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Abstract Macrophages hold vital roles in immune defense, wound healing, and tissue homeostasis, and have the exquisite ability to sense and respond to dynamically changing cues in their microenvironment. Much of our understanding of their behavior has been derived from studies performed using in vitro culture systems, in which the cell environment can be precisely controlled. Recent advances in miniaturized culture platforms also offer the ability to recapitulate some features of the in vivo environment and analyze cellular responses at the single‐cell level. Since macrophages are sensitive to their surrounding environments, the specific conditions in both macro‐ and micro‐scale cultures likely contribute to observed responses. In this study, we investigate how the presence of neighboring cells influence macrophage activation following proinflammatory stimulation in both bulk and micro‐scale culture. We found that in bulk cultures, higher seeding density negatively regulated the average TNF‐α secretion from individual macrophages in response to inflammatory agonists, and this effect was partially caused by the reduced cell‐to‐media volume ratio. In contrast, studies conducted using microwells to isolate single cells and groups of cells revealed that increasing numbers of cells positively influences their inflammatory activation, suggesting that the absolute cell numbers in the system may be important. In addition, a single inflammatory cell enhanced the inflammatory state of a small group of cells. Overall, this work helps to better understand how variations of macroscopic and microscopic culture environments influence studies in macrophage biology and provides insight into how the presence of neighboring cells and the soluble environment influences macrophage activation. 

Macrophages play a key role in the innate immune system, and their activation is tightly regulated to avoid excess and harmful inflammation. Studies have revealed the roles of soluble and adhesive cues in the regulation of macrophage polarization. Furthermore, recent studies also show that macrophage signaling within a population is critical for coordinating a collective response [1, 2]. However, how such coordination arises from groups of cells, and how the collective behavior of small groups of cells compared to single, isolated cells, remains to be investigated. In this study, we attempt to address this problem by utilizing a microwell-based platform to probe the responses of cells in isolation versus cells in small groups following a pro-inflammatory stimulation. Our study suggests that expression of the inflammatory marker iNOS depends on the communication among groups of cells, and this regulation may also depend on the absolute cell numbers. This experimental platform may help further explore different mechanisms utilized to regulate collective inflammatory responses.