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Cassandra Roberge1 David Kingsley1 Denzel Faulkner1 C.J. Sloat1 Xavier Intes1 David Corr1

1, Rensselaer Polytechnic Institute, Troy, New York, United States

3D tissue-engineered in vitro models, particularly multicellular tumor spheroids (MCTSs), are being increasingly used to explore disease progression and novel therapeutic strategies, especially for oncological applications. Once grown to 0.03-0.5mm3 in volume, these avascular constructs begin mimicking several key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. However, a lack of standardization of MCTS fabrication has led to a large variety of “spheroid” or “organoid” models, many of which are unable to achieve the necessary morphology (i.e., size, sphericity) for physiologically-representative behavior. Herein, we investigate the potential of laser direct-write (LDW) bioprinting to generate MCTSs in microcapsules and compare these to the gold standard technique in the field: liquid overlay.
Liquid overlay samples were prepared from MDA-MB-231 triple-negative breast cancer cells utilizing non-adherent, U-shaped 96-well plates, seeded either with (+) or without (-) the addition of 5% Matrigel growth factor (solubilized basement membrane from mouse sarcoma). The resulting cellular aggregates were imaged through maturity over a 4-day growth period via Optical Coherence Tomography (OCT) to assess construct morphology. Microcapsule fabrication was initiated by LDW printing of 400μm-diameter alginate microbead arrays containing MDA-MB-231 cells. These beads were incubated (~7min) in a chitosan-polysaccharide bath to create an alginate–chitosan polyelectrolyte complex shell around each bead, and then bathed briefly in sodium citrate to sequestrate the calcium and liquefy the bead cores. Following fabrication, the capsules were imaged via OCT over a 14-day maturation period.
By day 4, OCT imaging of the liquid overlay samples revealed disk-like aggregates in the absence of Matrigel, with low sphericity (0.597±0.01) and large volumes (0.390±0.011mm3) (n=6), as quantified by Imaris image analysis software. However, in the presence of Matrigel, cells formed highly spherical aggregates (sphericity=0.902±0.01) with compacted volumes (0.235±0.027mm3). Day 14 microcapsule imaging revealed the formation of spheroids with comparable sphericity to the Matrigel+ samples (0.862±0.04) with volumes of 0.061±0.008mm3.
Utilizing OCT, we monitored the development of 3D tumor models, and quantitatively assessed their evolving structures. Our findings show that while samples fabricated via liquid overlay reach physiologically-relevant sizes, the addition of Matrigel is essential for producing accurate 3D morphologies. Additionally, MCTSs formed via LDW were able to mimic this spheroidal morphology without the addition of exogenous factors. These findings have direct implications on the utility of direct-written MCTSs as in vitro tumor models for cancer research.

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