The Human Pancreas as a Source of Protolerogenic Extracellular Matrix Scaffold for a New-generation Bioartificial Endocrine Pancreas

Abstract

Objectives: Our study aims at producing acellular extracellular matrix scaffolds from the human pancreas (hpaECMs) as a first critical step toward the production of a new-generation, fully human-derived bioartificial endocrine pancreas. In this bioartificial endocrine pancreas, the hardware will be represented by hpaECMs, whereas the software will consist in the cellular compartment generated from patient's own cells.

Background: Extracellular matrix (ECM)-based scaffolds obtained through the decellularization of native organs have become the favored platform in the field of complex organ bioengineering. However, the paradigm is now switching from the porcine to the human model.

Methods: To achieve our goal, human pancreata were decellularized with Triton-based solution and thoroughly characterized. Primary endpoints were complete cell and DNA clearance, preservation of ECM components, growth factors and stiffness, ability to induce angiogenesis, conservation of the framework of the innate vasculature, and immunogenicity. Secondary endpoint was hpaECMs' ability to sustain growth and function of human islet and human primary pancreatic endothelial cells.

Results: Results show that hpaECMs can be successfully and consistently produced from human pancreata and maintain their innate molecular and spatial framework and stiffness, and vital growth factors. Importantly, hpaECMs inhibit human naïve CD4 T-cell expansion in response to polyclonal stimuli by inducing their apoptosis and promoting their conversion into regulatory T cells. hpaECMs are cytocompatible and supportive of representative pancreatic cell types.

Discussion: We, therefore, conclude that hpaECMs has the potential to become an ideal platform for investigations aiming at the manufacturing of a regenerative medicine-inspired bioartificial endocrine pancreas.

Figure 1

Decellularization process, fluoroscopy and DNA quantitation. Panels A and B show the set-up of the core for organ decellularization in a discarded human pancreas before and after the treatment. Red arrows in the first image show the arterial, represented by superior mesenteric artery (SMA) and splenic artery (SA) that share one of the two detergent inflows. Green arrow indicates the second inflow through the pancreatic duct (PD). Notably, during decellularization, the color of the organ macroscopically changed from a golden brown color to straw yellow but without reaching the whitish color described for porcine pancreas scaffolds [20]. Fluoroscopy of hpaECMs performed through SMA (Panel C), SA (panel D) and PD (panel E, F, G). Contrast media flows within the framework of the innate vasculature of hpaECMs without extravasation. Panel H confirms satisfactory cell and DNA clearance. Statistic was made by t-test of fresh vs. acellular tissue; *** p<0.001.

Figure 2

Basic histology of hpaECMs as compared to the native pancreas. H&E demonstrates complete loss of cellular elements with preservation the intercellular framework. The larger empty spaces on the left aspect of the section almost certainly reflect pathological lesions the nature of which cannot be ascertained with confidence without having examined the sections before treatment, although one can speculate that they represent focal fat necrosis. Masson trichrome shows the light green ECM framework. On the left aspect of the right panel there is evidence of scarring indicated by the dense area staining green. Alcian blue demonstrates preservation of the so-called ground substance in the connective tissue framework, although the process of digestion involved in decellularization has altered the faint blue color of Alcian blue into a faint green color as can be seen in the right panel. The van Gieson stain demonstrates preservation of elastine. The picrosirius red demonstrates abundant collagen in the right panel, reflecting significant scarring in the field photographed in contrast to the left panel.

Figure 3

Immunohistochemistry. Aside of nuclear staining, presence of possible immunogens was ruled out by immunostaining for major histocompatibility complex of class I and II (HLA I and II). Immunostaining for critical, ubiquitous components of pancreas ECM showed that laminin, fibronectin, collagen type I and IV remains well preserved in hpaECMs (nuclei were conterstained with DAPI) and displays a similar distribution of these proteins in the fresh and decellularized pancreas bioscaffold sections.

Figure 4

SEM of intact and acellular pancreas ECM. A. Low power magnification of a cross section through the decellularized pancreas demonstrating preservation of the ECM supporting the exocrine and endocrine elements as well as the vasculature. B. Three dimensional view of the ECM framework of an islet of Langerhans in the decellularized pancreas, identifiable by what is presumably its spherical microvascular framework. C. Cross-section through what we interpret as an arteriole with the lumen delimited by the internal elastic membrane and external to it the individual layers of basement membrane of dissolved smooth muscle fibers of the arteriole’s media. D. A presumed venule identifiable by the smaller number of layers of smooth muscle cells (identified by the layers of basement membrane) forming its wall and two small tributaries presumed to be contributing to its formation.

Figure 5

Immune properties of hpaECM. (A) Representative plots and (B) quantitation of CFSE dilution in CFSE-labeled human naïve CD4⁺ T cells stimulated with anti-CD3/anti-CD28 coated beads cultured with or without hpaECM. (C) Representative plots and (D) quantitation of Annexin-V and 7-AAD expression as a measure of apoptosis/necrosis in human naïve CD4⁺ T cells stimulated with anti-CD3/anti-CD28 coated beads cultured with or without hpaECM. (E) Representative plots and (F) quantitation of FoxP3 expression in CD4⁺CD25⁺ cells converted from human naïve CD4⁺ T cells stimulated with anti-CD3/anti-CD28 coated beads and IL2 cultured with or without hpaECM. All experiments were analyzed on day 5. Data are means+SD; four experiments). *P<0.05.

Figure 6

Evaluation of islets seeded onto decellularized scaffolds. (A) Decellularized scaffolds prior to seeding. (B) Scaffold immediately after seeding, black arrows indicate islets. (C) Dithizone stained islets after 4 days in culture on culture plates. (D) Dithizone stained islet located on periphery of scaffold, 4 days after seeding onto scaffolds. (E) Glucose stimulation index (GSI) for human islets after 4 days in culture. In order to determine the functionality of islets following 4 days of culture dynamic perifusion tests were conducted. 200 islets were placed into perifusion chambers, either seeded on scaffolds or free. The chambers were then perfiused with Krebs-Ringer-bicarbonate (KRB) solution containing 0.2% bovine serum albumin (BSA) and either 3.3 mM or 16.7 mM glucose at a flow rate of 0.3 mL/min with continuous gassing with 95% air/5% CO2 and effluent sample collection. The effluent samples were stored at −20°C until analyzed for insulin content by radioimmunoassay. The glucose stimulation index defined as the ratio of insulin secreted during the high glucose period normalized to the insulin secreted during the basal glucose period was then calculated. Both islets in standard culture conditions as well as islets seeded on scaffold demonstrated similar responses to changes in glucose concentration. Data represent mean + standard deviation, n=3. TCP denotes tissue culture plate.

Figure 7

Endothelial cell seeding. Matrix was seeded with human pancreatic endothelial cells and cultured for six days in a bioreactor, consisting in a closed circuit with one chamber for organ housing, a reservoir for medium oxygenation and a peristaltic pump (Ismatec), connected by tubing (ID 1/16", Pharmed BPT) Pancreatic tail was surgically isolated in order to obtain a smaller volume to seed keeping at the same time an inflow (SA – red connector) and an outflow (Splenic Vein -SV- blue connector) (Panel A). Panel B indicates a schematic representation of the perfusion circuit for seeded pancreatic scaffold culture. Panel C shows a representative image of H&E stain showing localization of infused cells in vessels. Boxes indicate areas reported with high magnification in the panel below. Panel D illustrates representative images of H&E (left), CD31 (middle) and Ki67 (right) matrix staining.