Decellularization of donor pancreatic fragment to obtain a tissue-specific matrix scaffold

Abstract

One of the pressing issues in tissue engineering is on how to obtain an artificial matrix that can simulate a biological microenvironment for cells. When creating a bioengineered pancreatic construct, a tissue-specific scaffold obtained from decellularized pancreatic tissue can serve as such matrix. Objective: to obtain and study the characteristic properties of a tissue-specific pancreas scaffold from decellularized human pancreatic fragments. Materials and methods. The decellularization protocol included 3 freeze/thaw cycles, followed by treatment with surfactants (sodium dodecyl sulfate and Triton X100). At each decellularization stage, samples were routinely stained with hematoxylin and eosin and for total collagen. In addition, immunohistochemical staining of decellularized human pancreas (DHP) for type I collagen and elastic fibers was performed. Cell nuclei in the original samples and the resulting matrix were visualized using DAPI fluorescent staining. DNA quantity in the native and decellularized pancreatic tissue was determined. The cytotoxicity of the tissue-specific matrix was evaluated in vitro by direct contact. The matrix properties of DHP samples were determined using mesenchymal stem cells (MSCs) of human adipose tissue. Results. A pancreatic decellularization method is proposed. This method allows to obtain a tissue-specific matrix in the form of a connective tissue scaffold completely free of detritus with preserved thin-fiber mesh-like structure, in which elastic and collagen fibers, including type I collagen, are identified. DAPI staining confirmed the absence of nuclear material in the decellularized matrix, while residual amount of DNA did not exceed 0.1%. Absence of matrix cytotoxicity and its ability to maintain adhesion and proliferation of human adipose tissue-derived MSCs was proved. Conclusion. As one of the stages in creating a bioengineered pancreatic construct, a method has been developed for producing a biocompatible (lack of cytotoxicity and immunogenicity) tissue-specific scaffold from decellularized human pancreatic tissue. In the scaffold, the morphofunctional properties of the native extracellular matrix-based scaffolds of the pancreas are preserved. Adhesion and proliferation of cell cultures are ensured.

Keywords

• pancreas
• decellularization
• tissue-specific scaffold
• tissue engineering