Microbial surveillance of human islet isolation, in vitro culture, and cryopreservation

Jonathan R.T. Lakey
Ray V. Rajotte
Garth L. Warnock

From the Departments of Surgery and Medicine and the Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta

(Original manuscript submitted 20/6/94, received in revised form 26/1/95; accepted 2/2/95)

Abstract

The aim of these studies was to investigate microbiological contamination during the isolation and preservation of islets of Langerhans in a low-temperature tissue bank. Islets were isolated from the pancreases of 117 organ donors, then cryopreserved. In the initial 47, microbial culture was completed only after thawing: Enterobacter cloacae (n = 4) and gram-negative bacilli (n = 9) were isolated for a positive culture rate of 27.6%. It was not possible to ascertain the source of the contaminants, since cultures were taken only at the conclusion. A total of 70 consecutive pancreases were then subjected to islet isolation and cryopreservation while prospectively culturing at these steps: step 1, from the pancreas transport media; step 2, after intraductal perfusion of collagenase; step 3, after dissociation; step 4, after purification; step 5, following a 6- to 48-h in vitro culture; step 6, post-thaw; step 7, after dilution of the cryoprotective agent; and step 8, after final 48-h in vitro culture. The transport fluid was infected with aerobic gram-negative (n = 6), gram-positive (n = 5), and yeast microorganisms (n = 2) for an overall step 1 contamination rate of 19%. These contaminants were found in 9% of our local program vs. 26% from distantly procured pancreases. Contaminants at subsequent steps were 10% (step 2), 9% (step 3), so that only 3% were infected at the conclusion of isolation (step 4), and 0% after initial culture (step 5). Amongst pancreases that were initially sterile, new contaminants could be identified in 12% at step 2 and 8% at step 3; however, these also became undetectable. Cryopreservation failed to introduce organisms during storage (step 6), but 2 instances of infection occurred at steps 7 and 8. We conclude that microbiological surveillance of human islet isolation and tissue banking facilitates identification of the sources of contamination. Significant contaminants present or introduced early in the process become undetectable by the conclusion of purification and cryopreservation.

Clin Invest Med 1995; 18 (3): 168-176

Table of contents: CIM vol. 18, no. 3