P-211 Poster of distinction

Comparative functional assessment of COPAS and manually sorted pancreatic islets

A. Steffen¹, S. Bornstein², M. Solimena¹, B. Ludwig^2
1 Molecular Diabetology, Dresden, Germany; ² Internal Medicine Dept. III, Dresden, Germany

Objective: Islet size has been attributed to functional potency, survival and transplantation outcomes. Large particle flow cytometry can be used to separate islets according to size. Here we aimed to validate the potential of the COPAS instrument (Complex Object Parametric Analyser and Sorter) for automated size-based sorting of islets, a crucial pre-requisite for the accurate study of size-specific pancreatic islet characteristics.

Methods: Human islets were isolated following the modified Ricordi method using SERVA collagenase and neutral protease. Purification was performed on continuous Biocoll gradient. Islets were cultured in M1A media supplemented with glutathione prior to experimentation. Using COPAS, islets were automatically sorted based on time of flight (TOF) relative to particle size in groups of small (<150µm) and large (>150µm) islets. As control, islets were handpicked and allocated to corresponding size-groups. Viability was assessed with FDA/PI staining. For determination of insulin secretion radioimmunoassay was performed following glucose stimulation (basal: 3.3mM; stimulated: 16.7mM glucose) for 1h under static conditions.

Results: Islets could successfully be separated automatically in groups smaller and larger than 150µm using COPAS instrument. Comparing automated and manually sorted islets we found no difference in viability and function irrespective of sorting method. However, insulin secretion was significantly different between large and small islets adjusted for islet particle number. S mall islets release a higher percentage of their total insulin in the basal state and during stimulation compared to large islets. Calculation of stimulation indices (SI) as surrogate marker for islet metabolic capacity showed a significant higher SI in small islets compared to large islets.

Conclusion: COPAS instrument provides a fast and reliable method to successfully sort islets automatically without causing morphological or functional damage and has no negative impact on viability and glucose responsiveness. Our data also demonstrated size-dependent differences in metabolic capacity in favour of small islet particles.

P-211 Poster of distinction

Comparative functional assessment of COPAS and manually sorted pancreatic islets

A. Steffen¹, S. Bornstein², M. Solimena¹, B. Ludwig^2
1 Molecular Diabetology, Dresden, Germany; ² Internal Medicine Dept. III, Dresden, Germany

Objective: Islet size has been attributed to functional potency, survival and transplantation outcomes. Large particle flow cytometry can be used to separate islets according to size. Here we aimed to validate the potential of the COPAS instrument (Complex Object Parametric Analyser and Sorter) for automated size-based sorting of islets, a crucial pre-requisite for the accurate study of size-specific pancreatic islet characteristics.

Methods: Human islets were isolated following the modified Ricordi method using SERVA collagenase and neutral protease. Purification was performed on continuous Biocoll gradient. Islets were cultured in M1A media supplemented with glutathione prior to experimentation. Using COPAS, islets were automatically sorted based on time of flight (TOF) relative to particle size in groups of small (<150µm) and large (>150µm) islets. As control, islets were handpicked and allocated to corresponding size-groups. Viability was assessed with FDA/PI staining. For determination of insulin secretion radioimmunoassay was performed following glucose stimulation (basal: 3.3mM; stimulated: 16.7mM glucose) for 1h under static conditions.

Results: Islets could successfully be separated automatically in groups smaller and larger than 150µm using COPAS instrument. Comparing automated and manually sorted islets we found no difference in viability and function irrespective of sorting method. However, insulin secretion was significantly different between large and small islets adjusted for islet particle number. S mall islets release a higher percentage of their total insulin in the basal state and during stimulation compared to large islets. Calculation of stimulation indices (SI) as surrogate marker for islet metabolic capacity showed a significant higher SI in small islets compared to large islets.

Conclusion: COPAS instrument provides a fast and reliable method to successfully sort islets automatically without causing morphological or functional damage and has no negative impact on viability and glucose responsiveness. Our data also demonstrated size-dependent differences in metabolic capacity in favour of small islet particles.