Optimization of an O2-balanced bioartificial pancreas for type 1 diabetes using statistical design of experiment

Abstract

A bioartificial pancreas (BAP) encapsulating high pancreatic islets concentration is a promising alternative for type 1 diabetes therapy. However, the main limitation of this approach is O₂ supply, especially until graft neovascularization. Here, we described a methodology to design an optimal O₂-balanced BAP using statistical design of experiment (DoE). A full factorial DoE was first performed to screen two O₂-technologies on their ability to preserve pseudo-islet viability and function under hypoxia and normoxia. Then, response surface methodology was used to define the optimal O₂-carrier and islet seeding concentrations to maximize the number of viable pseudo-islets in the BAP containing an O₂-generator under hypoxia. Monitoring of viability, function and maturation of neonatal pig islets for 15 days in vitro demonstrated the efficiency of the optimal O₂-balanced BAP. The findings should allow the design of a more realistic BAP for humans with high islets concentration by maintaining the O₂ balance in the device.

Figure 1

Screening experimental design. (A) The screening of the oxygenation strategies was done on MPIs encapsulated in alginate macrobeads using a full factorial design 2³. The three factors studied and their levels were: w/o or w/HEMOXCell (HEM), w/o or w/silicone-CaO₂ (S-O₂), and w/ 1% O₂ or 20% O₂ tension. The response variables assessed after 6 days of culture were the intracellular ATP content per well (RLU), ATP/LDH viability ratio per well (RLU/AU), and the insulin stimulation index. (B) Interaction plots of the screening DoE. Interactions involving HEMOXCell and silicon-CaO₂, HEMOXCell and O₂ tension, silicone-CaO₂ and O₂ tension are displayed concerning ATP content, ATP/LDH ratio, and insulin stimulation index.

Figure 2

Optimization experimental design. Based on (A) the silicone-CaO₂ O₂ production rate and (B) the MIN6 pseudo-islet O₂ consumption rate, (C) a central composite design was used to optimize the HEMOXCell concentration and islet seeding density in the BAP. The response variables, intracellular ATP content (RLU), and ATP/LDH viability ratio (RLU/AU) per device were assessed for MPIs encapsulated in alginate sheets after 1 day of culture. Results of O₂ production rate (n = 2) and consumption rate (n = 6) are presented as mean ± SEM of independent experiments. (D) Response surface analysis for the optimization DoE. The plots represent the effects of the HEMOXCell concentration, islet seeding density, and their interaction on the ATP content and the ATP/LDH ratio in the BAP after 1 day of culture under 1% O₂ tension.

Figure 3

Viability and function of MIN6 pseudo-islets (MPIs) embarked in O₂-balanced BAP. Alginate encapsulated MPIs (3000 IEQ/150 µL) were cultured for 3 days under 20% O₂ (positive control, white) or 1% O₂ condition without O₂ strategy (negative control, black) or with innovative strategy of oxygenation (ISO) composed of silicone-CaO₂ disk and 500 µg/mL HEMOXCell by alginate (1% O₂ + ISO, grey). (A) Total metabolic activity (ATP content, RLU), (B) viability (ATP/LDH ratio, RLU/AU), (C) Glucose plus theophylline (G + T) responsive insulin release by 30 min sequential incubations of alginate encapsulated MPIs in basal medium (black bar) and glucose plus theophylline stimulation (grey bar), (D) Stimulation index (ratio of high glucose plus theophylline stimulation over basal insulin secretion). Results from independent experiments (n = 5–9) are expressed as mean ± SEM. *p < 0.05, **p < 0.01 (unpaired parametric t-test).

Figure 4

Viability of neonate pig islets embarked in O₂-balanced BAP. Alginate encapsulated NPIs (3000 IEQ/150 µL) were cultured for 3, 8 and 15 days under normoxic condition (20% O₂, positive control, white bar) or hypoxic condition without O₂ strategy (1% O₂, negative control, black bar) or with the innovative strategy of oxygenation (ISO) composed of silicone-CaO₂ disk and 500 µg/mL HEMOXCell by alginate (1% O₂ + ISO, grey bars). Fold change in (A) total metabolic activity (ATP content, RLU) and in (B) viability (ATP/LDH ratio, RLU/AU) of encapsulated NPIs compared to the control cultured at day 3 under 20% O₂ without O₂ strategy. Results from independent experiments (n = 4–6) are expressed as mean ± SEM (A,B). *p < 0.05 (Non-parametric Wilcoxon apparatus test). (C) Histological analyzes of formalin-fixed paraffin-embedded NPIs 4 µm thick cross-sections stained with hematoxylin–eosin-saffron on pre-encapsulated NPIs, 24 h after isolation (day 1) and on decapsulated NPIs after 8 days of culture within the BAPs.

Figure 5

Function of neonate pig islets (NPIs) in O₂ balanced BAPs. Alginate encapsulated NPIs (3000 IEQ/150 µL) were cultured for 3, 8, and 15 days under 20% O₂ (positive control, white bar) or 1% O₂ conditions without the O₂ strategy (1% O₂, negative control, black bar), or with the innovative strategy of oxygenation (ISO) composed of silicone-CaO₂ disk and 500 µg/mL HEMOXCell by alginate (1% O₂ + ISO, grey bars). (A) Glucose plus theophylline (G + T) responsive insulin release by 30 min sequential incubations of alginate encapsulated NPIs in basal medium (close circle) and glucose plus theophylline stimulation (open circle) after 3, 8 and 15 days of culture. (B) Stimulation indexes (ratio of high glucose plus theophylline stimulation over basal insulin secretion) of alginate encapsulated NPIs after 3, 8, and 15 days of culture. Results from independent experiments (n = 4–6) are expressed (A) individual results matched in basal or stimulation media or as (B) mean ± SEM. * p < 0.05, ***p < 0.005 (Mann–Whitney test).

Figure 6

Effect of O₂ strategy on the maturation of neonate pig islets (NPIs) embarked in O₂ balanced BAPs. The analyses were performed on pre-encapsulated NPIs 24 h after isolation (day 1) and on encapsulated NPIs cultured for 8 or 15 days within BAPs in 20% O₂ (positive control) and 1% O₂ conditions without the O₂ strategy (1% O₂, negative control) or with the O₂ strategy composed of silicone-CaO₂ disk and 500 µg/mL HEMOXCell by alginate (1% O₂ + ISO) (n = 4–7 pigs). (A) Immunostaining of insulin ß cells (green, Alexa-Fluor 488) and glucagon α cells (red, Alexa-Fluor 555) staining of NPIs in 4 µm thick cross-sections. Views in white light are shown to the lower-left of each photo. The scale is indicated in each picture. (B) Percentage of mean insulin-positive area per islet (Insulin) and percentage of mean glucagon-positive area per islet (Glucagon). The percentages of insulin and glucagon were quantified within 150 islets in day 1 (n = 4 pigs) and an average of 30 islets per condition after day 8 of culture (n = 3 pigs). (C) Intracellular insulin by ATP content ratio. (D) Relative quantitative RT-PCR expression analysis of insulin (INS), glucagon (GCG), pancreatic progenitor transcription factor (PDX1) and NKX6.1 (NKX6.1). *p < 0.05, ****p < 0.0005 (Unpaired parametric t-test or Mann–Whitney test).

Figure 7

Hypoxic signature of neonate pig islets (NPIs) in O₂ balanced BAPs. Alginate encapsulated NPIs (3000 IEQ/150 µL) were cultured for 3, 8, and 15 days under 20% O₂ (20% O₂, positive control, white bar) or 1% O₂ condition without O₂ strategy (1% O_2, negative control, black bar) or with the innovative strategy of oxygenation (ISO) composed of silicone-CaO₂ disk and 500 µg/mL HEMOXCell by alginate (1% O₂ + ISO, grey bars). Fold change in VEGF secretion by total metabolic activity (AU/RLU) of encapsulated NPIs compared to the control cultured for 3 days under 20% O₂ without O₂ strategy. (B) Relative quantitative RT-PCR expression analysis of Heme oxygenase (HO-1) of decapsulated NPIs cultured for 15 days within BAPs (n = 4–7). Results from independent experiments (n = 4–7) are expressed as mean ± SEM. *p < 0.05 (Non-parametric Wilcoxon apparatus test).