Optimization in osmolality and range of density of a continuous ficoll-sodium-diatrizoate gradient for isopycnic purification of isolated human islets.
BACKGROUND
According to previous estimates from large animals and man, a minimum of approximately 5000 to 6000 engrafted islet equivalents (IEQ)/kg recipient weight is critical to establish insulin independence. Utilizing a single donor, this threshold yield of purified islets can be retrieved from approximately one third of all isolations. The aim of this study was to improve human islet purification by optimization of the osmolality and the density range of the continuous Ficoll-sodium-diatrizoate (FSD) gradient to facilitate consistent purities >80% of human islet preparations without considerable loss of islet yield.


METHODS
Aliquots of human pancreatic digests were placed on continuous density gradients. After centrifugation, sequential aliquots were extracted for amylase and insulin to determine the relative and cumulative density distribution of endocrine and exocrine tissue. We addressed the impact of two factors: (1) osmolalities (300 to 600 mosm/kg) in the gradient of FSD covering a density range of 1.070 to 1.100 g/cm(3); and (2) density (FSD 500/1.070 to 1.100) versus density-osmolarity gradient (DO-FSD 400-530/1.080 to 1.113).


RESULTS
The density of exocrine and endocrine tissue increased with rising osmolality. Differences in density of both tissues were highest at 450 and lowest at 300 and 600 mOsmol/kg. Purity and recovery were highest at 450 versus 400 or 500 mOsm/kg (NS). Exocrine but not endocrine tissue was more dense in DO-FSD than in FSD gradient (P < .05). The differences in density were 0.004 versus 0.013 g/cm(3) (P < .01), resulting in an increased islet purity and recovery.


CONCLUSIONS
The best osmolality for the FSD 1.070 to 1.100 g/cm(3) is at 450 mOsm/kg. Using the DO-FSD may improve human islet purification allowing successful clinical islet transplantation.
Eckhard
M
M
Third Medical Department and Policlinic, University of Giessen, 35932 Giessen, Germany. michael.eckhard@innere.med.uni-giessen.de
Brandhorst
D
D
Brandhorst
H
H
Brendel
M D
MD
Bretzel
R G
RG
D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods; D003287:Contrast Media; D003973:Diatrizoate; D005362:Ficoll; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation; D010179:Pancreas / Q000166:cytology; D020858:Tissue and Organ Harvesting / Q000379:methods; D014783:Viscosity
D003287:Contrast Media; D003973:Diatrizoate; D005362:Ficoll