Loczenski, Rabea
(2020)
Development of a supercritical CO2 decellularization technology.
PhD thesis, University of Nottingham.
Abstract
Decellularization of mammalian tissue describes the process by which cellular materials are removed from the extracellular matrix (ECM, Hinderer, Layland and Schenke-Layland, 2016). Current decellularization methodologies utilise a multitude of decellularization agents, such as detergents, solvents and biological enzymes, to achieve successful decellularization, which has been demonstrated with a number of different organs (i.e. heart (Ott et al., 2008), lung (Petersen et al., 2012), kidney (Song et al., 2013) and liver (Uygun et al., 2010; Mazza et al., 2015)). However, the use of these common decellularization agents causes damage to the derived ECM ultrastructure. Furthermore, these agents are often retained within the tissue following the decellularization process (Faulk et al., 2014; White et al., 2016), which may negatively impact potential downstream application.
In this thesis, a new scCO2 and scCO2 hybrid decellularization methodology was developed for liver and aorta tissue, respectively. ScCO2 effectively removed genomic material by -75% and -48% from liver and aorta tissue (respectively) as confirmed by DNA quantification and histological analysis. Interestingly, the required scCO2 exposure duration for decellularization was longer for aorta (72 h) compared to liver (48 h), suggesting that differences in tissue structure might affect decellularization efficacy by scCO2. Furthermore, decellularization by scCO2 was inhibited if tissue water content was removed, indicating that moisture content is likely mechanistically involved in the process of decellularization by scCO2. While 48 h scCO2 decellularization reduced DNA content of liver tissue by -75%, aorta tissue required an additional 24 h exposure to scCO2 and a further 1 h incubation in either Trypsin/EDTA, Triton-x-100, SDC or LS-54 to achieve a similar level of DNA reduction. ECM proteins, such as glycosaminoglycans and collagen, were retained following the scCO2 only method (liver) and the scCO2 hybrid method (aorta).
When tested for biocompatibility in vitro, the scCO2 decellularized liver and aorta ECM scaffold resulted in a non-cytotoxic response when exposed to HepG2 and 3T3 cells (respectively). In contrast, the hybrid scCO2 decellularization methodology caused cytotoxicity on both cell lines tested (HepG2 and 3T3) and requires further development/optimization to reduce residual toxicity caused by the addition of Trypsin/EDTA, Triton-x-100, SDC or LS-54.
This thesis demonstrates an in depth characterization of the response of mammalian tissue to scCO2, which resulted in the development of a novel, scCO2 based decellularization technology for liver and aorta tissue. The effects of scCO2 on liver tissue were more pronounced than those observed on aorta, suggesting that differences in tissue structure might influence the efficacy of scCO2 to decellularize mammalian tissue. Combinations of scCO2 with commonly used decellularization agents (i.e. hybrid method) further improved the removal of cellular material for aorta but not liver tissue. Taken together, this work demonstrates the potential utility for sCO2 as a deceullarization agent. However, the hybrid scCO2 method described herein requires further modification to improve biocompatibility in downstream applications.
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