Affinity flow fractionation of cells via transient interactions with asymmetric molecular patterns
Isolation of cells from complex mixtures such as blood and bone marrow is of immense importance in disease diagnosis1, 2, stem cell therapeutics3, genetic analysis4, and other applications. Microfluidic technologies for label-free separation of cells offer the advantages of simpler operation, lower...
| Main Authors: | , , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
Nature Publishing Group
2014
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| Online Access: | http://hdl.handle.net/1721.1/88239 https://orcid.org/0000-0003-0588-9286 https://orcid.org/0000-0002-5921-3436 |
| Summary: | Isolation of cells from complex mixtures such as blood and bone marrow is of immense importance in disease diagnosis1, 2, stem cell therapeutics3, genetic analysis4, and other applications. Microfluidic technologies for label-free separation of cells offer the advantages of simpler operation, lower cost, and faster time-to-result, and are emerging as important tools for point-of-care diagnostics5. Many of these technologies belong to a class of separations known as flow fractionations, wherein cells flowing in a microchannel are displaced perpendicular to the direction of flow under the action of a force, which results in continuous sorting. Currently, flow fractionation of cells is limited to long-range physical forces arising from dielectrophoresis, acoustophoresis, gravitational, magnetic, or inertial effects. The non-specific action of these long-range force fields limits the use of flow fractionation of cells to a few applications, while its extension to sorting based on molecular recognition requires pre-labeling of cells with magnetic or dielectric beads6. In contrast, transient interactions of molecules on the cell with adhesive molecules patterned asymmetrically on a surface can exert forces on the cell to deflect it perpendicular to the direction of fluid flow, without capture7, 8, 9, 10. This effect requires weak-affinity molecular interactions to avoid irreversible cell capture and provides a new paradigm for label-free flow fractionation of cells, called affinity flow fractionation (AFF).
Nature has evolved a number of molecules that exhibit weak, yet relatively specific adhesive interactions including bacterial adhesion molecules11, selectins involved in homing of circulating cells12, and MHC-II molecules on antigen presenting cells that exhibit weak affinity towards the T-cell receptor13. Among them, P-selectin is a model protein whose kinetics of interaction with its ligand are well characterized. P-selectin exhibits specificity for neutrophils over other leukocytes and is involved in recruitment of neutrophils during the early phase of inflammation14. A simple device for isolation of neutrophils from blood would be useful in a number of diagnostic applications including detection of sepsis15, discrimination between bacterial and viral infection16, and for HLA typing2.
Here we report affinity flow fractionation (AFF) of neutrophils from human blood using surfaces decorated with asymmetric patterns of P-selectin. The significance of the current method is that it is the first demonstration of flow fractionation of cells directly from blood purely based on molecular interactions, allowing very high rejection of non-interacting cells (> 5 log depletion of RBCs). The sorted leukocytes were highly enriched for viable, non-activated, and intact neutrophils with greater than 92% purity. Using a cell line that interacts with P-selectin to study the distribution of cells undergoing AFF, we developed a mathematical transport model that accounts for the key phenomena and accurately predicts the separation process. Finally, exploiting the activation-induced changes in interaction of neutrophils with P-selectin, we demonstrate the potential of using the current technology for developing quick point-of-care tests for detecting sepsis and other inflammatory conditions. |
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