Development of a platform for studying astrocyte mechanobiology in 3D culture

Abstract

Purpose : Optic nerve head (ONH) astrocytes are known to transition to a reactive phenotype in glaucoma, presumably as a response to mechanostimulation due to intraocular pressure (IOP). However, the mechanisms of how astrocytes respond to such stimulation are unclear. To date, ONH astrocyte mechanobiology has only been studied in 2D culture which primes astrocyte reactivity, confounding studies of astrocyte mechanobiology (East+, J Tissue Eng Reg Med 2009). We hypothesize that a 3D culture system, where biomechanical loads mimicking effects of IOP are applied, offers an improved system for studying astrocyte reactivity.

Methods : DI-TNC1 rat cortical astrocytes were used to establish proof of principle. Cells were seeded in gels (1x106 cells/ml in 2mg/ml Collagen Type I) and maintained in static culture for 7 days prior to testing. Gels were cyclically compressed by 10% for 24, 48 and 72 hrs at 1Hz. Western blot (WB) and flow cytometry (FC) analyses measured expression of GFAP, vimentin and s100β in compressed and control gels, and cells grown in 2D on tissue culture treated polystyrene. Microscopy was performed on phalloidin stained gels (n=3) to examine cell alignment. One-way ANOVA was used for statistical analysis.

Results : A significant difference in GFAP expression was found between 2D and 3D controls (fig A: WB *p=0.01, B: FC *p=0.03). By WB, GFAP expression was significantly increased in compressed vs. control gels at 24 (*p=0.02) and 72 hrs (**p<0.001); similarly by FC for 24 (p=0.03) and 72 hrs (p< 0.001). No statistical difference was found at 48 hrs. GFAP expression significantly increased between 24 and 72 hrs (WB p<0.001, FC p=0.02). Similar statistically significant trends were found for vimentin and s100β expression. Cells in compressed gels were found to align in a preferred direction compared to the random alignment in static controls (p<0.001).

Conclusions : Our 3D platform offers a less reactive environment for astrocytes compared to 2D. The applied compressive load, mimicking IOP loading, increased astrocyte reactivity vs. static controls, demonstrating mechanically-modulated astrocyte reactivity. Further morphological and protein quantifications are in progress to better understand 3D astrocyte mechanobiology.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

Fig (A) Percentage GFAP expression from WB (n=6) and (B) fluorescence intensity for GFAP from FC (n=3) for compressed (black) and control (grey) gels at 24, 48 and 72 hrs and for 2D culture (white)