Collagenases in pancreatic islet isolation

C H A P T E R

43 Collagenases in pancreatic islet isolation Ibrahim Fathi, Masafumi Goto Division of Transplantation and Regenerative Medicine, Tohoku University, Sendai, Japan O U T L I N E Introduction

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Basic structure Collagenases Neutral proteases Clostripain

529 529 533 534

ECM of the pancreas with emphasis on the peri-insular region

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Role of different enzyme fractions in pancreatic digestion

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Pancreatic dissociation enzymes in clinical islet isolation: Evolution, safety, and overview of commercial products

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Exogenous parameters affecting collagenase digestion

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Tailored approach to islet isolation

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Conclusions

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References

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Introduction

acteristic structural motif of collagen is formed by three polypeptide chains in a left-handed polyprolene type-II Bacterial collagenases are the cornerstone for pan- helical structure that winds together into a right-handed creatic islet isolation. A huge amount of experimental triple-helix conformation (Fig. 1). In this tightly packed and clinical research in association with technical re- conformation, each third residue must be glycine (Gly), 3 finements of the enzyme manufacturing process have resulting in the repetitive sequence Xaa-Yaa-Gly. While comprehensively improved our understanding of pan- Xaa and Yaa can be any amino acids, they are typically creatic digestion and also significantly improved the proline (Pro, 28%) and 4-hydroxyproline (Hyp, 38%), reisolation outcomes. In this chapter, we cover the basics spectively. The sequence Pro-Hyp-Gly is, therefore, the 4 and advances of pancreatic dissociation enzyme blends most common triplet in collagen (10.5%). These indiand outline the parameters leading to a better outcome, vidual triple-helical units (tropo-collagen) assemble tobased on the current understanding of the enzyme-­ gether in a hierarchical manner to form the macroscopic fibers and networks functioning as the main structural extracellular matrix (ECM)-islet interplay. component of the ECM.3 In all, 28 different collagen types have been identified with at least 48 distinct polypeptide chains.3, 5, 6 Thus far, Basic structure only collagen types I, III, IV, V, and VI have been detected in human pancreas (Table 1)7, 9; these types belong to the Collagenases classical fibrillar (I, III, and V) and network-­ forming Collagen is the most abundant protein in animals (IV and VI) categories.3, 5 In the network-forming type, and represents the major component of the ECM. The the classical helical Xaa-Yaa-Gly regions are sporadiword collagen is derived from κολλα (kolla), a Greek cally interrupted by non-helical regions, resulting in a word that means glue, as animal tissues rich in collagen non-fibrillar architecture.10 The presence of Pro and Hyp were historically used as a source for glue.1, 2 The char- in the Xaa and Yaa regions increases the stability of the

Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 1 https://doi.org/10.1016/B978-0-12-814833-4.00043-5

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© 2020 Elsevier Inc. All rights reserved.

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43.  Collagenases in pancreatic islet isolation

FIG.  1  Structure of the collagen triple-helix. Reproduced by permission from Springer Nature (Marini JC, Forlino A, Bächinger HP, Bishop NJ, Byers PH, Paepe AD, Fassier F, Fratzl-Zelman N, Kozloff KM, Krakow D, Montpetit K, Semler O. Osteogenesis imperfecta. Nature Reviews Disease Primers. 2017; 3: 17052), © 2017.

TABLE 1  Types of collagen demonstrated in mammalian pancreas Type

Classa

Compositiona

Ref.

I

Fibrillar

α1[I]2α2[I]

7, 8

b

II

Fibrillar

α1[II]3

7

III

Fibrillar

α1[III]3

7, 8

IV

Network

α1[IV]2α2[IV]α3[IV]α4[IV]α5[IV] α5[IV]2α6[IV]

7, 9

V

Fibrillar

α1[V]3α1[V]2α2[V]α1[V]α2[V]α3[V] 7, 8

VI

Network

α1[VI]α2[VI]α3[VI]α1[VI]α2[VI] α4[VI]

7, 9

aType-II was demonstrated in rat pancreas but not in human. bFrom Refs. 3, 5.

helical structure, in addition to the tightly packed conformation, the hydrogen bonds between the chains, and the extensive hydration network.4 A true collagenase (Col) in the strictest form is an enzyme that has the ability to hydrolytically cleave peptide bonds located within the helical regions of native un-denatured fibrillar collagen at physiological pH and temperature.11–13 According to this definition, enzymes that can digest denatured collagen (i.e., gelatinases) and enzymes that can digest terminal regions outside the helical folds (e.g., trypsin, chymotrypsin, pepsin, and pronase) are not considered true collagenases. Collagenases are classified into serine and metallo-­collagenases and can function within a wide range of temperature (20–40°C) and pH (6–8).14 Collagenases are produced by eukaryotes and prokaryotes.12, 15 Vertebrate collagenases are important contributors to the process of tissue remodeling and resorption and are not within the scope of this chapter.16, 17 In contrast, microbial collagenases facilitate the invasion and colonization of host tissue through the degradation of the host ECM, where they utilize degraded collagen as a carbon source.12, 18

Microbial collagenases are a part of the MEROPS peptidase family M9 (INTERPRO: IPR002169; PFAM: PF01752), which includes bacterial metalloproteinases from Vibrio and Clostridium spp. with presumable collagenolytic activity.17, 19 The classes I and II collagenases from Clostridium histolyticum fall in the subfamily types M09.002 and M09.003, respectively, which in turn belong to the M9B subfamily of the MEROPS M9 peptidase family.17 The inability to distinguish true bacterial collagenases from other forms of bacterial proteases sometimes leads to controversy in classification and nomenclature.17 Collagenases produced by C. histolyticum, the main model and the most extensively studied enzymes among bacterial collagenases, are the focus of this chapter, due to their utility in pancreatic islet isolation, either in crude, purified, or fractionated forms. Based on their molecular weights (MWs), at least six electrophoretically distinct collagenases with an MW ranging from 68 to 128 kDa were isolated from C. histolyticum.20 These collagenases were divided into two classes based on their chromatographic profiles, sequences, and relative activity against collagen and synthetic peptides (e.g., FALGPA), designated as classes I and II. Class I collagenases have high activity toward collagen (collagenolytic activity) and moderate activity toward FALGPA (peptidolytic activity), while class II collagenases have moderate activity toward collagen and high activity toward synthetic peptides.17, 20 Matsushita et  al.21 described the genes for collagenase G (ColG) and collagenase H (ColH) in the C. histolyticum chromosome that encode one class I and one class II collagenase, respectively. These genes may represent divergence from a common origin. Only slight differences in the gene structures were observed in data from two different laboratories, with four differences in the amino acid sequence of ColG and 13 differences in that of ColH.21–24 Thus, the previously reported high number of enzyme forms was ascribed to auto-proteolysis.21, 25, 26 The distinct domain structure and organization are characteristic of classes I or II and aid in classification based on the domain homology (Fig. 2). Both ColG and ColH are multidomain (or multi-­ modular) proteins containing a signal peptide, a putative pro-domain, a collagenase (catalytic domain, S1) unit, up to two linking domains (collagen recruitment units, S2) such as the polycystic kidney disease (PKD)like domains, and up to three collagen-binding domains (CBDs, S3).27 ColG (class I) is composed of segments S1, S2, S3a, and S3b,21, 24, 27 while ColH (class II) contains S1, S2a, S2b, and S3. A minimum of one CBD unit was reported to be necessary for the collagenase to be able to bind to native collagen independently from the other enzyme domains.21, 28 ColG contains up to two CBDs while ColH contains only one CBD.17 In order for the collagenase to be able to degrade collagen, it should have an

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Basic structure

A280/mL

1.50 1.00 0.50

4

(A)

8

12

16

20

24

28

32

36

1.4 1.2 1 0.8 0.6 0.4 0.2 0

15

20

25

30

35

40

140000 120000 100000 80000 60000 40000 20000 0

CDA specific Activity

A280/mL

Retention time minutes

Fraction number

(B)

A280/mL

Collagenase specific activity

FIG.  2  (A) Analytical MonoQ anion-exchange high-performance liquid chromatogram showing the fractionation of 5 mg of a CI:CII mixture over a 1-mL column, demonstrating the following peaks from left to right: C2 peak, intact C1 peak, degraded forms of C1 containing a single CBD (C1b and C1c). (B) Analysis of 1-mL fractions for A280 and CDA specific activity. Figures reproduced by permission from Elsevier, Transplantation proceedings (McCarthy R, Spurlin B, Wright M, et  al. Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation. Paper presented at: Transplantation proceedings, 2008) © 2008.

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intact catalytic domain in addition to one CBD. Enzyme forms meeting this criterion can thus be termed “active enzymes” and include intact C1 (116 kDa, two CBDs), the 100-kDa C1 form (one CBD), and intact C2 (114 kDa, one CBD) (Fig. 3). In contrast, the truncated forms without CBD are considered “inactive enzymes” and cannot degrade helical collagen, but retain their activity against denatured collagen as gelatinases.29 The mode of action of vertebral collagenases is largely settled, allowing for precise differentiation from gelatinases.30 Vertebral collagenases attack collagen molecules at a single peptide bond across the three alpha chains of the triple helix, yielding specific fragments that corresponds to three quarters and one quarter of the tropocollagen molecule.31 Digestion by collagenases is subsequently very limited, and the hydrolysis of the resulting polypeptides is continued by other proteases, such as gelatinases.32, 33 Bacterial collagenases, by contrast, are thought to be less specific than vertebrate collagenases. Where the degradation of the native water-insoluble collagen depends on the collagen type and the species of origin for vertebrate collagenases,17 bacterial collagenases are believed to be active against almost all collagen types.34, 35 They also split every polypeptide chain in the triple helix at multiple sites36 and are able to digest both water-­ insoluble native collagen and water-soluble denatured collagen, showing a ­versatile nature.37 However, despite

FIG. 3  Domain organization and quaternary architecture of mature clostridial collagenases. ColG and ColH from Clostridium histolyticum and ColT from Clostridium tetanai (not within the scope of the chapter). Reproduced under CC-BY license from The American Society for Biochemistry and Molecular Biology, Inc., JBC (Eckhard U, Schönauer E, Brandstetter H. Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T. J Biol Chem 2013;288(28):20184–20194) © 2013. Protein Data Bank entry 2080 was used for the CBD model.

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43.  Collagenases in pancreatic islet isolation

this versatility, which is referred to as broad substrate specificity,38, 39 there are currently no other known substrates for bacterial collagenases, making them dedicated and efficient collagen degraders.18 Eckhard et al.40 demonstrated the functional cascade of ColG domains in the process of collagen fibril digestion. In this model, (1) the CBD attaches the enzyme to the collagen fibrils after recognizing the triple-helical confirmation, (2) the PKD-like domains prepare and swell the fibril without unwinding the helical structure, and (3) the collagenase module (composed of an activator and peptidase domains) then performs collagenolysis after unrolling the collagen microfibrils and unwinding the triple-helical structure, which are necessary steps before hydrolysis. ColG is considered to act through 40 a classical “chew and digest” model to achieve collagenolysis, where the enzyme alternates between and opened and closed states. In the opened state, the enzyme becomes attracted to collagen microfibrils owing to the triple-helical structure, and thus the collagenase module assumes a saddle-shaped conformation and clamps the collagen fibril. This allows access of the peptidase domain to the monomeric triple helices. A closed conformation is then assumed as a result of interaction between collagen and the activator domain of the collagenase module, which results in unwinding of the triple-helical collagen conformation and digestion of the polypeptide α chains. More recently, using high-speed atomic force microscopy, similarities were reported in enzyme-substrate and enzyme migration between collagen-collagenase and DNA-nuclease group.41 Interestingly, Eckhard et  al.34, 40, 42, 43 showed that only the N-terminal collagenase module (containing activator and peptidase domains) is indispensable for collagenolysis by ColG. Full collagenolytic activity was preserved in the Tyr119Gly790 segment (collagenase module) of ColG (Fig. 3).40 Deletion of the activator domain, however, resulted in the retention of activity against small peptidic substrates but showed complete inactivity against collagen substrates.40 These findings challenged the previous concept that a catalytic domain and at least one CBD are necessary for collagenolysis.21, 28 However, this should not be confused with an equal collagen degradation activity (CDA), as the intact form of class I (containing two CBDs) has sevenfold to 10-fold higher specific CDA than the truncated 100-kDa class I form (containing one CBD).44 In contrast, an analysis of the ColH peptidase domain structure revealed a calcium-binding site in close proximity to the active site of peptidase domain, an aspartate switch for zinc stabilization, and a conformational selectivity filter45; the latter regulated the substrate access to the active site through steric shielding, which may explain the opposing peptidolytic and collagenolytic efficiencies.45

Hypothetically, in a setting of pancreatic digestion for islet isolation, after binding of collagenases to collagen fibrils and unraveling their helical structure, collagen hydrolysis is completed by the action of collagenases, neutral proteases (NPs), and gelatinases (representing truncated forms of the collagenases), with a controversial contribution by endogenous pancreatic proteases.29 At the same time, this mixture of proteases also digests other ECM macromolecules (i.e., glycoproteins, proteoglycans, and reticular fibers), exposing more collagen fibrils, and the process is repeated until the islets are released from their ECM and acinar-cell attachments.29 Several assays for collagenases have been developed; however, the current ones are still limited to some degree. Generally, they include physical and activity assays. A number of different methods have been classically described for measuring the enzyme activity, including radioactive assays to measure the elaborated [14C] amino acids or peptides from radio-labeled collagen, collagen film/gel assays, ninhydrin-based assays, synthetic peptide assays measuring the hydrolysis of peptides with a similar structure to collagen (e.g., FALGPA and Wünsch assays), the Mandl assay for measuring the CDA, and the Azocoll assay.12, 29 Table  2 describes the commonly used activity assays, their principle, and their main disadvantages. The Wünsch assay is commonly used to determine the activities of commercial lots and to describe the dosage of enzymes to be used in islet isolation, owing to its reproducibility and simplicity. However, significant concerns have been raised against the sole use of the Wünsch assay, including its inability to differentiate molecular forms with and without CBD, as it reflects the catalytic domain function only, which results in the assay detecting both intact C2 (114 kDa) and truncated forms without a CBD.24 This was further demonstrated after degradation of C2 with chymotrypsin that led to only a 7% decrease in specific Wünsch activity despite a 95% loss of specific CDA.44 In addition, the Wünsch assay is biased toward the detection of C2 activity, with a 50-fold higher specific Wünsch activity of purified C2 compared with purified C1.29 To overcome the shortcomings of the original Mandl assay, including a poor precision and nonlinearity, the fluorescent microplate CDA assay was developed. This assay utilizes soluble type-I calf skin collagen fibrils coupled with fluorescein isothiocyanate (FITC fibrils) and requires a 60-min incubation period.29, 44 This modified CDA assay correlated with the number of CBDs in the molecular form with sixfold to 17-fold higher specific CDA activity of purified C1 compared with purified C2.44 Obtaining complete data regarding the molecular forms in the utilized partially purified enzyme, however, requires a combination of assays with physical methods. Such molecular details are necessary as long

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Basic structure

TABLE 2  Collagenase activity assays Assay

Substrate

Isoform Bias

Disadvantages

Colorimetric

Pz peptide (Wünsch) FALGPA

Mainly detects C2 activity (C2 > C1)

Unable to differentiate between intact and a,29, 44 degraded forms.

FRET-peptide

AGGPLGPPGPGG

No data

Cleaved by thermolysin and CHNP

b

Triple-helical peptides FRET

Triple-helical peptide

No data

Absent isoform data, further validation

c

Azocoll

Colorimetric

Azo-dye impregnated C2 − C1 Collagen (colorimetric)

Nonspecific protease assay

d

Collagen degradation activity (CDA)

Mandl assay

Collagen fibers C2 − C1 (colorimetric ninhydrin method)

Time consuming ( ̴5 h) Nonlinear End-point assay

e

FITC-CDA

FITC collagen fibers

Peptide assay

Correlates to CBD Fluorescent assay number in molecular form C1 > C2 (5–17-fold) Intact C1 > degraded C1

Ref.

44

aWünsch E, Heidrich HG. On the quantitative determination of collagenase. Hoppe-Seyler’s Zeitsch Physiol Chem. 1963;333(1):149–151. bAl-Abdullah IH, Bagramyan K, Bilbao S, Qi M, Kalkum M. Fluorogenic peptide substrate for quantification of bacterial enzyme activities. Scient Rep. 2017;7:44321. cTokmina-Roszyk M, Tokmina-Roszyk D, Bhowmick M, Fields GB. Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity. Anal Biochem. 2014;453:61–69. dChavira Jr R, Burnett TJ, Hageman JH. Assaying proteinases with azocoll. Anal Biochem. 1984;136(2):446–450. eMandl I, MacLennan JD, Howes EL, DeBellis RH, Sohler A. Isolation and characterization of proteinase and collagenase from Cl. histolyticum. J Clin Invest. 1953;32(12):1323–1329. CBD, Collagen binding domain; CHNP, Clostridia histolyticum neutral protease; FRET, Föster resonance energy transfer.

as the optimization of the digestion process continues for accurate correlation with outcomes. Commonly used physical methods are reviewed in Ref. 29. It should be noted, however, that the best available physical method (anion-exchange chromatography) cannot detect truncated forms of C2.29 Bacterial collagenases are considered exotoxins, and their highest yields are obtained from bacterial cultures grown to the late exponential or stationary growth phase.12 The culture media are typically supplemented with materials that induce collagenase production (e.g., casein hydrolysate, gelatin, polypeptones, or denatured collagen).12 The collagenase reported by Worthington in 1959 isolated from C. histolyticum was the first commercially available collagenase,46 and many further commercial products were subsequently developed, as discussed later. Classically,12 bacterial collagenases are purified after collecting the supernatant from 8 to 10 L of bacteria that are grown for 48 h at 25°C. Ammonium sulfate is then added to the supernatant to reach 60%–90% saturation. The solution is allowed to stand overnight at 4°C, and the precipitated protein is collected through filtration or centrifugation. A buffer is then used to dissolve the protein, and the ammonium sulfate is removed by dialysis at 4°C. The lyophilized dialysate is considered to be the crude enzyme including collagenases in addition to other impurities (e.g., NPs and clostripain). Further purification of collagenases can then be performed using different chromatographic techniques.12, 14 Factors influ-

encing the enzyme activity should be carefully considered during the purification process (e.g., Ca+2).

Neutral proteases The use of highly purified natural and recombinant collagenases instead of partially purified and crude preparations necessitates adding NPs to the enzyme blends for islet isolation, given its important complementary effect (discussed later). For this purpose, the well characterized 34.6-kDa metalloendopeptidase thermolysin, produced by Bacillus thermoproteolyticus,47 has gained wide interest compared to less well-­ characterized alternatives. The enzyme can be purified by crystallization48 or affinity chromatography,49 and the recombinant enzyme has been produced in Escherichia coli.50 The mature enzyme contains 316 residues and has a bilobal structure with a zinc atom in the deep cleft between the two lobes, which is necessary for the enzyme activity.49 Four Ca2+ atoms also bind the enzyme, supporting the enzyme stability by preventing autolysis.50, 51 Thermolysin preferentially cleaves peptides and proteins at the N-terminal side of Leu, Phe, Ile, and Val, although hydrolysis of bonds with Met, His, Tyr, Ala, Asn, Ser, Thr, Gly, Lys, Glu, or Asp at P10 has been reported,33 and it shows maximum proteolytic and esterolytic activity near a pH of 7.0.52–56 However, other NPs have since been better characterized and have, therefore, been investigated as

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43.  Collagenases in pancreatic islet isolation

r­ eplacements for thermolysin in islet isolation. These include C. histolyticum neutral protease (CHNP), a dispase equivalent from Paenibacillus polymyxa (BP protease). An examination of recombinant CHNP, cloned in Bacillus subtilis, showed that it had a higher substrate specificity and lower proteolytic activity than thermolysin.57 The purified protein had a molecular mass of 31.5 kDa with 313 amino acid residues in the mature enzyme and a low sequence identity with other known enzymes.57 The same study also showed that CHNP could digest azocoll with higher activity than ColG or ColH. The enzyme was also able to digest PzPLGPR (between Pro and Leu) but not FALGPA, which highlighted the difference in the cleavage sites of collagen I (azocoll) between NPs and collagenases with possible synergistic collagenolytic activity, as well as indicated the higher specificity of FALGPA for estimating collagenases activity in enzyme mixtures than PzPLGPR.57 Different assays have been used by enzyme manufacturers to measure protease activity,29, 58 including endpoint assays (e.g., azocasein) and the more recent kinetic fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) assay. The FITC-BSA assay utilizes the same format as the microplate FITC-CDA assay.58 The analysis of purified NPs using an FITC-BSA assay showed that BP protease and CHNP had around 40% and 15% specific activity, respectively, compared with thermolysin.58

Clostripain The role of the cysteine-activated proteinase clostripain in pancreatic ECM digestion has been generally ignored, despite clostripain being a well-known component of CH culture filtrate. However, the association between a higher “trypsin-like activity (TLA) to collagenase activity” ratio found in impure collagenase preparations and a faster digestion time in rat pancreas and significant reduction in recirculation time in human pancreas has brought attention to clostripain.59 This enzyme selectively hydrolyzes the carboxyl terminal side Arg bonds, with Lys bonds cleaved at a lower rate.60 The mature enzyme is a heterodimeric protein with 526 amino acids and a molecular mass of 43,000 and 15,398 kDa for the heavy and light chains, respectively.61 A cysteine thiol group is necessary for its activity, and Ca2+ is an absolute requirement with maximum hydrolytic activity at pH of 7.2–7.8.62, 63 Despite having different substrate specificity from trypsin,62, 64 the enzyme is considered to have TLA.65 Clostripain can be purified from C. histolyticum culture broth, and its expression in E. coli,66 B. subtilis,67 and a virulence-attenuated strain of Clostridium perfringens was reported to be higher than in C. histolyticum.68

Factors influencing collagenase, NP, and clostripain activities are summarized elsewhere13, 48, 62, 69, 70 and will not be discussed in the present text.

ECM of the pancreas with emphasis on the periinsular region There are approximately one million islets of Langerhans in the adult human pancreas, formed of clusters of endocrine cells and dispersed throughout the exocrine pancreas.71 The collagen content in human pancreas was estimated to be 26.5 ± 7.2 μg collagen/mg protein using a colorimentric assay,72 with the amount significantly increasing with age (18.2 ± 4.0 μg collagen/ mg protein under 50 years of age vs 31.9 ± 8.0 μg collagen/mg protein in older individuals).72 In a later study, the 4-hydroxyproline content in normal pancreatic tissue was found to be 2.2 ± 0.5 μg/mg of wet tissue73 (collagen = 17.3 ± 4.0 μg/mg of wet tissue, considering that collagen contains 12.5% 4-hydroxyproline74). The ECM located at the endocrine-exocrine interface (peri-insular region) has been the focus of many studies due to its relevance in islet isolation. The “islet capsule” is a single layer formed by fibroblasts and the collagen produced by these cells, which directly surrounds islets. The peri-insular basement membrane (BM) is composed of two separate layers of BM, representing that of the endocrine islets and exocrine cells in their vicinity.75, 76 The endocrine BM extends into the islet along the vasculature as the endothelial BM. Generally, the BM is mainly composed of laminin and collagen IV forming independent networks that are interconnected by nidogens and heparan sulfate proteoglycans (e.g., perlecan).7, 77 Collagen types I (7, 8), III (7, 8), V (7, 8), and VI (7, 9) and fibronectin (7) were also detected in islet BM. While collagen types II and VII were not detected in human or porcine pancreas, type II was detected in rat pancreas.7 Importantly, a semi-­quantitative analysis showed a significantly higher proportion of collagen VI in human pancreas than types I and IV (more than double their amounts), suggesting it was the most prevalent type in the pancreatic ECM.9 A previous report demonstrated the insensitivity of native (under non-­ reducing conditions) collagen VI to digestion by bacterial collagenase (purified bacterial collagenase, form III, Advance Biofactures, New York, USA), while collagen V was sensitive under both reducing and non-­reducing conditions, and fibronectin was insensitive in both conditions.78 While mouse islets endocrine cells were reported to interact directly with the vascular endothelial BM,79, 80 Virtanen et al.76 showed that in adult human islets, a separate layer of endocrine BM extends from the peri-insular BM into the islet along the vascular channels (Fig.  4). The important role of the BM as a barrier and

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FIG. 4  Confocal laser scanning microscope of human islets showing immunoreactivity for EHS Laminin (A) demonstrating two distinct base-

ment membrane layers around intra-islet blood vessels (inner vascular “arrow” and outer endocrine “arrowhead” layers). Lm α4 chain immunostaining is demonstrated only in the inner layer (B, and in orange after merging in C). Immunostaining for PECAM-1 (D) and EHS Laminin (E) show after merging in (F) that the inner vascular BM layer (arrow) is lined inside by a PECAM-1 positive endothelium (arrowhead). Bars a–c 30 μm and d–f 10 μm. Reproduced by permission from Springer Nature, Diabetologia (Virtanen I, Banerjee M, Palgi J, et al. Blood vessels of human islets of Langerhans are surrounded by a double basement membrane. Diabetologia 2008;51(7):1181–1191) © 2008.

for maintaining the islet function makes its preservation after isolation desirable. Several studies have demonstrated the loss of BM after completion of isolation with a gradual recovery after 5-day culture81 or after isotransplantation.82 To ensure preservation of the BM, it may be valuable to design gentler isolation protocols. The prominence of the BM layer varies considerably among species,75 and accordingly, the extent of direct exocrine-endocrine cell-to-cell contact also varies (Fig.  5). Immunohistochemistry (IHC) showed that the BM almost completely surrounds the islets in dogs, whereas scarce BM surrounds porcine islets, with integration mainly relying on cell-to-cell adhesion. Rat and human pancreata demonstrated an intermediate state, with an increased contribution of cell-to-matrix adhesion. However, within islets, cell-to-cell adhesion is the predominant adhesion form in dogs, pigs, rats, and humans.75 A comparative study8 examined the relative prominence of collagens I, III, and V in rats, dogs, pigs, and humans in the peri-insular region as well as other regions of the pancreas. Meyer et al.7 studied the porcine islet capsule and compared it to that of humans and rats. The results showed that collagens I, III, and IV as well as laminin and fibronectin were the main components in pigs, while collagens I, III, IV, and VI as well as laminin and fibronectin were the predominant ECM proteins in

humans. Rats showed a predominance of collagens I–IV as well as laminin. Significant differences in the ECM protein expression were also noted among different breeds of pigs.7, 83

Role of different enzyme fractions in pancreatic digestion The optimization of the digestion process requires understanding the specific contribution of enzyme blend components in the pancreatic dissociation/islet release and their combined effect. Further tailoring of the digestion process based on the matrix structure requires identifying the exact ECM targets of each enzyme fraction. The addition of NPs to purified collagenases enhances the pancreatic digestion and the degradation of collagen and hastens the degradation of proteoglycans, glycoproteins, and elastin in rat pancreas.84 The enhanced collageneolytic activity in the presence of proteinase can be ascribed to two elements: enhanced accessibility to collagen due to degradation of the surrounding coating proteoglycans,85 and the contribution of the protease itself to completing the process of collagen degradation after initial partial degradation by collagenase, as NPs are active against denatured collagen.86 Under complete

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43.  Collagenases in pancreatic islet isolation

FIG. 5  Pancreas exocrine-endocrine interface in the rat (A), pig (B), dog (C), and human (D). Islets are well separated from ductular (A) or acinar (B, C) tissue by basement membrane (arrowheads). Extensive contact by interdigitations (arrows) is noted in (D). In the pig (B), endocrine cells are in direct contact to exocrine cells. Bar: 0.5 μm. Reproduced by permission from Springer Nature, Cell, and Tissue Research (Van Deijnen J, Hulstaert C, Wolters G, Van Schilfgaarde R. Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat, dog, pig, and man. Cell Tissue Res 1992;267(1):139–146) © 1992.

­ lockage of protease activity, purified collagenase reb quired 99 ± 10 min to complete the dissociation of rat pancreas as opposed to 36 ± 1 min with increasing amounts of purified protease.84 Prolonging the dissociation in the presence of protease resulted in significant deterioration of the islet yield,84 possibly due to the promotion of islet and acinar cell disintegration by protease.84, 87 A study obtained maximum islet yield at DMC-U/PZ-U = 1.2% coupled with collagenase NB1 (Serva, Heidelberg, Germany) in rat pancreas. That study highlighted the influence of NPs on islet morphology, as an increasing NP concentration reduced the number of trapped islets while simultaneously increasing the number of fragmented islets.88 Comparing 18 batches of collagenase in rat islet isolation showed that manufacturer’s caseinase activity was significantly correlated with the isolation grade.89 Furthermore, supplementing poorly performing batches with dispase (2.5 mg/mL), but not papain or trypsin, significantly improved the islet yields. However, dispase alone failed to digest the pancreas.89

In human pancreas, increasing the concentration of NPs from 2.6% to 4.5% (DMC-U/PZ-U) with purified Serva collagenase NB1 resulted in increasing the packed volume of digested tissue and decreasing that of undigested tissue, thereby enhancing the prepurification islet yield. However, equal islet yields with less purity resulted after purification using higher NP concentrations. Furthermore, higher NP concentrations decreased the islet stimulation index and viability.87 In an isolation series of 251 human islets at the University of Alberta,90 optimizing the thermolysin dosage based on the manufacturer’s suggested caseinase units/g of pancreas markedly improved the isolation outcome, where the post-­purification islet yield showed a logical inverted U-shaped curve when dosages were categorized into quartiles. The authors set the optimum dosage range at 624.3–987.9 NP/g pancreas.90 Isolations performed within this range had significantly higher pre-­purification and post-purification islet yields, with the odds of successful isolation 3.45-fold higher in such cases.90 However, no significant differences in the function were observed in recipients (insulin requirement) after islet transplantation at 1 month after the first or subsequent infusions.90 Balamurugan et  al.91 compared the results of using a dissociation enzyme mixture containing CIzyme collagenase HA (VitaCyte, Indianapolis, IN, USA) and either CHNP (Serva NB) or thermolysin (VitaCyte) in split human pancreata (n = 3). The use of CHNP resulted in a higher islet yield than that of thermolysin in these experiments, and the mixture also proved superior to six other enzyme combinations.91 The correlation of the collagenase activity (as WU/g pancreas) with the post-purification yield also showed an inverted U-shaped pattern.90 The optimum range was set at 21.0–32.3 WU/g pancreas. Comparing isolations within the optimum range with those outside the range showed significant differences in the post-purification islet yield but not the pre-purification yield, with odds of success of isolation 2.15-fold higher within the range.90 Analyzing the thermolysin and collagenase activities together showed that the isolation success rate was highest with the optimum thermolysin dose in combination with over-dosage of collagenase, while the worst result was achieved with under-dosage of thermolysin and over-dosage of collagenase (35.7% and 0%, respectively).90 Only the thermolysin dosage proved to be a significant predictor of the isolation success in a logistic regression equation.90 To assess the different roles of classes I and II in the CH collagenase, Gerrit et  al.92 compared the purified and fractionated classes (separately and together) with purified non-fractionated collagenase in rat islet isolation (all with suppression of endogenous proteolytic activity and 100 U of CHNP). Dissociation with class I was extremely slow and incomplete, releasing few islets,

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Pancreatic dissociation enzymes in clinical islet isolation: Evolution, safety, and overview of commercial products

while ­adequate dissociation was achieved with class II in 50 min with a high islet yield; a mixed I/II fraction showed similar results to class II. However, adding the two fractionated classes I and II together resulted in faster dissociation than either alone and a higher yield than either of them or non-fractionated enzyme (7.1 ± 0.8 μL/g vs 5.0 ± 0.4 μL/g pancreas for non-fractionated enzyme). Rat pancreatic digestion using highly purified recombinant ColG and ColH collagenases (with a fixed thermolysin dose of 0.3 mg) demonstrated the crucial role of ColH, as a considerable islet yield was retrieved using ColH alone (2811 ± 581 IEQ), while no yield resulted from using ColG alone.93 Combining both classes resulted in the highest yield (4101 ± 460 IEQ), further supporting a synergistic effect. In a trial to explain the superiority of class II, Vos-Scheperkeuter et al.94 studied the effects of both classes on rat pancreatic ECM, with or without protease, using selective histochemical staining. The two classes had similar effects on glycoproteins, represented by a degradation by one-third after 120 min of incubation. Approximately 80%–95% of collagen was degraded after 120 min with better efficiency with class II. No selective regional differences were observed, and combining both classes resulted in faster and more complete degradation after 120 min incubation than class II alone, highlighting their synergistic effect. Determining the optimum ratio was then attempted by comparing the effect of 20 PZ-U of collagenase containing class II/I ratios of 0.5, 1, and 1.5 in a rat model, with the highest yield and lowest deterioration in viability after 24-h culture achieved at a ratio of 1.95 However, different ratios minimally affected the digestion time and islet purity.95 A mass spectrometric analysis identified collagen types I and III to be the main targets of ColH, and IHC showed greater amounts of collagen III than collagen I in pancreatic tissue, suggesting that collagen III is the main substrate for ColH during pancreatic digestion.93

Pancreatic dissociation enzymes in clinical islet isolation: Evolution, safety, and overview of commercial products The introduction of Liberase HI (Roche Diagnostics, Indianapolis, IN, USA) in 1994, containing a standardized mixture of highly purified C. histolyticum collagenases (class I and II isoforms) and thermolysin, significantly improved the human islet yield compared to traditional collagenases (e.g., collagenase type P and Sevac collagenase) and decreased the lot-to-lot variability while resulting in similar in vitro and in vivo functional profiles.96, 97 The risk of causing bovine spongiform encephalopathy (BSE) due to possible contamination of the enzyme with BSE prions as a result of incorporating brain heart infusion (BHI) broth in the bacterial culture medium was

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identified and reported in April 2007 by the FDA (BHI is made from bovine brain and porcine heart).98, 99 This led to abandoning the use of Liberase HI for clinical islet isolation.98 Thus, a shift occurred toward using the “good manufacturing practice (GMP)” collagenase NB1 (Serva), which despite the use of animal products in the manufacturing process, did not involve material with any specified risk and was certified by the European Directorate for the Quality of Medicines (EDQM). As opposed to Liberase HI, the use of Serva collagenase NB1 required adding NP to the enzyme blend (e.g., Serva protease NB1). However, isolation using Serva collagenase NB1 achieved lower purified islet yield than Liberase HI, as shown by large-scale studies.100–102 Brandhorst et al.100 compared isolation with Liberase (n = 101) and Serva NB1 (n = 96) using identical procedures for isolation and assessment. The authors reported significantly more digested tissue and purified islet yield with Liberase HI and significantly higher purity and glucose stimulation index with NB1 with a lower tissue factor expression. Optimization of the isolation protocols using Serva NB1 was thus performed in order to obtain equivalent yields to those previously attained with Liberase HI.101 A mammalian tissue-free (MTF) version of Liberase (Roche Diagnostics GmbH, Mannheim, Germany) was later developed. In the subsequent period, state-of-the-art blends used in human islet isolation included Serva collagenase NB1, Liberase MTF, and the Vitacyte CIzyme collagenase HA. In 2010, Shimoda et al.103 compared the results of these three enzymes with Liberase HI (using a modified protocol for the newer enzymes). The authors reported a shorter phase I time with NB1 than with CIzyme HA. Furthermore, the pre-purification IEQ/g pancreas was significantly lower with Liberase HI than with the other enzymes, while the post-purification IEQ/g was significantly higher with Liberase MTF than with the other enzymes. In addition, Serva NB1 resulted in a significantly better viability than with Liberase HI. In 2014, Rheinheimer et  al.104 performed a mixed treatment comparison (MTC) meta-analysis to compare different digestion enzymes. The analysis included 15 studies that met the eligibility criteria. The compared enzymes were Liberase HI, Serva NB1, CIzyme HA, Liberase MTF, Collagenase P (Boehringer Mannheim, Indianapolis, IN, USA), Sevac (Crescent Chemical, Hauppauge, NY, USA), Sigma V (Sigma, St. Louis, MO, USA), Recombinant (Roche, Penzberg, Germany), and Collagenase Custom (Roche, Indianapolis, IN, USA). No significant differences were noted among the compared enzymes regarding the islet yield, purity, or viability percentages except for a small increase in the yield (IEQ/g pancreas) associated with Vitacyte CIzyme HA and Liberase MTF when compared to Sevac enzyme. Furthermore, Vitacyte HA and Serva NB1 showed

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TABLE 3  Examples of Good Manufacturing Practice (GMP) tissue dissociation enzymes for pancreatic islet isolation Enzyme

Manufacturer

Source

Isoform details

Activity assay

Wünsch assay >2.80 U/mg >1900 U/bottle, for CDA check44

Animal origin free Endotoxin

Neutral protease

No

<10.0 EU/ mg

Separate

No

≤10 EU/mg protein

Thermolysin MTF (separate vial).

Natural collagenase blends Natural blend Collagenase HAGMP grade

VitaCyte, Indianapolis, IN, USAa

Clost. histolyticum

60% ColI +40% ColII

Liberase MTF C/T GMP grade

Roche Diagnostics GmbH, Mannheim, Germanyb

Clost. histolyticum

ColI: 258–386 mg Wünsch assay ColII: 172–258 mg 2172–3617 U/vial HPLC purity: ≥85% ColIb+c: ≤10 area % ColII/total col. = 0.3–0.5

Collagenase AF-1 GMP grade

Serva/ Nordmark Arzneimittel GmbH & Co. KG, Uetersen Germanyc

Clost. histolyticum

Wünsch assay ≥3.00 U/mg ≥2000 U/vial

Yes

Separate

Recombinant collagenases d

e

Wünsch = 0.11 U/mg, Yes CDA = 125,839 U/mg. Yes Wünsch = 9.16 U/mg, CDA = 5170 U/mg

rC1 rC2

VitaCyte, Indianapolis, IN, USA

E. coli E. coli

Meiji Collagenase G (ColG) Meiji Collagenase H (ColH)

Meiji Seika Pharma Co., Ltd., Tokyo, Japan

E. coli E. coli

Solution type (10 mg/1.0 mL) Solution type (10 mg/0.5 mL)

Wünsch = 2.5 U/mL, Yes Azocoll = 34.0 U/mL Yes FALGPA = 13.9 U/mL Wünsch = 375 U/mL, Azocoll = 1.4 U/mL FALGPA = 478 U/mL

<1.0 EU/mL <1.0 EU/mL

VitaCyte, Indianapolis, IN, USAa

Bacillus polymyxa

Total protein = Lot specific

FITC-BSA Yes >90,000 Units/mg >800,000 Units/bottle

<50.0 EU/ mg

Serva/ Nordmark Arzneimittel GmbH & Co. KG, Uetersen Germany.c

Clost. histolyticum

Neutral protease activity (DMC) ≥0.50 U/mg ≥100 U/vial

Yes

≤100.0 EU/ mg

VitaCyte, Indianapolis, IN, USA.a Roche Diagnostics GmbH, Mannheim, Germanyb

Bacillus thermoproteolyticus ‘rokko’

FITC-BSA 1,200,000–2,000,000 Units/vial Casein 130,500–234,000 U/ vial

Yes No

<50.0 EU/ mg ≤50 EU/mg protein

e

Neutral proteases BP protease BP protease GMP grade CHNP Neutral Protease AFGMP grade

Thermolysin Thermolysin AFGMP grade Thermolysin MTF MPB-GMP grade

Total protein = lot specific Total protein = 12.0–18.0 mg/bottle HPLC purity ≥85%

ahttps://www.vitacyte.com bhttp://www.nordmark-pharma.de chttp://www.custombiotech.roche.com dInformation obtained from Ref. 105. eSingle lot information. CHNP, Clostiridium histolyticum neutral protease; NP, neutral protease. Information obtained from corresponding website for each product as follows (last accessed June 2018):

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Pancreatic dissociation enzymes in clinical islet isolation: Evolution, safety, and overview of commercial products

an ­increased stimulation index compared to Liberase MTF. In a study (“IPITA poster”) not included in the ­meta-analysis, Balamurugan et al.91 compared eight collagenase/protease combinations, including three SERVA collagenase products (GMP, premium grade, and hightryptic-like activity), two Vitacyte collagenase products (CIzyme collagenase HA and Vitacyte high-collagen-­ degrading-activity collagenase), and the Roche Liberase MTF collagenase in combination with either CHNP (Serva NP NB) or thermolysin (Vitacyte thermolysin, or Roche custom blend thermolysin). The best IEQ/g pancreas and morphology were achieved with Vitacyte collagenase (containing high intact C1 collagenase) in combination with SERVA NB (CHNP). The superiority of CHNP to thermolysin was further demonstrated by split-pancreas experiments using the same Vitacyte collagenase. Table  3 shows the composition of commonly used “GMP” commercial enzymes. The clear correlation of such comparative observations with the biochemical profile of dissociation enzymes was, for long, difficult to determine. However, several enzymatic contributors to successful islet isolation have been identified in the last two decades. These findings were fostered by the introduction of recombinant enzymes into human islet isolation. With these efforts, the influence of intact C1 proportion (i.e., C1a), NP selection, collagenase activity assay, and clostripain/ TLA is now appreciated. In 2010, Balamurugan et al.106 analyzed collagenases from Roche (Liberase HI), Serva (NB1, GMP grade), and VitaCyte (CIzyme HA) using high-performance liquid chromatography (HPLC) and CDA. Combining the data from HPLC and SDS-PAGE analyses showed that intact C2 with a MW of 112 kDa was the earliest eluting peak, followed by intact C1 with a MW of 116 kDa and another two molecular forms of C1 (C1b and C1c resulting from proteolytic cleavage of the C-terminal CBD of intact C1) with MWs of approximately 100 kDa. Compared with intact C2, C1b, and C1c, all of which have one CBD, the presence of two CBDs in intact C1 results in an approximately sevenfold to 10-fold higher specific CDA,44 rendering the assay a reflection of the proportion of intact C1. Analyses of the commercial enzymes showed that degraded C1 is the predominant form among C1 molecular forms in Serva NB1, resulting in a low specific CDA. Liberase HI had a higher proportion of intact C1 and accordingly a higher specific CDA, while VitaCyte HA showed the predominance of intact C1 and thus attained the highest specific CDA. Furthermore, comparing human islet isolations using VitaCyte HA and thermolysin to earlier results obtained by Serva NB1 and NB NP showed a significantly better yield with the VitaCyte mixture (4147 ± 1759 vs 2134 ± 1524 IEQ/g pancreas, respectively). This finding was in accordance with that of a previous report by the same group showing the relative superiority of Vitacyte

539

in combination with Serva NP compared to seven other enzyme mixtures91 (IPITA poster). A previous report by Barnett107 also showed that the fraction of degraded C1 (C1b) in Liberase HI was increased relative to the chronological order of manufacture, and its increase was associated with reduced islet yields in 12 human islet isolations. A recent report by Green et al.108 showed no marked difference in the isolation results using either intact C1 or truncated C1 (100 kDa) in adult porcine split-pancreas experiments when a fixed CDA dosage of 25,000 CDA U/g tissue was used. However, to achieve the same CDA dose, a 19-fold higher mass of truncated C1 was required.108 This suggests that the amount of CDA (as a measure of the C1 activity) rather than the molecular form of C1 is the main factor that should be considered in C1 preparation. Kin et al.109 examined different ratios of CII:CI using purified enzymes from Roche in human islet isolation. The ratios used were 1:1, 1:2, and 1.5:1. The worst yield and islet morphology was obtained with a high CII proportion (i.e., 1.5:1 ratio). Despite having the highest total collagenase activity represented by Wünsch units/ pancreas, a comparison with previous results obtained with mixtures having similar collagenase activities but different CII/CI portions suggested that the utilized ratio rather than the collagenase activity was the reason for the inferior results. In contrast, testing different combinations of recombinant C1 and recombinant C2 represented as high and low CDA for rC1 and high and low Wünsch activity for rC2 showed no significant difference in digestion results or islet functions [oxygen consumption rate/DNA and glucose-stimulated insulin release (GSIR)] among different combinations with a fixed NP dosage.105 Furthermore, the study105 showed a better islet yield with a comparable function to a natural collagenase blend consisting of collagenase HA (VitaCyte) and NB NP (Serva) despite the recombinant blend having approximately 40%–75% of the total collagenase dose (IEQ/g pancreas = 5209 ± 522 with the recombinant blend vs 3616 ± 739 with the natural blend). In addition, cumulative recombinant enzyme isolation results revealed the superiority of both the pre- and post-purification islet yield when compared to historical data using either Serva collagenase NB1 + Serva NB NP or Vitacyte CIzyme Collagenase HA + VitaCyte CIzyme Thermolysin.105 Two important implications of this study105 are the non-profound impact of the mass ratio of C1 and C2 on the isolation outcome and the comparable results achieved with lower C2 and C1 target activities (roughly 60% and 33%, respectively, of those utilized in traditional 60:40 natural enzyme blends). This supports the finding of a previous report by Friberg et al. 110 that showed that only 30% of FALGPA activity is absorbed by the pancreas during perfusion.

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43.  Collagenases in pancreatic islet isolation

The choice of NP was also shown to influence the results of islet isolation. Surprisingly, few studies have directly compared different NPs in human islet isolation. In split human pancreas experiments (n = 5), CHNP (NB, Serva) proved superior to thermolysin (VitaCyte CIzyme) with a significantly higher average total IEQ (219,003 ±  45,298 vs 151,153  ± 19,042; p = 0.047) and significantly higher percentage of islets with diameter > 200 μm (19.7 ± 4.7 vs 8.3 ± 2.5%, p = 0.021).111 A similar experiment (n = 4) showed comparable results of both CHNP (NB Serva, 1.75 dimethyl casein U/g) and an equivalent amount of CIzyme BP protease (VitaCyte, Indianapolis, IN), a dispase equivalent purified from Paenbacillus polymyxa culture supernatants (24,000 FITCBSA U/g).105 The role of clostripain has become better appreciated in the recent years. In 1989, McShane et  al.89 reported that the tryptic and clostripain activities in enzyme mixtures had no significant impact on the isolation results. However, Brandhorst et  al.59 examined the effect of TLA, as measured by the N-benzoyl-L-argininge-ethyl ester-specific assay, relative to the collagenase activity (TLA-ratio) in rat and human islet isolation using different Serva NB1 batches and found that increasing the ratio from 1.3% to 10% in rats resulted in a 50% decrease in the dissociation time without affecting the yield, viability, or in  vivo function, while an increase from 1.3% to 12.6% for human pancreata significantly shortened the recirculation time and incrementally increased the islet yield without affecting the purity, in vitro function, or recovery after culture. A similar effect was reported in juvenile pig pancreas dissociation,112 where a high efficacy of islet release from pancreas corresponded to a high specific TLA and collagenase activity and low levels of NP. As a result, the addition of clostripain to enzyme mixture before human islet isolation significantly increased the total IEQ, IEQ/g pancreas, purified tissue volume, and proportion meeting the criteria for transplantation without any adverse effects on the GSIR or islet size distribution.65 TLA was found to exert no detrimental effects on the yield, islet integrity, or GSIR,59 as opposed to those ascribed to exogenous or pancreatic trypsin.113, 114 This may be due to differences in the specificity of TLA and pancreatic trypsin toward islet and non-islet pancreatic tissue.59 A recent study115 examined the toxic effects of CHNP, thermolysin, and clostripain on isolated islets and found that both CHNP and thermolysin have similar toxic potency toward islet integrity, while a higher threshold is required for clostripain to cause adverse effects than thermolysin. When clostripain was used, no significant decrease in the viability or intracellular insulin loss was noted until reaching an activity of 6.7 benzoyl-L-­arginine-ethyl-ester units/mL, which is equivalent to 10-fold the common supplementation, and no adverse effects on mitochondria were noted within the examined concentration

range. Thus, the lot-to-lot variability of purified enzyme blends can be a consequence of the underestimated contributions of some mixture components (e.g., intact C1 proportion or CDA, C1/C2 ratio, clostripain/TLA). However, this issue can be resolved by expanding the use of highly purified recombinant enzymes and standardizing activity assays. In addition to their reported comparable116-to-­superior isolation results105 and effects of eliminating inconsistency,116 recombinant enzymes also offer the chance to select endotoxin-free species for the cloning process. In a recent report, the dose of recombinant enzyme blend was 42% of that of the natural enzyme blend required to digest the same amount of tissue. Reducing the dose in this manner resulted in a better in  vitro and in  vivo function after transplantation.117 In 1998, Vargas et al.118 showed that the main source of endotoxins during islet isolation and in the final preparation was the purified collagenase preparations, despite the presence of detectable endotoxin levels in other reagents (e.g., Ficoll). At that time, the endotoxin level ranged from 100 to 500 EU/mg in commonly used collagenase preparations using the chromogenic Limulus amebocyte lysate assay.118 Although the level of contamination was considerably reduced with repeated washing and centrifugation, the level remained higher than that acceptable for human transplantation (in 10 out of 13 isolations).118 Liberase HI, by contrast, was found to be almost endotoxin-free (0.1 ng/mg).119 The contaminated collagenase preparations strongly induced IL-1α, IL-1β, TNF, and IL-6 mRNA in macrophages.118 In addition to their effects on immune activation through toll-like receptors,120 endotoxins also damage islets, resulting in their apoptosis through the activation of mitogen-activated protein kinases (MAPKs),121 and decrease the in vitro GSIR through TLR4 signaling.122 The use of the endotoxin scavenger polymyxin-B improved the islet yield, ATP content, and glucose stimulation index while reducing the TNF-α expression in mouse islets.123 Another study124 showed slightly higher endotoxin contamination than the levels stated in the manufacturers’ datasheets (to be <1 EU) in three out of five recombinant protein products (not including collagenases, produced by E. coli). This study showed that even minimal endotoxin contamination (0.02–2  ng of lipopolysaccharide (LPS)/mL of protein preparation) was able to activate the NF-κB pathway and resulted in cytokine expression in different immune cells, the sensitivity of which to the LPS concentration was found to closely correlate with the expression of CD14, a pattern-recognizing receptor expressed by myeloid lineage cells.124 CD14 is expressed in human and rat islets, and LPS was shown to influence the GSIR of rat islets in vitro.122 As a result, ensuring undetectable levels of endotoxin contamination in the enzyme manufacturing process is desirable.

B.  Islet allo-transplantation



Exogenous parameters affecting collagenase digestion

Another study also showed that fluorescein-­ conjugated Liberase HI can be traced in freshly isolated and 3-day cultured mouse islets and acinar cells after intra-ductal injection as well as in isolated human islets and acinar cells after 1-h incubation, despite extensive washing.125 That study suggested several harmful effects of the retained collagenase; however, endotoxin contamination may be partly responsible for these findings. In contrast, a study using immune labeling in human islets showed that despite collagenase reaching intra-islet locations after trans-ductal injection,126 it is washed out during the isolation process, becoming undetectable in almost all islet samples starting from the digestion collection phase (detected in only one sample of one preparation) and in no samples from later phases.127 Another refinement to the collagenase production process is the use of animal tissue-free (AF) culture media and utilizing plant-based ingredients to avoid prion transmission, even from distantly related species, although this potential risk is still being investigated.128 Studies of plant-based enzymes, namely Collagenase AF-1 and NP AF (SERVA/Nordmark Arzneimittel GmbH & Co. KG, Uetersen, Germany), showed comparable efficiency in human islet isolation to that of ­premium-grade collagenase NB-1129 and high efficiency in a recent multi-centric study.130

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Exogenous parameters affecting collagenase digestion The isolation of islets requires their separation from the surrounding acinar tissue and ECM. This in turn requires the active digestion enzymes reach the peri-­ insular region, in addition to the proper digestion of the non-insular tissue to facilitate their liberation. The retrograde intra-ductal injection of digestion enzymes is the preferred method in human and mammalian islet isolation because of the uniform distribution of the enzymes and, as a result, access to more islet regions, half of which lie in a peri-ductal location.131 Indeed, using collagenase containing 1% marking dye showed a significantly lower number of unreached islets in human pancreas after ductal injection than without ductal injection (Fig. 6).132 A previous study by Cross et al.126 showed that collagenase injected transductally is distributed throughout the pancreas in association with collagen VI, being found in 67% ± 2% of islets (intra-islet) with a significantly higher frequency in the tail region and larger islets (>150 μm), regardless of the injection modality (manual syringe-­ loading vs recirculating perfusion). The penetration of collagenases into islets may contribute to their loss and fragmentation during isolation.126 Pressure monitoring during injection is thus crucial for preventing damage

FIG.  6  H&E histology showing Ink distribution in relation to pancreatic islets after ductal injection “DI”: (A) unreached; (B) reaching islet

surface; and (C) penetrating into islet. (D) shows proportion of each patter in different parts of the pancreas with or without DI. Magnification: × 400. Scale bars: 20 μm, *p, 0.05. Reproduced by permission from Taylor & Francis, Islets (Shimoda M, Itoh T, Sugimoto K, et al. Improvement of collagenase distribution with the ductal preservation for human islet isolation. Islets 2012;4(2):130–137) © 2012.

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43.  Collagenases in pancreatic islet isolation

of the duct system, which is necessary for the optimum distribution of enzymes along the pancreatic tissue and allowing extravasation into the neighboring peri-insular regions. Lakey et al.133 showed that controlled automated perfusion is significantly superior to syringe loading with regard to the amount of undigested tissue, the islet yield, and the post-purification islet recovery without any influence on the in vitro islet function. The use of dye (metyltioninklorid) to detect collagenase leakage during injection and a tissue adhesive (indermil) resulted in a significantly better islet yield and isolation success rate, without an adverse effect on the collagenase activity or in vitro islet function.134 Following distension, the pancreas is cut and transferred to the modified Ricordi chamber for a combination of enzymatic and mechanical digestion. The Ricordi chamber, originally introduced by Ricordi et al. in 1986, allows for tight temperature control, dilution, and collection of the liberated islets. These features have allowed this chamber to maintain its superiority among static digestion methods.135 The choice of solution for pancreas preservation and as a solvent for collagenase is also important. As calcium ions are necessary for collagenase stability,136 the use of solutions with components that bind calcium can decrease the collagenase activity. University of Wisconsin (UW) solution can inhibit collagenase activity owing to its content of glutathione and lactobionate, which bind to divalent metal ions.137, 138 Thus, UW was demonstrated to inhibit the collagenase digestion phase of human pancreata,139 and a higher collagenase concentration was required to liberate the maximum amount of islets in porcine (5-weeks old) pancreata when UW was used as a solvent.140 The UW content of magnesium, allopurinol, hydroxyethyl starch and adenosine as well as the Na/K ratio were also implied to have inhibitory effect on the collagenase activity during digestion of human pancreata.139 An analysis showed that this effect is more prominent toward class II collagenases than class I collagenases.141 Histidine/tryptophane/ketoglutarate (HTK) solution, another common organ preservation solution, also exerted an inhibitory effect on the metalloproteinase activity owing to its histidine content.138 Studies on Liberase HI at the University of Alberta showed that filtration through 0.22-μm porosity membranes (four types examined: nylon, nitrocellulose, cellulose acetate, and polysulfone) was associated with a substantial decrease in the collagenase, but not protease, activity.142 Loss of activity was also noted in reconstituted aliquots stored at −80°C, reaching 35% at 6 weeks for collagenase and 54% at 6 weeks for protease.142

Tailored approach to islet isolation The long-recognized problem of inconsistency in the outcome of islet isolation, although commonly ascribed to lot-to-lot variability, can also be confounded by do-

nor pancreatic tissue variability. A tailored donor-tissue specific islet isolation approach may, therefore, be the next step for enzyme blend progression. Obvious examples of these donor-related factors include the age, body mass index, and gender. A study showed that despite advances in isolation techniques and enzyme blends, islet yields from younger pancreata are still significantly lower when the BMI is fixed (<25), with no successful isolation achieved from donors <30 years of age.143 That study also highlighted a gender discrepancy, with significantly higher yields from female donors than from males. Variation in the pancreatic tissue composition in relation to these factors has not yet been clearly outlined. Attempts to optimize isolation protocols for younger donors in order to obtain a better post-purification yield have been reported, such as performing rescue purification after continuous gradient purification in select cases.144 However, a similar approach regarding optimizing the enzyme blend is not yet feasible due to the lack of a definition between tissue elements and corresponding enzyme variables. A thoughtful study recently presented at the 2017s IPITA compared different conditions for optimizing the yield from pancreata of younger donors.145 The factors included enzyme concentrations with a fixed ratio, enzyme vendors, NP choice, and the ratio of NP to collagenase. Interestingly, only an increased NP-to-collagenase ratio was associated with a better yield and fewer entrapped islets,145 a finding that agrees with the high percentage of entrapped islets observed in isolations from pancreata of younger donors. The study also reported a high yield from the tail region in all examined conditions, which also accords with the higher frequency of intra-islet collagenase in the tail region mentioned previously. A study showed a more than threefold increase in collagen VI and increased collagen IV in cardiac ECM of aging mice compared with younger mice.146 As previously mentioned, pancreata of older individuals showed a higher total collagen content than that of younger ones72; however, no significant difference in collagen VI or mean islet area was demonstrated in pancreata from younger and older individuals.9 Our results show that tailoring the digestion blend based on the collagen III proportion in different rat species resulted in superior outcomes.147

Conclusions Investigations into the mechanism underlying islet release and the factors determining efficient enzyme blends are expected to robustly proceed until complete/near-complete retrieval of islet content in human pancreata with a good functional profile is achieved. Such studies are accompanied by advances in the manufacturing process to offer matching enzymes and as-

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References

says that are both feasible and cost effective. Research over the last three decades has contributed massively to our understanding of pancreatic dissociation and improving the clinical isolation outcome. A consensus regarding efficient enzyme parameters based on updated understanding is necessary to translate the craftsmanship of pancreatic digestion into standardized protocols.

References 1. Chamberlain  P, Drewello  R, Korn  L, et  al. Construction of the Khoja Zaynuddin mosque: use of animal glue modified with urine. Archaeometry. 2011;53(4):830–841. 2. Snedeker JG, Gautieri A. The role of collagen crosslinks in ageing and diabetes-the good, the bad, and the ugly. Muscles, Ligament Tendons J. 2014;4(3):303. 3. Shoulders MD, Raines RT. Collagen structure and stability. Annu Rev Biochem. 2009;78:929–958. 4. Ramshaw  JA, Shah  NK, Brodsky  B. Gly-XY tripeptide frequencies in collagen: a context for host–guest triple-helical peptides. J Struct Biol. 1998;122(1–2):86–91. 5. Brinckmann J. Collagens at a glance. Collagen: Springer; 2005:1–6. 6. Veit  G, Kobbe  B, Keene  DR, Paulsson  M, Koch  M, Wagener  R. Collagen XXVIII, a novel von Willebrand factor A domain-­ containing protein with many imperfections in the collagenous domain. J Biol Chem. 2006;281(6):3494–3504. 7. Meyer  T, Chodnewska  I, Czub  S, et  al. Extracellular Matrix Proteins in the Porcine Pancreas: A Structural Analysis for Directed Pancreatic Islet Isolation 1. In: Paper presented at: Transplantation proceedings; 1998. 8. Van Deijnen J, Van Suylichem P, Wolters G, Van Schilfgaarde R. Distribution of collagens type I, type III and type V in the pancreas of rat, dog, pig and man. Cell Tissue Res. 1994;277(1):115–121. 9. Hughes  SJ, Clark  A, McShane  P, Contractor  HH, Gray  DW, Johnson  PR. Characterisation of collagen VI within the islet-­ exocrine interface of the human pancreas: implications for clinical islet isolation? Transplantation. 2006;81(3):423–426. 10. Hulmes DJ. Building collagen molecules, fibrils, and suprafibrillar structures. J Struct Biol. 2002;137(1–2):2–10. 11. Harrington  DJ. Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun. 1996;64(6):1885. 12. Lim  DV, Jackson  RJ, Pull-VonGruenigen  CM. Purification and assay of bacterial collagenases. J Microbiol Methods. 1993;18(3):241–253. 13. Seifter S, Harper E. 18 The Collagenases. The Enzymes. 1971;3:649– 697. Elsevier. 14. Daboor SM, Budge SM, Ghaly AE, Brooks S, Dave D. Extraction and purification of collagenase enzymes: a critical review. Am J Biochem Biotechnol. 2010;6(4):239–263. 15. Nordwig  A. Collagenolytic enzymes. Adv Enzymol Relat Areas Mol Biol. 1971;34:155–205. 16. Cawston TE. Chapter 152—Matrix Metallopeptidase-1/Interstitial Collagenase. In: Rawlings  ND, Salvesen  G, eds. Handbook of Proteolytic Enzymes. 3rd ed. Academic Press; 2013:718–725. 17. Duarte AS, Correia A, Esteves AC. Bacterial collagenases—a review. Crit Rev Microbiol. 2016;42(1):106–126. 18. Eckhard U, Huesgen PF, Brandstetter H, Overall CM. Proteomic protease specificity profiling of clostridial collagenases ­reveals their intrinsic nature as dedicated degraders of collagen. J Proteomics. 2014;100:102–114. 19. Rawlings ND, Barrett AJ, Bateman A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2011;40(D1):D343–D350.

543

20. Bond  MD, Van Wart  HE. Characterization of the individual collagenases from Clostridium histolyticum. Biochemistry. 1984;23(13):3085–3091. 21. Matsushita  O, Jung  C-M, Katayama  S, Minami  J, Takahashi  Y, Okabe  A. Gene duplication and multiplicity of collagenases in Clostridium histolyticum. J Bacteriol. 1999;181(3):923–933. 22. Ambrosius  DD, Hesse  FD, Burtscher  HD. Rekombinante Collagenase Typ II aus Clostridium histolyticum und ihre Verwendung zur Isolierung von Zellen und Zellverbänden. Google Patents; 1995. 23. Burtscher H, Ambrosius D, Hesse F. Recombinant collagenase type I from Clostridium histolyticum and its use for isolating cells and groups of cells. Google Patents; 2002. 24. Yoshihara  K, Matsushita  O, Minami  J, Okabe  A. Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase. J Bacteriol. 1994;176(21):6489–6496. 25. Teramura N, Tanaka K, Iijima K, et al. Cloning of a novel collagenase gene from the gram-negative bacterium Grimontia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis. J Bacteriol. 2011;193(12):3049–3056. 26. Vaitkevicius  K, Rompikuntal  PK, Lindmark  B, Vaitkevicius  R, Song T, Wai SN. The metalloprotease PrtV from Vibrio cholerae. FEBS J. 2008;275(12):3167–3177. 27. Matsushita  O, Koide  T, Kobayashi  R, Nagata  K, Okabe  A. Substrate recognition by the collagen-binding domain of Clostridium histolyticum Class I Collagenase. J Biol Chem. 2001;276(12):8761–8770. 28. Matsushita  O, Jung  C-M, Minami  J, Katayama  S, Nishi  N, Okabe  A. A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase. J Biol Chem. 1998;273(6):3643–3648. 29. 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. 30. Fasciglione GF, Marini S, D’Alessio S, Politi V, Coletta M. pH-and temperature-dependence of functional modulation in metalloproteinases. A comparison between neutrophil collagenase and gelatinases A and B. Biophys J. 2000;79(4):2138–2149. 31. Gross  J, Nagai  Y. Specific degradation of the collagen molecule by tadpole collagenolytic enzyme. Proc Natl Acad Sci. 1965;54(4):1197–1204. 32. Gross J, Harper E, Harris Jr ED, et al. Animal collagenases: specificity of action, and structures of the substrate cleavage site. Biochem Biophys Res Commun. 1974;61(2):605–612. 33. Welgus  HG, Jeffrey  J, Stricklin  G, Eisen  A. The gelatinolytic activity of human skin fibroblast collagenase. J Biol Chem. 1982;257(19):11534–11539. 34. Eckhard  U, Schönauer  E, Ducka  P, Briza  P, Nüss  D, Brandstetter  H. Biochemical characterization of the catalytic domains of three different Clostridial collagenases. Biol Chem. 2009;390(1):11–18. 35. Mookhtiar K, Van HW. Clostridium histolyticum collagenases: a new look at some old enzymes. Matrix (Stuttgart, Germany) Suppl 1992;1:116–126. 36. Goldberg GI, Wilhelm SM, Kronberger A, Bauer EA, Grant GA, Eisen  AZ. Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein. J Biol Chem. 1986;261(14):6600–6605. 37. Mookhtiar K, Steinbrink DR, Van Wart HE. Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium h ­ istolyticum collagenases: evidence for both e­ ndopeptidase and tripeptidylcarboxypeptidase activities. Biochemistry. 1985;24(23):6527–6533. 38. Bond  MD, Van Wart  HE. Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography. Biochemistry. 1984;23(13):3077–3085.

B.  Islet allo-transplantation

544

43.  Collagenases in pancreatic islet isolation

39. Toyoshima T, Matsushita O, Minami J, Nishi N, Okabe A, Itano T. Collagen-binding domain of a Clostridium histolyticum collagenase exhibits a broad substrate spectrum both in  vitro and in vivo. Connect Tissue Res. 2001;42(4):281–290. 40. Eckhard  U, Schönauer  E, Nüss  D, Brandstetter  H. Structure of collagenase G reveals a chew-and-digest mechanism of bacterial collagenolysis. Nat Struct Mol Biol. 2011;18(10):1109. 41. Watanabe-Nakayama T, Itami M, Kodera N, Ando T, Konno H. High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils. Sci Rep. 2016;6:28975. 42. Eckhard  U, Brandstetter  H. Polycystic kidney disease-like domains of clostridial collagenases and their role in collagen recruitment. Biol Chem. 2011;392(11):1039–1045. 43. Eckhard  U, Nüss  D, Ducka  P, Schönauer  E, Brandstetter  H. Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008;64(5):419–421. 44. McCarthy R, Spurlin B, Wright M, et al. Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation. In: Paper presented at: Transplantation proceedings; 2008. 45. Eckhard U, Schönauer E, Brandstetter H. Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T. J Biol Chem. 2013;288(28):20184–20194. 46. Worthington  K, Worthington  V. Worthington Enzyme Manual. 2011; http://www.worthington-biochem.com/pap/default.html. Accessed May, 2018. 47. Endo S. Studies on protease produced by thermophilic bacteria. J Ferment Technol. 1962;40:346–353. 48. Matsubara H. [46] Purification and assay of thermolysin. Methods Enzymol. 1970;19:642–651. Elsevier. 49. Walsh  K, Burstein  Y, Pangburn  M. [50] Thermolysin and other neutral metalloendopeptidases. Methods Enzymol. 1974;34:435– 440. Elsevier. 50. Yasukawa K, Kusano M, Inouye K. A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Protein Eng Des Sel. 2007;20(8):375–383. 51. Roche RS, Voordouw G, Matthews BW. The structural and functional roles of metal ions in thermolysin. CRC Crit Rev Biochem. 1978;5(1):1–23. 52. de Kreij  A, van den Burg  B, Venema  G, Vriend  G, Eijsink  VG, Nielsen  JE. The effects of modifying the surface charge on the catalytic activity of a thermolysin-like protease. J Biol Chem. 2002;277(18):15432–15438. 53. Feder  J, Schuck  JM. Studies on the Bacillus subtilis neutral-­ protease-and Bacillus thermoproteolyticus thermolysin-catalyzed hydrolysis of dipeptide substrates. Biochemistry. 1970;9(14): ­ 2784–2791. 54. Holmquist  B, Vallee  BL. Esterase activity of zinc neutral proteases. Biochemistry. 1976;15(1):101–107. 55. Kunugi S, Hirohara H, Ise N. pH and temperature dependences of thermolysin catalysis. FEBS J. 1982;124(1):157–163. 56. Pangburn  MK, Walsh  KA. Thermolysin and neutral protease. Mechanistic considerations. Biochemistry. 1975;14(18):4050–4054. 57. Maeda  H, Nakagawa  K, Murayama  K, et  al. Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate. Appl Microbiol Biotechnol. 2015;99(24):10489–10499. 58. Breite A, Dwulet F, McCarthy R. Tissue dissociation enzyme neutral protease assessment. Paper presented at: Transplantation proceedings 2010.

59. Brandhorst H, Friberg A, Andersson HH, et al. The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation. Transplantation. 2009;87(3):370–375. 60. Labrou  E. N. Chapter  521—Clostripain. In: Rawlings  ND, Salvesen G, eds. Handbook of proteolytic enzymes. 3rd ed. Academic Press; 2013:2323–2327. 61. Gilles AM, Lecroisey A, Keil B. Primary structure of N-clostripain light chain. FEBS J. 1984;145(3):469–476. 62. Mitchell  WM. Hydrolysis at arginylproline in polypeptides by clostridiopeptidase B. Science. 1968;162(3851):374–375. 63. Ogle JD, Tytell AA. The activity of Clostridium histolyticum proteinase on synthetic substrates. Arch Biochem Biophys. 1953;42(2):327–336. 64. Mitchell WM, Harrington WF. Purification and properties of clostridiopeptidase B (Clostripain). J Biol Chem. 1968;243(18):4683–4692. 65. Ståhle M, Foss A, Gustafsson B, et al. Clostripain, the missing link in the enzyme blend for efficient human islet isolation. Transplant Direct. 2015;1(5). 66. Dargatz H, Diefenthal T, Witte V, Reipen G, von Wettstein D. The heterodimeric protease clostripain from Clostridium histolyticum is encoded by a single gene. Mol Gen Genet MGG. 1993;240(1):140–145. 67. Witte V, Wolf N, Diefenthal T, Reipen G, Dargatz H. Heterologous expression of the clostripain gene from Clostridium histolyticum in Escherichia coli and Bacillus subtilis: maturation of the clostripain precursor is coupled with self-activation. Microbiology. 1994;140(5):1175–1182. 68. Tanaka H, Nariya H, Suzuki M, et al. High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens. Protein Expr Purif. 2011;76(1):83–89. 69. Griffin  PJ, Fogarty  WM. Physicochemical properties of the native, zinc-and manganese-prepared metalloprotease of Bacillus polymyxa. Appl Microbiol. 1973;26(2):191–195. 70. Sparrow  L, McQuade  A. Isolation by affinity chromatography of neutral proteinase from Clostridium histolyticum. Biochim Biophys Acta (BBA)-Enzymol. 1973;302(1):90–94. 71. Carroll PB. Anatomy and physiology of islets of Langerhans. In: Ricordi C, ed. Pancreatic Islet Cell Transplantation, 1892–1992: One Century of Transplantation for Diabetes. Austin, TX: RG Landes Co; 1992:7–18. 72. Bedossa P, Lemaigre G, Bacci J, Martin E. Quantitative estimation of the collagen content in normal and pathologic pancreas tissue. Digestion. 1989;44(1):7–13. 73. Imamura T, Iguchi H, Manabe T, et al. Quantitative analysis of collagen and collagen subtypes I, III, and V in human pancreatic cancer, tumor-associated chronic pancreatitis, and alcoholic chronic pancreatitis. Pancreas. 1995;11(4):357–364. 74. Edwards  C, O’Brien Jr W. Modified assay for determination of hydroxyproline in a tissue hydrolyzate. Clin Chim Acta. 1980;104(2):161–167. 75. Van Deijnen  J, Hulstaert  C, Wolters  G, Van Schilfgaarde  R. Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat, dog, pig, and man. Cell Tissue Res. 1992;267(1):139–146. 76. Virtanen I, Banerjee M, Palgi J, et al. Blood vessels of human islets of Langerhans are surrounded by a double basement membrane. Diabetologia. 2008;51(7):1181–1191. 77. Timpl R, Brown JC. Supramolecular assembly of basement membranes. Bioessays. 1996;18(2):123–132. 78. Heller-Harrison RA, Carter W. Pepsin-generated type VI collagen is a degradation product of GP140. J Biol Chem. 1984;259(11):6858–6864. 79. Nikolova  G, Jabs  N, Konstantinova  I, et  al. The vascular basement membrane: a niche for insulin gene expression and β cell proliferation. Dev Cell. 2006;10(3):397–405. 80. Nikolova  G, Strilic  B, Lammert  E. The vascular niche and its basement membrane. Trends Cell Biol. 2007;17(1):19–25.

B.  Islet allo-transplantation



References

81. Wang RN, Paraskevas S, Rosenberg L. Characterization of integrin expression in islets isolated from hamster, canine, porcine, and human pancreas. J Histochem Cytochem. 1999;47(4):499–506. 82. Irving-Rodgers H, Choong F, Hummitzsch K, Parish C, Rodgers R, Simeonovic C. Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: impact for allograft rejection. Cell Transplant. 2014;23(1):59–72. 83. Meyer  T, Czub  S, Chodnewska  I, et  al. Expression pattern of extracellular matrix proteins in the pancreas of various domestic pig breeds, the Goettingen Minipig and the Wild Boar. Ann Transplant. 1997;2(3):17–26. 84. Wolters G, Vos-Scheperkeuter G, Van Deijnen J, Van Schilfgaarde R. An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation. Diabetologia. 1992;35(8):735–742. 85. Linares HA, Larson DL. Proteoglycans and collagenase in hypertrophic scar formation. Plast Reconstr Surg. 1978;62(4):589–593. 86. McQuade A, Crewther W. Peptide substrates for a proteinase of Clostridium histolyticum. Biochim Biophys Acta (BBA)-Enzymol. 1968;167(3):619–620. 87. Brandhorst H, Brendel M, Eckhard M, Bretzel R, Brandhorst D. Influence of neutral protease activity on human islet isolation outcome. In: Paper presented at: Transplantation proceedings; 2005. 88. Bucher P, Bosco D, Mathe Z, et al. Optimization of neutral protease to collagenase activity ratio for islet of Langerhans isolation. In: Paper presented at: Transplantation proceedings; 2004. 89. McShane  P, Sutton  R, Gray  DW, Morris  PJ. Protease activity in pancreatic islet isolation by enzymatic digestion. Diabetes. 1989;38(suppl1):126–128. 90. Kin  T, Zhai  X, Murdoch  T, Salam  A, Shapiro  A, Lakey  JR. Enhancing the success of human islet isolation through optimization and characterization of pancreas dissociation enzyme. Am J Transplant. 2007;7(5):1233–1241. 91. Balamurugan  A, Loganathan  G, Anazawa  T, et  al. Improved method of human islet isolation for clinical transplantation using combination of clostridium histolyticum neutral protease [serva] and high proportion of intact c1 collagenase [vitacyte]. Xenotransplantation. 2009;16(6):545. 92. Wolters  GH, Vos-Scheperkeuter  GH, Lin  H-C, van Schilfgaarde R. Different roles of class I and class II Clostridium histolyticum collagenase in rat pancreatic islet isolation. Diabetes. 1995;44(2):227–233. 93. Fujio  A, Murayama  K, Yamagata  Y, et  al. Collagenase H is crucial for isolation of rat pancreatic islets. Cell Transplant. 2014;23(10):1187–1198. 94. Vos-Scheperkeuter  GH, Van Suylichem  PT, Vonk  MW, Wolters GH, Schilfgaarde RV. Histochemical analysis of the role of class I and class II Clostridium histolyticum collagenase in the degradation of rat pancreatic extracellular matrix for islet isolation. Cell Transplant. 1997;6(4):403–412. 95. Brandhorst D, Huettler S, Alt A, et al. Adjustment of the ratio between collagenase class II and I improves islet isolation outcome. In: Paper presented at: Transplantation proceedings; 2005. 96. Linetsky  E, Bottino  R, Lehmann  R, Alejandro  R, Inverardi  L, Ricordi C. Improved human islet isolation using a new enzyme blend, liberase. Diabetes. 1997;46(7):1120–1123. 97. Olack BJ, Swanson CJ, Howard TK, Mohanakumar T. Improved method for the isolation and purification of human islets of Langerhans using Liberase™ enzyme blend. Hum Immunol. 1999;60(12):1303–1309. 98. Saito  T, Anazawa  T, Gotoh  M, et  al. Actions of the Japanese Pancreas and Islet Transplantation Association regarding ­transplanted human islets isolated using Liberase HI. In: Paper presented at: Transplantation proceedings; 2010.

545

99. Therapy ISfC. Risk of Bovine Spongiform Encephalopathy (BSE) in Collagenase Enzymes. 2007. http://www.celltherapysociety.org/ files/PDF/Resources/Risk_BSE_in_Collagenase_Enzymes.pdf. Accessed April 2018. 100. Brandhorst H, Friberg A, Nilsson B, et al. Large-scale comparison of Liberase HI and collagenase NB1 utilized for human islet isolation. Cell Transplant. 2010;19(1):3–8. 101. Kin  T, O’Gorman  D, Zhai  X, et  al. Nonsimultaneous administration of pancreas dissociation enzymes during islet isolation. Transplantation. 2009;87(11):1700–1705. 102. Szot GL, Lee MR, Tavakol MM, et al. Successful clinical islet isolation using a GMP-manufactured collagenase and neutral protease. Transplantation. 2009;88(6):753. 103. Shimoda M, Noguchi H, Naziruddin B, et al. Assessment of human islet isolation with four different collagenases. In: Paper presented at: Transplantation proceedings; 2010. 104. Rheinheimer J, Ziegelmann PK, Carlessi R, et al. Different digestion enzymes used for human pancreatic islet isolation: a mixed treatment comparison (MTC) meta-analysis. Islets. 2014;6(4):e977118. 105. Balamurugan AN, Green ML, Breite AG, et al. Identifying effective enzyme activity targets for recombinant class I and class II collagenase for successful human islet isolation. Transplant Direct. 2016;2(1):e54. 106. Balamurugan A, Breite AG, Anazawa T, et al. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplantation. 2010;89(8):954–961. 107. Barnett  MJ, Zhai  XW, LeGatt  DF, Cheng  SB, Shapiro  AMJ, Lakey JR. Quantitative assessment of collagenase blends for human islet isolation. Transplantation. 2005;80(6):723–728. 108. Green  ML, Breite  AG, Beechler  CA, Dwulet  FE, McCarthy  RC. Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets. Islets. 2017;9(6):177–181. 109. Kin T, Zhai X, O’Gorman D, Shapiro AMJ. Detrimental effect of excessive collagenase class II on human islet isolation outcome. Transpl Int. 2008;21(11):1059–1065. 110. Friberg AS, Korsgren O, Hellgren M. A vast amount of enzyme activity fails to be absorbed within the human pancreas: implications for cost-effective islet isolation procedures. Transplantation. 2013;95(6):e36–e38. 111. Balamurugan  A, Loganathan  G, Mellina  DB, et  al. A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products. Transplantation. 2012;93(7):693. 112. Klöck  G, Kowalski  MB, Hering  BJ, et  al. Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas. Cell Transplant. 1996;5(5):543–551. 113. Krause U, Puchinger H, Wacker A. Inhibition of glucose-induced insulin secretion in trypsin-treated islets of Langerhans. Horm Metab Res. 1973;5(05):325–329. 114. Lecroisey  A, Keil-Dlouha  V, Woods  D, Perrin  D, Keil  B. Purification stability and inhibition of the collagenase from Achromobacter iophagus. FEBS Lett. 1975;59(2):167–172. 115. Brandhorst  H, Johnson  PR, Korsgren  O, Brandhorst  D. Quantifying the effects of different neutral proteases on human islet integrity. Cell Transplant. 2017;26(11):1733–1741. 116. Brandhorst  H, Brandhorst  D, Hesse  F, et  al. Successful human islet isolation utilizing recombinant collagenase. Diabetes. 2003;52(5):1143–1146. 117. Loganathan  G, Subhashree  V, Breite  AG, et  al. Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield. Am J Transplant. 2018;18(2):478–485.

B.  Islet allo-transplantation

546

43.  Collagenases in pancreatic islet isolation

118. Vargas F, Vives-Pi M, Somoza N, et al. Endotoxin contamination may be responsible for the unexplained failure of human pancreatic islet transplantation. Transplantation. 1998;65(5):722–727. 119. Jahr  H, Pfeiffer  G, Hering  B, Federlin  K, Bretzel  R. Endotoxinmediated activation of cytokine production in human PBMCs by collagenase and Ficoll. J Mol Med. 1999;77(1):118–120. 120. Geisler  F, Algül  H, Riemann  M, Schmid  RM. Questioning current concepts in acute pancreatitis: endotoxin contamination of porcine pancreatic elastase is responsible for experimental pancreatitis-associated distant organ failure. J Immunol. 2005;174(10):6431–6439. 121. Paraskevas  S, Aikin  R, Maysinger  D, et  al. Activation and expression of ERK, JNK, and p38 MAP-kinases in isolated islets of Langerhans: implications for cultured islet survival. FEBS Lett. 1999;455(3):203–208. 122. Vives-Pi M, Somoza N, Fernandez-Alvarez J, et al. Evidence of expression of endotoxin receptors CD14, toll-like receptors TLR4 and TLR2 and associated molecule MD-2 and of sensitivity to endotoxin (LPS) in islet beta cells. Clin Exp Immunol. 2003;133(2):208–218. 123. Park  SG, Kim  JH, Oh  JH, et  al. Polymyxin B, scavenger of endotoxin, enhances isolation yield and in vivo function of islets. Transpl Int. 2010;23(3):325–332. 124. Schwarz  H, Schmittner  M, Duschl  A, Horejs-Hoeck  J. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells. PLoS One. 2014;9(12):e113840. 125. Balamurugan  A, He  J, Guo  F, et  al. Harmful delayed effects of exogenous isolation enzymes on isolated human islets: relevance to clinical transplantation. Am J Transplant. 2005;5(11):2671–2681. 126. Cross  SE, Hughes  SJ, Partridge  CJ, Clark  A, Gray  DW, Johnson  PR. Collagenase penetrates human pancreatic islets following standard intraductal administration. Transplantation. 2008;86(7):907–911. 127. Cross SE, Hughes SJ, Clark A, Gray DW, Johnson PR. Collagenase does not persist in human islets following isolation. Los Angeles, CA: SAGE Publications Sage CA; 2012. 128. Málaga-Trillo E, Salta E, Figueras A, Panagiotidis C, Sklaviadis T. Fish models in prion biology: underwater issues. Biochim Biophys Acta (BBA)-Mol Basis Dis. 2011;1812(3):402–414. 129. Parnaud  G, Lavallard  V, Meier  R, Bedat  B, Bosco  D, Berney  T. Human Islet Isolation Using Animal-Free Collagenase and Neutral Protease. In: Paper presented at: Transplantation; 2013. 130. Brandhorst  D, Parnaud  G, Friberg  A, et  al. Multicenter assessment of animal-free collagenase AF-1 for human islet isolation. Cell Transplant. 2017;26(10):1688–1693. 131. Watanabe  T, Yaegashi  H, Koizumi  M, Toyota  T, Takahashi  T. The lobular architecture of the normal human pancreas: a computer-assisted three-dimensional reconstruction study. ­ Pancreas. 1997;15(1):48–52. 132. Shimoda M, Itoh T, Sugimoto K, et al. Improvement of collagenase distribution with the ductal preservation for human islet isolation. Islets. 2012;4(2):130–137.

133. Lakey JR, Warnock GL, Shapiro A, et al. Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. Cell Transplant. 1999;8(3):285–292. 134. Goto  M, Eich  TM, Felldin  M, et  al. Refinement of the automated method for human islet isolation and presentation of a closed system for in  vitro islet culture. Transplantation. 2004;78(9):1367–1375. 135. Matsumoto S, Noguchi H, Naziruddin B, et al. Improvement of pancreatic islet cell isolation for transplantation. In: Paper presented at: Baylor University Medical Center Proceedings; 2007. 136. Ohbayashi  N, Yamagata  N, Goto  M, Watanabe  K, Yamagata  Y, Murayama  K. Enhancement of the structural stability of fulllength clostridial collagenase by calcium ions. Appl Environ Microbiol. 2012;78(16):5839–5844. 137. Burgmann  H, Reckendorfer  H, Sperlich  M, Doleschel  W, Spieckermann P. The calcium chelating capacity of different protecting solutions. Transplantation. 1992;54(6):1106. 138. Upadhya  GA, Strasberg  SM. Glutathione, lactobionate, and histidine: cryptic inhibitors of matrix metalloproteinases contained in University of Wisconsin and histidine/tryptophan/ketoglutarate liver preservation solutions. Hepatology. 2000;31(5):1115–1122. 139. Contractor  HH, Johnson  PR, Chadwick  DR, Robertson  GS, London  NJ. The effect of UW solution and its components on the collagenase digestion of human and porcine pancreas. Cell Transplant. 1995;4(6):615–619. 140. Heald K, Jay T, Topham D, Downing R. The influence of collagenase solvent on the isolation of islets from 5 week old pigs: a comparison of TCM-199 and UW. J Mol Med. 1999;77(1):83–86. 141. Johnson  P, Button  C, Roberts  D, et  al. University of Wisconsin solution inhibits the class II collagenase within crude Clostridium histolyticum collagenase. In: Paper presented at: Transplantation proceedings; 1995. 142. Kin  T, Johnson  PR, Shapiro  AMJ, Lakey  JR. Factors influencing the collagenase digestion phase of human islet isolation. Transplantation. 2007;83(1):7–12. 143. Bateman  PA, Cross  SE, Gray  DW, Hughes  SJ, Johnson  PR. Islet yields from younger pancreas donors with lower BMIs are still sub-optimal using current enzyme methods. In: Paper presented at: Transplantation; 2013. 144. Miki A, Ricordi C, Messinger S, et al. Toward improving human islet isolation from younger donors: rescue purification is efficient for trapped islets. Cell Transplant. 2009;18(1):13–22. 145. Gopalakrishnan Loganathan  SV, Tucker  W, Narayanan  S, et  al. Identifying optimal enzyme blend of collagenase and protease for obtaining high islet yield from young donor pancreases. In: 16th IPITA, Oxford, UK; 2017. 146. de Castro Brás  LE, Toba  H, Baicu  CF, et  al. Age and SPARC change the extracellular matrix composition of the left ventricle. Biomed Res Int. 2014;2014:. 147. Miyazaki  Y, Murayama  K, Fathi  I, et  al. Strategy towards tailored donor tissue-specific pancreatic islet isolation. PLoS One. 2019;14(5):e0216136.

B.  Islet allo-transplantation