A new method to valued efficiency of enzyme blend for pancreas tissue digestion.

Abstract

One of the best successful example of cell therapy is represented by islet transplantation since the '90. However islet isolation methods are not completely standardized yet. More than half of isolation procedures failed to isolate adequate islets for transplantation, due to variable pancreas condition, and to unpredictable enzymatic blend efficiency. Enzymes used for pancreas digestion are purified from Clostridium histolyticum; these enzymes has a broad substrate specificity and potent collagenolytic activity compared to vertebrate collagenases. However, a major obstacle in human islet isolation successful is due to the variability in composition and concentration of the collagenases used during digestion phase‏. Another protease involved in islet isolation is the neutral protease, it plays an important role, however, should be considered as a double-edged sword: it accelerates tissue dissociation but, on the other hand, it could result in a decrease in islet yield, through islet fragmentation and breakdown‏. Therefore, protease activity should be carefully adjusted in a narrow range, in fact, there are not sufficient studies showing optimal dosage and composition of proteases that must be use in human pancreas islets extraction procedure. The current parameters available to characterize enzymatic blend are not predictive their efficiency in pancreas digestion and therefore are not useful to select which enzyme bathes can be successfully used for islet isolation purpose. Our goal is in direction to maximize the yield of functional islets; therefore we set up a new in vitro method to better in vitro characterize enzymatic blend before its use in human pancreas. In our experimental approach we have growth human immortalized cells (ECV-304) or human islets, obtained using a canonical extractive approach, within a 3-D type-I collagen gel in 96 wells plate. After one culture day, cells and/or islets were treated with different commercial enzymes (Liberase, Serva NB1 premium grade, Collagenase type P, Thermolysin, Neutral proteases) from different batches at different concentrations and for different times. Digestion of 3-D type-I collagen fibril gels were monitored by optical dense absorption to a fix l; while, morphology of released cells and/or islets were valued by confocal microscopy analyses. Cells were immunostained about expression of some adhesion molecules, like: integrins, cadherins and associated molecules, catenins, to appreciate cells morphology and islets aggregation modifications. Moreover, using SYTO 13/Et/Br in viability assayes we have quantify the toxiticy or not of tested enzymes in the different experimental conditions. We found that Neutral proteases is less pure and more toxic than Thermolysin: it has collagenase activity and it significantly decrease cell viability. Isolated cells by Neutral proteases appeared to be 77,5 % died. Even viable cells showed an alterated morphology with an impairment of cell to cell communications. We, also, observed an higher efficiency in extraction by Serva NB1 compared to Liberase and Collagenasi type P, used in the same experimental conditions. By this in vitro method we were also able to compare the minimal active enzyme concentration of different enzyme blends: we found that Liberase has its best value as nuber of extracted cell/alive cells in 130 mg/ml; while, in actual protocols is used to 1,3 mg/ml. Preliminary results showed a correlation between data achieved in cell lines with those of human islets, thus confirming the predictive role of this method for the selection of enzymes for human pancreas digestion purpose.