Cold-perfusion decellularization of whole-organ porcine pancreas supports human fetal pancreatic cell attachment and expression of endocrine and exocrine markers

Whole-organ porcine pancreas isolation and harvest

Following local ethical guidelines, pancreata (n = 3) were harvested from euthanized, heparinized (400 IU/kg, LEO Pharma, Denmark) young normal pigs (30–50 kg) used for other studies or surgical training. To further ensure good heparinization, cold phosphate-buffered saline (PBS, pH 7.4) with heparin (17 IU/mL) was administrated through portal vein until whole pancreas felt cold. Intact pancreata with all three lobes (connective, duodenal, and splenic lobe) were excised together with duodenal segment stretching from duodenal to connective lobe. Both the aorta and the portal vein were harvested along with each pancreas. During backbench work, one side of the aorta was cannulated, while all other vasculature outlets except the portal vein were ligated. Organs were flushed with 5 mM disodium ethylenediaminetetraacetic acid (EDTA, Alfa Aesar, Germany) and 0.02% (w/v) sodium azide (Sigma-Aldrich, USA) in cold (4°C) ultrapure water for 1 h at 1.2 L/h speed using peristaltic pump (Oina, Sweden) in series with degasser (Biotech, Sweden) to remove blood. Flushed pancreata were frozen at −20°C in PBS with 5 mM EDTA and 0.02% sodium azide until start of DC.

Cold-perfusion DC

The frozen pancreas was gradually thawed and connected to perfusion system (peristaltic pump and degasser connected in series) at 4°C and perfusion of 1.2 L/h at 4°C started. To further wash out blood remnants, the organ was perfused with distilled water containing 5 mM EDTA and 0.02% (w/v) sodium azide overnight. Briefly, cold-perfusion DC was achieved by consecutive flow-through perfusion of 4% (w/v) sodium deoxycholate (SDC, Sigma-Aldrich, Italy) combined with 6% (v/v) Triton X-100 (Alfa Aesar, Germany) and 0.4 mM PMSF (Roche, Germany) for 8 h followed by washing with distilled water for 96 h, 40 IU/mL deoxyribonuclease I (DNase I, Worthington, USA) in Dulbecco’s PBS with calcium chloride and magnesium chloride (Sigma-Aldrich, UK) for 4 h at 37°C, and finally, 120 h of wash with distilled water. All DC solutions contained 0.02% (w/v) sodium azide to prevent bacterial growth. Decellularized pancreata were kept in PBS with 0.02% (w/v) sodium azide, at 4°C for short time or at −20°C for long-time storage.

Culture of hFPSC

Cells isolated from pancreas of legally aborted human fetus as described by us earlier were expanded. ²⁰ The isolation protocol was approved by the local ethics committee. The cells were cultured in human placental collagen-coated culture flasks with pancreas differentiation medium (Dulbecco’s Modified Eagle Medium with high glucose (Lonza, Belgium), 1% l-glutamine (Gibco, UK), 1% antimycotic-antibiotic (Gibco, USA), 10% heat-inactivated human AB serum (Sigma-Aldrich, USA), 50 μM ethanolamine (Sigma-Aldrich, USA), 50 μM O-phosphoethanolamine (Sigma-Aldrich, USA), 0.1 nM liothyronine (Sigma-Aldrich, USA), 20 ng/mL recombinant human IGF-1 (PeproTech, USA), and 10 ng/mL human recombinant prolactin (Peprotech, USA)).

Recellularization of cold-perfusion decellularized porcine pancreas scaffolds

Approximately 1 cm² pieces of cold-perfusion decellularized porcine pancreas scaffolds were cut perpendicular to splenic lobe prolongation for preparation for RC. Pieces were sterilized in 0.1% peracetic acid (Sigma-Aldrich, Germany) in PBS for 70 min at 37°C and washed repeatedly in PBS with 1% antimycotic-antibiotic (Gibco, USA). Scaffolds were placed in 12-well Transwell^® permeable supports (0.4 μm pore size, Costar, USA), and 0.5 million hFPSC (at passage 6) were seeded onto each scaffold. Scaffolds were incubated at 37°C for 1 h after cell seeding followed by addition of pancreas differentiation media. Tissue pieces were harvested for characterization at day 5 (n = 3) and day 14 (n = 3).

Histology, immunohistochemistry, and immunofluorescence

Tissue biopsies (normal, DC and RC) were processed according to standard procedure. Biopsies were fixed in 4% phosphate-buffered formaldehyde (Histolab, Sweden) for 24 h and dehydrated with ascending concentrations of ethanol (Histolab, Sweden) and X-TRA-Solv (Medite, Germany) before embedding in paraffin (Histolab, Sweden). Samples were cut in 5 μm thickness cross sections using a microtome (Thermo Fisher Scientific, USA).

To analyze general histology of all biopsies, sections were stained with hematoxylin & eosin (H&E, Histolab, Sweden) and 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies, USA) according to standard procedures. H&E staining of sections from porcine pancreata (n = 2) decellularized with a protocol similar to the one used in this study, but at room temperature was also performed for comparison of ECM structure. Biopsies from normal and decellularized tissue were stained with Masson’s trichrome (MT, Polysciences, USA) and Verhoeff van Gieson (VVG, Polysciences, USA) according to manufacturer’s instructions.

Immunohistochemical analysis of sections from recellularized scaffold was done with primary antibodies specific to pancreatic α-amylase (1:1000, ab21156, Abcam, UK). Normal human pancreas (Abcam, UK) was included as reference. For identification of specific staining horseradish peroxidase–conjugated broad-spectrum secondary antibody (Invitrogen, USA) was used. Briefly, sections were rehydrated, antigen retrieved, endogenous peroxidases blocked, and protein blocked before incubating with primary antibodies overnight at 4°C. Slides were washed and incubated with secondary antibody for 10 min at room temperature. Colorimetric identification was performed using 3,3′-diaminobenzidine (DAB, Dako, USA) and counter-stained with hematoxylin before dehydration and then cover-slipped. Sections with only secondary antibody were included as negative control.

To further analyze function of cells in recellularized scaffolds, immunofluorescent stainings were performed with antibodies against C-peptide (1:500, ab8297, Abcam, UK), pancreatic and duodenal homeobox 1 (PDX1, 1:150, ab84987, Abcam, UK), glucagon (1:750, ab10988, Abcam, UK), and proliferating cellular nuclear antigen (PCNA, 1:1000, ab184660, Abcam, UK). Normal human pancreas (Abcam, UK) and human fetal kidney were included as reference. Alexa Fluoro 568 conjugated immunoglobulin G (IgG) goat anti-mouse secondary antibody (A11031, Thermo Fisher Scientific, USA) was used. Briefly, sections were rehydrated, antigen retrieved, and blocked with 5% goat serum before incubating with primary antibodies overnight at 4°C. Slides were washed and incubated with secondary antibody for 45 min at room temperature and then counter-stained with DAPI before cover-slipping. Sections with only secondary antibody were included as negative control. Unspecific background staining was reduced digitally in software (Leica, Germany).

DNA and ECM protein quantifications

For DNA quantification, biopsies were taken from splenic lobe of normal (n = 3) and decellularized (n = 3) pancreata and after 5 and 14 days of RC (n = 3, respectively). Normal biopsies were taken from newly euthanized pigs and biopsies were placed in liquid nitrogen immediately for storage until use. Before the start of DNA extraction, biopsies were placed on wipes to remove excess fluid and weighed. DNA was extracted using GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, USA) according to manufacturer’s instructions (RNase step included). Isolated double-stranded DNA was quantified using Qubit^® dsDNA HS Assay Kit (Thermo Fisher Scientific, USA).

ECM components were quantified in duplicate biopsies of splenic lobe from normal (n = 3) and decellularized (n = 3) pancreata. Collagen (soluble and insoluble), elastin, and glycosaminoglycans (GAGs) were quantified with Sircol, Fastin, and Blyscan assays (Biocolor, UK), respectively. All assays were performed according to manufacturer’s instructions.

Qualitative analysis of damaging effect from pancreatic extract

To further understand the damaging effect of pancreatic exocrine enzymes on cells and tissue, normal porcine pancreas tissue was homogenized in ice-cold PBS and then centrifuged at 2000 r/min at 4°C for 20 min. Supernatant was collected, re-centrifuged at 4000 r/min at 4°C for 20 min, and lyophilized. Porcine kidney isolate extracted with same procedure was included as a low-enzymatic activity control to exclude xenogeneic effects. The protein content in each powder was measured with standard Bradford method (Bio-Rad, USA). Isolates were resuspended in standard cell culture media (1 mg protein/mL media) and sterile filtered. Prepared cell culture media (with pancreas and kidney powder, n = 2, respectively) were added to cultures of human mesenchymal stem cells (MSCs) in 6-well format along with negative (only media) and positive (0.05% trypsin, Gibco, UK and 1% sodium dodecyl sulfate (SDS), Sigma-Aldrich, USA, respectively) controls. Photos were taken before addition and at 10 min 1 h and 4 h time points.

Statistics

All values and graphs represent group mean, while error bars represent the standard error of the mean. The graphs were plotted using GraphPad Prism 7.0. Welch’s t-test was used to analyze significant differences between groups after normality for each group had been confirmed with Shapiro–Wilk test. A p < 0.05 was considered significant.

Morphology after cold-perfusion DC

Intact decellularized porcine pancreas was obtained by perfusion of 4% SDC and 6% Triton X-100 at 4°C followed by DNase I treatment. A distinct color change could be observed as a result of DC. Washed normal porcine pancreas has a pinkish color (Figure 1(j)), whereas after DC, pancreas was totally white in appearance (Figure 1(k)). The color change of duodenum indicates an extensive vascular connection between pancreas and intestine. During DC process, a complete perfusion could be seen with aorta as inlet and portal vein as outlet.

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Figure 1.

Histological and morphological evaluation of cold-perfusion decellularization. Histological comparison of normal and cold-perfusion decellularized porcine pancreas by (a) and (b) H&E, (d) and (e) DAPI, (f) and (g) MT, and (h) and (i) VVG stainings showing complete nuclei and cytoplasm removal while preserving ECM and vasculature (arrows). With H&E stainings of (b) cold-perfusion and (c) room-temperature decellularized porcine pancreata, it could be concluded that cold-perfusion preserved ECM structure more. Small circular structures, believed to be cytoplasmic remnants, could be seen in pancreata decellularized in room temperature. Gross evaluation revealed a distinct change in appearance (j) before and (k) after decellularization. Graphic representation of duodenum, connective lobe, duodenal lobe, and splenic lobe can be seen.

Characterization of decellularized porcine pancreas scaffolds

H&E staining of cold-decellularized porcine pancreas (Figure 1(b)) showed absence of nuclei as compared to normal porcine pancreas (Figure 1(a)), where purple-stained nuclei were seen. H&E staining of porcine pancreas decellularized in cold environment also showed a more intact ECM structure as compared to H&E staining of porcine pancreas decellularized with a comparable protocol at room temperature (Figure 1(c)). Pancreas decellularized at room temperature had a more ruptured ECM structure and staining patterns reminiscent of cytoplasmic residues. DAPI staining of normal (Figure 1(d)) and DC porcine pancreas (Figure 1(e)) confirmed complete washout of intact nuclei. MT stainings (Figure 1(f) and (g)) showed absence of cytoplasm (red) after DC, while collagen fibers (blue) were retained. Retention of collagen fibers (pink) was confirmed with VVG stainings (Figure 1(h) and (i)). Histological stainings of DC tissue also confirmed preservation of vasculature structures (arrows, Figure 1(b) and (g)).

DNA quantification of biopsies from normal and DC tissues showed a significant (p = 0.0005) decrease in the double-stranded DNA with DC (Figure 2(a)). Double-stranded DNA decreased from 840 ± 18 to 1.67 ± 0.4 ng/mg with DC.

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Figure 2.

DNA and ECM quantification. Quantification of (a) double-stranded DNA (dsDNA) demonstrated a significant (p = 0.0005) decrease after decellularization compared to normal porcine pancreas and a significant (p = 0.0137) increase after 5 days of recellularization when compared to cold-perfusion decellularized tissue. Recellularized tissue harvested after 5 and 14 days showed significantly (p < 0.0001 and p = 0.0264) lower DNA content compared to normal porcine pancreas. Quantification of ECM proteins (b) collagen (soluble and insoluble combined), (c) elastin, and (d) GAGs showed no significant change of collagen, while both elastin and GAGs decreased significantly (p = 0.0021 and 0.0034, respectively).

ECM quantification demonstrated no significant change in collagen (soluble and insoluble combined) content (Figure 2(b)) after DC, while elastin (p = 0.0021, Figure 2(c)) and GAGs (p = 0.0034, Figure 2(d)) decreased significantly. The collagen, elastin, and GAGs contents in decellularized pancreas were 40.4 ± 5.8, 3.54 ± 0.42 and 0.96 ± 0.25 µg/mg, respectively, as compared to 31.5 ± 1.7, 21.2 ± 1.2 and 3.5 ± 0.11 µg/mg for normal porcine pancreas, respectively.

Harmful effect of pancreatic extract

Isolated porcine pancreatic extract showed a clear cytotoxic effect (Figure 3(a)) on two-dimensional (2D) culture of human MSCs, more comparable to the addition of trypsin (Figure 3(d)) than SDS (Figure 3(e)). The effect was acute already after 10 min and almost all cells detached from the cell culture plate. At 4 h, a visible difference in cell numbers could be noted between wells treated with pancreatic extract and wells treated with trypsin. More cells were seen in trypsin control. In controls treated with SDS, a complete cell disintegration could be seen already after 10 min. Similar isolate from porcine kidney tissue showed no or minimal effect (Figure 3(b)) on cultured cells, akin to negative control (Figure 3(c)).

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Figure 3.

Damaging effect of pancreatic isolate. Survival of mesenchymal stem cells after addition of pancreas isolate was studied qualitatively at 10 min, 1 h and 4 h after addition. Addition of (a) porcine pancreas isolate had an acute effect more comparable to (d) trypsin than (e) SDS treatment, where cell disintegration could be seen already after 10 min. With addition of (b) porcine kidney isolate, no effect could be seen even after 4 h. Also, for (c) negative control, no effect could be seen after 4 h.

Recellularization of cold-perfusion decellularized scaffolds with hFPSC

Expanded hFPSC (Figure 4(a)) were seeded onto sterilized pieces of cold-perfusion decellularized porcine pancreas scaffold (Figure 4(c)). Scaffolds were cultured in Transwell^® permeable supports (Figure 4(b)), and pieces were harvested at day 5 (Figure 4(d)) and day 14 (Figure 4(e)). Shrinkage in tissue volume could be seen with RC (compare Figure 4(c) with Figure 4(d) and (e)). H&E staining of recellularized scaffolds revealed presence of cells (purple nuclei in Figure 4(f) and (g)). For none of the pieces, complete cell coverage was seen. Instead, the coverage of cells varied within each piece; in some areas, cells were well-spread (Figure 4(f)), but in other areas, no cells could be seen. In some of the pieces, cells had aggregated together into cluster-like structures (arrow, Figure 4(g)). No significant difference regarding cell numbers and coverage could be seen between scaffolds harvested at day 5 and day 14.

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Figure 4.

Recellularization of acellular pancreas scaffolds with human fetal pancreatic stem cells. Human fetal pancreatic stem cells were (a) expanded and (b) seeded onto acellular pancreas scaffolds and cultured in permeable supports. A slight increase in the tissue density could be seen when comparing scaffolds (c) before and scaffolds harvested at (d) day 5 and (e) day 14. H&E stainings of harvested scaffolds showed areas where cells were (f) well-spread (purple nuclei) and other areas where no cells could be seen. In some of harvested scaffolds (g) cluster-like aggregations of cells could be seen.

Characterization of recellularized pancreatic scaffolds

Contents of double-stranded DNA in recellularized scaffolds harvested at day 5 and day 14 were 159 ± 32 and 278 ± 171 ng/mg, respectively (Figure 2(a)). A significant (p = 0.0137) increase in the double-stranded DNA could be seen when comparing acellular tissue and recellularized tissue harvested after 5 days, but not for 14 days of RC. However, as expected DNA contents of recellularized samples were lower than for normal porcine pancreas (p < 0.0001 and p = 0.0264, respectively).

Immunological stainings for pancreas function showed both endocrine and exocrine tissue properties. Immunofluorescence stainings revealed both C-peptide (red, Figure 5(b)), that is, insulin and glucagon (red, Figure 5(f)) secretions as well as transcription factor PDX1 expression (red, Figure 5(d)) in all recellularized scaffolds. Also exocrine function through α-amylase (brown, Figure 5(h)) secretion was seen in all recellularized scaffolds. No significant difference regarding endocrine or exocrine function between scaffolds harvested at day 5 or day 14 was seen. Expression of PCNA (red, Figure 5(j)) in nuclei was seen in fraction of cells, indicating proliferative capacity for these cells. No significant difference in PCNA expression could be seen between day 14 and day 5. Staining of normal human pancreas as positive control for pancreas markers C-peptide, PDX1, glucagon, and α-amylase (Figure 5(a), (c), (e), and (g), respectively) and human fetal kidney as positive control for PCNA (Figure 5(i)) confirmed specificity of primary antibodies. Negative controls (recellularized scaffolds without primary antibodies, Figure 5(k) and (l)) showed no or weak background staining.

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Figure 5.

Endocrine and exocrine markers expression in recellularized scaffolds. Immunostainings showed expression of (b) C-peptide (red), (d) PDX1 (red), (f) glucagon (red), and (h) α-amylase (brown) in recellularized scaffolds. Recellularized scaffolds also expressed (j) PCNA (red) in nuclei in fraction of cells. No significant difference between scaffolds harvested at day 5 and day 14 was seen. Positive control stainings could be seen for (a) C-peptide (red), (c) PDX1 (red), (e) glucagon (red), and (g) α-amylase (brown) in normal human pancreas and for (i) PCNA (red) in human fetal kidney. No or less background stainings were seen in (k) immunofluorescence and (l) immunohistochemistry stainings of recellularized scaffolds without primary antibodies.