DE Collagenase Optimization Kit: a fresh approach to defining enzyme composition and dose for maximal cell recovery Part 1: Determination of an optimal DE Collagenase product and dose for recovery of primary cells from tissue or adherent cells from in vitro culture substrates White Paper June 2, 2017 Introduction 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 Optimzation kit are defined enzyme mixtures, containing intact forms of C. histolyticum collagenase and purified BP Protease (a Dispase- equivalent enzyme). Each DE Collagenase product is manufactured to ensure consistent enzyme activity from lot-to-lot. This consistency enables users to confidently translate DE Collagenase Optimization kit results to selection of a DE Collagenase, or purified collagenase-BP Protease products to recover cells from tissue or after in vitro culture. Current state of the art Limitations of crude collagenase Crude collagenase products from Clostridium histolyticum have been used since the 1960’s for isolating cells from tissue. These products are minimally purified, C. histolyticum culture supernatants, containing a broad range of enzyme activities including collagenase, endoprotease, exoprotease, phospholipase, neuraminidase, hyaluroinidase, -D-galactosidase, -N-acetyl-D-glucosoamidase, and -L-fucosidase.1 This organism is a saprophyte that secretes these enzymes to break down organic matter into simpler components that can be used as nutrients for bacterial growth. The diversity of enzyme activities in crude collagenase is beneficial for sustenance of the organism in nature, but is problematic when used as a biochemical reagent to recover cells. Crude collagenase contains all the enzyme activities listed above, and little to no attempt is made to manipulate the enzyme activities contained in the product. Each lot of product is unique, since enzyme activities reflect the characteristics of the bacterial culture supernatant from which the product was derived. Manufacturers of crude collagenase quickly became aware of this problem after customers had difficulty using the product for cell isolation because of high lot to lot variability. To address this issue, manufacturers developed lot qualification programs where small samples of specific lots of crude collagenase products were sent to customers so they could assess whether the reagent successfully isolated cells in their laboratory. At the time of sampling, customers requested that a specific amount of product from each lot be held in reserve for a short period of time, allowing them to assess the performance of these lots for use in their isolation procedure. If the product worked, then the reserved amount of a specific lot (i.e., “good” lot) was purchased by the customer, and the non-selected lots VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 2 returned to inventory. If none of the lots worked (i.e., “bad” lots), all the reserved lots were returned to inventory and the process repeated again with product from the same or different supplier until the customer identified a good lot of crude collagenase. This “trial and error” approach has been the only path forward to use crude collagenase because of lot to lot variability and the unique requirements of the user in recovering or isolating a specific cell population. For some cell recovery applications there may be little or no problem in obtaining a good lot of collagenase, but for others this may not be the situation. In either case, the biochemical composition of these reagents is a “black box" so if the quality or supply of product is disrupted, then the downstream ability to isolate cells is compromised. How can you identify key characteristics of a critical material in your cell recovery process if the biochemical characteristics of the material is unknown? To overcome these potential problems, a brief overview of the key enzymes responsible for releasing cells from the extracellular matrix is presented, along with a theoretical model of how these enzymes release cells from the matrix. This knowledge is relevant for understanding the application of the DE Collagenase Optimization kit to the problem of identifying an optimal enzyme formulation and dose for recovery of primary cells from tissue or adherent cells from tissue culture substrate. New knowledge of collagenase and proposed mechanism for tissue dissociation In the last twenty years, the key enzymes in crude collagenase responsible for release of cells from tissue were identified as collagenases (C. histolyticum class I and class II collagenases) and neutral protease (C. histolyticum neutral protease and clostripain). Class I (C1) has strong gelatinase activity (i.e., gelatin substrate, denatured collagen) but low peptidase activity (i.e., Pz peptide or FALGPA peptide substrate) whereas the converse is true for class II (C2) collagenase. As will be noted below, these two classes work cooperatively to degrade native collagen. Subsequent studies showed that C. histolyticum class I (C1) and class II (C2) collagenases are expressed by separate genes. Each class contains about 1000 amino acids, subdivided into 4 protein domains of ≈ 670, 110, 110, and 110 amino acids. The function of each domain was shown by further analysis of purified recombinant C1 or C2 collagenase from bacteria that expressed each gene product. The largest domain is the catalytic domain found at the amino terminal end of the protein followed by linking (intervening) domain(s) and collagen binding domain(s) (CBDs) at the carboxy end of the molecule. Each form is identified by its class and molecular weight expressed in kilo Daltons (kDa). Intact C1 has a catalytic domain, one linking domain and two CBDs (C1116 kDa), whereas intact C2 has a catalytic domain, two linking domains and one CBD (C2114 kDa). The truncated form of C1 (C1100 kDa) is generated after proteolysis of a short segment linking the two CBDs together. This can occur if the bacterial culture supernatant has high protease activity or if the product is “mishandled” after fermentation. The figure below is the crystal structure of each domain found in intact C1 with a schematic outline of the protein domain structure below. If the structure were determined for the entire enzyme, all of the protein domains would be linked together. NH Catalytic Linking Binding Binding COOH VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 3 Three forms of collagenase, containing a catalytic domain and at least one CBD, are effective in degrading native collagen. These three enzymes ― C1116 kDa, C2114 kDa, and C1100 kDa ― are termed “functional collagenase” since they possess the unique ability to degrade the fundamental unit of all collagen forms: a tightly wound, triple helical protein chain. Subsequent studies showed purified intact C1 with two CBDs had 7-10 fold higher collagen degradation activity than purified intact C2 or purified truncated C1. Collagenase-protease enzyme mixtures containing primarily intact C1 and C2 with an optimal dose of protease are the highest quality and most efficient collagenase enzyme products because of a high specific collagen degradation activity (CDA U/mg protein) at the lowest amount of protein. This is seen in an earlier report comparing the efficiency of purified collagenase from VitaCyte and another supplier to correlate yield of human islets to quality of collagenase. The use of VitaCyte’s product containing primarily C1116 kDa, and C2114 kDa gave significantly higher islet yields and shorter digestion times than those isolations performed using another supplier’s enzymes that contained primarily C1100 kDa, and C2114 kDa. “Non-functional collagenases” are defined as collagenase with a functional catalytic domain and no CBDs. The inability of these enzymes to bind native collagen means they are active only on those substrates that require a functional catalytic domain. These substrates include collagenase-specific peptide substrates (Pz or FALGPA peptide) or gelatin. The latter enzyme activities are termed gelatinases, based on the substrate that detects this activity. Gelatin is a non-specific substrate, since other proteases also degrade this protein. Functional collagenase also has gelatinase and peptidase activities, so these substrates cannot distinguish functional from non-functional collagenase. Although C1 and C2 collagenase have very unique specificities for degrading native collagen, their narrow specificity does not enable them to degrade other proteins. Both purified forms (alone or together) are ineffective in tissue dissociation, leading to poor recovery of cells from tissue. This problem is overcome when neutral protease activity is part of the collagenase-containing, enzyme mixture. The role proteases play to accelerate cell release from tissue is summarized in the hypothetical mechanism below Theoretical