Microfluidic-gradient generator for studying amoeboid cell chemotaxis (Hammer, DA)

Purpose: Microfabricated chambers have been used for the study of cell motility, particularly the motility of ameoboid cells of the immune system.(Irimia, 2006, Jeon, 2002) These cells display directional motion, known as chemotaxis, in defined gradients of chemokines, which results from the differential occupancy of receptors. The purpose of the chambers is to make well-defined spatial gradients that do not change with time, and to measure the directional motion of cells in defined gradients. We have been employing chambers with three inlet ports in which a different concentration of chemokine was delivered, originally developed by Jeon and co-workers. (Jeon, 2002, Jannat, 2011, Jannat, 2010, Ricart, 2011). Through mixing in serpentine channels, the gradient is well defined in the cell channel. The channel is bonded to a glass coverslip to which adhesion molecules have been coated, or which force array detectors are embedded, and the motion of cells is visualized by video microscopy.

Microfluidic gradient chamber (Ricart BG, John B, Lee D, Hunter CA, Hammer DA. ://000285688700012">Dendritic Cells Distinguish Individual Chemokine Signals through CCR7 and CXCR4. Journal of Immunology Jan 2011; 186 (1): 53-61). A. Design of the chamber, verified by COMSOL modeling. The chamber was used to make counter gradients of two chemokines. B and C show measurements of green and red fluorescent dyes in the channel, and D. illustrates the comparison between modeled and measured profiles.

Microfluidic gradient chamber (Ricart BG, John B, Lee D, Hunter CA, Hammer DA. Dendritic Cells Distinguish Individual Chemokine Signals through CCR7 and CXCR4. Journal of Immunology Jan 2011; 186 (1): 53-61). A. Design of the chamber, verified by COMSOL modeling. The chamber was used to make counter gradients of two chemokines. B and C show measurements of green and red fluorescent dyes in the channel, and D. illustrates the comparison between modeled and measured profiles.

Method of Fabrication: The microfluidic-gradient generator is fabricated in polydimethylsiloxane (PDMS) using soft lithography, as described previously (Jeon et al., 2002). A high-resolution printer is used to generate a mask from a CAD file. The mask was used in 1:1 contact photolithography with a photoresist to generate a negative master, consisting of patterned photoresist on a 3-inch Si wafer. Positive replicas with embedded channels were fabricated by molding PDMS against the master. The PDMS replica and a glass microscope slide were activated by oxygen plasma treatment and then irreversibly bound. Inlet and outlet ports are punched out of the PDMS using a 20-gauge blunt-end needle. Inlet flows is controlled by a syringe pump.

DC migration in countergradients of CCL19 (red) and CxCL12 (blue) show that cells can ignore gradients when concentrations are too high, leading to directional motion. Cells that experience equistimulation from each gradient (black) meander back and forth in response to each chemokine (Ricart BG, John B, Lee D, Hunter CA, Hammer DA. ://000285688700012">Dendritic Cells Distinguish Individual Chemokine Signals through CCR7 and CXCR4. Journal of Immunology Jan 2011; 186 (1): 53-61.

DC migration in countergradients of CCL19 (red) and CxCL12 (blue) show that cells can ignore gradients when concentrations are too high, leading to directional motion. Cells that experience equistimulation from each gradient (black) meander back and forth in response to each chemokine (Ricart BG, John B, Lee D, Hunter CA, Hammer DA. Dendritic Cells Distinguish Individual Chemokine Signals through CCR7 and CXCR4. Journal of Immunology Jan 2011; 186 (1): 53-61.

Results/Use: The Hammer laboratory has used the microfluidic gradient generator to study the directed motion of both neutrophils and dendritic cells. The microfluidic gradient chamber has been integrated with various devices to measure forces during cell crawling, including bead based traction force microscopy (Jannat, 2011) and microfabricated post array detectors (mPADs). (Ricart, 2011)  Furthermore, the device has been used to interrogate the effects of chemokine countergradients on the motility of dendritic cells. (Ricart, 2011) These cells possess two types of chemokine receptors, and countergradients of chemokine allow investigation of which chemokine is more potent, and at what concentration and gradient magnitude.

References

Irimia D, Liu SY, Tharp WG, Samadani A, Toner M, Poznansky MC. Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients. Lab Chip Feb 2006; 6 (2): 191-198.

Jannat RA, Dembo M, Hammer DA. Traction forces of neutrophils migrating on compliant substrates. Biophys J Aug 3 2011; 101 (3): 575-584.

Jannat RA, Robbins GP, Ricart BG, Dembo M, Hammer DA. Neutrophil adhesion and chemotaxis depend on substrate mechanics. J Phys Condens Matter May 19 2010; 22 (19): 194117.

Li Jeon N, Baskaran H, Dertinger SK, Whitesides GM, Van de Water L, Toner M. Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device. Nat Biotechnol Aug 2002; 20 (8): 826-830.

Ricart BG, John B, Lee D, Hunter CA, Hammer DA. Dendritic cells distinguish individual chemokine signals through CCR7 and CXCR4. J Immunol Jan 1 2011; 186 (1): 53-61.

Ricart BG, Yang MT, Hunter CA, Chen CS, Hammer DA. Measuring traction forces of motile dendritic cells on micropost arrays. Biophys J Dec 7 2011; 101 (11): 2620-2628.

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