P-249

Development of a new analytical method and new surface modification method to optimize microencapsulation of human islets

J. SoRelle¹, B. Naziruddin², T. Itoh³, S. Matsumoto³, R. Kane^1
1 Institute of Biomedical Studies, Waco, USA; ² Baylor University Medical Center, Dallas, TX, USA; ³ Baylor Reserach Institute Fort Worth Campus, Fort Worth, TX, USA

Background: Immunoisolation by poly-ethylene glycol (PEG) has been shown to offer both short and long term protection to islets. We describe a novel surface modification method for PEGylation of human islets and also a sensitive analytical tool to determine the density and uniformity of PEG coating of islets.

Method: We performed PEGylation of human islets by a newly developed reaction of maleimide-PEG to reduced thiols on islet surface proteins (RED group) and compared it to the established NHS esterification (NHS group) and by reacting hydrazide-LC-biotin with oxidized surface sugars (OX group) as described previously. DyLight488 labeled streptavidin was used to label each PEG molecule. Bulk fluorescence was captured with a fluorescent microscope and the mean fluorescent intensity per islet was measured using ImageJ software. Confocal microscopy was used to obtain surface outlines of PEGylated islets and uniformity was determined as percent of the total surface covered. PEGylated Islets were analyzed at days 0, 1, 2, and 7 to determine the longevity. The viability was determined by Hoe342/PI staining and function by glucose stimulated static incubation.

Results: Fluorescence intensity of the RED group was higher than NHS and OX groups at 0h (76.5±25.3 vs. 63.9±8.8 and 63.2±11.0 MFI) and 24h (67.4±19.1 vs. 51.9±13.9 and 28.6±8.5 MFI). The uniformity data was similar for the NHS and RED groups for days 1, 2, and 7. The viability and function of the islets was not affected by all three chemical modifications (>90% after 48h, S.I. >1.0 at 24h).

Conclusion: Novel targeting of reduced thiols yields higher PEG density versus the established NHS method. A new analytical method has been established that measures the density, uniformity, and longevity of PEG coatings. This will allow optimization and comparison of various surface modifications techniques on human and animal islets.

P-249

Development of a new analytical method and new surface modification method to optimize microencapsulation of human islets

J. SoRelle¹, B. Naziruddin², T. Itoh³, S. Matsumoto³, R. Kane^1
1 Institute of Biomedical Studies, Waco, USA; ² Baylor University Medical Center, Dallas, TX, USA; ³ Baylor Reserach Institute Fort Worth Campus, Fort Worth, TX, USA

Background: Immunoisolation by poly-ethylene glycol (PEG) has been shown to offer both short and long term protection to islets. We describe a novel surface modification method for PEGylation of human islets and also a sensitive analytical tool to determine the density and uniformity of PEG coating of islets.

Method: We performed PEGylation of human islets by a newly developed reaction of maleimide-PEG to reduced thiols on islet surface proteins (RED group) and compared it to the established NHS esterification (NHS group) and by reacting hydrazide-LC-biotin with oxidized surface sugars (OX group) as described previously. DyLight488 labeled streptavidin was used to label each PEG molecule. Bulk fluorescence was captured with a fluorescent microscope and the mean fluorescent intensity per islet was measured using ImageJ software. Confocal microscopy was used to obtain surface outlines of PEGylated islets and uniformity was determined as percent of the total surface covered. PEGylated Islets were analyzed at days 0, 1, 2, and 7 to determine the longevity. The viability was determined by Hoe342/PI staining and function by glucose stimulated static incubation.

Results: Fluorescence intensity of the RED group was higher than NHS and OX groups at 0h (76.5±25.3 vs. 63.9±8.8 and 63.2±11.0 MFI) and 24h (67.4±19.1 vs. 51.9±13.9 and 28.6±8.5 MFI). The uniformity data was similar for the NHS and RED groups for days 1, 2, and 7. The viability and function of the islets was not affected by all three chemical modifications (>90% after 48h, S.I. >1.0 at 24h).

Conclusion: Novel targeting of reduced thiols yields higher PEG density versus the established NHS method. A new analytical method has been established that measures the density, uniformity, and longevity of PEG coatings. This will allow optimization and comparison of various surface modifications techniques on human and animal islets.