(F-205) Engineering a co-cultured 3D in vitro model of cervical dysplasia for testing novel therapeutics

Introduction:: Cervical cancer is highly prevalent in low and middle-income communities, causing an estimated 604,000 new cases and 342,000 deaths each year. Due to inadequate access to early detection and treatment, women in these regions are at a higher risk of developing cervical cancer and have lower survival rates. We tackled this issue by devising a low-cost ethyl cellulose-ethanol ablation therapy. However, no existing animal or in vitro models evaluate its effectiveness, microenvironment impact on ethanol diffusion, or healthy cell preservation. Thus, we created a 3D in vitro model of cervical dysplasia containing cancerous and healthy cells to replicate the microenvironment's architecture and geometry. This model will enable future tests of this novel therapy.




Materials and Methods:: Constructs were fabricated in 24-well plates with human fibroblasts, keratinocytes, and a human cervical cancer cell line, SiHa (Fig 1A). To form the outside portion of the construct, we designed a silicone mold, added fibroblasts suspended in GelMa (8.7% w/v, 17 mg/mL LAP) and photo-crosslinked the construct at 405 nm (Figure 1B). The construct was then placed in a well and the inside portion was formed by adding SiHa cells suspended in GelMa (8.7% w/v, 17 mg/mL LAP) and photo-crosslinked at 405 nm (Figure 1C). Lastly, keratinocytes were added on top to simulate normal epithelium, and the constructs were covered in media. To capture the morphology of the 3D model and calculate cell coverage for each cell type, we obtained images at 0, 24, and 48 hours post-culture using a z-stack montage.


Results, Conclusions, and Discussions:: Results. Our 3D model captured the architecture and geometry of cervical dysplasia, with a central lesion and healthy fibroblasts and keratinocytes surrounding it. We observed even cell coverage both radially and vertically throughout the construct in both the center and the outside regions of the model. After 48 hours of culture, fibroblasts, and keratinocytes were the predominant cell types in the outside region, mimicking healthy tissue, with minimal invasion of SiHa cells (Figure 1D). In contrast, in the inside region mimicking a cervical lesion, keratinocytes, and SiHa cells were the predominant cell types with minimal fibroblasts present (Figure 1E).

Conclusion. We successfully developed a 3D in vitro model of cervical dysplasia that closely mimics the tumor microenvironment, allowing for the evaluation of novel therapeutics such as ethyl cellulose-ethanol ablation. In future studies, we plan to use this model to evaluate the efficacy of our novel therapeutic and identify patient-specific parameters that must be accounted for to preserve the surrounding healthy tissue.


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