Liberase™ DH Research Grade

Description

• General description: The most commonly used method for decellularization procedures, such as tissue dissociation and cell harvesting, is based on the use of proteolytic enzymes. Proteases disrupt the extracellular matrix of tissue to allow the release of individual cells or the harvesting of cultured cells for transfer. The goal of cell isolation is to maximize the yield of dissociated cells that are viable and functionally active.[1]Liberase enzyme technology comprises methods for purifying clostridial collagenase isoforms to high specific activity, and for blending them together with high-specific-activity neutral protease in optimal ratios for effective dissociation of primary tissues and cultured cells. Liberase DH Research Grade contains highly purified Collagenase I and Collagenase II. These two collagenase isoforms are blended in a precise ratio to each other, and with a high concentration of Dispase®, a non-clostridial neutral protease.[1]Liberase enzymes are designed to increase the quality and reproducibility of tissue dissociation, and improve the viability and functionality of isolated cells. Liberase Purified Enzyme Blends replace traditional collagenase, which is a crude and variable fermentation by-product of Clostridium histolyticum.[1]
• Application: Liberase™ DH (Dispase High) Research Grade is used for the dissociation of a broad range of tissue types, where high purity of the enzyme blend is necessary for high cell yield and viability. Bacterial by-products, such as endotoxins, are reduced up to several thousand-fold.It has been used for the digestion of metastatic tumor fragments.[2] It has been used for the enzymatic detachment of human embryonic stem cells colonies.[3]
• Features and Benefits: Maximize viability and yield of isolated cells with an enzyme blend that has less clostripain and trypsin activity, as well as reduced endotoxin content.Count on higher specific activity of the enzyme blend as a result of higher Collagenase I + II purity (determined by HPLC analysis).Obtain higher experimental reproducibility due to higher lot-to-lot consistency.
• Preparation Note: Stabilizers: CalciumWorking concentration: Liberase Research Grade Enzyme Working ConcentrationLiberase enzymes have significantly higher specific activities than traditional collagenases. This means that the working concentration of Liberase Research Grade Purified Enzymes, expressed in mg/ml, will be lower than that of traditional collagenase.When the application is on the Roche list of applications at www.collagenase.com, use the Liberase Research Grade concentration recommended for that application.When the application is not included on this list, first use Liberase TM Research Grade at a concentration of 0.08–0.28 Wünsch units/ml.The goal is to determine the best starting concentration of Liberase Research Grade Enzyme Blends. This is a starting point, and the final concentration may vary due to differences in procedure and lot-to-lot differences in traditional collagenase.Collagenase Working ConcentrationMultiply your previous collagenase working concentration (mg/ml) by its specific activity (Wünsch units/mg, [as determined above]), to obtain Wünsch units/ml. To determine how much Liberase Research Grade Enzyme Blend to use, first multiply your collagenase working concentration (in Wünsch units/ml) times the total volume of your working enzyme solution to obtain the total collagenase activity needed (Wünsch units). Divide the total collagenase activity required by the Liberase Research Grade stock concentration ( “Reconstitution and Storage”). This indicates how many milliliters of Liberase Research Grade Enzyme Blend stock solution to use in your working enzyme solution.Storage conditions (working solution): Store unused stock solution in single-use aliquots at -15 to -25 °C. For further information on product stability, please visit the Roche Liberase Enzyme website at www.collagenase.com.Note: Avoid repeated freezing and thawing!
• Reconstitution: Reconstitute the lyophilized enzyme with injection-quality sterile water or tissue-dissociation buffer. Do not add serum or other components (e.g., albumin or protease inhibitors) to the dissociation buffer. Enzyme stability is reduced at higher concentrations and warmer temperatures (4 °C). Avoid both the above conditions.Reconstitute the entire vial. Do not weigh individual aliquots of the lyophilizate. The introduction of moisture into the vial results in a decline in enzymatic activity.Place vial on ice to rehydrate the lyophilized enzyme.Gently agitate the vial at 2 to 8 °C until the enzyme is completely dissolved (max. 30 min).Depending on the type of tissue-dissociation buffer used to dissolve Liberase Research Grade Purified Enzyme Blends, slight precipitations may be observed which readily dissolve in the diluted working solution, and have no influence on enzyme activity.Remove an aliquot of the stock solution to prepare the working solution.Reconstitution volume2 ml (1 vial with 5 mg–10 mg pack size)10 ml (1 vial with 50 mg–100 mg pack size)Collagenase Wünsch (units/ml)13 (1 vial with 5 mg–10 mg pack size)26 (1 vial with 50 mg–100 mg pack size)Total Collagenase concentration [mg/ml]2.5 (1 vial with 5 mg–10 mg pack size)5.0 (1 vial with 50 mg–100 mg pack size)
• Storage and Stability: Store dry
• Other Notes: This product is intended for life science research and in vitro use only. These products are not to be used for diagnostic or clinical applications, such as human islet transplantation. All 1st generation Liberase and Liberase Blendzyme products (Liberase HI, CI, RI, PI, Liberase Blendzyme 1, 2, 3, 4) have been replaced by the 2nd generation Liberase Research Grade and Liberase GMP grade enzyme blend portfolios. For instructions concerning the transition from previously used Liberase EnzymesFor life science research only. Not for use in diagnostic procedures.
• Legal Information: Dispase is a registered trademark of Godo Shusei Co., Ltd.