Following the first recorded description of a cell in Robert Hooke’s Micrographia in 1665, microscopy has empowered the study of life in miniature. Modern cell culture devices, including culture plates and flasks, are designed to meet the demands of high-resolution imaging, employing surface-treated glass coverslips and optically transparent plastics as cell-adherent substrates. These relatively simple devices, when deployed alongside contemporary techniques including computational imaging, fluorescent labeling, and super-resolution microscopy (SRM), have enabled researchers to investigate cellular mechanisms ex vivo with remarkable precision. However, mounting evidence has indicated that the infamous rift between in vivo and ex vivo cellular responses is a consequence of the monolayer itself; cells grown in cover culture on a rigid substrate often do not reflect their in vivo analogues.
In contrast to monolayer culture which spreads in an often-predictable fashion, cells and tissues seeded into 3D culture remodel their microenvironment and adopt fascinating morphologies as they adapt to their culture conditions. This is readily observable even in rudimentary cell aggregate culture where samples will depart from their typical spherical morphologies to prevent the formation of a necrotic core as a result of diffusion-limited transport. More complex resected tissue cultures, exemplified here by mouse colorectal explants, illustrate the dynamism of 3D culture by responding to distinct media compositions with varying degrees of stem cell differentiation, E-Cadherin expression, and structural reorganization.
The potency of these imaging techniques is further expanded with the introduction of collagen-1 bioconjugated LLS™ microgel particles which facilitate cellular adhesion, as demonstrated by the spreading of malignant mouse glioma (KLuc) cells from a BioPelle-printed spheroid in a field of LLS™particles. Fluorescently labels can be added to LLS™ microgel particles in addition to surface conjugations which can support spatial mapping of experiments and improve observation of cell-particle interactions. Metastatic events and migratory behaviors can easily be visualized in LLS™ while retaining perfusion and mechanical stability.
Continuous in situ imaging in LLS™ cell culture medium presents an attractive platform for observing co-culture interactions in a 3D environment. Immune cell killing assays, here exemplified by checkpoint inhibitor-treated autologous immune cells attacking a cancerous tumoroid, are facilitated by the transparency of the LLS™ microgel particles and the stability of experiments composed within. Continuous live-cell imaging of these systems enables modeling of immune cell proliferation and killing rates,3D-tracking of markedly active cells, and in situ cytokine concentration gradient profiling within the LLS™ medium. These analyses provide valuable metrics for the efficacy of immune cell-mediated therapies, and could ultimately inform patient treatment protocols for immunotherapy.
