A one-step, operator-independent method for isolating islets of Langerhans from the porcine pancreas.
Large-scale isolation of islets of Langerhans is one of the major obstacles in islet transplantation. Until now, isolation methods relied on enzymatic digestion, the duration of which relies on a decision dictated by the operator's experience. This approach has always hindered development of an automated method. The aim of this study was to develop a one-step method based on complete digestion of the pancreas. The original aspect of the technique (derived from the Ricordi method) is use of the University of Wisconsin (UW) solution in the digestion medium and a continuous flow collagenase processing circuit with local cooling and rewarming to allow tissue digestion to proceed at 37 degrees C while settling of the cell suspension takes place at 4 degrees C. A stopcock system permits the alternate use of two settling chambers so that while one is in the circuit, the other can be removed for centrifugation, resuspension of the crude islet preparation in collagenase in free UW solution, and further purification in a density gradient system. Ten experiments were performed, and 545,750 +/- 48,670 purified pig islets were obtained per totally digested pancreas. Histological studies showed cell integrity. Insulin secretion in response to double glucose stimulation under perfusion conditions demonstrated the functional viability of the isolated islets. In conclusion, this one-step method makes it possible to obtain a high number of viable islets of Langerhans in the absence of any decision by an operator, and it should therefore provide basis for an automated method.
Arbet-Engels
C
C
Service de Diabétologie, Hopital de l'Hotel-Dieu, Paris, France.
Darquy
S
S
Capron
F
F
Pueyo
M E
ME
Dimaria
S
S
Poitout
V
V
Reach
G
G
D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002470:Cell Survival; D046508:Culture Techniques; D005947:Glucose / Q000494:pharmacology; D005978:Glutathione; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019188:Organ Preservation Solutions; D011887:Raffinose; D013552:Swine; D014021:Tissue Preservation
D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D005947:Glucose; D000241:Adenosine; D011887:Raffinose