Development of a Simple In Vitro Assay to Assess Digestion of the Extracellular Matrix of the Human Pancreas by Collagenase Enzyme Blends
Spiers RM., Cross SE., Brown HL., Bateman PA., Vaughan RH., Hughes SJ., Johnson PRV.
Despite huge advances in the field of islet transplantation over the last two decades, current islet isolation methods remain suboptimal, with transplantable yields obtained in less than half of all pancreases processed worldwide. Successful islet isolation is dependent on the ability of collagenase-based enzyme blends to digest extracellular matrix components at the islet–exocrine interface. The limited availability of donor pancreases hinders the use of full-scale islet isolations to characterize pancreas digestion by different enzyme components or blends, or allow the influence of inter-pancreatic variability between donors to be explored. We have developed a method that allows multiple enzyme components to be tested on any one pancreas. Biopsies of 0.5 cm3 were taken from seven standard (age ≥45) and eight young (age ≤35) pancreases. Serial cryosections were treated with Serva collagenase, neutral protease (NP), or the two enzymes together at clinically relevant concentrations. Following digestion, insulin and either collagen IV or laminin-α5 were detected by immunofluorescent labeling. Protein loss at the islet–exocrine interface was semi-quantified morphometrically, with reference to a control section. Differential digestion of the two proteins based on the enzyme components used was seen, with protein digestion significantly influenced by donor age. Treatment with collagenase and NP alone was significantly more effective at digesting collagen IV in the standard donor group, as was the NP mediated digestion of laminin-α5. Collagenase alone was not capable of significantly digesting laminin-α5 in either donor group. Combining the two enzymes ameliorated the age-related differences in the digestion of both proteins. No significant differences in protein loss were detected by the method when analyzed by two independent operators, demonstrating the reproducibility of the assay. The development of this simple yet reproducible assay has implications for both enzyme batch testing and identifying inter-donor digestion variability, while utilizing small amounts of both enzyme and human tissue.