Aggregates-on-chip: a microfluidic device for crowding plant cells in culture.

Introduction: Microfluidic devices enable the study of cellular properties in a highly controlled environment while allowing close observation. One important aspect of a plant cell's environment is the influence of neighboring cells on cell growth through chemical signals and mechanical forces. However, while the use of microfluidics in plant cell biology has expanded in recent years, very few systems provide cell-cell contact together with long-term growth and imaging. Methods: Here, we develop a microfluidic device that uses arrays of cylindrical cups to gently capture and confine multiple protoplasts into stable aggregates, generating a constrained microenvironment with cell-to-cell contact. Results: The device addresses a key challenge wherein aggregating cells can create hypoxic, media-starved environments that rapidly lead to cell death. The porous micro-post design supported sustained media flux through confined aggregates, reducing limitations typically associated with dense cell clustering. The platform maintained sterile culture conditions and enabled convenient media switching, as demonstrated by hormone treatment and salt-stress assays. Protoplasts from Arabidopsis thaliana and Zea mays remained viable for more than two weeks. Over this period, cells showed growth, cell wall formation, and cell divisions, with divisions occurring significantly more frequently in crowded aggregates than in isolated cells. Discussion: This aggregates-on-chip platform provides a controlled and live-imaging system for studying how cell-cell contact and microenvironmental cues influence plant protoplast behavior. By integrating physical confinement, continuous perfusion, and chemical perturbation, the device offers a technical advance for long-term studies of plant cell growth, regeneration, division, and stress responses.