DE Collagenase Optimization Kit: a fresh approach to defining composition and dose for maximal 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 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 ) 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 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 . 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 mechanism of tissue dissocation To understand the mechanism of how bacterial collagenase-protease enzyme mixtures dissociate tissue, it is important to understand the structure of the extracellular matrix. This matrix is a collection of molecules secreted by cells into the intercellular spaces, providing structural and biochemical support to surrounding cells. The matrix contains collagen, elastin, proteoglycans, glycosoaminoglycans, and other proteins that anchor cells to the matrix. Collagen fibrils (10-300 diameter) or fibers (0.5 to 3 μm diameter) are the predominant proteins found in the matrix. The fibril or fiber structures act as backbone for the extracellular matrix, holding other macromolecules in a tight structure, resistant to proteolysis. The fibers and fibrils are composed of collagen monomers (1.5 nm diameter) that associate in an overlapping pattern to form larger multimeric structures. Included in the matrix are proteins that indirectly or directly hold cells to the matrix. These cell-anchoring proteins must be degraded to release cells from tissue. When a tissue is exposed to an optimal amount of functional collagenase and neutral protease activities, C1 collagenase starts collagen degradation by binding to the amino or carboxy terminal end of monomeric collagen. It appears that C2 collagenase binds and cuts the middle portion of collagen. Once bound, the catalytic domain cuts collagen’s triple helical structure. Since the fibers and fibrils contain many collagen unit structures, each catalytic domain can cut many collagen molecules in close proximity to the enzyme. Once the triple helical structure is cut, the three strands unravel, leading to the formation of denatured collagen (i.e., gelatin). These small portions of denatured collagen are now susceptible to further degradation by unbound C1 and C2 collagenases and neutral proteases. Presumably, the collagenolytic and proteolytic activities lead to release of sections of collagen from the fiber or fibril, leading to exposure of new binding sites for collagenase, thereby re-initiating the process described above. Concomitantly, as the extracellular matrix is “loosened”, proteins associated with the extracellular matrix change conformation, leading to exposure of new sites that can be cut by neutral protease. When a sufficient number of protein “anchors” that hold cells to each other or to the matrix are broken, cells are

VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 4 released from the tissue. Cell recovery does not require complete degradation of the extracellular matrix, but only an amount sufficient to free cells from their anchoring molecules.

A simple and direct method to optimize tissue dissociation enzyme mixtures

Key features of DE Collagenase-protease enzyme mixtures used in the Optimization Kit Each DE Collagenase product contains a consistent collagenase preparation of > 85% (w/w) collagenase, with no C. histolyticum neutral protease activity and minimal clostripain contamination [< 0.2 % (w/w) of total collagenase]. This collagenase is added in increasing amounts to a fixed amount of purified BP Protease (i.e., a Dispase equivalent enzyme). This enzyme cocktail is supplemented with an inert, low hygroscopic, peptide excipient that stabilizes the product during storage at ≤ 2-8 C. This protein crystalline powder (Figure 1) is easily weighed out on a balance. Repetitive sampling of the product yields a coefficient of variation for collagen degradation or neutral protease activity of < 5%. Internal accelerated stability studies show this material is extremely stable: 2 years at 4 to 8º C. The material can be shipped at ambient temperature.

Table 1: DE Collagenase Product Line

Collagenase: Product Amount per FALGPA NPA U/mg protease Name bottle U/mg ratio x 103

DE 10 100 mg ≈ 4.9 0.1/mg 20 DE 100 1 g U/mg

DE 20 100 mg ≈ 4.9 0.2/mg 41 DE 200 1 g U/mg

DE 40 100 mg ≈ 4.9 0.4/mg 82 DE 400 1 g U/mg

DE 60 100 mg ≈ 4.9 0.6/mg 122 DE 600 1g U/mg

DE 80 100 mg ≈ 4.9 0.8/mg 163 U/mg DE 800 1g The DE Collagenase Product line is summarized in Table 1. The 5 DE Collagenase products are labelled as the targeted amount of collagenase activity (in FALGPA units) per bottle, each with two different pack sizes: 100 mg and 1 g of crystalline powder. Each product pair has the same specific collagenase activity (FALGPA U/mg dry weight). A fixed amount of purified BP Protease is added to each product so that the specific NPA remains constant across all DE products. Throughout the range of DE Collagenase products, an increasing he amount of collagenase is added to a fixed amount of NPA. This results in an increasing collagenase:protease ratio, providing users greater flexibility in reducing the amount of protease in their cell isolation procedure. To simplify the calculation of the collagenase:protease ratios, the ratio in this table is multiplied by 1000.

VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 5 Purpose and rationale for the kit This white paper provides a defined method to identify the best DE Collagenase product and dose for use in your cell isolation or recovery process. If, at the end of this process, you want to further optimize the enzyme formulation and dose for your application, request the white paper “DE Collagenase Optimization Kit: a fresh approach to defining enzyme composition and dose for maximal cell recovery; Part 2: Advanced application to define enzyme composition and dose for maximal cell recovery”. The rationale for this approach assumes that collagenase has restrictive enzyme activity, degrading only native or denatured collagen. Adding excess collagenase activity to an enzyme mixture will have little effect on the rate of tissue dissociation, since its effectiveness is limited by the number of binding sites for collagenase on native collagen. Functional or non-functional collagenase has a narrow specificity for gelatin and does not degrade other proteins. In contrast, neutral protease activity in the presence of sufficient collagen degradation activity has a pronounced effect on the rate of cell release. If too little protease activity is added, digestion of partially proteolyzed collagen and other extracellular matrix proteins is slowed, leading to poor release and sub-optimal recovery of cells. If too much protease activity is added, tissue dissociation is overly rapid, causing potential damage of cell surface proteins, and leading to lower viability and loss of cells.

Prerequisites prior to using kit Prior to using this kit to identify an optimal enzyme formulation, the user must have specific prerequisites in place. These include:  A defined protocol for the digestion of the tissue or recovery of cells from in vitro culture. It is known that the optimal collagenase and protease concentrations required for cell release/recovery are dependent upon digest conditions. Some of these parameters are listed below.  Temperature  Buffer  Excipients (DNase, Albumin, etc.)  Tissue weight to buffer ratio  Agitation rate and energy  Is a reference product available?  Yes, if a crude collagenase product has been used previously. . Go to Table 2 below to identify the corresponding DE Collagenase product that is similar to the reference product. . Record the corresponding DE Collagenase (DE #, hereafter refered to as Designated DE product) & concentration where: Concentration = (Y) mg/mL used in the protocol.  No, if a crude collagenase or purified collagenase reference product has not been used. . Start with DE 200 Collagenase at a concentration of 2 mg/mL. Table 2. DE Collagenase vs Corresponding Collagenase

VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 6 DE Product FALGPA U/mg Corresponding Product DE 10 0.1 Worthington collagenase Types 1-4 Sigma Type I collagenase DE 20 0.2 Worthington collagenase Types 1-4 Sigma Type I collagenase DE 40 0.4 Sigma Type V

DE 60 0.6

DE 80 0.8 Liberase HI

 Sufficient tissue or adherent cells to perform a minimum of ten (10) or preferably fifteen (15) digest experiments to complete experiments 1 & 2 and 1 ,2 , & 3, respectively  A measurement method for characterizing and quantitating the cells recovered from the digestion process. This will be called the “digestion readout” and could be (for example) cell number, viability, cell cluster size, cell surface receptor density, or assay of relevant cell function.

Screening cell isolations to determine optimal DE product for use. Experiment 1: 1. The first experiment is designed to determine an acceptable protease activity used for cell recovery, while holding the collagenase activity constant. 1.1. Each DE Collagenase product in the sampler kit contains the same amount of purified protease activity (4.9 NPA U/mg dry weight) and increasing amounts of FALGPA collagenase activity per mg dry weight as indicated by the label on the bottle: 0.1, 0.2, 0.4, 0.6, and 0.8 FALGPA U/mg dry weight for DE10, DE20, DE40, DE 60, and DE 80, respectively. 1.2. If a Designated DE Collagenase and dose of product is identified above, this is the target collagenase concentration. 1.2.1. For example, the following formula below is used [Designated DE (X0 U/mg)][Concentration(Y mg/mL)] = Collagenase concentration (Z U/mL) Performing the calculation assuming 2 mg/mL of DE 20 (X) is the Designated DE product: 0.2 FALGPA U/mg (X) x 2 mg/mL (Y)= 0.4 FALGPA U/mL (Z) 1.2.2. Dividing the target collagenase concentration (U/mL) by the collagenase specific activity (U/mg) finds the collagenase concentration (mg/mL) in the final solution. By keeping the FALGPA Units constant for each DE product tested (red box), only the protease input will vary in each enzyme solution (blue box) beacause each DE Collagenase product has 4.9 NPA U/mg as shown in the following calculations:

DE 10 (0.4 U/mL) /(0.1 U/mg) = 4 mg/mL x 4.9 NPA U/mg = 19.6 NPA U/mL DE 20 (0.4 U/mL) /(0.2 U/mg) = 2 mg/mL x 4.9 NPA U/mg = 9.8 NPA U/mL DE 40 (0.4 U/mL) /(0.4 U/mg) = 1 mg/mL x 4.9 NPA U/mg = 4.9 NPA U/mL DE 60 (0.4 U/mL) /(0.6 U/mg) = 0.67 mg/mL x 4.9 NPA U/mg = 3.3 NPA U/mL DE 80 (0.4 U/mL) /(0.8 U/mg) = 0.50 mg/mL x 4.9 NPAU/mL = 2.4 NPA U/mL

VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 7

1.3. By keeping the DE Collagenase activity constant, you are testing the effect of a broad range of neutral protease activity in the isolation procedure. Given a constant collagenase activity for each DE collagenase tested, as the DE # increases (from DE 10 to DE 80), there is a corresponding decrease in the amount of protease activity in the digest mixture. The table above shows the range of neutral protease concentrations (in U/mL) for a fixed collagenase input of 0.4 FALGPA U/mL. 1.4. If no Designated DE Collagenase is identified in step 1.2 above, assume using a dose of 2 mg/mL of DE 20 as the Designated DE Collagenase since this is a common collagenase activity for most applications. 1.5. Use each of the 5 different enzyme mixtures in your cell isolation or cell recovery experiments and plot the digestion readout data (e.g.,cell yield, cell viability, or cell function, etc…) on the y axis and the lowest to highest protease activity on the x axis. 1.5.1. The graphed data below are from hypothetical isolations of primary cells where cell yield is plotted on the y axis. 1.6. Each result is independent of the other and are used for illustrative purposes. 1.7. When Collagenase input is held constant and NPA input is varied, four possible results scenarios may be observed. They are shown in graphs A, B, C, D below: Graph A: Fixed collagenase; optimal NPA identified Graph A illustrates a case in which an optimal dose A of 4.9 NPA U/mL and 0.4 FALGPA U/mL yielded 550 cells per g tissue Further studies will be performed in experiments 2 & 3 to optimize collagenase activity input (U/mL) and overall product dose (mg/mL) to use in the isolation procedure

Graph B: Fixed collagenase; optimal NPA not identified Graph B illustrates a case in which the upper limit B of protease activity (19.6 NPA U/mL) gave highest cell yields when using 0.4 FALGPA U/mL, but optimal neutral protease activity was not defined. Further studies will be performed in experiments 2 & 3 to optimize collagenase activity input (U/mL) and overall product dose (mg/mL) to use in the isolation procedure, where NPA input is 19.44 U/mL. User can also consider further optimization of protease activity by consulting the method in Part 2 of this white paper: “Advanced application to define enzyme composition and dose for maximal cell recovery”.

VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 8 Graph C: Fixed collagenase, optimal NPA not identified Graph C illustrates a case in which the lower limit of protease activity (2.4 NPA U/mL) gave highest cell yields when using 0.4 FALGPA U/mL, but optimal neutral protease activity was not defined Further studies will be performed in experiments 2 & 3 to optimize collagenase activity input (U/mL) and overall product dose (mg/mL) to use in the isolation procedure, where NPA input is 2.43 U/mL. User can also consider further optimization of protease activity by consulting method in Part 2 of this white paper: “Advanced application to define enzyme composition and dose for maximal cell recovery”.

Graph D: Fixed collagenase; NPA does not affect isolation Graph D illustrates a case in which neutral protease activity has no effect on cell isolation, given collagenase held constant at 0.4 FALGPA U/mL Further studies will be performed in experiments 2 & 3 to optimize collagenase activity input (U/mL) and overall product dose (mg/mL) to use in the isolation procedure, where NPA input is 2.43 U/mL.

Conclusions: The results from Experiment 1 provide the user specific information on the effect of neutral protease activity on the success of their cell isolation procedure. In the examples above, isolations A & D indicated an optimal protease activity for recovering primary cells from tissue or adherent cells from tissue culture vessels. In contrast, results from isolations B & C were unable to define a truly optimal protease activity for cell recovery. The user can take two different paths forward:  Accept as optimal the dose of protease which yielded the highest cell yield, and go to experiment 2 to determine the effect of different collagenase activities on cell isolation.  Consult part 2 of this white paper “DE Collagenase Optimization Kit: Advanced application to define enzyme composition and dose for maximal cell recovery”. Part 2 outlines a method to determine an optimal enzyme formulation by using supplemental Collagenase Gold or BP Protease. The choice depends upon how exacting the user’s need is for an optimal enzyme formulation. Experiment 2: 2. In this experiment, the effect of collagenase activity (FALGPA U/mL) is evaluated. To perfom experiment 2, the user must have completed Experiment 1 and identified an optimal (or provisional) neutral protease activity (NPA U/mL).

VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 9 2.1. For the purpose of illustration, we will use 4.9 NPA U/mL as a fixed, optimal protease activity. Since each DE product has 4.9 NPA U/mg, using 1.0 mg of DE product per mL will provide 4.9 NPA U/mL. 1.0 mg/mL of DE 10, 20, 40, 60 or 80 will provide 0.1, 0.2, 0.4, 0.6, or 0.8 FALGPA U/mL, respectively 2.2. As above, there are four potential, independent outcome scenarios. Representative cell isolation data are illustrated in the 4 graphs (E, F, G, and H) below Graph E: Fixed NPA, optimal collagenase activity identified Graph E results indicate that DE Collagenase 40 (0.4 FALGPA U/mL) is optimal when enzyme mixture also contains 4.9 NPA U/mL. The broad plateau of cell yield using the DE 40 or DE 60 products (0.4 and 0.6 FALGPA units/mL, respectively) is consistent with belief that excess collagenase does not have an adverse effect on cell recovery. Experiment 3 will titer the dose of this formulation for maximal cell recovery.

Graph F: Fixed NPA, optimal collagenase activity not identified Graph F results indicate that although optimal collagenase activity is not defined, the highest dose of FALGPA activity tested (0.8 FALGPA U/mL) gave the highest cell yields with 4.9 NPA U/mL. Experiment 3 will titer the dose of this formulation for maximal cell recovery . Users can consider further optimization of collagenase activity by consulting method in Part 2 of this white paper: “Advanced application to define enzyme composition and dose for maximal cell recovery”.

Graph G: Fixed NPA; Optimal collagenase input not identified Graph G shows that although the optimal collagenase activity is not defined, the lowest dose of FALGPA activity tested (0.1 FALGPA U/mL) gave the highest cell yields with 4.9 NPA U/mL. Experiment 3 will titer the dose of this formulation for maximal cell recovery. F Users can consider further optimization of collagenase activity by consulting method in Part 2 of this white paper: “DE Collagenase Optimization Kit: Advanced G application to define enzyme composition and dose for maximal cell recovery”.

VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 10 Graph H: Collagenase input has no effect at fixed NPA input Graph H shows that collagenase activity had no effect on cell isolation when neutral protease was held constant at 4.9 NPA U/mL. For collagenase, this result may not be atypical since excess collagenase will have minimal adverse effect on cell recovery. Experiment 3 will titer the dose of this formulation for maximal cell recovery .

Conclusions: If acceptable results were obtained in experiments 1 & 2, then the optimal enzyme formulation has been determined, and the only remaining task is to define the optimal dose (mg/mL) to use in the isolation procedure (see Experiment 3 below). This step is equivalent to those used to pre-qualify a new lot of crude collagenase, except that in this case, the enzyme composition enzyme mixture has been determined.

Experiment 3: This experiment is recommended to determine optimal DE Collagenase concentration and performance plateau. The range of concentrations assessed should change by 20% increments of the dose determined in experiment 2. Assuming the dose of chosen formulation used in experiment 2 is 1.0 mg/mL, then dose titration will yield 0.6, 0.8, 1.0, 1.2, and 1.4 mg/mL in experiment 3. The digestion readout parameter (cell yield, cell function, etc) for each dose should be plotted and an optimal mg/mL dose determined. If there is a peak and no plateau, the user can further titer the dose around the highest response using a tighter dose range (e.g., 5 or 10% increments) to determine if a dose plateau exists for the cell recovery procedure.

Additional Benefits when using DE Collagenase The broader range of collagenase to protease ratios provided by DE Collagenase products enables users to explore new enzyme compositions using lower amounts of protease, potentially improving cell viability or function. The graphs below (I, J) plot collagenase type along a Y axis of percentage of dry weight and X axis of the ratio of collagen degradation activity (CDA) to neutral protease activity. Enzyme activities for commonly used crude or enriched collagenase products in Figure I were determined by assays developed at VitaCyte, enabling estimation of collagenase mass. Graph I: % Collagenase mass and CDU/NPA activity for crude and enriched collagenase products Graph I plots each crude or enriched collagenase product against their %collagenase (CDA U/mg dry wt) and ratio of CDA to NPA per mg dry weight. The percentage of collagenase in the product varies depending on the manufacturer (Sigma, Worthington) and the form of product (enriched Type XI, crude Type 1 and Type IV).

It is important to note the narrow range of collagenase:protease ratios, which is limited by the C. histolyticum fermentation process.

VitaCyte White Paper: DE Collagenase Optimization kit, Part 1 June 2, 2017 • page 11 Graph J: Collagenase-protease enzyme compositions used to isolate different cell types The breadth of purified collagenase-protease enzyme compositions used to isolate different cell types is shown in Graph J. This plot summarizes results from use of VitaCyte’s purified collagenase-neutral protease mixtures to isolate specific cell types. As noted in the graph, all of these enzyme mixtures have a high percentage of purified collagenase (>70%). For several of these compositions (rodent islets and rat hepatocytes), the collagenase: protease ratios is higher than those found when using crude or enriched collagenase products. This is consistent with the mechanism of cell isolation described above where the primary function of the protease is to accelerate degradation of collagen cut by collagenase subsequently leading to loosening of the extracellular matrix and degradation of anchoring proteins that hold cells to the matrix. Graph K: % Collagenase mass and CDU/NPA activity for five DE Collagenase products Graph K plots representative lots of the five DE Collagenase products. Of note on this Graph is the increasing percentage of collagenase per dry weight of product as the DE Collagenase number increases. Also, since a highly enriched collagenase is added to a fixed amount of purified BP Protease, the collagenase to protease ratios are tightly controlled. This enables manufacture of a broader range of low cost collagenase products than previously available.

Conclusions and optional path forward The introduction of DE Collagenase products creates a new paradigm for selecting a lot of collagenase. Once an optimal formulation and dose of DE Collagenase is determined, the user can easily make adjustments when switching to a new lot of DE Collagenase product by consulting the Certificate of Analysis, and determining if an adjustment is required change in the mass of product used per mL in the isolation procedure. If your goal is to validate the enzyme parameters used in a cell isolation procedure, then you may require additional guidance apart from the instructions in this document. Such guidance may be found in the white paper entitled “DE Collagenase Optimization Kit: Advanced application to define enzyme composition and dose for maximal cell recovery”.

1. Cavanagh T, Dwulet F, Fetterhoff T, Gill J, SC L, McCarthy R. Collagenase Selection. In: RP Lanza WC, ed. Pancreatic Islet Transplantation. Austin: RG Landes; 1994: 39-50.

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