An automated pipeline for tracking and measuring cell spheroids encapsulated in 3D hydrogel systems

Additionally, we established a method for identifying and tracking migration trajectories of protruded cell clusters that detached from spheroids into the hydrogel. For a comparative hydrogel analysis, fluorescent beads were encapsulated in the ionically crosslinked xanthan gum–alginate (XG–Alg) and photocrosslinked methacrylate hyaluronic acid (HAMA). For pipeline validation, human mesenchymal stem cell spheroids were encapsulated in XG–Alg hydrogel.

By employing our pipeline, a high dynamicity and intense swelling effect were detected within XG–Alg, while HAMA remained stable, without noticeable movements up to 60 h. Accurate imaging and tracking detected several spheroid morphological changes, including reversible spheroid-ellipsoid shapes, axis rotational motion, outermost layer movements, spheroid fusion, and a spheroid migration speed of approximately 1.3 μm h−1.

Protruded cell clusters were detected in high numbers (83–173 per spheroid), migrating arbitrarily into the hydrogel (16◦–311◦), with an average speed of approximately 11.4 μm h−1. Our results indicate that this automated pipeline can facilitate the understanding of several cellular dynamic events with high accuracy and low manual interference, which are essential for scaling up tissue engineering and other advanced applications such as drug screening platforms.