A simple method using a polymethylpenten chamber for isolation of human pancreatic islets.
OBJECTIVE
Isolation of large numbers of intact and functional human islets remains difficult and expensive. We describe a novel method using a polymethylpenten chamber (PMPC) and compare its efficacy to the classic method using a stainless steel chamber (SSC).


METHODS
Five pancreases obtained from cadaveric donors were processed with the SSC method, and the islets were purified with a Cobe cell separator. The next 15 pancreases (similar donor characteristics) were distended with Liberase HI, minced, and digested in a PMPC whose thermic properties did not require continuous heating to maintain temperature of the prewarmed medium at 37 degrees C. The digestion was done in 2 phases to avoid damaging the first freed islets. Digested tissue was filtered on a column of 6-mm glass beads and 500-microm mesh screen, so that tissue volume was small enough to permit purification on discontinuous Ficoll gradients in tubes.


RESULTS
With the PMPC method, the extent of digestion (+/-70%), yield (approximately 5000 IEQ/g), and final purity (73%) and viability (84%) of the islets was similar to those with the SSC, but the proportion of large islets (>150 microm in diameter) was higher. Cell composition (beta vs. non-beta cells) of isolated islets was not different from that of islets in situ in the same pancreas. Islet function, assessed by perifusion, showed an excellent average stimulation index of approximately 13-fold (15 vs. 1 mmol/L glucose, without cAMP-raising agent).


CONCLUSIONS
This new method for isolation of human islets uses simple, low-cost, and potentially disposable material and requires a team of only 2 persons. The technique is as efficient as the classic SSC method and provides islets with excellent integrity and insulin-secreting capacity.
Dufrane
Denis
D
Laboratory of Experimental Surgery, Université Catholique de Louvain, Faculty of Medicine, Brussels, Belgium. denis.dufrane@chex.ucl.ac.be
Goebbels
Rose-Marie
RM
Guiot
Yves
Y
Squifflet
Jean-Paul
JP
Henquin
Jean-Claude
JC
Gianello
Pierre
P
D000328:Adult; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D017364:Collagenases; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation; D008875:Middle Aged; D013193:Stainless Steel; D013696:Temperature; D013820:Thermolysin; D014019:Tissue Donors
D013193:Stainless Steel; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin