Human pancreatic tissue dissociation enzymes for islet isolation: Advances and clinical perspectives

Diabetes & Metabolic Syndrome: Clinical Research & Reviews 14 (2020) 159e166

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Review

Human pancreatic tissue dissociation enzymes for islet isolation: Advances and clinical perspectives Gopalakrishnan Loganathan a, b, Appakalai N. Balamurugan a, Subhashree Venugopal b, * a b

Clinical Islet Cell Laboratory, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, USA School of Biosciences and Technology, Vellore Institute of Technology, Vellore, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 October 2019 Received in revised form 26 January 2020 Accepted 27 January 2020

Background and aims: Successful clinical human allo or auto-islet transplantation requires the recovery of a sufficient number of functional islets from either brain-dead or chronic pancreatitis pancreases respectively. Methods: In the last two decades (2000e2019), significant progress has been made in improving the human islet isolation procedures and in standardizing the use of different tissue dissociation enzyme (TDE; a mixture of collagenase and protease enzymes) blends to recover higher islet yields. Results and Conclusions: This review presents information focusing on properties and role of TDE blends during the islet isolation process, particularly emphasizing on the current developments, associated challenges and future perspectives within the field. © 2020 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Keywords: Islet isolation Islet transplantation Collagenase Thermolysin Neutral protease BP-Protease

1. Introduction Pancreatic extracellular matrix and basement membrane composition: Pancreas is an organ composed of a vast network of endocrine and exocrine tissue. Islets comprise the endocrine portion and are connected to the surrounding exocrine tissue by an extensive extracellular matrix (ECM) [1,2]. A large percentage of human islets reside near the exocrine ducts [3]. For successful islet isolation procedures, the tissue dissociation enzymes (TDEs) must effectively breakdown this ECM that keep islets embedded in the surrounding tissue, without impairing their function. All ECM consist of both fibrillar and non-fibrillar proteins. The predominant fibrillar proteins include collagen types I, III, and V [4]. These proteins all share a common triple-helix structure that contribute to the strength of the protein but also serve as an important target for collagenases used in islet isolation [5]. Collagen type VI has recently been identified as another key constituent in pancreatic ECM that is abundant at the islet and the surrounding exocrine acinar interface.

Abbreviations: TDE, Tissue dissociation enzyme; WU, Wunsch Units; NP, Neutral protease; MTF, Mammalian Tissue Free; CDA, Collagen degradation assay; Wunsch, Measurement of C2 activity. * Corresponding author. School of Biosciences and Technology, Vellore Institute of Technology, Vellore, India. E-mail address: [email protected] (S. Venugopal). https://doi.org/10.1016/j.dsx.2020.01.010 1871-4021/© 2020 Diabetes India. Published by Elsevier Ltd. All rights reserved.

It differs from other types of collagen in that it does not form fibrils. It is also resistant to cleavage by bacterial proteases due to its uniquely high levels of disulfide bridge-forming cysteine residues [6]. Closer to the surface of the islets and lining the exocrine ducts, there is a distinct area of ECM referred to as the basement membrane (BM) that has a significantly altered composition compared to the rest of the ECM. While it does contain some of the fibrillar proteins, it has a much higher concentration of collagen type IV and laminin [7]. Several factors affect the composition of the pancreatic ECM making each organ unique. This variability in the ECM has drastic implication for islet isolation, in that the ECM of pancreases from certain patient groups have an increased resistance to standard TDE mixtures. It has been well-observed that some TDEs more easily digest the tissue and release islets from pancreatic tissue of older donors, when compared to young donors [1]. One study identified that a disruption in the BM of acinar tissue in pancreases from donors of age 50 [6], by allowing enzyme solutions to retrogradely perfuse through the pancreatic ducts, permitted more easy access to free the desired islets. Though Bedossa et al., reported higher amounts of total collagen in patients [8], a recent study noticed a significant increase in the amounts of collagen type IV and laminin, the main elements of the BM, in pancreases from younger patients [9]. This distinctive BM structure could be the cause of decreased digestion in younger pancreases. Advanced chronic pancreatitis

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(CP) has also been shown to decrease yields during islet isolations (Fig. 1B). It has also been observed, that pancreatic tissue from patients with CP has significantly higher amounts of collagen than normal pancreases [10,11]. 1.1. Tissue dissociation enzymes Historical perspectives: Collagens are a major component of connective tissue ECM throughout the human body. As implied by its name, collagenase has been the standard component in many enzyme blends used for cell isolation, having the unique ability to sever the triple-helical structure of native collagen [12]. It was first commercially available from Worthington Biochemical Corporation in 1959. By the 1970’s many other suppliers offered a “crude collagenase” produced from Clostridium histolyticum which, apart from collagenase, contained many other enzymes, such as phospholipase, clostripain, elastase, aminopeptidase, galactosidase, and other proteases [13]. This crude product was widely used to isolate specific cell types from different tissue sources such as pancreas [14], liver [15], cartilage [16], heart [17], adipose [18], and bone [19]. Collagenases are Zn-metalloproteinases with endopeptidase capacities produced from the fermentation broth of C. histolyticum. Considering most other proteases are ineffective at degrading collagen’s triple helix, the unique specificity that collagenases share has been the motive for extensive research into its biological and enzymatic properties [20]. Collagenase is produced by two distinct genes, ColG and ColH, coding for Class I (C1) and Class II (C2) collagenases having molecular weights of 116 kDa and 114 kDa, respectively [21]. C1 and C2 collagenases appear to digest most forms of fibrillar collagen since the triple helical structure is a key component of these molecules. The C1 and C2 enzymes work synergistically to hydrolyze these helical structures into smaller peptides [22,23]. Collagenase has been commonly used for human pancreas digestion. Its effectiveness in isolating pancreatic islets has made it the standard base of almost every enzyme mixture for clinical and research islet isolation protocols. Up until the 1990’s, the use of “crude” or enriched collagenase for pancreatic islet isolation [24] came with some problems, such as lot-to-lot variability of enzyme activity and endotoxin contamination. To overcome these hurdles, a purified enzyme blend from Boehringer Mannheim Biochemicals

(now sold by Roche), “Liberase™-HI Purified Enzyme Blend”, was introduced in 1994. This enzyme was offered as one vial with a fixed enzyme dose. It contained the C1/C2 collagenase from C. histolyticum and thermolysin (TL), a thermostable neutral protease from Bacillus thermolyticus rokko, and a low endotoxin contamination. Introducing this enzyme in the market immediately improved islet yields from clinical islet isolation procedures [25]. As a result, crude enzyme usage declined for human islet isolation. However, purification of collagenase did not eliminate batch variability, as many laboratories were still obtaining inconsistent islet isolation results. In recent years, isolation enzyme blends have begun to contain purified C1/C2 collagenases. C1 is more stable and has a greater activity towards native collagen; whereas, C2 has the ability to digest a broader range of peptide substrates when compared to C1. Among the three well-known enzyme suppliers (VitaCyte, Serva, and Roche), only the VitaCyte and Roche C1/C2 collagenase ratio is known (60:40) [26]. Some studies have indicated that the ratio between C1 and C2 collagenases is important for optimal islet isolation in rat and human pancreas [27,28]; conversely, previous studies have demonstrated that different C1/C2 ratios and doses of purified collagenase, when tested for human islet isolation, had no significant effect on digestion and islet yield [29]. Collagenase: In order to separate the islets from the rest of the pancreatic islet tissue, the interface between islets and exocrine tissue needs to be digested. Collagen is the main component of this interface. Collagenases are proteolytic enzymes, which break the long chain-like molecules of proteins into peptides. Proteolytic enzymes are classified based on their target site of cleavage. There are two major groups, exopeptidases, which target protein terminal ends, and endopeptidases, which target sites within a protein. Proteolytic enzymes are present in mammals, bacteria, and some viruses and plants Collagenases bind specifically to, and cut, amino acid sequences containing Pro-X-Gly-Pro, where X is usually an amino acid that is neutral, such as arginine or lysine. The combination of these sequences is unusual in proteins other than collagens. Therefore, collagenases are highly specific in their activity and are also the only enzymes that are able to degrade native collagen [30e32]. There are two isoforms of collagenase, class I and class II, also called ColG and ColH, respectively. Two homologous but separate genes, colG and colH, encode the respective isoforms [33].

Fig. 1. Islet isolation difficulties due to donor-to-donor variability, severity of fibrosis and embedded islets morphology from young donor pancreas: Representative image of (A) Brain-dead donor pancreas, (B) Chronic pancreatitis pancreas and (C) image of embedded islet (islet surrounded by exocrine tissue) morphology from young donors resulting with large pellet size (D).

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The two classes have both endopeptidase and exopeptidase activity. They have similar specificity but complement each other in terms of their mode of action on the collagen triple helix. Class I (C1) collagenase hydrolyze loci at the triple helix ends and then continues to cleave all three strands simultaneously close to the Cterminus. Class II (C2) collagenase, on the other hand, begins to cleave the helix interiors [34e36]. There is also diversity of form (i.e., different isoforms) within each collagenase class. Not much is known about how these isoforms function [37], but they seem to be cross-reactive [38]. The ratio between collagenase classes I and II also seems to affect the efficacy of the enzyme blend in digesting the pancreas. However, no definite conclusions can be drawn because some studies have indicated an increased success in human isolations by reducing class I activity [39], while the opposite has been found in rodents, where only collagenase class II is needed for pancreatic digestion if the collagenase is supplemented with neutral protease [40,41]. Earlier collagenases from C. histolyticum were commonly called crude collagenase, because C. histolyticum produces several different collagenases that are able to degrade different types of collagen. In addition to producing collagenases, this bacterial strain also produces other proteases such as aminopeptidase, clostripain, phospholipase, and neutral protease, all of which have been present in the collagenase batches commercially available. Taken together, these factors strongly contribute towards the variability in enzyme activity between different batches of collagenase, and sometimes even from vial to vial because of the instability of the enzymes [42] and dosing of product in the vials. Over these years, the collagenase purification has improved. Using purified collagenase alone without supplementing the enzyme blend, however, can result in insufficient digestion of the pancreas. The presence of these other proteases seems to be essential for proper release of the islets from the pancreatic parenchyma, and these additional enzymes synergize with collagenase to produce effective pancreas digestion. Caution is needed in the digestion, since excessive exposure to proteases can disintegrate and fragment islets and thus reduce islet yields [43,44]. Collagenases containing higher concentrations of tryptic-like activity (TLA) seem to facilitate significantly higher islet yields than do batches with lower amounts of this activity, an observation that may be true both in rodents and humans [45]. Proteases: Since collagenase alone may not sufficient for isolating human islets, current purified collagenases are supplemented with proteases to achieve higher islet yields (Table-1). Neutral protease (NP) from C. histolyticum and thermolysin (TL) have been extensively utilized in clinical islet isolations. In addition, BP protease (BPP), or dispase, from Paneibacillus polymyxa and clostripain from C. histolyticum have also successfully been used for human islet isolations [29,46]. NP, TL, and BPP are all Znmetalloproteases ranging from 30 to 35 kDa. TL has been a topic of interest for many years due to its thermally stable structure as a result of four spectator calcium ions [47] and its very broad substrate specificity, which includes many components of the interstitial ECM and the basement membrane [48e52]. Though NP and BPP appear to have narrower specificities than TL [53e55], all three proteases have been shown to preferentially cleave the N-terminal bonds of hydrophobic residues [56e58]. Another protease used by some human islet isolation laboratories is clostripain, a heterodimeric cysteine-protease also isolated from C. histolyticum. It is produced in an inactive form before an autocatalysis reaction removes a linker peptide [59]. The final active enzyme consists of a 43 kDa heavy chain and a 15 kDa light chain [60]. It has been shown to possess amidase and esterase properties, as well as the ability to cleave laminin, an important component of the basement membrane [61].One study has suggested the possibility that clostripain may provide improved efficiency of islet isolations when used in

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conjunction with both collagenase (C1 and C2) and NP [62]. Collagenase Enzyme Activity Assessment: Clinical islet isolation enzyme suppliers (VitaCyte, Serva/Nordmark, and Roche), express their enzyme (collagenase) activity in ‘Wunsch units’. The Wunsch and FALGPA assays use a peptide substrate that preferentially detects collagenase activity; however, these peptidase assays primarily detect C2 activity and assess only the function of the catalytic domain. In contrast, the true functional collagenase activity is detected by using a native collagen substrate. VitaCyte’s fluorescent microplate collagen degradation activity (CDA) assay measures enzyme that contains a catalytic domain and a collagen binding domain. The collagen must first securely bind to native collagen before the catalytic domain in the same molecule can cut collagen’s triple helical structure. Treatment of collagenase with chymotrypsin leads to removal of collagen binding domains and loss of nearly all the CDA activity with minimal change in the Wunsch activity. Studies at VitaCyte LLC have shown the specific CDA activity of C1116kDa is about 7e8 times higher than the C1100kDa or C2114kDa forms of collagenase [63]. 1.2. Clinical perspectives Liberase-HI has been used for many years around the world for clinical islet transplant. In 2007, the purified enzyme Liberase-HI, was withdrawn from the market because of safety concerns (the use of bovine brain-derived materials during the fermentation process). Identifying a suitable replacement enzyme blend became a top priority for the continued success of clinical islet transplant programs across the globe [64]. During this time, the only available enzyme for clinical isolation was Serva/Nordmark collagenase NB1 [65]. The purified clinical grade collagenase and neutral protease enzymes were supplied in separate vials. Lacking viable alternatives, many centers struggled to establish standardized isolation protocols with these enzymes, which resulted in lower islet yields eventually leading to fewer islet transplantation procedures [64,66]. Szot et al. used the Serva/Nordmark enzyme to isolate islets and used a high percentage of these preparations for clinical allotransplantations [67]. As part of the islet group at the University of Minnesota, our group also reported that the same enzyme could be successfully utilized for autologous islet isolation from CP pancreases. The results from the autologous islet isolations using the Collagenase NB1 were comparable with the traditional Liberase-HI in terms of islet yield and clinical outcomes [68]. However, the Minnesota group could not obtain high enough islet yields from cadaveric donor pancreases when using the Serva/Nordmark enzyme to improve the percentage of successful islet isolation that went on to be used for transplant [69], VitaCyte Collagenase HA was used in place of the Serva NB Collagenase. Biochemical comparison of these products showed that the Serva/Nordmark enzyme contained a higher fraction of truncated class I collagenase than was found in the VitaCyte collagenase, which contained primarily intact C1 collagenase. As noted above, intact C1 collagenase has a higher specific CDA than truncated C1 or intact C2 collagenase. The use of the Serva/Nordmark’s NB-1 collagenase enzyme affected islet yields as it poorly digested tissue resulting in mantled islets (islets embedded in exocrine tissue), which in turn lead to difficulty during the purification process [70]. Purified tissue dissociation enzyme blends (TDEs) are critical to successful human islet isolation, but little was known about the key enzymes-class I (C1) and class II (C2) collagenase characteristics. We were the first group to demonstrate unique differences between the C1 collagenase found in purified collagenase products manufactured by three main suppliers and evaluated the impact of C1 differences between two suppliers on human islet yield. Our results emphasized the

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Table 1 Characteristics of islet isolation enzymes. Enzyme

Bacteria

Collagenase G (ColG, C1)

Clostridium histolyticum

Collagenase H (ColH, C2)

Clostridium histolyticum

Neutral protease (NP)

Clostridium histolyticum

Substrates

Structure

Zn-metalloprotease (155 kDa) containing two collagen binding domains, CBDs. The CBDs located on the C-terminal end of the protein are able to identify the triple helix of collagen molecules. The peptidase active site plays a crucial role in, not only cleaving the specific peptides, but also recognizing them [85]. To the right is a ribbon depiction [87]. To the left is domain structure of C1 [63]. Like C1, it preferentially cleaves the Zn-metalloprotease (155 kDa) containing one collagen binding site. N-terminal bonds of Gly residues within typical collagen motif triplets. Like C1, its CBD, which recognizes The highest specificity is for Gly-Pro- collagen’s triple helix, is present on the C-terminal end, and, similarly, X and Gly-X-Hyp, which are very the peptidase active site plays a role characteristic of collagen [85]. Its in its specificity. To the right is a known ECM substrates include: ribbon depiction [87]. To the left is Collagen types I, II, III [41] domain structure of C2 [63].

It preferentially cleaves the Nterminal bonds of Gly residues within typical collagen motif triplets. The highest specificity is for Gly-ProX and Gly-X-Hyp, which are very characteristic of collagen [85]. Its known ECM substrates include: Collagen types I, III, V [41,86].

Similar to thermolysin, it preferentially cleaves N-terminal bonds of hydrophobic and bulky residues and Tyr residues, depending on the adjacent residue [88]. Its known ECM substrates include: Collagen type I, Ln [88,89].

Thermolysin Bacillus It preferentially cleaves N-terminal (TL) thermoproteolyticus bonds of hydrophobic residues with larger side [91]. Its known ECM substrates include: Collagen types I, II, III, IV, Fn, Ln, elastin [89,92e97].

BP protease

Bacillus polymyxa

Cleaves the N-terminal bonds of hydrophobic residues [103]. Its known ECM substrates include: Collagen types I, IV, VII, Fn, En [104,105].

Clostripain (CP)

Clostridium histolyticum

Specificity is similar to that of trypsin, though it preferentially cleaves C-terminal bonds of Arg. residues [108]. It also contains tryptic-like activity, TLA. As a cysteine protease, it should also possess some elastase-activating capabilities [63]. Its known ECM substrates include: Ln [89]

Role in pancreatic ECM digestion C1 has a lower capacity for digesting Type III Collagen and produces a significantly different pattern of digestion than its accompanying collagenase, ColH. Prior loosening of the ECM by ColH cleaving of these fibrillar molecules may allow ColG access to the components for which it is better fitted for cleaving, such as Type V Collagen [41].

As shown by Fujio, C2 appears to be an important first step in pancreatic ECM degradation. Possibly, due to its higher affinity for Type III Collagen and distinct pattern of digestion compared to C1 [41], it seems to be better suited for initial degradation of the ECM, allowing for further degradation with subsequent addition of other enzymes. Zn-metalloprotease (32 kDa) The His residues at positions Like other neutral proteases, studies have shown that NP seems to be an 364 and 368 are believed to be the binding sites for Zn, while Glu365 is believed to be the catalytic site [88]. No effective supplement to the already proven ColG/ColH islet isolation ribbon depiction was available for this enzyme. combination. Because of its ability to degrade Fn and Ln, It may be essential in degrading the basement membrane. Though it has a lower rate of reaction than its popular counterpart, TL, this may be due to its less aggressive nature, making it ideal for preserving islet function during isolation [89,90]. Thermally-stable, coiled zincBecause of its broad binding metalloprotease (35 kDa) with high capabilities, TL is able to help ColH amount of tyrosine, serine, and ColG in degrading Collagen threonine, and hydrophobic residues types I and II while also degrading [98]. Four calcium atoms contribute key components of the basement to the stability of the protein [99]. membrane: type IV collagen, Fn, and The protein has two domains, with Ln[102]. Though this broad His, Asp, Glu, Tyr, and Arg residing at specificity does help to increase the the active site [100]. To the right is yield during islet isolation, it may the ribbon depiction [101]. also cause increased damage to the cells, decreasing their function [89]. Zn-metalloprotease (30 kDa) [106] Like other neutral proteases, its composed of two domains with a ability to effectively degrade key central a-helix containing several of components of the basement membrane could make [104,105] it the residues that aid in catalysis. The very effective supplement to presence of four calcium atoms collagenase mixtures used to isolate stabilizes the molecule. To the right pancreatic islets. is the ribbon depiction [107]. Heterodimeric cysteine protease Because of its elastase-activating and with amidase and esterase TLA capabilities, CP seems to be a properties. consisting of a heavy very good additional tool in the chain (43 kDa) and a light chain standard mix of islet isolation (15 kDa) [109]. Low a-helical enzymes. Though high TLA has been shown to degrade collagenases content. 25% of amino acids are needed for ECM degradation, acidic. Asp229 is responsible for the previous studies demonstrated that Arg. specificity. Calcium is required a precise amount of CP is able to for auto-activation. The site of the improve islet isolation [89,112]. peptidase catalytic activity is His176 [110]. [111]

importance of intact C1 collagenase that is necessary for successful human islet isolation and transplantation [71]. Interestingly, non-simultaneous administration of the same enzymes has also been shown to improve isolation outcomes [72]. Brandhorst et al. performed detailed enzyme activity analyses for the current enzyme lots [57]. The group determined the high TLA is

a key parameter for the efficacy of pancreatic dissociation enzymes. In order to identify suitable enzyme combinations, we also evaluated many different enzyme products containing different levels of intact or truncated collagenases, used in combination with thermolysin or neutral protease. We incorporated biochemical analysis of the collagenase enzymes as part of our evaluation criteria and

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identified a new blend containing intact C1/C2 collagenase and neutral protease, both from C. histolyticum, and called it the new enzyme mixture (NEM). Isolations with our NEM were considerably more effective than any other enzyme combination, recovering consistently high yields of quality islets from human pancreata [71]. Additionally, clinical autologous and allo-transplantations with islets isolated from the NEM achieved greater success over previous transplantations [73]. During this course of enzyme standardization, Roche introduced Liberase MTF, a new preparation of purified collagenase and purified thermolysin. HPLC analysis confirmed that their product had intact C1 collagenase. This enzyme has successfully been utilized for clinical islet isolations [74,75]. As previously mentioned, three different enzyme combinations (from Serva, Vitacyte, and Roche) have been implemented for clinical preparations. Though all these enzymes were successfully utilized for processing clinical isolations, only an average of 50% yielded sufficient islet numbers for transplantation [66]. Despite successful utilization of enzyme combinations from different enzymes vendors for isolating human islets intended for clinical islet transplantation, one of the persistent challenges which requires immediate attention is the variability in islet yield. Several factors contribute to this variability. Total islet number within whole pancreases has not yet been determined. Further optimization of the enzyme type or dose and better understanding of the ECM of human pancreas is required to improve islet yield. 1.3. Tissue dissociation enzymes-new developments Recombinant enzymes for human islet isolation: Manufacturing purified recombinant collagenases may overcome problems commonly encountered with purified natural collagenase. Recombinant class I (rC1) and class II (rC2) collagenase are expressed from single genes in the Escherichia coli strain containing low protease activity. This minimizes proteolysis of collagenase enzymes, leading to the generation of intact rC1 and rC2 enzymes. Clostripain contamination is eliminated, improving the control of protease activity during the islet isolation procedure. In our previous study [29], we successfully utilized purified recombinant rC1 and rC2 and assessed 4 different collagenase formulations to recover islets from human pancreases (n ¼ 12, 3 per group). Varying amounts of rC1 and rC2 collagenase activity, supplemented with a fixed amount of P. polymyxa NP activity (BPProtease [VitaCyte LLC, Indianapolis, IN], a dispase-equivalent NP), were tested using a statistically designed experiment in a two splitpancreas model. We observed that the low-dose collagenase-protease enzyme mixture (Recombinant collagenase enzyme mixture [RCEM], 100 000 CDA U, 12 WU/g tissue, in combination with 23 400 U of BP-Protease/g tissue) digested the pancreas and recovered islets as effectively as the 3 other enzyme mixtures that contained 1.38- to 1.79-fold higher mass of recombinant collagenase enzymes per g tissue. These RCEMs contained about 42%e75% of the mass of purified natural collagenase commonly used for digesting a 100-g pancreas. Recombinant collagenase is an effective replacement for natural enzyme when used for human islet isolation. A lower-dose recombinant collagenase in combination with BP protease was successfully used to recover greater than 5000 IEQ/ g pancreas. Results from this study indicate the effectiveness of this animal-free enzyme mixture to recover islets at doses of collagenase that are approximately 50% of those used commonly for human islet isolation. In our seminal study [1] we used a low RCEM dose (12 WU/g) to successfully isolate islets, with good functionality, from whole human pancreata. These results, compared with islet isolation performed using natural collagenase enzyme mixture (60:40C1:C2 mixture containing about 530 mg collagenase to digest a 100-g

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pancreas) (NCEM) with equivalent (12 WU/g) or standard (20 WU/ g) amounts, demonstrated better islet recovery, tissue digestion efficiency, insulin secretion, and transplantation outcomes. This is the first report indicating that islet functionality can be improved with low-dose enzyme. Overall, the quality and functionality assessments suggest that this RCEM dose has potential to be used for human islet isolations to improve clinical outcomes. The results summarized here strongly suggest the recombinant collagenases possess an intrinsic superiority to the naturally derived collagenases, but the biochemistry responsible for this has yet to be identified. This improvement is aligned with the evolving regulatory challenges that will likely demand the use of animal originefree materials in the islet isolation process and economic considerations regarding the cost effectiveness of islet transplantation that justifies reimbursement by third party payers. Enzyme blends for young donor pancreatic islet isolation: Islets isolated from young donor pancreas (YDP) remain mantled and embedded by surrounding acinar cells (Fig. 1C) resulting with large pellet size (Fig. 1D) after enzymatic digestion of the pancreas [76]. Though the surrounding layer of acinar cells may protect islet integrity [77], mantled islets are often lost during the purification process [78]. The release of mantled islets from YDP pancreases is likely due to the composition of the ECM. Accepting this assumption, one approach to reduce the percentage of mantled islets from young donors is to modify the composition of collagenase-protease enzyme mixtures used to free cells from the ECM. Several studies have attempted to improve islet morphology and purification from YDP [76,78e82] with limited success. However, achieving high islet recovery after islet isolation from YDP remains a challenge. In our recent study, we identified the unique role of protease for digesting the peri-islet ECM proteins to recover mantle-free islets from young donor pancreases. We developed a novel approach to this problem by using a “trisected” pancreas model to compare the effectiveness of different enzyme mixtures to reduce the percentage of mantled islets recovered from YDP. Each pancreas was split into three lobes: the head, body, and tail. Three different enzyme mixtures were evaluated within a single pancreas, with each lobe receiving one of the three enzyme mixtures. The lobes were altered between experiments to minimize anatomical bias. Application of this novel methodology showed that increased protease dose was the critical variable in increasing the release of mantle-free islets and improving islet yields from YDP. These results were further confirmed when 13 consecutive whole pancreas islet isolations from YDP were performed using collagenase-increased protease enzyme mixtures from 3 different suppliers (SERVA, Roche, and VitaCyte). Results from this study have immense clinical benefits; utilizing YDP increases the number of acceptable donors for allo-islet transplantation and, based on earlier reports [82,83], could improve clinical outcomes [1]. Enzymes for research islet isolation: Purified enzyme formulation has become available for clinical islet isolation, and is now used by many centers, including those in the Clinical Islet Transplantation consortium. These highly purified enzyme collagenase and proteases are manufactured by well-known companies such as SERVA, Roche, and VitaCyte, and are provided in separate vials as described in previous section. In addition to clinical use, human islets are routinely isolated from brain-dead donor pancreases (Fig. 1A) for various research studies. Currently, most of these laboratories tend to utilize the aforementioned highly purified enzyme blends for isolating islets from research pancreases. The major drawback in utilizing these enzyme blends for research preparation is that the cost can be prohibitive. At the same time, the crude enzymes available from Sigma and Worthington have been used for both human and other animal pancreatic islet isolations, but these have not gained

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widespread application due to factors such as low digestion efficacy related to enzyme impurity, imbalanced combination of key active components, significant batch-to-batch and vial-to-vial enzyme variation and high endotoxin levels. The crude collagenase lots are inherently variable since they reflect the unique biochemical composition of their source, C. histolyticum culture supernatants, and the active enzymes responsible for cell release from tissue, collagenase and neutral protease, comprise only 3e8% of their dry weight. The remainder of dry weight is bio-pigment, other enzymatic activities, undefined contaminants, and endotoxins. This biochemical variability between different lots of the same product makes it very difficult to apply crude collagenase either for human research or for clinical islet preparations. In our recent study [113], a new enriched collagenase product from VitaCyte was successfully utilized to improve the islet isolation process, mainly focusing on the research pancreas. This DE 800 enriched collagenase (>85% purity) product containing primarily intact class I (C1) and class II (C2) C. histolyticum collagenase, at a C1:C2 ratio of z75:25, and with minimal clostripain contamination. In each vial a fixed dose of 800 Wunsch units was present. In addition, a purified Dispase™ equivalent enzyme was added at either 19,000e48,000 or 32,500e78,800 neutral protease U (NP U) per g tissue, and hereafter referred to as low or high protease enzyme mixtures. This new product was tested at three different islet processing centers to confirm the efficacy of low-cost enriched collagenase on islet yield and functions. The use of a low cost enriched collagenase products with biochemical characteristics comparable to purified collagenase enzymes were shown to be as effective in human islet isolation as higher priced products. 2. Conclusions Collagen is the most abundant protein within the human pancreas. Variability in human donor pancreases can be attributed to genetic and environmental factors. Critical analysis of how TDEs may act on known and novel tissue substrates present within the ECM for better tissue dissociation is also equally critical. This could facilitate the improvement of islet yields and also advance the cellular and molecular therapies associated with clinical islet transplantation [2,84]. Further evaluation is necessary to optimize different enzyme doses and combinations to maximize islet yields for clinical transplantations. An enzyme blend which core islet facilities across the world can utilize as a consensus mixture would be ideal, with this becoming one of the ultimate goals within clinical islet research requiring collaboration between islet centers and core facilities, enzyme companies and clinicians. Acknowledgements The authors thank the Jewish Heritage Fund for Excellence for providing a generous support to our program. The authors also sincerely thank the Kentucky Organ Donor Affiliates for a constant supply of donor pancreases. A special thanks to Dr. Siddharth Narayanan for editing and proof-reading the manuscript. References [1] Loganathan G, Subhashree V, Narayanan S, Tweed B, Andrew Goedde M, Gunaratnam B, et al. Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix. Am J Transplant : Offc J Am Soc Transplant Am Soc Transplant Surg 2019;19(3):831e43. [2] Narayanan S, Loganathan G, Dhanasekaran M, Tucker W, Patel A, Subhashree V, et al. Intra-islet endothelial cell and beta-cell crosstalk: implication for islet cell transplantation. World J Transplant 2017;7(2): 117e28.

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