Solubilized Pancreatic Extracellular Matrix from Juvenile Pigs Protects Isolated Human Islets from Hypoxia-Induced Damage: A Viable Option for Clinical Islet Transplantation

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Introduction
Te requirement to extract a subpopulation of heterogeneous cell clusters from an organ whilst preserving their native structure and organization is a unique challenge faced during pancreatic islet isolation compared with other types of tissue separation [1]. Tis requires a combination of enzymatic and mechanical techniques. During this process, the pancreatic matrix is digested, and the islets released from the dispersed acinar tissue before the two tissues are separated by density-gradient purifcation. A key aspect of islet extraction, therefore, is the enzymatic detachment or cleavage of the islet basement membrane from the surrounding pancreatic matrix [2]. As a consequence, the bidirectional communication between the integrins expressed on the islet cell surface and the basement membrane is lost [3][4][5][6]. Tis, in turn, interrupts the signaling of vital physiological functions from the proximal microenvironment to the islet cells sufering from increased rates of apoptosis and severe loss during islet culture [7,8]. Te absence of an extracellular matrix (ECM) [9,10], in combination with hypoxic conditions [11] and a shortage of essential nutrients [12], is the key factor resulting in 73% of islet recipients currently requiring two or more islet grafts to achieve improved glycaemia and elimination of life-threatening hypoglycemia unawareness [13].
Numerous attempts have been made to identify an ECM substitute for islet culture. Tese have mainly involved in the use of individual ECM proteins [14][15][16][17][18][19]. However, a small number of studies used combinations of diferent ECM proteins and found a synergistic efect on islet function and viability when compared with individual ECM proteins [20,21]. In contrast, our group observed no signifcant protection of isolated human islets when combining collagen type-IV (COL-4), Laminin-521 (L-521), and Nidogen-1 (NID-1) compared with using individual basement membrane proteins. In fact, we detected that a concentration-related detrimental impact on islet integrity was noted after combining ECM proteins [22]. Llacua et al. also had similar fndings of harmful efects of overdosed ECM proteins in their study [23]. Te importance of identifying the most suitable ratios and concentrations of ECM proteins was emphasised in another study where COL-4, L-521, and NID-1 had been combined [24]. As demonstrated by analyzing the pancreatic and islet matrisome, the pancreatic ECM is enormously complex [25,26], and the data from all these studies underline the challenges of identifying the optimal combination of ECM proteins for promoting survival and function of isolated islets before and after transplantation. To achieve this, consideration of the correct composition, structure, and stoichiometric ratios of the pancreatic ECM components is required [27].
As a consequence of these requirements, the current gold standard of scafolds for tissue-engineering that mimic the natural environment of cells is decellularized matrices [28]. Although human pancreatic tissue is deemed as ideal ECM source for protection of isolated human islets, the shortage of young healthy human organ donors is a viable argument to select other species as tissue donors, such as the pig, particularly when the production of hydrogel-related products is intended [29][30][31].
Te aim of this study therefore was to compare the efciency of a preassessed and efective combination of human COL-4, L-521, and NID-1 [22] to solubilized ECM manufactured from decellularized porcine pancreases, which represents the most relevant preclinical model and that is already in use for numerous FDA-approved clinical applications [28,32].

Materials and Methods
An overview of the experimental study design is shown in Figure 1.

Manufacturing of Solubilized ECM from Porcine
Pancreases. Te manufacture of solubilized ECM from decellularized porcine pancreases (ppECM) was performed as previously described in detail [33]. Briefy, the pancreases were retrieved from 6 months old slaughterhouse pigs, trimmed, and dissected prior to alternating treatment with hyper-and hypotonic solution prepared as 1.1 and 0.7% (w/ v) of sodium chloride dissolved in double-distilled water. Afterwards, the tissue was incubated at 37°C in 0.05% (w/v) of trypsin supplemented with 0.02% (w/v) of EDTA (Sigma-Aldrich, Rehovot, Israel) prior to a treatment with 1% (v/v) of Triton-X-100 (Sigma-Aldrich) and ammonium hydroxide in phosphate-bufered saline (PBS). Acellularity of the treated tissue was confrmed by hematoxylin and eosin staining of 10 μm-tissue sections. After overnight lyophilization, 500 mg aliquots of ground extracellular matrix (ECM) were immersed in 20 mL of 0.1 M HCl supplemented with 100 mg of pepsin (Sigma-Aldrich) and stirred for 48 hours at room temperature. After solubilization had been completed, pepsin was inactivated by adjusting the pH to 7.4. Finally, the material was aliquoted (500 mg/mL) and stored at −20°C.

Islet Isolation and Culture.
Eight human donor pancreases within a range 40 to 59 years were voluntarily donated with written consent according to the Declaration of Istanbul. Ethical approval for using isolated human islets for research was given by the NHS National Research Ethics Service (09/H0605/2). After a cold ischaemia time of 6.1 ± 1.6 hours (mean ± standard deviation [SD]), islets were isolated and purifed as previously described [2]. After isolation, islets were cultured for 4-5 days in hypoxic atmosphere at 2% oxygen and 5% CO 2 . Aliquots of approximately 1000 (range 1300−700) islet equivalents (IEQ) were incubated per well in 12-well plates (Greiner Bio-One, Stonehouse, U.K.) and suspended in 800 μL/well of CMRL 1066 supplemented with 20 mmol/L HEPES, 2 mmol/L Lglutamine, 200 units/mL penicillin, 200 μg/mL streptomycin (all reagents from Life Technologies, Paisley, U.K.) and 2% fetal calf serum (PAA Laboratories, Pasching, Austria). In one of the treatment groups, ppECM, manufactured as described above, was added in a concentration of 180 μg/mL in order to prevent the solidifcation of the ppECM into a hydrogel [34]. Tis experimental group was compared with islets cultured in the presence of a mixture of human ECM proteins (hEPM) assessed in a previous study [22] and composed of 80 μg/mL COL-4 (Sigma-Aldrich, Dorset, U.K.), 10 μg/mL L-521 (Biolamina, Uppsala, Sweden), and 10 μg/mL NID-1 (R&D Systems, Abingdon, United Kingdom). Sham-treated islets, cultured under identical conditions in supplemented CMRL 1066 without the addition of any ECM-components, served as controls. All experimental groups were performed as free-foating culture.

Islet Characterization.
After the culture in hypoxic atmosphere, islet-preconditioned supernatants were collected and assessed in duplicate for production of hypoxia-and infammation-related chemokines. Release of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor A (VEGF-A) was detected utilising enzyme immunoassays specifc for human chemokines (Invitrogen/Termo Fisher, Rochford, U.K.). After retrieval of the supernatants islets of a well were collected in a sample volume of 1 mL and assigned to a certain quality assessment parameter measured for the corresponding treatment group. All samples were measured in duplicate except the counting samples which were assessed in triplicate. Islets for in vitro function were collected from the counting samples.
Before and after islet culture, islet number was quantifed and expressed as islet particle number (IN) and converted to islet equivalents (IEQ) as previously described in detail [35]. Islet yield, expressed as a percentage, was calculated by normalizing IEQ to preculture yield of IEQ. Islet morphological integrity was determined as a fragmentation index by calculating the ratio of IN over IEQ. Islet viability was assessed using 0.67 μmol/L fuorescein diacetate (FDA, Sigma-Aldrich) and 4.0 μmol/L propidium iodide (PI, Sigma-Aldrich) for staining of viable and dead cells, respectively [36]. Islet overall survival was used to calculate the recovery of living cells only. For this variable, normalized postculture islet yield was multiplied by the proportion of viable cells. Apoptosis in hypoxic islets was determined by simultaneous staining with Annexin-V FITC (Becton-Dickinson Biosciences, Oxford, U.K.) and PI used at a concentration of 450 ng/mL and 4.0 μmol/L, respectively. Intraislet generation of reactive oxygen species (ROS) was determined in duplicate by measuring the intracellular conversion of dichlorofuorescein diacetate (DCFH-DA) into fuorescent dichlorodihydrofuorescein (DCFH) as previously described in detail [37].
In vitro function of 20 hand-picked islets of similar size (150-200 μm) was assessed in duplicate during static glucose incubation as previously described in detail [38]. Islets were seeded on 8 μm-pore size flter inserts, transferred into 24-well plates, and sequentially incubated for 45 min in 1 mL Krebs-Ringer bufer supplemented with 2.0 mmol/L glucose followed by 45 min at 20 mmol/L followed by a second period of 45 min at 2 mmol/L glucose. Afterwards, islets were recovered and sonicated in distilled water for disintegration prior to subsequent determination of DNA content measured by the Pico Green assay (Life Technologies). To minimize sample contamination with DNA released by dead and fragmenting cells, every sample was washed twice to carefully remove the supernatant with cell debris and released DNA. An aliquot of disintegrated islet cell suspension was mixed with acid ethanol at a ratio of 1 : 4 followed by overnight insulin extraction at 4°C. Prior to performance of the enzyme immunoassay samples were diluted and neutralized by Krebs-Ringer-bufer. Intracellular and secreted insulin was determined utilizing an enzyme immunoassay for human insulin (Mercodia, Uppsala, Sweden). Chemokine as well as insulin release was normalized to ng of DNA. Furthermore, insulin secretion was expressed as percentage of intracellular insulin content [39]. Te glucose stimulation index (-----) was calculated by dividing the insulin release at 20 mmol/L glucose by the mean of the two basal periods. In order to evaluate the total secretory potency of an entire treatment group, the postculture recovery of IEQ was multiplied by the glucose stimulation index and expressed as proportion of sham-treated islets.
Te fuorescence intensity (FI) of FDA, PI, Annexin-V, and DCFH was quantifed in duplicate by means of a fuorometric plate reader as previously described [40]. As performed for the cytokine and insulin release and early apoptosis, ROS production was normalized to islet DNA content as well.

Results
Te major characteristics of human islet morphological integrity after culture in hypoxic atmosphere of 2% oxygen are shown in Figure 2(a)-2(c). Nearly 60% of the shamtreated islets were lost during 4-5 days of hypoxic culture (p < 0.001 vs. preculture) (Figure 2(a)). Nevertheless, when the islets were cultured in the presence of a mixture of human COL-4, L-521, and NID-1 (hEPM), the recovery was signifcantly increased (p < 0.05 vs. sham-treated;p < 0.01 vs. preculture). Islet recovery could be even further improved by adding ppECM to the culture medium (p < 0.001 vs. shamtreated; NS vs. preculture) (Figure 2(a)). Loss of islets during hypoxic culture was closely associated with an increase of the preculture fragmentation index of initially 0.44 ± 0.17 (p < 0.01 vs. sham-treated;p < 0.05 vs. hEPM) which was lowest in the ppECM group (NS vs. preculture) (Figure 2(b)). Another indicator of islet fragmentation is islet purity refecting the accumulation of cell fragments and tissue debris during hypoxic culture. As demonstrated in Table 1, islet purity was found to be signifcantly higher when culture media were supplemented with hEPM or ppECM (NS vs. preculture; p < 0.01 vs. sham-treated). Viability of sham-treated islets as refected by FDA-PI staining was reduced during hypoxia by approximately 20% in average when normalized to the preculture islet viability of 66.0 ± 8.9% (p < 0.01 vs. sham-treated) (Figure 2(c)). In contrast, the presence of hEPM minimized the loss of viability in hypoxic islets (NS vs. preculture; p < 0.05 vs. shamtreated). Moreover, the addition of ppECM completely preserved the initial preculture viability during 4-5 days of hypoxia (NS vs. preculture; p < 0.001 vs. sham-treated). When simultaneously considering recovery and viability by calculating islet overall survival, it became obvious that this variable was signifcantly increased by adding hEPM (p < 0.05 vs. sham-treated) ( Table 1). Furthermore, using ppECM nearly doubled the overall survival compared with sham-treatment (p < 0.001) and was also signifcantly more efcient in comparison with hEPM (p < 0.05).
As expected, the lack of oxygen during culture triggered a proinfammatory state in hypoxic islet cells as refected by the production of 6 diferent chemokines. As shown in Table 2, sham-treated islets released the signifcantly highest levels of chemokines (p < 0.01 vs. hEPM; p < 0.05-p < 0.01 vs.ppECM). Despite the diferences between the amounts of individual chemokines that were produced, the accumulated release of the diferent chemokines followed a very similar pattern. In general, the chemokine production in the treatment groups was at least 50% lower compared with sham-treated islets. Te close interrelationship between the individual chemokines is also refected by the high correlations that were found between TNF-α, the central and dominant component of the chemokine network, and IL-1β, IL-6, MCP-1, and VEGF-A as shown in Figure 3. Te calculated correlation coefcient was always r ≥ 0.80. Tis applies also to IL-8 which is not included in Figure 3 (r � 0.80; p < 0.001).
A tight correlation was also detected between TNF-α and ROS (r � 0.90, p < 0.001) which serve as signaling molecules for TNF-α activity [41]. As demonstrated in Table 1, the intraislet generation of ROS in sham-treated islets increased almost threefold during hypoxic culture when compared with the preculture level of ROS production (21.4 ± 15.8 FI/ ng DNA, p < 0.001 vs. sham-treated). Te ROS production in hEPM-treated islets reached approximately only half of that measured in sham-treated islets (NS vs. preculture; p < 0.01 vs. sham-treated). However, when hypoxic islets were cultured in the presence of ppECM, the level of ROS generation could be stabilized on the preculture level (NS vs. preculture; p < 0.01 vs. sham-treated).
Although the diferences between the experimental groups in terms of islet viability were signifcant, the discrepancy between sham-treated islets and the treatment groups was considerably more distinct with respect to apoptosis (Table 1). Remarkably, the extent of apoptosis in islets cultured in the presence of ppECM was signifcantly lower when compared with the preculture level of apoptosis (19.5 ± 13.0 FI/ng DNA, NS vs. hEPM; p < 0.05 vs. shamtreated, vs. ppECM).
In order to estimate the infuence of ROS on diferent variables of islet cell death, matrix correlation analysis was performed. As shown in Figure 4, the analysis revealed that islet ROS production had a strong inverse efect on postculture islet yield (r � −0.73, p < 0.001) but did surprisingly not show any correlation with islet viability (r � 0.11, NS). A signifcant correlation was found between ROS and apoptosis (r � 0.72, p < 0.001). A similar observation was made for the production of TNF-α that correlated with apoptosis (r � 0.60, p < 0.01) and postculture islet yield (r � −0.59, p < 0.001) but no correlation was seen with islet viability (r � −0.01, NS). Other proinfammatory cytokines, such as IL-1β, IL-6, IL-8, and MCP-1, followed the same pattern as TNF-α regarding their correlation with islet yield, apoptosis, and islet viability (data not shown). Even VEGF, a representative of islet-protective chemokines, showed a similar correlation with islet yield (r � −0.59, p < 0.01), apoptosis (r � 0.62, p < 0.01), and islet viability (r � 0.15, NS).
As seen in several previous studies, the lack of oxygen during 4-5 days of culture had a strong inhibitory efect on glucose-stimulated insulin release when islets were cultured without the presence of ECM-components. As demonstrated in Figure 5, sham-treated islets had a signifcantly higher basal insulin release than islets exposed to hEPM (p < 0.05) or ppECM (p < 0.01). In addition, sham-treated islets failed to up-regulate insulin release after glucose challenge and also failed to down-regulate insulin release after switching from high to low glucose concentration (p < 0.01 vs. ppECM). As a consequence, the glucose stimulation index dropped below the clinically relevant threshold of 1.0 [42] in contrast to islets treated with hEPM (p < 0.05 vs. sham-treated) or ppECM (p < 0.001 vs. shamtreated) ( Table 1). In contrast to insulin release, intracellular insulin content was not signifcantly altered by treatment with hEPM or ppECM ( Figure 5). When estimating the secretory potency of the experimental groups, a parameter that considers not only the stimulatory capacity but also the postculture recovery of islets, it became clear that islet treatment with hEPM (p < 0.05) or ppECM (p < 0.001) roughly triplicated the total secretory potency in comparison with sham-treated islets (Table 1).

Discussion
To the best of our knowledge, this is the frst study that compares the protective potency of solubilized ECM from decellularized porcine pancreas (ppECM) on isolated human islet to a mixture of human COL-4, L-521, and NID-1 (hEPM). Under certain biochemical conditions, these ECM proteins assemble to form suprastructures [43,44] that represent the most relevant constructs in the native human islet basement membrane [45]. In comparison with shamtreated islets, the mixture of human ECM proteins (hEPM) Journal of Tissue Engineering and Regenerative Medicine  signifcantly protected the functional and structural integrity of human islets exposed to hypoxia with respect to yield, viability, and insulin secretory capacity. Nevertheless, islet protection was further increased by culturing hypoxic islets in the presence of ppECM. Te extent of protection clearly correlates with the complexity of the materials. Te mixture of COL-4, L-521, and NID-1 may potentially serve as a substitute for the enzyme-cleaved islet basement membrane, provided that the ideal ratio between the individual components can be identifed [21,22,46]. In contrast, the physiological function of the ECM can be attributed to its hierarchical and orchestrated organization rather than to its individual components [45]. It is, therefore, very unlikely that the abundance of functions can be fully replaced by individual ECM components or limited constructs [34]. Te same applies to synthetic compounds, despite the fact that their characteristics and properties can be tightly controlled [47][48][49]. In contrast, decellularized ECM provides a nearly endless variety of signals that are required to maintain tissue and cell integrity, growth as well as diferentiation thus representing the current gold standard of scafolds for tissue engineering that mimic the natural environment of cells [27].
Tough the ideal source for such an ECM supplement for human islet protection postisolation would be a human source, the low availability of young healthy human pancreas donors is a substantial argument to consider other species as tissue donors. Porcine ECM from diferent organs has been shown to be a safe choice for clinical application, conserving its original bioactivity through the diferent species while evading an immune reaction. As such, the application of porcine ECM in diverse biomedical devices has been extensively investigated, including several FDA-approved clinical applications [28,32].
Te composition of decellularized ppECM was addressed in our previous work [50], revealing that the ppECM is a complex mixture, comprised of 84% collagen. Particularly, type I and type III collagen are the most abundant, accounting for 98% of the ppECM proteins, while other types of collagen, including II, V, VI, and IV are present in lower quantities. Proteins such as fbronectin, laminin, and others are also present in very low percentages but, apparently, highly contribute to the molecular structure and the biological activity of the material, thus equipping it with pancreas-specifc attributes. In accordance, a quantitative histological comparison with other species revealed a lower periislet expression of collagen-IV, Laminin, and Fibronectin in the pig than observed in human pancreases [51]. Nevertheless, compared with the hEPM treatment in the present work (human collagen-IV, Laminin-521, and Nidogen-1), only collagen type IV (COL-4) is present in both treatments in diferent concentrations.
Te interspecies biocompatibility of the solubilized ppECM, used in our study, can be demonstrated by the protection of human islet morphological integrity in a hypoxic environment and by a substantially decreased production of chemokines and ROS when compared with sham-treated islets. Te reduction of proinfammatory mechanisms has also implications for the functional integrity of isolated islets particularly when islets had been exposed to a hypoxic environment. Tis not only concerns islet in vitro function but may also be of importance for islet posttransplant function. As discussed above, the enormous  variety of prosurvival signals provided by the whole ECM may be one reason that diferent stages of cell death could be prevented or ameliorated by ppECM. Apart from its antiinfammatory efect, we found that ppECM minimized apoptosis to a level that was signifcantly lower compared with preculture values. Interestingly, ROS, as well as TNF-α, signifcantly correlated with apoptosis and islet yield but did not show any correlation with islet viability. Tis is consistent with the fndings of previous studies showing that ROS are serving as signaling molecules for TNF-α [41,52]. It has been demonstrated in isolated human islets that cytokines can induce apoptosis but are not the primary triggers of cell necrosis [53,54]. Remarkably, VEGF, generally categorized as a survival factor for human islets in a detrimental environment [55], followed the same pattern as TNF-α by correlating positively with apoptosis and inversely with islet yield. Tis can be explained by the decisive role that is played by TNF-α in the cytokine network controlling the release of IL-8 and VEGF amongst other chemokines [56].
However, when considering islet overall survival the utilization of ppECM was signifcantly more efective than the triple mixture of human ECM proteins indicating that the manufacturing process of ppECM resulted in a product that provides a human islet-compatible environment. Te efective application of ppECM may be partially attributed to its use as a media supplement, rather than coating culture plastic containers with this material. It was previously shown, that the adsorption of ECM structures to synthetic surfaces can signifcantly alter conformation and bioactivity of adsorbed ECM [57].
Nevertheless, the fnal proof of efciency by presenting data from transplant experiments was not provided which clearly is a limitation of our study. In vivo studies would also reveal how long the protective efect of human islet pretreatment with ECM proteins may last after implantation. So far, we can only speculate whether the positive efect on human islets' functional and morphological integrity may improve survival at least during the early phase of islet engraftment.

Conclusion
Tis study shows that solubilized ECM produced from decellularized porcine pancreases is highly efcient for protecting isolated human islets from hypoxically induced cell damage. Te interspecies biocompatibility of ppECM is demonstrated by its inhibitory efect on proinfammatory and proapoptotic mechanisms in hypoxic human islets, which exceeds the protective potency of a mixture of three human human islet basement proteins suggesting ppECM as viable alternative to limited combinations of human ECM proteins. Nevertheless, transplant experiments have to be undertaken in the future to provide the fnal proof of ppECM efciency.

Data Availability
Te data used to support the study are available from the corresponding author upon reasonable request.

Ethical Approval
Human islet isolation and utilization of isolated islets for research purposes have been approved by the NHS National Research Ethics Service (09/H0605/2).

Consent
Pancreases from human multiorgan donors were retrieved and processed after informed consent was given by the relatives of the donor.

Disclosure
Te views expressed are those of the authors and not necessarily those of the NHS, NIHR, or the Department of Health.

Conflicts of Interest
Te authors declare that there are no conficts of interest.