Date of Award

Summer 2024

Language

English

Embargo Period

8-9-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Nanoscale Science and Engineering

Program

Nanoscale Engineering

First Advisor

Susan Sharfstein

Second Advisor

Yubing Xie

Committee Members

Susan Sharfstein; Yubing Xie; Melinda Larsen; Andres Melendez; Nathaniel Cady

Keywords

fibrosis, senescence, radiation, aging, salivary gland, spheroid co-culture

Subject Categories

Molecular, Cellular, and Tissue Engineering

Abstract

Patients with head and neck cancer often suffer collateral radiation damage to the salivary glands, leading to fibrosis. Histological examinations indicate that radiation encourages fibrosis by promoting cellular changes leading to both senescent and myofibroblast phenotypes, coupled with a significant loss of regenerative and acinar cells. Chronic hypofunction remains a primary concern, even with modified radiation approaches to sparing salivary glands. Current treatments primarily offer symptomatic relief without rectifying irreversible glandular tissue damage. This limitation arises, in part, from a deficiency in predictive preclinical models to study this multifaceted problem.

We introduce a 3D in vitro microtissue model to address this limitation, employing a scaffold-free co-culture of stromal cells, such as NIH-3T3 fibroblasts and salivary epithelial cells, such as SCA-9 cells. Our methods prioritize generating salivary gland spheroids less than 200 µm in diameter, ensuring optimal nutrient diffusion and preventing necrotic core development. The formation process involves precise controls on parameters such as initial cell count, cell solution volume, culture duration, and spheroid formation technique (e.g., hanging drops, non-adhesive microwells). We have also devised assays tailored for 3D spheroids, encompassing various staining techniques for cell viability, apoptosis, senescence, fibrosis, and immunocytochemistry analysis of specific markers.

When exposed to clinically relevant X-ray radiation doses, these microtissues showed cell death and apoptosis, especially on the spheroid's surface, and manifested changes in collagen type I fibers and α-smooth muscle actin (α-SMA) within a sixteen-day frame.

Further, we sought to examine the effects of a preexisting senescent cell burden on our 3D microtissue model by exposing it to senescence-conditioned media before radiation exposure. This experiment incorporated additional assays, including 3D assays of cell viability, apoptosis, DNA damage, and senescence and additional assays such as immunocytochemistry analysis of fibrosis markers and picrosirius red staining of collagen fibrils to evaluate fibrosis-related ECM changes.

In conclusion, we have made considerable progress in creating a 3D in vitro model to study radiation-induced salivary gland fibrosis and the role that senescent cell burden plays in its development. This novel tool promises to enhance our comprehension of salivary gland fibrosis mechanisms and pave the way for more effective therapeutic interventions, drug evaluations, and exploration of new treatment approaches.

License

This work is licensed under the University at Albany Standard Author Agreement.

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