Low-temperature culture of human islets isolated by the distention method and purified with Ficoll or Percoll gradients.
Islets were isolated from human pancreases by the distention method and purified by centrifugation on Ficoll or Percoll gradients. The purity of the final preparations was 60% to 90% islets, making it possible to culture the islet preparations in vitro. Perifusion of the islet preparations after 1, 3, or 7 days of culture at 24 degrees C indicated that the islets were viable and would respond to glucose stimulation. If the islets were returned to 37 degrees C culture for 1 day, then the insulin secretory response at 7 days was comparable with the response during the first day of culture at 37 degrees C. Extending the culture period at 24 degrees C to 14 days damaged the islets as indicated by the lack of response to glucose stimulation. The Ficoll technique was a much more effective method for purifying the islets, since it provided approximately twice the yield of islets and insulin compared with the Percoll procedure. The mean yield of purified islets by the Ficoll technique was 2180 +/- 325 islets/gm of pancreas with a calculated islet mass of 3.7 +/- 0.8 mm3/gm. The findings that massive numbers of purified human islets can be obtained by centrifugation on Ficoll gradients and that the purified islet preparations remain morphologically and functionally intact during 7 days' culture at 24 degrees C make it possible to assess the function of donor islets before human islet transplantation, transplant the islets via the portal vein, and use low-temperature culture as a possible approach for altering the immunogenicity of donor human islets.
Scharp
D W
DW
Department of Pathology, Washington University School of Medicine, St. Louis, Mo. 63110.
Lacy
P E
PE
Finke
E
E
Olack
B
B
D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000032:analysis; D007515:Islets of Langerhans / Q000032:analysis / Q000166:cytology*; D013696:Temperature
D007328:Insulin