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 processes^1,2,3,4. 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-dimension....

Protocol

This research study was approved by the human research committee of Wake Forest Baptist Medical Center. Human pancreases were ethically obtained from organ donors through Carolina Donor Services. Organ donors were screened for infectious diseases relevant to humans, according to UNOS regulations. Organs were received in sterile preservation solution where they were kept until use. Upon delivery to the lab, all organs were inspected, and samples of the native pancreas were collected for histological purposes. The organs were then frozen and stored in sterile conditions at -20 °C until further use.

1. Surgical preparation of the human panc....

Results

Native and acellular pancreatic samples were processed for histological staining with H&E, MT, and AB. The H&E staining showed complete absence of nuclear material and cells, confirming successful decellularization. MT and AB stainings showed the framework of the ECM, highlighting qualitatively collagenous and stromal components, respectively (Figure 1).

This method enabled the consistent generation of an ECM powder from the human pancreas. DNA quantificat.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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