2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study

Abstract

A physiologically relevant in vitro human-based model could be the 'gold standard' to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D.

Fig 1. 2D- and 3D-HTMC oxidative stress sensitivity.

Panel A- Confocal microscopy analyses of nucleus and cytoskeletal markers were performed on untreated and H₂O₂-treated 2D- and 3D HTMC cells after 72 hrs of experimental procedures. Representative images are related to immunoreactivity for To-Pro^TM and Phalloidin, as nuclear and cytoskeleton markers, respectively. Merged images showed cytoskeleton plus Nucleus. Fluorescence signals were captured at 60x magnification. Panel B- DCF assay. DCF assay was performed on untreated and H₂O₂ (U.T.)-treated 2D- and 3D HTMC cells and the fluorescence was recorded at 1, 2 and 4 hrs of experimental procedures. Data are expressed as % of ROS production in untreated HTMCs after 1hr and represent the mean ± SD of 3 independent experiments, each performed six times. White and grey bars = 2D and 3D cultures, respectively ***p <0.001 vs respective untreated cultures (1h); §§§p<0.001 3D-permanent H₂O₂ vs 3D-pulse H₂O₂; @@@p<0.001 3D permanent H₂O₂ vs 2D permanent H₂O₂; ++p<0.01, +++p<0.001 3D pulse H₂O₂ vs 2D pulse H₂O₂ (Two-way ANOVA followed by Bonferroni posttest).

Fig 2. Effects of chronic H₂O₂ treatment on HTMC metabolic activity.

Panel A- Mitochondrial respiratory functionality. MTT assay was performed in 2D- and 3D-HTMC exposed to H₂O₂ (500μM) for 24, 48 and 72 hrs. Panel B- Metabolic state of untreated and H₂O₂ treated 2D and 3D HTMC cultures, during experimental treatments, was evaluated by Alamar blue assay. Data are expressed as A.U. of MTT test and of resazurin reduction of each HTMC cultures, and represent the mean ± S.D of 3 separated experiments, in triplicate. *p <0.05 treated vs respective untreated cultures;°p<0.05,°°p<0.01 untreated 48 and 72 hrs vs untreated HTMCs 24 hrs; ^@@p<0.01 untreated 72 hrs vs untreated HTMCs 48 hrs; +p<0.01 treated 48 and 72 hrs vs treated HTMCs 24 hrs; ^ap<0.05, ^aap<0.01; ^aaap<0.001 treated 72 hrs vs treated HTMCs 48 hrs. (Two-way ANOVA followed by Bonferroni posttest).

Fig 3. Apoptosis array.

Analysis of anti- apoptotic and pro- apoptotic protein levels in 2D- and 3D-HTMC were performed after 48 (panel A) and 72h (panel B) of 500μM H₂O₂, by Human Antibody Array C1 (RayBio^® C-series). The light blue dotted line represents the protein level of untreated HTMC for each of the 43 proteins examined. For each time point twelve individual models were arrayed (six 2D-HTMC plus six 3D-HTMC) and per experiment the intensity of Positive Control Spot was used to normalize signal responses for comparison of results across multiple arrays. *p<0.05; **p<0.01; ***p<0.001 vs. respective untreated cultures (One-way ANOVA followed by Bonferroni posttest).

Fig 4. Induction of pro inflammatory factors by chronic H₂O₂ treatment.

Quantitative PCR gene expression analysis of 2D- and 3D-HTMC subjected 500μM for 48 h and 72 h. IL-1α IL-1β, and IL-6 (Panels A, B and C, respectively). Data are expressed as fold-increase relative to the 2D control at the same end-point and normalized to Ubiquitin housekeeping gene expression. Each bar represents the mean ± S.D. of three independent experiments performed in triplicate. (Panel D) The figures depicted are representative of at least three similar immunoblot analysis of NF-kB (p65), p-NF-kB (p65) protein levels in untreated HTMCs and treated HTMCs (H₂O₂) whole protein lysates at indicated time points. GAPDH was used as an internal control for equal protein loading on the gel. (Panel E) NF-kBp65 activation was evaluated in HTMC cells subjected to chronic treatment with H₂O₂ for 24, 48 and 72 hrs. The analysis was performed by immunoblotting and the bars represent the ratio of phosfoNF-kBp65/NF-kBp65, and are expressed as fold vs. untreated HTMC cultures. Data represent the mean ± S.D. of 3 independent experiments. ***p <0.001,* p <0.05 treated 3D-HTMCs vs. untreated 3D-HTMCs cells; ### p<0.001, # p <0.05 untreated 3D-HTMC vs. untreated 2D-HTMC cells; §§§p<0.001 treated 3D-HTMC vs. treated 2D-HTMC cells (Two-way ANOVA followed by Bonferroni posttests).