Elsevier

Biomaterials

Volume 198, April 2019, Pages 37-48
Biomaterials

3-D physiomimetic extracellular matrix hydrogels provide a supportive microenvironment for rodent and human islet culture

https://doi.org/10.1016/j.biomaterials.2018.08.057Get rights and content

Abstract

Organ-on-a-chip platforms serve as cost-efficient testbeds for screening pharmaceutical agents, mimicking natural physiology, and studying disease. In the field of diabetes, the development of an islet-on-a-chip platform would have broad implications in understanding disease pathology and discovering potential therapies. Islet microphysiological systems are limited, however, by their poor cell survival and function in culture. A key factor that has been implicated in this decline is the disruption of islet-matrix interactions following isolation. Herein, we sought to recapitulate the in vivo peri-islet niche using decellularized extracellular matrix (ECM) hydrogels. Sourcing from porcine bladder, lung, and pancreas tissues, 3-D ECM hydrogels were generated, characterized, and validated using both rodent and human pancreatic islets. Optimized decellularization protocols resulted in hydrogels with distinctive viscoelastic properties that correlated to their matrix composition. The in situ 3-D encapsulation of human or rat islets within ECM hydrogels resulted in improved functional stability over standard culture conditions. Islet composition and morphology were also altered, with enhanced retention of islet-resident endothelial cells and the formation of cord-like structures or sprouts emerging from the islet spheroid. These supportive 3-D physiomimetic ECM hydrogels can be leveraged within microfluidic platforms for the long-term culture of islets.

Introduction

Engineering human microphysiological systems (MPS) capable of intimately characterizing the influence of various parameters on organ function and viability, as well as its impact on “downstream” organs, would not only advance scientific knowledge, but also minimize animal research and improve the safety and efficacy of potential new drugs. Multiple MPS platforms mimicking the heart, lung, liver, and vasculature have been developed [[1], [2], [3], [4]]. For the pancreas, unique islet-on-a-chip systems have been designed to permit ease in short-term islet assessments and characterization [[5], [6], [7], [8], [9]] (for recent review see Ref. [10]). Leveraging MPS for the long-term culture of primary pancreatic islets, however, has been limited by their poor stability ex vivo.

In the pancreas, islets of Langerhans are surrounded by a layer of extracellular matrix (ECM) defined as the peri-insular basement membrane (BM), comprised predominately of collagen type IV, laminin, and fibronectin [11,12]. Interactions between the multicellular islet and its peripheral ECM serve a critical role in the maintenance of cell viability, the preservation of β cell insulin production and glucose responsiveness, and the coordination of intercellular signaling [13,14]. During the enzymatic isolation procedure, islets are stripped of this native BM. The subsequent reduction of cell adhesion signaling triggers β cell apoptosis via anoikis, as well as declines in insulin secretion [11,15,16]. The islet isolation procedure also disrupts other islet-resident nonhormonal cells, such as endothelial and mesenchymal cells, which serve to support endocrine cell function [[17], [18], [19]].

Previous in vitro studies have shown that the replenishment of individual ECM components found in the pancreatic basement membrane, such as collagen type IV, laminin, and fibronectin, improves islet viability and restores glucose responsive insulin secretion [[20], [21], [22]]. Moreover, 3-D co-culture of islets within hydrogels co-mixed with ECM components (e.g. collagen type IV and laminin) aids in the preservation of islet viability [11,23,24]. While these approaches are promising, supplementation with selected ECM proteins will not fully recapitulate the multiple islet adhesion motifs of the native peri-islet niche.

The use of decellularized tissues enables a diverse spectrum of biochemical and biophysical cues, adapted from nature, to be presented to the encapsulated cells. The composition of the ECM can be tailored via selection of the tissue source and the decellularization approach [[25], [26], [27]]. While whole organ decellularization provides retention of both ECM composition and the organ's 3-D architecture, loading islet or even β cells into a cell-free organ is complex, requiring multi-step infusions, and incomplete, with only partial cell repopulation into the non-conduit tissue spaces [28,29]. Alternatively, a decellularized ECM hydrogel permits in situ gelation to uniformly encapsulate cells or organoids within a physiological 3-D microenvironment. ECM hydrogels are prepared via a lyophilizing, milling, and enzymatic digestion process [30]. This approach provides an easily adaptable system that is amendable to in vivo injection, 3-D printing, and loading within porous biomaterial platforms (e.g. polypropylene mesh) [[30], [31], [32], [33]].

In this study, we sought to explore the potential of decellularized porcine ECM hydrogels for supporting islet culture (Fig. 1). Tissues selected for study were the pancreas and BM-rich lung and bladder. Tissue-specific decellularization protocols were optimized and resulting materials were characterized for a cellularity, protein composition, endotoxin content, and mechanical properties. Resulting hydrogels were subsequently used to encapsulate rat and human pancreatic islets. Islet viability, functionality, cytokine/hormone production, and morphology were evaluated.

Section snippets

Porcine tissue decellularization and ECM solubilization

Porcine pancreas, lung, and bladder were harvested from the UF Department of Animal Studies under IACUC protocols approved by the University of Florida. The decellularization protocol was modified from published methods to maximize host cell removal and retention of ECM proteins [[34], [35], [36]]. Specifically, freshly procured tissues were rinsed under water, trimmed of peripheral connective tissue, and sectioned into small pieces (5 mm × 5 mm) to increase surface area for processing. Next,

Characterization of acellurity and ECM composition in decellularized porcine tissue

To obtain high-quality decellularized tissues for cell culture, the decellularization process must be carefully optimized. While removal of donor cellular components is needed to mitigate immunogenicity, the integrity and bioactivity of the ECM are subject to degradative loss if a harsh decellularization process is used [[43], [44], [45]]. To optimize this balance for this study, a combination of published decellularization approaches, including osmotic burst, freeze and thaw cycles, mechanical

Discussion

In this study, 3-D physiomimetic porcine ECM hydrogels were developed to recapitulate critical islet adhesion junctions in an effort to preserve islet stability in culture. Given the anatomical and physiochemical complexities of the different tissues tested, distinct decellularization strategies were employed to remove highly immunogenic cells with minimal protein loss [26,50]. Low remnant DNA found in decellularized tissues demonstrated effective cellular removal, thereby reducing potential

Conclusion

ECM hydrogels, sourced from decellularized bladder or pancreas tissues, supported long-term human and rat islet culture, with preservation of glucose stimulated insulin release. These multi-cellular mini-organs further exhibited dynamic interactions with the 3-D ECM hydrogel, forming protrusions during culture, comprised of fibroblast-like sprouts that served as conduits for endothelial cell migration. Decellularized ECM hydrogels provide a unique supportive 3-D microenvironment that can be

Acknowledgement

This work was supported by the NIDDK-supported Human Islet Research Network (HIRN, RRID:SCR_014393; https://hirnetwork.org; UC4 DK104208) and JDRF (SRA-2017-347-M-B). Human pancreatic islets were provided by the NIDDK-funded Integrated Islet Distribution Program (IIDP) at City of Hope (2UC4DK098085). We thank Irayme Labrada for her excellent technical assistance during rat islet procurement. We thank Dr. John Driver from the UF Department of Animal Science for assistance in procurement of

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