P-196

Improving young porcine islet isolation and maturation in culture

M. Lamb, K. Laugenour, O. Liang, M. Alexander, S. Li, H. Ichii, J. Lakey
University of California Irvine Medical Center, Surgery, Orange, USA

Objectives: Porcine pancreatic islets have been explored as a source of tissue for xenotransplantation. The isolation of islets from mature market weight pigs is costly since the islets often fragment during and after the tissue dissociation and islets recovered from neonatal pigs take time to mature before becoming insulin responsive. The aim of this study was to develop a reproducible and scalable procedure for isolating porcine islets that have high viability and are insulin responsive.

Methods: Pancreases from Yorkshire pigs (mean age 18 days, range 14 to 28 days) were recovered using a rapid surgical procurement (< 5 min). Gentle partialdigestion of the pancreas was performed using low dose CIzyme collagenase MA/BPprotease (VitaCyte LLC) and then cultured at 37^o C and 5% CO ₂ for up to 14 days with media changes every 48 hrs. Viability was assessed usingFDA/PI or Newport Green and function was assessed using glucose stimulated insulin release (GSIR) assay.

Results: Islet yield immediately following dissociation was 12.6x10³ ± 183IE (mean ± sem) per organ which then increased to 30.6x10³ ± 170 and 33.3x10³ ± 136 after 5 and 7 days of culture, respectively. At day 7 yields were 6.4 x10³ ± 77 IE/gm tissue (average tissue5.18 ± 0.1g).Viability was >98 ± 0.03%(FDA/PI) and >90 ± 0.4% (NewportGreen) at day 7 of culture. Islet function (GSIR) initially after isolation was1.3 ± 0.1(SI, ratio of insulin during high glucose (28mM) stimulation/low glucose(2.8mM)). At day 2, insulin response increased to an SI of 1.9 ± 0.5 which further improved to 2.6 ± 0.2after 7 days of culture.

Conclusions: This model provides a reproducible and scalable method of isolating and maturing pig islets in culture. Partial pancreas dissociation, followed by extended culture allows for islets to gently separate from the surrounding exocrine matrix and may be a key factor in this protocol success.

P-196

Improving young porcine islet isolation and maturation in culture

M. Lamb, K. Laugenour, O. Liang, M. Alexander, S. Li, H. Ichii, J. Lakey
University of California Irvine Medical Center, Surgery, Orange, USA

Objectives: Porcine pancreatic islets have been explored as a source of tissue for xenotransplantation. The isolation of islets from mature market weight pigs is costly since the islets often fragment during and after the tissue dissociation and islets recovered from neonatal pigs take time to mature before becoming insulin responsive. The aim of this study was to develop a reproducible and scalable procedure for isolating porcine islets that have high viability and are insulin responsive.

Methods: Pancreases from Yorkshire pigs (mean age 18 days, range 14 to 28 days) were recovered using a rapid surgical procurement (< 5 min). Gentle partialdigestion of the pancreas was performed using low dose CIzyme collagenase MA/BPprotease (VitaCyte LLC) and then cultured at 37^o C and 5% CO ₂ for up to 14 days with media changes every 48 hrs. Viability was assessed usingFDA/PI or Newport Green and function was assessed using glucose stimulated insulin release (GSIR) assay.

Results: Islet yield immediately following dissociation was 12.6x10³ ± 183IE (mean ± sem) per organ which then increased to 30.6x10³ ± 170 and 33.3x10³ ± 136 after 5 and 7 days of culture, respectively. At day 7 yields were 6.4 x10³ ± 77 IE/gm tissue (average tissue5.18 ± 0.1g).Viability was >98 ± 0.03%(FDA/PI) and >90 ± 0.4% (NewportGreen) at day 7 of culture. Islet function (GSIR) initially after isolation was1.3 ± 0.1(SI, ratio of insulin during high glucose (28mM) stimulation/low glucose(2.8mM)). At day 2, insulin response increased to an SI of 1.9 ± 0.5 which further improved to 2.6 ± 0.2after 7 days of culture.

Conclusions: This model provides a reproducible and scalable method of isolating and maturing pig islets in culture. Partial pancreas dissociation, followed by extended culture allows for islets to gently separate from the surrounding exocrine matrix and may be a key factor in this protocol success.