Reproducible high yields of rat islets of Langerhans.
Isolation of islets of Langerhans from the pancreas by action of collagenase, a major breakthrough for physiological studies in vitro, has long appeared empirical, and the results were sometimes unpredictable. Isolation yields (number of islets obtained per pancreas) and their reproducibility, purification from exocrine remnants and vitality of the islets obtained, can improve owing to precise techniques, adapted to the architecture of collagen in the pancreas. We have tested four isolation-purification techniques in the rat pancreas. The best results were obtained by combining intra-ductal collagenase injection, with complete but moderate distension of the gland, avoiding leakage, multistep digestion (in situ and then in vitro), followed by purification on a discontinuous bovine serum albumin (BSA) gradient. Average yields were 670 +/- 40 islets per pancreas (range 570-800), versus 170 +/- 9 (range 40-350) with the technique used initially. The use of BSA discontinuous gradient improved the purification yield: 90-96% of islets obtained were concentrated in the 26/29% BSA interface. Furthermore, this technique shortened the duration of purification step: 1 hr (centrifugation gradient) vs 3 hrs (handpicked). It was verified that islets were morphologically free of exocrine tissue. Islet structure was well preserved either in conventional histology or insulin and glucagon immunoperoxidase staining. Islet vitality, as assessed by trypan blue exclusion test, was 100% of freshly isolated islets, and 89% after 24 hrs culture. Insulin secretory responses to a given stimulus were stronger than in the case of islets isolated by former techniques: 10-12 times the basal release (vs 5 times) with clear dose-response proportionality.(ABSTRACT TRUNCATED AT 250 WORDS)
Delaby
J
J
Diabetes Department, Hôpital Bichat, Paris, France.
Campana
M
M
Laborie
C
C
Assan
R
R
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D003850:Deoxyribonuclease I; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D003012:Microbial Collagenase; D051381:Rats; D011919:Rats, Inbred Strains; D013806:Theophylline / Q000494:pharmacology
D007328:Insulin; D001120:Arginine; D013806:Theophylline; D003850:Deoxyribonuclease I; D003012:Microbial Collagenase; D005947:Glucose