The study was carried out in compliance with the ARRIVE guidelines.
Animals and ethical concerns
The data were the collective results gathered from eight neonate pigs. Mini pigs aged 2 to 12 days were obtained from the INRAE PEGASE unit (Rennes, France). Experiments using pigs were approved by the Pays de la Loire Ethic Committee (Approval 01074.01/02) and were carried out in accordance with the relevant French (2013-118) and European regulations (2010/63 EU Directive). All efforts were made to minimize animal suffering and to restrict the number of experimental animals. Analgesia and anesthesia were provided by IM injection of methadone, midazolam and ketamine, and maintained with 2% isofluran. Piglets were subjected to laparotomy, and the pancreas was removed after exsanguination via the aorta causing euthanasia.
Pancreatic islet isolation
NPIs were isolated from mini-pig pancreas as described previously57. Briefly, the pancreas was cut into 1 to 2 mm3 small pieces and digested with 25 mg/mL collagenase type V-S (Sigma-Aldrich, Saint-Louis, MO, USA) with gentle agitation for 14 to 16 min at 37 °C. The digest was filtered through a 500 µm pore size filter, washed in HBSS buffer (Biowest, ref L0612) supplemented with 0.5% (w/w) bovine serum albumin (BSA, Sigma-Aldrich), and then cultured in Ham’s F10 (Dutscher, Brumath, France) supplemented with 10 mM glucose, 50 mM IBMX, 2 mM l-glutamine, 10 mM nicotinamide, 100 IU/mL penicillin, and 100 mg/mL streptomycin. NPIs were cultured for at least 24 h in a normoxic condition (37 °C, 20% O2, 5% CO2) before encapsulation into alginate sheets.
Pseudo-islet formation
The mouse MIN6 beta cell line was kindly provided by Pr. Jun-ichi Miyazaki (Osaka University Medical School, Japan)58. MIN6 cells were expanded in DMEM medium (Dutscher, Brumath, France) supplemented with 10% heat-inactivated calf serum (Invitrogen, Villebon-sur-Yvette, France), 1% penicillin/streptomycin/neomycin, and 50 µM 2-mercaptoethanol. To generate MIN6 pseudo-islets (MPIs), 106 MIN6 cells/mL were cultured in non-treated culture petri dishes for 3 days at 37 °C in normoxic conditions.
Alginate encapsulation
Clinical grade low viscosity and high guluronate sodium alginate 2.2% (w/v) (PRONOVA UP LVG, Novamatrix, Sandvika, UK), later called “hydrogel” was used for islet (NPIs and MPIs) encapsulation. Alginate was solubilized in 0.9% NaCl (w/v) by gentle stirring overnight at 4 °C and sterilized using 0.2 µm filtration. NPIs and MPIs were quantified using canonical standardized Islets Equivalent Quantities (IEQ)59. For encapsulation in macrobeads, islets were gently mixed in the hydrogel at 2500 IEQ/mL alginate. Macrobeads 3 mm in diameter were obtained by alginate extrusion through a 23 G needle using a syringe driver into a 100 mM CaCl2 gelation bath for 5 min. Alginate sheets (680 µm thickness, 1.2 cm diameter) containing varying islet concentrations (2000 to 46,667 IEQ/mL alginate) were prepared in 48-well plate (ref 150787, Thermofisher) lids by pouring 75 µL of the alginate suspension on the well surface on the lid. Crosslinking of flat alginate sheets was achieved by covering the lid wells with 0.22 µm filters (Merck Millipore, Burlington, MA, USA) associated with sintered glass filters (DWK Life Sciences, Wertheim, Germany) previously soaked in a 100 mM CaCl2 solution. A volume of 5 mL of CaCl2 solution was added to the top of the glass filter before a 7 min incubation at room temperature. After crosslinking, alginate beads or sheets were washed twice in 0.9% NaCl and then in NPI or MPI culture medium.
Encapsulated islet culture
Eight macrobeads were placed in each P48-well plate well containing 500 µL of culture medium. Alginate sheets were cultured in 12-well plate inserts in 3 mL of culture medium (two sheets per insert) or in 24-well plates in 1.5 mL of medium (one sheet per well). Encapsulated islets were incubated either in a normoxic O2 tension environment (20% O2, 5% CO2, 37 °C) or in a hypoxia chamber (STEMCELL Technologies, Grenoble, France) filled with 1% O2 and 5% CO2 in N2 (37 °C) by purging the chamber at a rate of 20 L/min for 5 min as recommended by the supplier. Culture media and hypoxic atmosphere were renewed every 2 to 3 days of culture.
Pseudo-islet viability assessment
Viable cell content in encapsulated islets was determined by intracellular ATP quantification (RLU, relative light unit) using the CellTiter-Glo® 3D Cell Viability kit (Ref 69682, Promega, Charbonnieres-les-Bains, France) following the manufacturer’s recommendations. The ATP content standard curve for MPIs was obtained from pseudo-islets immediately after encapsulation in alginate patches at several densities. A linear correlation was observed between the luminescent signal of the CellTiter-Glo® 3D Cell Assay and the fluorescent Cyquant DNA Assay (Ref C7026, Waltham, MA USA) regardless of the culture conditions. Cell death within the encapsulated islets was evaluated by quantifying the lactate dehydrogenase activity (Ref 11644793001, LDH, absorbance unit (AU), Roche, Meylan, France) in culture supernatants according to the manufacturer’s recommendations. ATP luminescence and LDH absorbance were evaluated on a FLUOstar OPTIMA luminometer (BMG Labtech, Champigny-sur-Marne, France).
Insulin secretion assay
The capacity of encapsulated islets to secrete insulin following metabolic stimulation was evaluated by 30 min sequential incubations of alginate encapsulated islets in basal medium (RPMI [Ref 10043CV, PAA, Velizy-Villacoublay, Franc] containing 2 mM l-glutamine, 0.5% BSA, and 2.8 mM glucose), stimulation medium [basal medium supplemented with 20 mM glucose and 10 mM theophylline (Ref T1633, Sigma-Aldrish)], and basal medium. Secreted insulin concentrations were assessed in culture supernatants by ELISA (Ref 10124701 porcine insulin ELISA, Ref mouse insulin ELISA 10120001, Mercodia, Uppsala, Sweden,). Insulin secretion stimulation indexes were calculated as the ratio of the glucose + theophylline-stimulated insulin secretion level over the basal level of the encapsulated islets. Theophylline was used to potentiate insulin secretion as NPIs are immature islets containing insulin precursor cells, whose spontaneous secretion is notoriously low57.
Intracellular insulin content
NPIs were recovered from the alginate sheet by incubation for 20 min at 37 °C in a decapsulating solution by calcium chelation in 5 mM citrate and 1 mM EDTA in PBS followed by mechanical dissociation and centrifugation. Proteins from naked or decapsulated NPIs were extracted by repeated pipetting and incubation steps at − 20 °C in 50 µL of an ethanol-HCl solution. Protein extracts were neutralized by adding 25 µL of Tris–HCl 1 M (pH = 7.5). Pig intracellular insulin was assayed in protein extracts by ELISA Absorbance was evaluated using a FLUOstar OPTIMA luminometer.
Vascular endothelial growth factor (VEGF) quantification
VEGF secretion was assayed by ELISA in islet culture supernatants (Clinisciences, Nanterre, France).
Transcriptomic analysis
Islets were recovered from alginate sheets as previously described and frozen at − 80 °C in NucleoZOL reagent (Macherey–Nagel, Düren, Germany). Total RNA was isolated according to the manufacturer’s instructions and reverse transcribed using MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Pig primer sequences as described earlier24,60 were purchased from Eurogentec (Angers, France). Validated TaqMan Gene Expression Assays (Thermofisher) were used for targets listed in Table 7. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed on a CFX 96 Touch instrument (Bio-Rad, Hercules, CA, USA) using Hot FirePol qPCR reagents (Solis BioDyne, Tartu, Estonia). No template or samples processed without reverse transcriptase were included as negative controls. For each sample, the relative quantity was inferred from a standard curve created through the amplification of serial dilutions of a pool of representative samples. Whenever necessary, the target gene expression in the standard pool was artificially increased by spiking 5 µL of amplification product sequences diluted 1:1250. The expression of porcine genes encoding RPL19 (ribosomal protein L19) and PPIA (peptidylprolyl isomerase A) were used to normalize the expression of porcine PDX1 (pancreatic and duodenal homeobox 1), HO1 (heme oxygenase 1), NKX6-1 (NK6 homeobox I), INS (Insulin) and GCG (glucagon).
Immunohistological analyses
Slide preparation
NPIs were recovered from alginate sheets as previously described, centrifuged, and fixed in paraformaldehyde (PFA; 4% (v/v)) for 30 min before being embedded in HistoGel™ (Thermo Fisher Scientific, Waltham, MA, USA). Morphology of formalin-fixed paraffin-embedded (FFPE) NPIs in cross-sections (3 µm) were analyzed using Hematoxylin–Eosin–Saffron stain.
Insulin and glucagon immunostaining
FFPE cross-sections of NPIs (3 µm) were incubated overnight with rabbit anti-insulin at 1:400 dilution (C27C9; Cell Signaling Technology, Beverly, MA, USA) and mouse anti-glucagon at 1:500 dilution (G2654; Sigma-Aldrich) IgGs, followed by 1 h with secondary Alexa-Fluor 488 donkey anti-rabbit IgG at 1:2000 dilution and Alexa-Fluor 555 donkey anti-mouse IgG at 1:1000 dilution as previously described54.
PDX-1 immunostaining
Slides were incubated overnight with horseradish peroxidase-conjugated anti-PDX-1 antibody at 1:500 dilution (219207-Abcam, Cambridge, UK). PDX-1 was visualized using EnVision + System-HRP, rabbit (DAB+) (K4011; Agilent, Santa Clara, CA, USA) and counterstained with hematoxylin. Neonate-pig-pancreatic sections were used as positive controls. Analyses without primary antibodies were performed as a negative control.
Insulin and glucagon stained area quantification
Images were acquired using an AxioVert microscope and Zen lite software (Carl Zeiss, Jena, Germany). Photos of representative fields of the slices were taken under both white light and fluorescence using the same exposure time for all images taken with the same staining. The percentage of insulin and glucagon staining per NPI area was quantified using ImageJ software (NIH, Bethesda, MD, USA).
Pseudo-islet O2 consumption rate
The O2 consumption rate (OCR) by MPIs was measured in bioreactor experiments. OCR was determined on the first day of culture by placing 1000 IEQ MPIs in a P48 well (non-treated culture dish) in 500 µL of culture medium. Dissolved O2 concentration was assessed to the medium with a Clark electrode (InPro 6850i, Mettler-Toledo, Viroflay, France) inserted in the P48 plate wells and the Rhapsody software (Pierre Guerin Technologies, Niort, France). MPI OCR (pmol/min.IEQ) was calculated as the initial slope of the dissolved O2 concentration curve in the medium. A negative control without islets was performed in parallel.
O2 supply strategies
The O2 carrier HEMOXCell hemoglobin (Hemarina, Morlaix, France) was mixed with the pseudo-islets in the alginate before crosslinking into macrobeads or sheets. The O2-generating biomaterial was prepared by mixing calcium peroxide (Sigma-Aldrich) in polydimethylsiloxane (silicone, Sylgard® 184, Sigma-Aldrich) in a 1:3 ratio (weight/weight) as previously described by Pedraza27. A volume of 100 µL per well of silicone-CaO2 was degassed using vacuum bell and cross-linked in a P48 plate for 24 h at 60 °C.
Silicone-CaO2 O2 production rate
The O2 production rate (OTR) of the silicone-CaO2 disks was followed during culture for 12 days by placing four silicone-CaO2 disks in 200 mL of PBS (Eurobio, Courtaboeuf, France) at 37 °C. The PBS was first deoxygenated by stirring at 200 rpm in a hypoxic atmosphere (N2). After reaching 0% O2, the container was sealed and the dissolved O2 concentration was measured in the PBS using a Clark electrode and Rhapsody software. OTR (nmol/min/disk) was calculated as the initial slope of the dissolved O2 concentration curve in the PBS. A negative control without a silicone-CaO2 disk was performed in parallel.
Design of Experiment (DoE)
Screening
Screening of the oxygenation strategies was performed on MPIs encapsulated in alginate macrobeads using a full factorial design 23 (Fig. 1). The influence of the main factors and their first-order interactions was analyzed. The three factors studied and their levels were: without/with HEMOXCell, without/with silicone-CaO2 and normoxic/hypoxic O2 tension. The response variables were intracellular ATP content per well (RLU), ATP/LDH viability ratio per well (RLU/AU), and insulin stimulation index. These response variables were assessed after 6 days of culture under the different conditions defined by the experimental design (Table 2).
Response surface method (RSM)
Based on the screening of the O2 strategies, RSM was used to optimize the BAP configuration concerning the O2 balance in the alginate sheet device. The objective was to maximize the density of viable MPIs in the BAP by tuning the HEMOXCell concentration and the islet seeding density in the hypoxic environment in the presence of the silicone-CaO2 disk. A central composite design (22 factorial design with 4-star points and four replicates of the central point) was used to fit a second-order polynomial model (Fig. 2C). The model validation was performed by analyzing the lack-of-fit test results, the determination coefficient R2 value, and the diagnostic plots. The two factors studied were HEMOXCell concentration and islet seeding density, and their ranges were determined according to the literature and the determination of silicone-CaO2 OTR and pseudo-islet OCR. The response variables were intracellular ATP content (RLU) and ATP/LDH viability ratio (RLU/AU) per device. These response variables were assessed on pseudo-islets encapsulated in alginate sheets cultured for 24 h under the different conditions defined by the experimental plan (Table 5). The optimum values were determined by solving the regression equations and analyzing the response surface plots. A multi-response optimization was performed to achieve the best compromise to maximize viability and function of the encapsulated pseudo-islets.
Statistical analyses
The experimental design for the screening and the optimization steps were repeated independently at least three times. Analysis of variance (ANOVA), regression analysis, and graphical display of DoE results were performed using the Statgraphics Centurion software 18.1.06. Assessment of the optimal BAP design on NPIs was performed on a minimum of four independent experiments. The significance of differences between groups was evaluated using a non-parametric test (Mann–Whitney or paired Wilcoxon tests) or a parametric unpaired t-test. A p-value < 0.05 was considered statistically significant. Graphs’ formatting was performed using Graphpad Prism software 8.0.2.
