31⁺ (red-fluorescent staining, C) cells. Microscopy images were taken at ×40 objective lens

Fig. 7

Z-stack confocal analysis of the human pancreatic β-1.1B4 cell lines and HUVEC constructs obtained by gelatine-based PhenoDrive-Y. Bright light microscopy (A) and single analysis of CD31⁺ (red-fluorescent staining, B) and PDX-1⁺ (green-fluorescent staining, C) cells. Microscopy images were taken at ×40 objective lens