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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.
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This content will become publicly available on July 1, 2025
CellMag‐CARWash: A High Throughput Droplet Microfluidic Device for Live Cell Isolation and Single Cell Applications
Abstract The recent push toward understanding an individual cell's behavior and identifying cellular heterogeneity has created an unmet need for technologies that can probe live cells at the single‐cell level. Cells within a population are known to exhibit heterogeneous responses to environmental cues. These differences can lead to varied cellular states, behavior, and responses to therapeutics. Techniques are needed that are not only capable of processing and analyzing cellular populations at the single cell level, but also have the ability to isolate specific cell populations from a complex sample at high throughputs. The new CellMag‐Coalesce‐Attract‐Resegment Wash (CellMag‐CARWash) system combines positive magnetic selection with droplet microfluidic devices to isolate cells of interest from a mixture with >93% purity and incorporate treatments within individual droplets to observe single cell biological responses. This workflow is shown to be capable of probing the single cell extracellular vesicle (EV) secretion of MCF7 GFP cells. This article reports the first measurement of β‐Estradiol's effect on EV secretion from MCF7 cells at the single cell level. Single cell processing revealed that MCF7 GFP cells possess a heterogeneous response to β‐Estradiol stimulation with a 1.8‐fold increase relative to the control.
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- Award ID(s):
- 1921677
- PAR ID:
- 10539875
- Publisher / Repository:
- Advanced Biology
- Date Published:
- Journal Name:
- Advanced Biology
- Volume:
- 8
- Issue:
- 7
- ISSN:
- 2701-0198
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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