Multicellular spheroids have emerged as a promising tool for drug delivery, cancer therapy, and tissue engineering. Compared to 2D monolayers, spheroids provide a more realistic representation of the 3D cellular environment, enabling better understanding of the signaling cascades and growth factors involved in vivo. The formation of in vitro spheroids involves the aggregation of several cells that proliferate to grow into larger spheroids. Biophysical cues provide crucial information for the cells to assemble into 3D structures. We used suspended fiber networks to demonstrate a new way to form and spatially pattern spheroids comprised of human pericytes. We show that fiber architecture (aligned vs. crosshatched), diameter (200, 500, and 800 nm), and contractility influence spheroids in their spontaneous formation, growth, and maintenance, and report a dynamic trade of cells between adjacent spheroids through remodeled fiber networks. We found that aligned fiber networks promoted spheroid formation independent of fiber diameter, while large-diameter crosshatched networks abrogated spheroid formation, promoting growth of 2D monolayers. Thus, a mixture of diameters and architectures allowed for spatial patterning of spheroids and monolayers within a single system. We further quantified various dynamic interactions and describe the forces involved during spheroid formation, cell efflux from spheroids, and show the loss and recovery of spheroid forces with pharmacological perturbation of Rho-associated protein kinase (ROCK). Thus, we develop new insights on the dynamics of spheroids using suspended fiber networks of varying diameters and architectures, with the potential to connect matrix biology with developmental, disease, and regenerative biology. / Master of Science / In recent years, studies involving multicellular spherical aggregates or 'spheroids' have gained popularity since they capture the 3D cellular environments seen within the body more realistically when compared to 2D cell culture systems (such as monolayers) traditionally used for biological studies. These spheroids resemble organs and tissues in terms of their structure and function better and are increasingly being studied for an array of applications such as drug delivery, cancer therapy, as implants and in tissue regeneration and tissue engineering. The cellular microenvironment consists of fibrous proteins of varying diameter arranged in various geometric patterns, which can influence the growth and culture of spheroids. Here, we use our Spinneret-Based Tunable Engineered Parameters (STEP) technique to fabricate fibrous networks with precise control over fiber diameter and architecture and study how biophysical cues can influence the formation and culture of spheroids. Using aortic pericytes, we show that fiber architecture (aligned vs. crosshatched) and diameter (200, 500, and 800 nm) can control how pericytes aggregate into either 2D monolayers or 3D spheroids. We study the effect of each of these parameters to show that stiffer, denser fibers are robust networks which the cells refrain from remodeling, and thus lead to monolayers while more compliant and sparser networks are easily remodeled to promote spheroid formation. Thus, we spatially pattern a mixture of 3D spheroids and 2D monolayers in a single system by varying the parameters at different regions. We quantify various interactions such as spheroid formation, spheroid merging, spheroid migration, cell efflux from spheroids and the dynamic contractile forces exerted on the matrix by spheroids during these interactions. We also show that contractility has a major role in spheroid formation and to maintain their structure and look at the changes in the gene expressions associated with contractility during the formation and growth of spheroids. Thus, we develop new knowledge in controlling the growth of pericytes into 2D and 3D structures and show that our fiber networks can be an essential platform for studying spheroids.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/115851 |
| Date | 25 July 2023 |
| Creators | Sharma, Sharan |
| Contributors | Mechanical Engineering, Nain, Amrinder, Phillippi, Julie A., Paul, Mark R. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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