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Droplet encapsulation improves accuracy of immune cell cytokine capture assays
Yuan Yuan1, Julie Brouchon, J Mauricio Calvo-Calle
1Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.
Insights
Encapsulating immune cells in microfluidic droplets improves cytokine capture assays. This method significantly reduces false positives and negatives, enhancing the accuracy of immune response characterization and enabling precise cell isolation.
Area of Science:
- Immunology
- Microfluidics
- Cell Biology
Background:
- Quantifying cell-secreted molecules like cytokines is crucial for understanding immune responses.
- Existing cytokine capture assays can suffer from cytokine diffusion, leading to inaccurate results.
- This diffusion causes false positives (identifying non-secreting cells as responders) and false negatives (missing true responders).
Purpose of the Study:
- To develop a method to limit cytokine diffusion during capture assays.
- To improve the accuracy and sensitivity of single-cell immune response detection.
- To enable the isolation of activated immune cells post-assay.
Main Methods:
- Encapsulation of single immune cells (NK-92 MI) and target cells (K562) within microfluidic droplets.
- Performing cytokine capture assays (IFN-γ) within these droplets to confine secreted molecules.
- Utilizing droplet release for subsequent Fluorescence-Activated Cell Sorting (FACS) isolation.
Main Results:
- Droplet encapsulation effectively prevents cytokine diffusion, reducing background noise.
- Demonstrated significant reduction in false positives (98% to 1%) and false negatives (94% to 2%).
- Enabled successful isolation of highly enriched populations of activated effector immune cells via FACS.
Conclusions:
- In-droplet cytokine capture assays using microfluidics offer superior accuracy over traditional methods.
- This technique dramatically enhances the detection of true positive and negative cells in immune assays.
- Droplet encapsulation is a versatile approach for improving various single-cell secretion assays.
Abstract:
Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay.
