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Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.

ABSTRACT: Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches.

SUBMITTER: Goh SK

PROVIDER: S-EPMC3748589 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.

Goh Saik-Kia SK Bertera Suzanne S Olsen Phillip P Candiello Joseph E JE Halfter Willi W Uechi Guy G Balasubramani Manimalha M Johnson Scott A SA Sicari Brian M BM Kollar Elizabeth E Badylak Stephen F SF Banerjee Ipsita I

Biomaterials 20130617 28

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic co ...[more]

PMID: 23787110

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Project description:Introduction: Whole-organ decellularization is an attractive approach for three-dimensional (3D) organ engineering. However, progress with this approach is hindered by intra-vascular blood coagulation that occurs after in vivo implantation of the re-cellularized scaffold, resulting in a short-term graft survival. In this study, we explored an alternative approach for 3D organ engineering through an axial pre-vascularization approach and examined its suitability for pancreatic islet transplantation. Methods: Whole livers from male Lewis rats were decellularized through sequential arterial perfusion of detergents. The decellularized liver scaffold was implanted into Lewis rats, and an arteriovenous bundle was passed through the scaffold. At the time of implantation, fresh bone marrow preparation (BM; n = 3), adipose-derived stem cells (ADSCs; n = 4), or HBSS (n = 4) was injected into the scaffold through the portal vein. After 5 weeks, around 2,600 islet equivalents (IEQs) were injected through the portal vein of the scaffold. The recipient rats were rendered diabetic by the injection of 65 mg/kg STZ intravenously 1 week before islet transplantation and were followed up after transplantation by measuring the blood glucose and body weight for 30 days. Intravenous glucose tolerance test was performed in the cured animals, and samples were collected for immunohistochemical (IHC) analyses. Micro-computed tomography (CT) images were obtained from one rat in each group for representation. Results: Two rats in the BM group and one in the ADSC group showed normalization of blood glucose levels, while one rat from each group showed partial correction of blood glucose levels. In contrast, no rats were cured in the HBSS group. Micro-CT showed evidence of sprouting from the arteriovenous bundle inside the scaffold. IHC analyses showed insulin-positive cells in all three groups. The number of von-Willebrand factor-positive cells in the islet region was higher in the BM and ADSC groups than in the HBSS group. The number of 5-bromo-2'-deoxyuridine-positive cells was significantly lower in the BM group than in the other two groups. Conclusions: Despite the limited numbers, the study showed the promising potential of the pre-vascularized whole-organ scaffold as a novel approach for islet transplantation. Both BM- and ADSCs-seeded scaffolds were superior to the acellular scaffold.

Project description:Development of systems allowing the maintenance of native properties of mesenchymal stromal cells (MSC) is a critical challenge for studying physiological functions of skeletal progenitors, as well as towards cellular therapy and regenerative medicine applications. Conventional stem cell culture in monolayer on plastic dishes (2D) is associated with progressive loss of functionality, likely due to the absence of a biomimetic microenvironment and the selection of adherent populations. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow cells within the pores of 3D scaffolds in a perfusion-based bioreactor system, followed by enzymatic digestion for cell retrieval. The 3D-perfusion system supported MSC growth while maintaining cells of the hematopoietic lineage, and thus generated a cellular environment mimicking some features of the bone marrow stroma. As compared to 2D-expansion, sorted CD45- cells derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7-8 doublings) maintained a 4.3-fold higher clonogenicity and exhibited a superior differentiation capacity towards all typical mesenchymal lineages, with similar immunomodulatory function in vitro. Transcriptomic analysis performed on MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability as well as a significant upregulation of multipotency-related gene clusters following 3D-perfusion as compared to 2D expansion. The described system offers a model to study how factors of a 3D engineered niche may regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems. Nucleated cells were isolated from 5 fresh human bone marrow aspirates by means of red blood cells lyses buffer and then were seeded into a 3D perfusion bioreactor system using a pure hydroxyapatite 3D scaffold and in conventional Petri dishes (2D). After culture for 19 days, cells from both systems were enzymatically retrieved and sorted using anti-CD45-coated magnetic beads. Total RNA was extracted from CD45- cells, QCed and hybridized to Affymetrix microarrays.

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Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.

Publications

Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.

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