In Vitro Maturation of Viable Islets from Partially Digested Young Pig Pancreas

Abstract

Isolation of islets from market-sized pigs is costly, with considerable islet losses from fragmentation occurring during isolation and tissue culture. Fetal and neonatal pigs yield insulin unresponsive islet-like cell clusters that become glucose-responsive after extended periods of time. Both issues impact clinical applicability and commercial scale-up. We have focused our efforts on a cost-effective scalable method of isolating viable insulin-responsive islets. Young Yorkshire pigs (mean age 20 days, range 4-30 days) underwent rapid pancreatectomy (<5 min) and partial digestion using low-dose collagenase, followed by in vitro culture at 37°C and 5% CO2 for up to 14 days. Islet viability was assessed using FDA/PI or Newport Green, and function was assessed using a glucose-stimulated insulin release (GSIR) assay. Islet yield was performed using enumeration of dithizone-stained aliquots. The young porcine (YP) islet yield at dissociation was 12.6 ± 2.1 × 10(3) IEQ (mean ± SEM) per organ and increased to 33.3 ± 6.4 × 10(3) IEQ after 7 days of in vitro culture. Viability was 97.3 ± 7% at dissociation and remained over 90% viable after 11 days in tissue culture (n = ns). Glucose responsiveness increased throughout maturation in culture. The stimulation index (SI) of the islets increased from 1.7 ± 2 on culture day 3 to 2.58 ± 0.5 on culture day 7. These results suggest that this method is both efficient and scalable for isolating and maturing insulin-responsive porcine islets in culture.

Method measuring oxygen tension and transport within subcutaneous devices

“…[1][2][3][4][5] To this end, we have developed a scalable islet of Langerhans (islet) isolation method wherein partially digested piglet pancreatic tissue is matured in vitro over 8 days by incubation in a novel cell culture media. 6,7 During this period, exocrine tissue dies and isolated islets remain, which we have shown to be responsive to glucose challenges. Isolated islets can be encapsulated within permeable hydrogels such as alginate to provide an immunoprotective barrier that may preclude pharmacological immunosuppression.…”

Noninvasive evaluation of the vascular response to transplantation of alginate encapsulated islets using the dorsal skin-fold model

“…The islets were cultured for 8–10 days using protocols developed in our laboratory as previously described [24] and counted under 25 ×magnification after staining with dithizone [39]. Their viability was assessed using fluorescence microscopy with a mixture of Newport Green DCF diacetate (Life Technologies, NY) and propidium iodide (Life Technologies, NY) [40].…”

“…Also, porcine insulin being structurally similar to human insulin [19], has been used to treat T1D patients for decades [20]. While pigs of various strains and ages have been used as islet sources [21], we have expanded on the work done by Korbutt et al on neonatal pigs [22,23] and developed a simple and effective method of isolating islets from young pigs [24]. Islets isolated using this method were used in our noninvasive imaging studies.…”

Xenotransplantation for Islets from Clinical Side

“…The mean digestion time was around 16 min. The islet tissue clusters (50-500 μm) isolated using the method above were allowed to mature into complete islets during culture in vitro at 37°C, 5% CO 2 at first in recovery maturation media (Optatio LLC) supplemented with 10% porcine serum, 417 mM dornase α, 215 mM aprotinin, 0.5 mM pefabloc, and then in a novel maturation media (Optatio LLC) 48 hours later, supplemented with 10% porcine serum [23,24].…”