Maintenance of Mouse, Rat, and Pig Pancreatic Islet Functions by Coculture with Human Islet-Derived Fibroblasts

Abstract

Development of an efficient preculture system of islets is ideal. Toward that goal, we constructed a human pancreatic islet-derived fibroblast cell line MNNK-1 for a source as a coculture system for freshly isolated islets to maintain islet functions. Human pancreatic islet cells were nucleofected with a plasmid vector pYK-1 expressing simian virus 40 large T antigen gene (SV40T) and hygromycin resistance gene (HygroR). One of the transduced cell lines, MNNK-1, was established and served as a feeder cell in the coculture for freshly isolated mouse, rat, and pig islets. Morphology, viability, and glucose-responding insulin secretion were analyzed in the coculture system. MNNK-1 cells were morphologically spindle shaped and were negative for pancreatic endocrine markers. MNNK-1 cells were positive for α-smooth muscle actin and collagen type I and produced fibroblast growth factor. Coculture of the mouse, rat, and pig islets with MNNK-1 cells maintained their viability and insulin secretion with glucose responsiveness. A human pancreatic isletderived fibroblast cell line MNNK-1 was established. MNNK-1 cells were a useful means for maintaining morphology and insulin secretion of islets in the coculture system. Key words: Islets; Simian virus 40 large T antigen; Coculture INTRODUCTIONtransplant program through a collaborative relationship with a remote islet isolation center (4,12). The advantage of islet culture before transplantation Since the year 2000, a dramatic turnaround has been made in the field of clinical islet transplantation by the is to decrease total volume of islet mass, to allow adequate assessment of transplantable islets and preparation introduction of the Edmonton Protocol. International multicenter trials of the Edmonton Protocol demonstrate of the patient with immunosuppressive agents. One of the major drawbacks of preculture of islets is a decrease successful replication. The success of the Edmonton protocol has spurred expectations and interests about isin viability and transplantable islet number. Thus, the development of an efficient preculture system of freshly let transplantation, raising people's awareness that islet transplantation is an ideal therapy for patients with type isolated pancreatic islets is one of the important issues to facilitate islet transplantation. 1 diabetes mellitus (33). Current developments in islet transplantation include: 1) islet culture prior to transToward that goal, we have focused on the use of coculture system of islets with pancreatic islet-derived fiplantation (preculture), 2) pancreas shipment using twolayer method, 3) islet infusion using a bag instead of a broblasts, because heterotypic cell interactions between parenchymal cells and nonparenchymal neighbors have syringe (3), 4) achievement of a single donor islet transplant success (15,16) Immunochemical staining for α-SMA and collagen type 1 was carried out using an ENVISION+ Kit/HRP Here we present the effect of MNNK-1 cells on islet functions in the coculture system. Because of the diffi-(DAB) (Dak...

Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft

“…Fibroblasts can also improve islet cell viability. The essential role of fibroblasts in islet physiological competence has previously been reported, and it was shown that some fibroblast-produced factors can promote islet survival in culture (35,36). In addition, availability of syngeneic or autologous fibroblasts eliminates the risk of immune response against these cells.…”

“…Here, we engineered a three-dimensional composite islet allograft equipped with IDO-expressing fibroblasts and examined whether local expression of IDO, conferred by adenoviral-mediated gene transfer to bystander syngeniec fibroblasts, prevents the rejection of islet allograft. Our approach here is novel compared with other studies that examined the suppressive effect of IDO in islet transplantation (28,29) because 1 ) bystander syngeneic fibroblasts were used as the target of gene transfer instead of islets to avoid deleterious effects of adenovirus infection on islets (30–32), 2 ) islets were embedded within an extracellular matrix that by itself improves islet function and viability (33,34), and 3 ) cotransplanted fibroblasts are more than just a source of IDO and can enhance islet physiological competence (35,36). …”

Mouse Pancreatic Islets Are Resistant to Indoleamine 2,3 Dioxygenase-Induced General Control Nonderepressible-2 Kinase Stress Pathway and Maintain Normal Viability and Function

“…51 Miki et al recently showed that coculture of the mouse, rat, and pig islets with isletderived fibroblasts maintained islet viability, insulin secretion and glucose responsiveness. 52 Cocultured fibroblast would not cause any concern in terms of overproliferation since it was shown that fibroblasts in floating collagen matrices have low levels of DNA synthesis and become quiescent. [53][54][55] Thus, bystander fibroblasts will not have any negative impact on islet viability and function.…”

Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells

“…3 Therefore, different strategies were investigated for improving the results of experimental and clinical islet isolation. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 Among these attempts, the antiapoptotic and protective effects of some compounds, functioning as cytoprotectives, such as nicotinamide, 3 lisofylline, 5 exendine-4, 6 interleukin (IL)4, 10 prolactin 12 and protein kinase C epsilon, 13 were examined on islet cells in vivo and/or in vitro . In other studies, many approaches of cellular application were used.…”