Most collagenase and protease products stand on a firm foundation of decades of research and use in labs across the world. VitaCyte’s unique contributions are simple. The first is the consistency of our formulations. Our manufacturing processes give us better control over key enzyme fermentation processes, which means no need for lot testing because our products are designed and developed for superior consistency.
Second, we know the science. VitaCyte’s co-founders were key contributors on the project team that developed the first commercially-available, purified collagenase-protease enzyme blend for clinical use. Today, the VitaCyte team continues to push the boundaries of the science of cell isolation and contribute to the ongoing research, design, and development of these critical reagents and their uses.
McCarthy RC, Breite AG, Green ML, Dwulet FE. Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria. Transplantation. 2011;91(2):137-45.
McCarthy RC, Spurlin B, Wright MJ, Breite AG, Sturdevant LK, Dwulet CS, et al. Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation. Transplant Proc. 2008;40(2):339-42.
Holdcraft RW, Green ML, Breite AG, Circle L, Meyer ED, Adkins H, et al. Optimizing Porcine Islet Isolation to Markedly Reduce Enzyme Consumption Without Sacrificing Islet Yield or Function. Transplantation Direct. 9000;Online Now.
Lot qualification: a common but costly pathway to select collagenase products for cell isolation.
A common approach for adopting or maintaining a consistent cell isolation procedure is to qualify lots of traditional (i.e., crude or enriched) collagenase products prior to use. This is a standard practice for most laboratories since it is an effective approach to help identify a lot of collagenase that works acceptably for the user’s cell isolation application. Unfortunately, most scientists do not realize the hidden costs of using traditional collagenase products because they are unaware of the basic biochemistry and function of collagenase reagents as it relates to their performance in recovering cells from tissue. This white paper is written to address and answer those questions.
Overcoming the limitations of traditional collagenase products
Traditional collagenase products (i.e., crude or enriched collagenase) have been used for over 50 years to isolate cells from tissue. Little has changed in the selection and use of these products since the original introduction of crude collagenase in the early 1960’s for use in cell isolation procedures. Many laboratories pre-qualify lots prior to purchase of a larger amount of product. This procedure is repeated once the supply of the specific product lot is depleted. In the mid-1990’s, the need for development of defined collagenase products was driven by clinical research scientists who needed to increase human islet yields for use in islet transplantation into adult patients with type 1 diabetes. These and subsequent studies showed that Clostridium histolyticum collagenase and neutral protease were the key enzymes responsible for degrading the extracellular matrix, leading to the release of cells from tissue. The knowledge gained from development of purified collagenase-protease enzyme mixtures has not been translated into improvements in low cost collagenase products. This white paper focuses on defining characteristics of an ideal, low cost collagenase product that overcomes the limitations of traditional collagenase products, and shows how VitaCyte’s DE Collagenase products are aligned with these ideal characteristics.
DE Collagenase Optimization Kit: a fresh approach to defining enzyme composition and dose for maximal cell recovery
Currently, there is no simple method for defining optimal collagenase enzyme formulations for recovery of primary cells from tissue, or adherent cells from in vitro culture substrates. Crude or enriched collagenase products commonly used for these applications are minimally purified, ill-defined enzyme mixtures in which the collagenases and neutral proteases responsible for releasing cells from the extracellular matrix have variable enzyme activities and comprise only 3 to 20% of product dry weight. The DE Collagenase Optimization kit is designed to help determine the best DE Collagenase product and dose for primary cell isolation or cultured cell recovery. The products in the DE Collagenase Optimization kit are defined enzyme mixtures, containing intact forms of histolyticum collagenase and purified BP Protease (a Dispaseä-equivalent enzyme). Each DE Collagenase product is manufactured to ensure consistent enzyme activity from lot-to-lot. This consistency enables users to confidently translate results after using the DE Collagenase Optimization kit to selection of a DE Collagenase, or purified collagenase-BP Protease products to recover cells from tissue or after in vitro culture
What is an acceptable rodent islet yield?
Isolation of rodent islets is a unique skill that usually requires a person interested in adopting this skill to receive advice or training from an islet isolation expert who will provide a demonstration of the method but will also share often unwritten recommendations that are often critical to the success of the procedure. A complication arises when either individual is faced with isolating islets from a new and different rodent strain. The key question asked is what islet yields should I expect? It has been known for many years that islet yields vary by the age, sex, and strain of the animal. These expected islet yields are rarely published in the scientific literature and if mentioned, they are briefly noted in the materials and methods or in a table in the results section. Commonly, the primary way an individual obtains this information is to ask their colleagues about their islet isolation experience using animals with similar characteristics: age, sex, and strain. To provide a resource to the rodent islet isolation community, VitaCyte surveyed its users and asked them to submit rodent islet yields isolated from different strains of mice and rats. These results are restricted to those isolations performed using purified collagenase and protease for reasons outlined in the white paper.
Green M, Beechler C, Breite A, Dwulet F, McCarthy R. 12th Congress of the International Xenotransplantation Society 2013 – Osaka, Japan #250 “Optimization of a Porcine Islet Isolation and Purification Procedure that Utilizes Recombinant Collagenase”
Breite AG, Dwulet FE, McCarthy RC. 13th World Congress of IPITA 2011 – Prague, Czech Republic O-038 “Characterization and functional assessment of Clostridium histolyticum class I (C1) collagenases and the synergistic degradation of native collagen in enzyme mixtures containing class II (C2) collagenase”
McCarthy RC, Breite AG, Dwulet FE. 8th Annual IFATS Conference 2010 – Dallas, TX “Biochemical Analysis of Crude Collagenase Products Used in Adipose Derived Stromal Cell (ADSC) Isolation Procedures and Development of a Purified Tissue Dissociation Enzyme (TDE) Mixture”
Breite AG, McCarthy RC, Dwulet FE. International Pancreas and Islet Transplantation Association – International Xenotransplantation Association Joint Meeting 2009 – Venice, Italy Presentation IPITA O 3.7 “Assessing Neutral Protease Activity in Tissue Dissociation Enzyme Mixtures”
McCarthy RC, Breite AG, et al. MSC Regenerative Medicine and Adult Stem Cell Therapy 2009 – Cleveland, OH “Analysis of the Enzyme Composition of Crude Collagenase Products Used in Adipose Derived Stromal Cell Isolation Procedures”