Decellularized Tobacco Hairy Roots as Novel Tissue Culture Platform

Rahman, Mohammed Masrur

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Decellularized Tobacco Hairy Roots as Novel Tissue Culture Platform

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The use of human mesenchymal stem cells (hMSCs) is gaining increased traction in clinical research and therapies. However, current tissue culture platforms for the large-scale expansion of hMSCs, such as microcarrier beads in bioreactors, result in lowered viability of the cells and affect differentiation potential by reducing surface antigen expression when detached using traditional dissociation agents such as trypsin. Microcarrier beads, commonly made from polystyrene, are well characterized in the expansion of other cell types. It is known that plant-derived scaffolds can be used to culture mammalian cells. Our lab has previously shown that decellularization of bright yellow-2 (BY-2) cell cultures, a tobacco (Nicotiana tabacum) plant cell, can yield matrices for the culture of human fibroblasts (hFFs). Tobacco hairy roots are plant-based tissue cultures from the same transgenic Nicotiana tabacum plant. In this thesis, we evaluated decellularized tobacco hairy roots as a novel culture platform for hFFs, which served as an analog for other human cell types such as hMSCs. The hairy roots were genetically modified to express enhanced green fluorescent protein (EGFP). The hairy roots were mechanically decellularized via lyophilization followed by chemical treatment with DNase I to remove the DNA. The DNase I treatment resulted in 99% DNA removal, resulting in a DNA content less than 50 ng/mg tissue. This threshold level is considered to be sufficiently decellularized for reseeding with human cells in clinical and tissue engineering applications. Quantification of protein content showed 20% protein retention after decellularization of the hairy roots. Fluorescence microscopy confirmed removal of nuclei and retention of EGFP within the matrices. To realize this matrix as a mammalian cell expansion platform, we evaluated matrix degradation using cellulase, the in vitro biocompatibility of cellulase, and the ability to detach viable hFFs from the matrix. First, a degradation protocol for the tobacco hairy root matrices was developed using cellulase as the hydrolytic enzyme as an alternative means of cell dissociation. The enzymatic degradation resulted in approximately 30% mass loss of the hairy root matrices in physiological conditions. In vitro biocompatibility experiments showed no adverse effects on hFF viability. Fluorescence imaging confirmed that the hFFs attached to the hairy root matrices. Subsequently, cells could be detached from the matrices using trypsin or the plant degrading enzyme, cellulase. The detached cells remained viable and could continue to be cultured on standard tissue culture plastic. Our studies show that decellularized tobacco hairy root matrices could be used for the support of human cell culture.

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