Reversible Intracellular Gelation of MCF10A Cells Enables Programmable Control Over 3D Spheroid Growth

In nature, some organisms survive extreme environments by inducing a biostatic state wherein cellular contents are effectively vitrified. Recently, a synthetic biostatic state in mammalian cells is achieved via intracellular network formation using bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions between functionalized poly(ethylene glycol) (PEG) macromers. In this work, the effects of intracellular network formation on a 3D epithelial MCF10A spheroid model are explored. Macromer-transfected cells are encapsulated in Matrigel, and spheroid area is reduced by approximate to 50% compared to controls. The intracellular hydrogel network increases the quiescent cell population, as indicated by increased p21 expression. Additionally, bioenergetics (ATP/ADP ratio) and functional metabolic rates are reduced. To enable reversibility of the biostasis effect, a photosensitive nitrobenzyl-containing macromer is incorporated into the PEG network, allowing for light-induced degradation. Following light exposure, cell state, and proliferation return to control levels, while SPAAC-treated spheroids without light exposure (i.e., containing intact intracellular networks) remain smaller and less proliferative through this same period. These results demonstrate that photodegradable intracellular hydrogels can induce a reversible slow-growing state in 3D spheroid culture. Intracellular crosslinking in MCF10A spheroids is achieved via transfection of reactive poly(ethylene glycol) macromers, inducing a biostatic state. The intracellular network increases the proportion of quiescent cells and decreases the growth of Matrigel-encapsulated spheroids. These effects can be reversed using photodegradation, enabling a user-directed method to control proliferation and cell state in 3D spheroid culture.image