Circulation perfusion culture of insulin-producing cells using rat decellularized pancreatic scaffolds

Objective To cultivate mouse bone marrow mesenchymal stem cells (mBMSCs) drived insulin-producing cells (IPCs) in rat decellularized pancreatic scaffolds and evaluate the growth and function of cells. Methods Decellularized pancreatic scaffolds were obtained by continuous perfusion of detergent by the splenic artery. HE staining, Masson staining, DNA content, glycosaminoglycan (GAG) content, extracellular matrix (ECM), biological compatibility were adopted to evaluate the result. mBMSCs were differentiated into IPCs via infected by pancreatic and duodenal homeobox-1 (PDX-1), neurogenic differentiation-1 (NeuroD1) and V-mar musculoaponeurotic fibrosarcoma oncogene homologue A (MafA). Immunofluorescence and real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) were detected of insulin expression of the IPCs. The IPCs were exposed to a glucose gradient and insulin release was measured by enzyme linked immunosorbent assay (ELISA) assay. The IPCs were cultivated in the decellularized pancreatic scaffolds in the circulation perfusion device. Hematoxylin-eosin staining (HE) and immunofluorescence were implemented to evaluate the growth and function of cells. The insulin gene expression was compared with traditional culture by FQ-PCR. Results After perfused of detergent, HE staining and Masson staining demonstrated no residual cells remained and most of the collagen fiber components were well retained. DNA content of decellularized scaffolds was (44.92±3.89) ng/mg (t=15.160, P=0.001)and GAG content was (30.27±2.70) ng/mg, (t=2.862, P=0.046). Immunohistochemical staining showed collagen I and fibronectin were retained. Scaffolds were desirable biocompatibility. Immunofluorescence and FQ-PCR showed gene and protein expression of insulin after infected by PDX-1, NeuroD1 and MafA. The IPCs were glucose responsiveness to different glucose concentration according to ELISA assay. After cultivated in the decellularized pancreatic scaffolds, HE and immunofluorescence demostrated that the IPCs grew well in the scaffolds. FQ-PCR showed that insulin gene expression was increased compared with the traditional two-dimensional culture (t=15.030, P=0.004). Conclusion The rat decellularized pancreatic scaffolds not only preserved the ECM and structure of vascular, but also good biocompatibility. The IPCs differentiated from mBMSCs had the gene expression of insulin expression. The decellularized pancreatic scaffolds not only supported the growth and function of IPCs, but provided a favourable platform compared with the two-dimensional culture. Key words: Pancreas; Decellularized scaffolds; Diabetes; Tissue engineering