PeloNews with Focus on Liver/Islets

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PELONews Islet Isolation Reducing the Cost of Human Islet Isolation Clostridium histolyticum collagenase is a critical reagent for isolating islets from human pancreatic tissue. The development of Liberaseä HI Purified Enzyme Blend overcame the problem of repetitive evaluation of different crude collagenase lots to identify an acceptable product. The potential threat of transmission of spongiform encephalopathy from bovine tissue led NIH to disallow the use of Liberase HI in the Clinical Islet Transplantation Consortium (CITC) trial. Initially, Nordmark/Serva was chosen as the sole enzyme vendor for this trial, but VitaCyte and Roche were added as suppliers after several experienced islet transplant laboratories were unable to achieve human islet yields comparable to those obtained when using Liberase HI. All of the enzyme formulations in the CITC trial used about 20 Wünsch Units of collagenase per g of trimmed tissue, the same dose used in the Immune Tolerance Network Trial for human islet transplantation. Human islet isolation is an inefficient process since ≈ 53% of the islet isolations had a sufficient number of islets for subsequent transplantation. The use of more consistent collagenase reagents may increase the efficiency of this process. A new era of understanding enzyme-mediated tissue dissociation began when Matsushita’s laboratory reported the gene sequence and protein domain structure of C. histolyticumcollagenase. His laboratory showed class I (C1) and class II (C2) collagenase were expressed by separate genes, validating earlier observations that showed these two classes had different enzyme activity profiles and were separated by chromatographic techniques. They also showed the degradation of native collagen only occurred <strong>with</strong> those molecular forms of collagenase that contained a catalytic domain and at least one collagen binding domain. VitaCyte correlated the specific collagen degradation activity (CDA) of purified natural collagenase to the molecular form of the enzyme. Intact C1 <strong>with</strong> two collagen binding domains was 7-10% more active in degrading native collagen when compared to “truncated” C1 or intact C2, each having only one collagen binding domain. The new information on collagenase structure-function led to the development of a hypothetical model for collagenase-protease digestion of tissue. The six key principles from this model are: 1. Differences in the efficiency of intact C1 vs truncated C1 or intact C2 likely reflects the ability of intact C1 <strong>with</strong> two collagen binding domains to stick tighter to the collagen monomer during the degradation process than those forms <strong>with</strong> a single collagen binding domain. 2. Collagenase appears to degrade collagen by moving from the amino terminal end of the collagen monomer to carboxy terminal, degrading a collagen monomer in a fiber or fibril. 3. Excess collagenase CDA must be used to ensure rapid degradation of collagen; excess purified collagenase will have a minimal adverse effect on cells since it has a narrow specificity for native collagen and gelatin. 4. Collagenase products that contain predominantly intact collagenase will have the highest specific CDA, and can be used at lower doses in the islet isolation procedure than those products that contain multiple forms of collagenase. 5. The ratio of C1 to C2 is unlikely to impact the efficiency of degradation of collagen in the extracellular matrix since these two enzymes work cooperatively to degrade native collagen. 6. Choice and dose of neutral protease used for the isolation procedure is the critical variable that must be controlled if excess intact collagenase is used in the isolation procedure. Read full paper here: https://www.vitacyte.com/news/reducing-cost-human-islet-isolation
Human Islet Read here common protocols used in human islet isolation. Includes a table listing factors to consider when focusing on enzymes used in these isolation procedures: https://www.vitacyte.com/product-applications/humanislet-applications/ Hepatocytes Rodent Hepatocytes Overview. Overview discussion of methods for isolating rodent hepatocytes. Includes a table of recommended products and doses for use <strong>with</strong> rat and mouse islet isolations. Rodent HEPATOCYTE APPLICATIONS We recommend: Defined Enriched Collagenases DE Collagenase 60 or 600 DE Collagenase 10 or 100 These recommendations are dependent on the protocol used in the isolation procedure. (Recommended method: Seglen’s Two -step Isolation Method 1,2. F To read more, please go to: https:// www.vitacyte.com/product-applications/ rodent-hepatocyte-applications/ 1,2: Seglen P.O. (1976) Preparation of isolated rat liver cells. Methods in Cell Biology 13, 29-83.; 2: Seglen PO. Isolation of hepatocytes. In: Celis JE, ed. Cell biology : a laboratory handbook. San Diego :: Academic For Hepatocytes: Learn more
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