Efficient release of immunocaptured cells using coiled-coils in a microfluidic device

Abstract

Label-free and affinity-based cell separation allows highly specific cell capture through simple procedures, but it remains a major challenge to efficiently release the captured cells without changing their structure, phenotype, and function. We report a microfluidic platform for label-free immunocapture of target cells and efficient release of the cells with minimal biochemical and biophysical perturbations. The method capitalizes on self-assembly of a pair of heterodimerizing coiled-coils, A and B. Target cells are captured in microchannels functionalized with an antibody and A and efficiently released by a liquid flow containing B-PEG (a conjugate of B and polyethylene glycol) at a controlled, low shear stress. The released cells have no antibodies attached or endogenous surface molecules cleaved. In a model system, human umbilical vein endothelial cells (HUVECs) were isolated from a mixture of HUVECs and human ovarian carcinoma cells. The capture selectivity, capture capacity, and release efficiency were 96.3% ± 1.8%, 10 735 ± 1897 cells per cm², and 92.5% ± 3.8%, respectively, when the flow was operated at a shear stress of 1 dyn cm^-2. The method can be readily adapted for isolation of any cells that are recognizable by a commercially available antibody, and B-PEG is a universal cell-releasing trigger.

Fig. 1. Schematic of surface modification and cell separation in a microfluidic channel.

Fig. 2. Capture of HUVECs (red) from a mixture of HUVECs and OVCAR3 cells (green). (a) The mixture was injected into a microchannel functionalized with an anti-CD31 antibody and coiled-coil A. (b) Non-captured cells in (a) was rinsed off with a slow flow of medium imposing a shear stress of 0.5 dyn cm⁻² for 4 min, revealing selective capture of HUVECs. (c) In a control microchannel functionalized with A alone, few cells of either type adhered after rinsing. (d) In a control microchannel with an unmodified surface, both cell types adhered after rinsing. The scale bars are 200 μm.

Fig. 3. Capture selectivity (a) and capture capacity (b) of HUVECs in microchannels functionalized with an anti-CD31 antibody and coiled-coil A when non-captured cells were removed with a flow of medium imposing various shear stresses. The durations of the flow were 4, 2, and 1 min for 0.5, 1, and 2 dyn cm⁻², respectively, to keep the volume of the liquid used to remove non-captured cells the same in different conditions. Error bars represent standard deviations, n = 4.

Fig. 4. Release of HUVECs captured in microchannels functionalized with an anti-CD31 antibody and coiled-coil A. The release experiments were conducted at a flow rate imposing a shear stress of 1 dyn cm⁻². The durations for the release experiments were 10 min for 400 μM B-PEG or PEG and 20 min for 200 μM B-PEG. (a) and (c) HUVECs captured in microchannels. (b) The captured HUVECs in (a) were efficiently released with a flow of medium containing 400 μM B-PEG. (d) The captured HUVECs in (c) were not efficiently released with a flow of medium containing 400 μM PEG. (e) Quantitative analysis of the release efficiency of captured HUVECs. Error bars represent standard deviations, n = 3. **p < 0.01, Student's t-test. The scale bars are 200 μm.

Fig. 5. Release efficiency of captured HUVECs with a flow of medium not containing B-PEG at various shear stresses. The duration of the release experiments was 1 min. Error bars represent standard deviations, n = 3.

Fig. 6. Culture of released HUVECs. (a–c) The released cells 1 h (a), 3 h (b), and overnight (c) after replating. (d) The fluorescence image of the released cells cultured overnight and stained with a live cell tracker CellTracker™ Orange CMTMR dye. The scale bars are 200 μm.