Flexible Management of Enzymatic Digestion Improves Human Islet Isolation Outcome from Sub‐Optimal Donor Pancreata

Abstract

Worldwide growing interest in reproducing the result of the Edmonton protocol in islet transplantation trials poses the problem of paucity of donors to supply sufficient amount of islets for clinical use. Improved outcomes include finding better ways to obtain higher yields from every donor organ processed and the possibility of extending islet isolation processing to glands of suboptimal quality. In order to optimize enzymatic digestion of marginal donor organs, we have modified the technique of tissue collection following enzymatic digestion of human pancreatic organs, allowing for reduced time of exposure of free islets to warm Liberase TM solution. Our results indicate that better controlled exposure to enzyme yields: (i) higher islet numbers; (ii) complete dissociation of all parts of pancreatic tissue; (iii) successful islet harvest from organs otherwise excluded. We also show that by limiting the exposure of free islets to enzyme solution, islet fragmentation and loss of insulin content are reduced. We further support evidence that enzymatic digestion may contribute to impairment of insulin secretory capacity of the islets in vitro during culture.

Suitability of human juvenile pancreatic islets for clinical use

“…Rescue procedures involving subsequent collagenase or trypsin incubation to reduce the thickness of the exocrine mantle failed to yield free islets. Based on this observation, and on the basis of studies indicating collapse of the ducts in young pancreases [16], we favoured enzymatic, thus chemical digestion, at the cost of a reduced mechanical digestion, without increasing the overall total time of exposure of the pancreatic tissue to exogenous enzymes which are potentially harmful [22,27]. In order to inject the enzyme solution, typically, in adult organs, the pancreatic neck is cut to expose the two-sectioned parts of the gland as well as the pancreatic duct.…”

“…Our approach involves the following steps: delivery of enzyme solution and initial digestion using the recirculation chamber designed by R. Rajotte [21], further digestion in a Ricordi chamber equipped with additional filtration devices, minimisation of mechanical shaking, and collection of islets through a tissue sieve filter. A collection phase with warm and cold recirculation was also followed in some isolations, as described previously [22].…”

“…Morphological and histological studies The histo-anatomical structure of pancreatic islets (from the whole pancreas prior to isolation and from the islet samples after isolation) was assessed in formalin-fixed tissue/cells after haematoxylin and eosin (H&E) staining as well as insulin immunostaining (DakoCytomation, CA, USA), as described [22]. Islet size measurement was performed using an Axiplan 2ie MOT Research Microscope (Carl Zeiss Micro imaging, NY, USA).…”

“…Insulin concentration in the culture medium, or after islet insulin extraction, was measured by ELISA (ALPCO, Windham, NH, USA) [22,24]. DNA content of islets was determined as described [24].…”

Prolongation of islet allograft survival following ex vivo transduction with adenovirus encoding a soluble type 1 TNF receptor–Ig fusion decoy

“…Pancreata were obtained from at least five cadaveric donors of different ages and sex, and subjected to digestion, isolation and purification as described. 77,78 For each experiment, islets from all donors were used. The purity of islets was usually greater than 80% (mantled islets).…”