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Mitochondrial damage occurs in human trabecular meshwork (HTM) cells as a result of normal aging and in open angle glaucoma. Using an HTM cell model, we quantified mitochondrial function and ATP generation rates after dexamethasone (Dex) and TGF-β2 treatments, frequently used as in vitro models of glaucoma. Primary HTM cells were assayed for metabolic function using a Seahorse XFp Analyzer. We additionally assessed the mitochondrial copy number and the expression of transcripts associated with mitochondrial biogenesis and oxidative stress regulation. Cells treated with Dex, but not TGF-β2, exhibited a significant decrease in total ATP production and ATP from oxidative phosphorylation relative to that of the control. Dex treatment also resulted in significant decreases in maximal respiration, ATP-linked O2 consumption, and non-mitochondrial O2 consumption. We did not observe significant changes in the level of mitochondrial genomes or mRNA transcripts of genes involved in mitochondrial biogenesis and oxidative stress regulation. Decreased mitochondrial performance and ATP production are consistent with the results of prior studies identifying the effects of Dex on multiple cell types, including HTM cells. Our results are also consistent with in vivo evidence of mitochondrial damage in open-angle glaucoma. Overall, these results demonstrate a decrease in mitochondrial performance in Dex-induced glaucomatous models in vitro, meriting further investigation. 

3D cell culture models have gained popularity in recent years as an alternative to animal and 2D cell culture models for pharmaceutical testing and disease modeling. Polydimethylsiloxane (PDMS) is a cost-effective and accessible molding material for 3D cultures; however, routine PDMS molding may not be appropriate for extended culture of contractile and metabolically active tissues. Failures can include loss of culture adhesion to the PDMS mold and limited culture surfaces for nutrient and waste diffusion. In this study, we evaluated PDMS molding materials and surface treatments for highly contractile and metabolically active 3D cell cultures. PDMS functionalized with polydopamine allowed for extended culture duration (14.8 ± 3.97 days) when compared to polyethylamine/glutaraldehyde functionalization (6.94 ± 2.74 days); Additionally, porous PDMS extended culture duration (16.7 ± 3.51 days) compared to smooth PDMS (6.33 ± 2.05 days) after treatment with TGF-β2 to increase culture contraction. Porous PDMS additionally allowed for large (13 mm tall × 8 mm diameter) constructs to be fed by diffusion through the mold, resulting in increased cell density (0.0210 ± 0.0049 mean nuclear fraction) compared to controls (0.0045 ± 0.0016 mean nuclear fraction). As a practical demonstration of the flexibility of porous PDMS, we engineered a vascular bioartificial muscle model (VBAM) and demonstrated extended culture of VBAMs anchored with porous PDMS posts. Using this model, we assessed the effect of feeding frequency on VBAM cellularity. Feeding 3×/week significantly increased nuclear fraction at multiple tissue depths relative to 2×/day. VBAM maturation was similarly improved in 3×/week feeding as measured by nuclear alignment (23.49° ± 3.644) and nuclear aspect ratio (2.274 ± 0.0643) relative to 2x/day (35.93° ± 2.942) and (1.371 ± 0.1127), respectively. The described techniques are designed to be simple and easy to implement with minimal training or expense, improving access to dense and/or metabolically active 3D cell culture models. 

Photoaging is an important extrinsic aging factor leading to altered skin morphology and reduced function. Prior work has revealed a connection between photoaging and loss of subcutaneous fat. Currently, primary models for studying this are in vivo (human samples or animal models) or in vitro models, including human skin equivalents (HSEs). In vivo models are limited by accessibility and cost, while HSEs typically do not include a subcutaneous adipose component. To address this, we developed an “adipose-vascular” HSE (AVHSE) culture method, which includes both hypodermal adipose and vascular cells. Furthermore, we tested AVHSE as a potential model for hypodermal adipose aging via exposure to 0.45 ± 0.15 mW/cm2 385 nm light (UVA). One week of 2 h daily UVA exposure had limited impact on epidermal and vascular components of the AVHSE, but significantly reduced adiposity by approximately 50%. Overall, we have developed a novel method for generating HSE that include vascular and adipose components and demonstrated potential as an aging model using photoaging as an example.