A novel technique for simultaneous whole‐body and multi‐organ decellularization: umbilical artery catheterization as a perfusion‐based method in a sheep foetus model

Abstract

The aim of this study was to develop a method to generate multi-organ acellular matrices. Using a foetal sheep model have developed a method of systemic pulsatile perfusion via the umbilical artery which allows for simultaneous multi-organ decellularization. Twenty sheep foetuses were systemically perfused with Triton X-100 and sodium dodecyl sulphate. Following completion of the whole-body decellularization, multiple biopsy samples were taken from different parts of 21 organs to ascertain complete cell component removal in the preserved extracellular matrices. Both the natural and decellularized organs were subjected to several examinations. The samples were obtained from the skin, eye, ear, nose, throat, cardiovascular, respiratory, gastrointestinal, urinary, musculoskeletal, central nervous and peripheral nervous systems. The histological results depicted well-preserved extracellular matrix (ECM) integrity and intact vascular structures, without any evidence of residual cellular materials, in all decellularized bioscaffolds. Scanning electron microscope (SEM) and biochemical properties remained intact, similar to their age-matched native counterparts. Preservation of the collagen structure was evaluated by a hydroxyproline assay. Dense organs such as bone and muscle were also completely decellularized, with a preserved ECM structure. Thus, as shown in this study, several organs and different tissues were decellularized using a perfusion-based method, which has not been previously accomplished. Given the technical challenges that exist for the efficient generation of biological scaffolds, the current results may pave the way for obtaining a variety of decellularized scaffolds from a single donor. In this study, there have been unique responses to the single acellularization protocol in foetuses, which may reflect the homogeneity of tissues and organs in the developing foetal body.

Evidence for continuity of interstitial spaces across tissue and organ boundaries in humans

“…The work of Franklin Mall over a century ago 1 , 2 , as well as modern-day decellularization techniques 3 , demonstrate that there are “reticular networks” made up of collagens, elastin, glycosaminoglycans, and other extracellular matrices (ECM) components surrounding, within, and between organs. These networks have biological and mechanical roles in defining the architecture and physiology of organs and, as a result, are now used as scaffolding for the creation of customized organ grafts for regenerative medicine 4 .…”

“…Continuity between mesenteric fascia and the connective tissues of the small intestinal and colonic walls has also been previously recognized 6 – 9 . More recently, the decellularization of entire fetal sheep shows that the connective tissue network is continuous throughout the body and that the connective tissue of nerves creates structural continuity between the nervous system and other tissues 3 . Dissection of human bodies likewise demonstrates continuity across large, multiorgan regions of the body, including the entirety of the dermis and the fascia of diverse organs and organ systems 10 – 13 .…”

Characterization of perfusion decellularized whole animal body, isolated organs, and multi‐organ systems for tissue engineering applications

“…Here, we demonstrate the perfusion decellularization of a whole rat body as a proof of concept that this technology can decellularize any perfusable solid organ in situ. Currently, the ability to decellularize scaffolds from different organs in situ remains limited (Gerli et al, 2018;Kajbafzadeh et al, 2015). Using both harvested (traditional) and non-harvested (in situ) perfusion, decellularized organs showed the preservation of the vascular conduits, organ capsules, ECM components, and structural architecture, and presented low levels of DNA, while preserving the glycosaminoglycan, chemical and mechanical components of the ECM.…”

Interstitial spaces are continuous across tissue and organ boundaries in humans

“…The work of Franklin Mall over a century ago 1,2 as well as modern day decellularization techniques 3 demonstrate that there are "reticular networks" made up of collagens, elastin, glycosaminoglycans, and other extracellular matrix (ECM) components surrounding, within, and between organs. These networks have biological and mechanical roles in defining the architecture and physiology of organs and, as a result, are now used as scaffolding for the creation of customized organ grafts for regenerative medicine.…”

“…5 More recently, decellularization of entire fetal sheep shows that the connective tissue network is continuous throughout the body and that the connective tissue of nerves creates structural continuity between the nervous system and other tissues. 3 Dissection of human bodies likewise demonstrates continuity across large, multiorgan regions of the body, including the entirety of the dermis and the fascia of diverse organs and organ systems. 6,7,8,9 Clinical disciplines including osteopathy have suggested that these connective tissue networks contain fluid and represent a body-wide communications network, akin to interstitial spaces, although this lacks detailed microscopic confirmation.…”