title: High yield of rodent islets with intraductal collagenase and stationary digestion--a comparison with standard technique.
abstract: Intraductal distention of the pancreas with collagenase followed by stationary warm incubation improves the recovery of islets of Langerhans in the rat, but controlled studies are needed for valid comparison with standard isolation methods. We have modified Gotoh's technique of stationary digestion for high-yield isolation in the rat (Stationary). The method is subjected herein to rigorous blinded comparison with the standard chopped tissue (Chopped) technique, based on Lacy et al., as performed in our laboratory for over 10 yr. Islet recovery was determined by a single observe 'blinded' to the method of isolation used, and only intact islets of diameter > or = 100 microns were included. Stationary gave 719 +/- 114 islets per pancreas (mean +/- SD, n = 21 isolations) vs. 487.5 +/- 69 for Chopped (n = 36 isolations), a 47.5% increment in yield (p < 0.0001). In vitro islet perifusion showed no statistical difference in stimulation index (SI) or stimulated area under the curve (SAUC) between the two methods, but Stationary showed a trend towards improved phase II insulin release. In vivo function was assessed by isogeneic transplantation of 2,000 islets beneath the renal capsule of streptozotocin diabetic recipients (65 mg/kg Sigma); Stationary recipients (n = 7) became normoglycemic (< or = 8 mmol/L) by 3.3 +/- 4.8 days vs. 1.6 +/- 1.5 days for Chopped recipients (p = 0.4 ns, mean +/- SEM). IVGTT performed at 1 mo posttransplant gave K-values for Stationary of 2.64 +/- 0.8 vs. 2.62 +/- 0.8 for Chopped (mean +/- SD, p = 0.9 ns, n = 6, unpaired t-test), which were not distinguishable from normal control rats (2.59 +/- 0.8) (p = 0.9 ns, n = 10). Graft function remained stable until graft bearing nephrectomy induced hyperglycemia uniformly within 1 day. Graft histology showed a healthy well-preserved structure on light microscopy, with well-granulated beta cells on EM. Economic costs of rat, collagenase, and Ficoll were 26% ($50.82) lower per recipient for Stationary. We conclude that modified stationary digestion significantly improves islet recovery with excellent in vitro and in vivo function, and is cost effective.
authors.affiliation: Department of Surgery, University of Alberta, Edmonton, Canada.
authors.forename: A M
authors.initials: AM
authors.lastname: Shapiro
authors.forename: E
authors.initials: E
authors.lastname: Hao
authors.forename: R V
authors.initials: RV
authors.lastname: Rajotte
authors.forename: N M
authors.initials: NM
authors.lastname: Kneteman
mesh_terms: D000818:Animals; D017364:Collagenases / Q000494:pharmacology; D003584:Cytological Techniques; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000098:blood supply / Q000166:cytology* / Q000187:drug effects; D016381:Islets of Langerhans Transplantation / Q000191:economics / Q000379:methods*; D008297:Male; D051381:Rats; D011920:Rats, Inbred WF; D016037:Single-Blind Method; D013997:Time Factors
chemical_list: D007328:Insulin; D017364:Collagenases; D005947:Glucose