"Introduction: Spinal cord injury (SCI) is a condition that has caused disability worldwide. However, progress in the development of effective therapies to treat SCI-associated neuropathologies has been slow so far. In particular, a few days after injury, a scar made of two components, an outer glial scar and an inner fibrotic scar composed of extracellular matrix (ECM) proteins, is formed and some inhibitory ECM molecules become upregulated. Changes in the composition and structure of the ECM primarily contribute to regeneration failure in the injured spinal cord. Hence, developing model systems that enable cells to produce ECM within in vitro assays is a critical need to test therapeutic strategies for targeting inhibitory molecules. One proposed strategy is to expose cells to an environment crowded with adequate macromolecules to mimic the physiological cellular milieu. In this study, astrocytes were cultured in the presence of macromolecular crowders (MMC) which can dramatically improve the deposition of a variety of ECM molecules. The ECM deposition can be modulated by the biophysical properties of the crowder. Herein, we assessed the influence of different concentrations of MMCs, dextran sulphate (DxS), and carrageenan (Ca), on Neu7 astrocyte cell morphology, metabolic activity, viability, and collagen type I (Col I) deposition. Furthermore, the effect of optimized MMCs concentration on the expression of different ECM proteins of the glial and fibrotic scar was investigated.
Methodology: Cells were cultured in DMEM-low glucose supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin. The cells were seeded at a density of 25,000 cells/cm2 in the conventional medium. After 24 hours, the medium was replaced with the same conventional medium (control group) and medium with 100 mM ascorbic acid and different concentrations of DxS and Ca. Cells were grown for 5 days in each condition. Phase-contrast images were captured using an inverted microscope at different time points (3 and 5 days) to evaluate the influence of DxS on cell morphology. In addition, the alamarBlueTM assay was used to assess the effect of DxS on cell metabolic activity. On day 5, the viability of cells was evaluated by Live/Dead staining and then analyzed by fluorescence microscopy. Immunocytochemistry (ICC) was performed to assess deposition of Col I and other proteins of the glial and fibrotic scar, such as glial fibrillary acidic protein (GFAP), chondroitin sulphate proteoglycan proteins (NG2 and CS56), fibronectin, and collagen IV.
Result and Conclusion: Phase contrast images revealed that MMCs did not have any impact on cell morphology, and the cells maintained their spindle-shaped morphology at all time points. Additionally, the metabolic activity and cell viability of cells cultured in medium with MMCs were identical to those cultured in conventional medium. ICC analysis demonstrated that at 100 mg/ml MMCs, the Col I deposition significantly increased compared to no MMCs. Ca and DxS enhanced the accumulation of cell-secreted molecules which formed a secreted matrix. This novel culture system opens up a new avenue to the screening of therapeutic compounds to modulate the environment of the glial and fibrotic scar in spinal cord repair."