Pancreatic islet isolation by mechanical-enzymatic separation, stationary collagenase digestion and dextran discontinuous density gradient purification: experimental study in dogs

The prospects for allotransplantation of pancreatic islets in man depend on the development of methods that provide sufficient quantities of pancreatic islets from a single donor, which are capable, when transplanted, of achieve the normalization of carbohydrate metabolism. Objective: Evaluate the efficacy of the isolation of Langerhans islets from dogs, by means of mechanical-enzymatic separation technique with stationary digestion using collagenase, and purification with a discontinuous dextran density gradient. Methods: The counting of islet numbers and evaluation of their sizes was accomplished by staining with diphenylthiocarbazone and using stereoscopic microscopes equipped with eyepiece reticule for the measurement of average diameters of stained islets. Results: The results disclosed that the average number of islets isolated was 81032.20 ± 24736.79 and the average number of islets isolated per kg of body weight was 6938.70 ± 1392.43. The average number of islets isolated per kg of body weight showed significant correlation with body weight and weight of the pancreas resected. Conclusion: The number of islets isolated, of a single donor, by mechanical-enzymatic separation, stationary collagenase digestion and discontinuous dextran density gradient purification can be sufficient to success of pancreatic islets transplant in dogs.

Preparation of Crude Subcellular Fractions by Differential Centrifugation

The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. Buoyant density gradient purification of peroxisomes or lysosomes for example is almost invariably carried out on a light mitochondrial fraction so as to eliminate smaller particles that may have similar densities. Unless they are first removed, large rapidly sedimenting particles in homogenates may also disturb shallow gradients designed to fractionate small low-density microsomes.

Assessment of Islet Isolation Efficacy in Dogs

The outcome of islet isolation is considered uncertain because of the large variability of islet and insulin yield, but comparison of the isolated and native islet population has not been attempted. We therefore addressed the efficacy of collagenase digestion, and density gradient purification of islets from the splenic dog pancreas (n = 31) by morphometry of the islet volume and size distribution, and by extraction of insulin and amylase, in samples from the pancreas, the digest, and gradient fractions. In contrast to a ~90% recovery of pancreatic insulin and amylase after digestion, islet yield amounted to 50% of the islet content of the pancreas. After density separation, islets were mainly found in the purified fractions, while half of the recovered insulin was located in the acinar fraction of the gradients — indicating a substantial proportion of islets entrapped in acinar fragments. The islet and insulin content of the pancreas correlated well with islet and insulin yield after digestion (r = 0.7, p < .0001). The insulin content of digest suspensions did neither correlate with islet nor insulin recovery in the purified fraction of the gradients (r = 0.4) as opposed to the islet content of digest suspensions, which correlated with both (r = 0.7, p < .0001). After density separation near 100% purity was obtained, and no loss of insulin from isolated islets was demonstrated by extraction and microscopy. Size distributions of native and isolated islets demonstrated no fragmentation. We conclude that the variability of isolation outcome may be attributed to a large extent to the variability of the native endocrine pancreas. Isolation efficacy was best documented by morphometry, because insulin extraction did not discriminate between free and entrapped islets. However, assessment by both morphometry and extraction allowed the quantitation of entrapped islets, and demonstrated preservation of β-cell granulation. Similar studies should facilitate the analysis of other factors affecting islet isolation in man.

Unexpected destruction of chitosomal chitin synthetase by an endogenous protease during sucrose density gradient purification

Because of their intrinsic low buoyant density, chitosomes can be separated from crude cell homogenates (1000 g or 35,000 g supernatants) of Mucor rouxii by isopycnic sedimentation in sucrose density gradients. To accelerate and simplify the isolation of chitosomes, we centrifuged the cell-free extracts at ultrahigh speed (in a fixed-angle rotor at forces up to 311,000 g Rav) and found that the duration of centrifugation was critical. Prolonged centrifugation at ultrahigh speed caused severe distortion of the chitin synthetase profile in the gradient as the peak of chitosomal chitin synthetase nearly disappeared. We traced the problem to a soluble protease(s) that moved into the chitosome band during protracted centrifugation and destroyed the chitin synthetase activity. The interfering protease was a soluble protein with a sedimentation coefficient of 4.6 S and a pH optimum of 7–7.5, and it was sensitive to PMSF (phenylmethylsulfonyl fluoride), indicating that it was a serine protease. Unlike other proteases, it destroyed chitin synthetase but did not activate the chitin synthetase zymogen. The interfering protease could be eliminated either by adding PMSF to the cells immediately after breakage or by removing the upper part of the sucrose gradient midway through the centrifugation of the cell-free extract and then completing the sedimentation with the ‘decapitated’ gradient.