Plant-Based Scaffolds Modify Cellular Response to Drug and Radiation Exposure Compared to Standard Cell Culture Models

Abstract

Plant-based scaffolds present many advantages over a variety of biomaterials. Recent studies explored their potential to be repopulated with human cells and thus highlight a growing interest for their use in tissue engineering or for biomedical applications. However, it is still unclear if these in vitro plant-based scaffolds can modify cell phenotype or affect cellular response to external stimuli. Here, we report the characterization of the mechano-regulation of melanoma SK-MEL-28 and prostate PC3 cells seeded on decellularized spinach leaves scaffolds, compared to cells deposited on standard rigid cell culture substrate, as well as their response to drug and radiation treatment. The results showed that YAP/TAZ signaling was downregulated, cellular morphology altered and proliferation rate decreased when cells were cultured on leaf scaffold. Interestingly, cell culture on vegetal scaffold also affected cellular response to external stress. Thus, SK-MEL-28 cells phenotype is modified leading to a decrease in MITF activity and drug resistance, while PC3 cells showed altered gene expression and radiation response. These findings shed lights on the decellularization of vegetal materials to provide substrates that can be repopulated with human cells to better reproduce a soft tissue microenvironment. However, these complex scaffolds mediate changes in cell behavior and in order to exploit the capability of matching physical properties of the various plant scaffolds to diverse physiological functionalities of cells and human tissue constructs, additional studies are required to better characterize physical and biochemical cell-substrate interactions.

Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds

“…Using moderate pressure and a PAA entrainer, the dissolving power of the fluid effectively removed plant cellular content to generate a biocompatible scaffold in mere hours. The gold standard chemical processing of plant material decellularization is time-consuming, taking several days (Table S1) 6,8,9,11,12,14 . Leaves are mechanically agitated in a bath of SDS/detergent for anywhere from 2 to 5 days.…”

“…Although we did not assess scaffold immunogenicity in this study, these results suggest that scCO 2 decellularization of vegetal material is indeed a non-toxic approach to provide biocompatible scaffolds for human cell culture. Several types of mammalian cells have been cultured on chemically decellularized vegetal scaffolds, including cancer cell lines, mesenchymal stem cells, mouse fibroblasts, human-induced pluripotent stem cell-derived cardiomyocytes, and more 6,8,9,11,12 . Although we foresee that scCO 2 decellularized scaffolds could also accommodate a diversity of cell types, additional investigations are required to confirm this assumption.…”

“…DNA and protein quantification. Leaf material (25 mg) was flash-frozen using liquid nitrogen and ground by mortar and pestle as previously reported 11 . Resulting material was transferred to a microcentrifuge tube for DNA and protein quantification.…”

“…were cultured in Eagle's Minimum Essential Medium, supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin and maintained at 37 °C in 5% CO 2 atmosphere. All leaf structures were functionalized with collagen and fibronectin proteins as previously described 11 . Briefly, scaffolds were incubated in 50 ug/ml of collagen I (A1048301, Thermo Scientific) in 20 mM acetic acid solution for 4 h, followed by two washes in PBS and a final wash in complete medium.…”

“…Interestingly, plant scaffolds have been tested in vivo and found to be pro-angiogenic, where blood vessels grew throughout the biomaterial demonstrating that the scaffold in vivo generates a low inflammatory response, while promoting cell invasion and extracellular matrix (ECM) deposition 7 . In addition, the stiffness of decellularized plant tissue has also been shown to match the stiffness of specific human anatomical tissue sites 11 .…”