Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production
Summary
The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets’ microenvironment in vitro and, potentially, in vivo settings.
Abstract
Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.
Introduction
The isolation of pancreatic islets is a meticulous process carried out through the enzymatic digestion of the connections between islets and their extracellular surrounding supportive structure. This destruction of the extracellular matrix (ECM) is one of the critical factors in characterizing islets' damage taking place during isolation processes1,2,3,4. The peri-islet ECM is an essential acellular component of the endocrine pancreas. It is composed of proteins and polysaccharides, which interact and cross-link to form a three-dimensional net that structurally and biochemically supports the physiological homeostasis, and helps in the recreation of this in vitro microenvironment2,5,6. The loss of fundamental signaling processes between islets and ECM is recognized as one of the contributing factors, which limits islets' survival in vitro and in vivo2,7,8,9,10.
Animal- and human-derived ECM have been widely used for the recapitulation of the pancreatic islets' microenvironment11,12,13,14,15,16,17,18,19. Since the ability of the ECM to enhance rat islet cells attachment, proliferation, and long-term culture maintenance was first reported in ref.20, many other studies have provided strong evidence that the restoration of native ECM interaction with human islets enhances islet function21,22. For instance, recent data has shown that islets encapsulated with ECM significantly improved glycemic control in diabetic mice, enhancing and facilitating the delivery of insulin in a novel cell-based insulin delivery platform23. Furthermore, studies have demonstrated that incorporation of critical components of pancreatic ECM can significantly improve the endocrine function of β-cells24,25,26,27.
ECM manufacturing protocols present in the literature are based on the application of chemical detergents, e.g., Triton X-100 or sodium dodecyl sulfate (SDS). Despite providing excellent DNA clearance, chemicals used in decellularization processes are cytotoxic, expensive, and residues on the decellularized tissue bring concerns in view of potential clinical application.
Based on these observations, the objectives of this study were three-fold: First, to develop a decellularization method for the human pancreas with minimal use of ionic or non-ionic chemical detergents; Second, to produce a soluble ECM scaffold for tissue culture; Third, to characterize the pancreatic ECM in order to assess its cytotoxicity and impact on islet cell function. The characterization is necessary for all the cell-culture-based applications, as it demonstrates that solubilized pancreatic ECM could be beneficial in recapitulating the pancreatic microenvironment for isolated islets. Described herein is an effective, detergent-free decellularization method for the human pancreas, characterization of the ECM, and the effect of ECM on viability and function of encapsulated isolated human pancreatic islets.
Protocol
Representative Results
Discussion
The aim of this work was to develop a gentler, detergent-free decellularization protocol to produce pancreatic ECM. Attention was paid to the preservation of ECM components of the pancreatic parenchyma and the avoidance of a lengthy tissue exposure to conventional ionic or non-ionic chemical detergents during the decellularization process.
The most innovative aspect of the developed decellularization method is the avoidance of classic ionic and non-ionic chemical detergents. Our previous exper…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This project has received funding from the European Union’s Horizon 2020 Research and Innovation Program under grant agreement No. 646272. Human Pancreatic Islets were obtained from Prodo Laboratories, Aliso Viejo, CA 92656.
Materials
| Corning 1L Easy Grip Polystyrene Storage Bottles with 45mm Caps | ThermoFisher | 430518 | Container use for decellularization |
| Cryogenic Mill | SPEX Certiprep | 6870-230 | |
| Deoxyribonuclease I from bovine pancreas | Sigma Aldrich | DN25 | |
| Distilled Water | ThermoFisher | 15230147 | |
| Falcon 50mL Conical Centrifuge Tubes | Corning | 352070 | |
| Human Pancreas | na | na | |
| Insulin-Elisa | Mercodia | 10-1113-01 | |
| Magnesium Chloride, 1M, Sterile | Bio-World | 41320004-1 | |
| Pepsin from porcine gastric mucosa | Sigma Aldrich | P7012-5G | |
| Placenta Basin w/o Lid, Sterile | DeRoyal | 32-881 | |
| Polypre chromatography tubes | Bio-rad | 731-1500 | Polypropylene columns |
| Quant-iT PicoGreen dsDNA Assay Kit | Invitrogen | P7589 | DNA Kit |
| SamplePrep Large-Capacity Freezer/Mill Accessory | SPEX | 6801 | Large Grinding Vial Set |
| Sephadex G-10 beads | Cytiva | 17001001 | Gel Filtration Resin |
| Surgical kit | na | na | |
| UltraPur 0.5M EDTA, pH 8.0 | ThermoFisher | 15575020 | |
| UltraPur 1 M Tris-HCI Buffer, pH 7.5 | ThermoFisher | 15567027 | |
| UltraPure DNase/RNase-Free Distilled Water | ThermoFisher | 10977023 |
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