Purified bacterial collagenase and protease enzymes are commonly used to recover human islets from clinical grade pancreata for subsequent clinical islet transplantation. The high cost of purified enzymes can be prohibitive for recovering human islets from research pancreata used for translational research or pre-clinical studies. In this study, we successfully isolated islets from human research pancreata using enriched collagenase products supplemented with BP Protease (DE Collagenase 800 or Collagenase Gold-BP Protease mixtures) that cost significantly less than purified collagenase-
Methods: Defined, enriched collagenase products (DE Collagenase 800 and Collagenase Gold) with a purity comparable to purified clinical collagenase blends (> 85% purity) were prepared from Clostridium histolyticum culture supernatants. These products contained primarily intact class I (C1) and class II (C2) at about a 75:25 C1:C2 ratio as determined by analytical anion exchange chromotography. Enriched collagenase-BP Protease mixtures were used at either 19,000-48,000 or 32,500-78,800 neutral protease U (NP U) per g pancreas, and hereafter referred to as low or high protease enzyme mixtures. Each enzyme mixture was diluted up to 350 mL of HBSS and used in a modified Ricordi procedure with isolations performed at three different islet processing centers.
Results: Comparison of the biochemical characteristics of enriched collagenase to the purified collagenase products showed the enriched product is comparable and in some cases superior to those higher priced purified collagenase products. The mean post-purification islet yields from three different centers were 4444 and 4261 IEQ/g pancreas when using the high and low protease enzyme mixtures, respectively. The mean stimulation indices calculated after analysis of glucose-stimulated insulin release were 2.68 and 2.11 when using the high and low protease enzyme mixtures, respectively.
Conclusions: The use of a low cost enriched collagenase products with biochemical characteristics comparable to purified collagenase enzymes were shown to be as effective in human islet isolation as higher priced products. These data indicate that using enzyme mixtures with high Wunsch activity (measurement of C2 collagenase) may not be required for successful human islet isolation. The amount of Wunsch activity/g tissue can be reduced to 25-50% of the amount currently used by some islet transplant centers for isolating human islets. Further studies are needed to validate enzyme compositions required for successful human islet isolation.
Citation: Loganathan G, Hughes MG, Szot GL, Smith KE, Hussain A, Collins DR, Green ML, Dwulet FE, Williams SK, Papas KK, McCarthy RC, Balamurugan AN. Low Cost, Enriched Collagenase-Purified Protease Enzyme Mixtures Successfully Used for Human Islet Isolation. OBM Transplantation 2019;3(2):14; doi:10.21926/obm.transplant.1902064.protease enzymes used for clinical islet isolation.
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