Controlled viable release of selectively captured label-free cells in microchannels

Abstract

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.

Figures

Fig. 1

Thermoresponsive microfluidic chip developed for releasing selectively captured cells from blood. (A) The microfluidic chip was composed of three parallel channels (4 mm × 22 mm × 80 μm), one of which (middle channel) was used as the temperature indicator channel. Blood was introduced into the top and bottom release channels with a manual pipette. The tubing connected to the outlet ports allowed the collection of the released cells in microcentrifuge tubes. (B) The middle channel was coated with temperature sensitive liquid crystal dye, which was responsive between 35 °C (red-orange) and 40 °C (blue-purple). At the target temperature of 37 °C, the middle channel displayed green color. (C) Schematic drawing of the working principle of label-free selective capture from whole blood and controlled release of cells in thermoresponsive microfluidic channels. Biotin binding protein (Neutravidin) and biotinylated antibody (Anti-CD4 or Anti-CD34) were immobilized on the PNIPAAm channel surface at 37 °C. (D) Pre-warmed blood sample (at 37 °C) was injected into the microfluidic channel, and the CD4+ cells or the CD34+ cells in blood were captured on the channel surface. (E) The non-captured cells in the channels were rinsed off and the red blood cells were lysed. (F) The microchip was then cooled down below 32 °C (in less than 5 minutes). The released cells were rinsed out of the channels and collected at the channel outlet.

Fig. 2

Release efficiency for the captured CD4+ cells in control and release microchannels. (A) Bright field image of all captured cells from blood (buffy coat) in control channels at 37 °C. (B) Unreleased cells remaining in control channels after the temperature was reduced below 32 °C and the channels were rinsed. (C) All captured cells from blood in release channels at 37 °C. (D) Unreleased cells in the release channels after the temperature was reduced below 32 °C and the channels were rinsed. (E) When the temperature of control channels was decreased below 32 °C, a statistically significant release of captured cells was not observed after rinsing. On the other hand, when the temperature of the release channels was decreased below 32 °C, a significant (paired t-test, p < 0.05) release of captured cells was observed after rinsing. (F) Control microchannels displayed a release efficiency of less than 2%, whereas the thermoresponsive release channels allowed release efficiency in excess of 59%. The difference in release efficiencies of control and release channels was statistically significant (n = 6 channels, 10 images per channel, t-test, p < 0.05). Brackets connecting groups indicate statistically significant difference. Error bars represent the standard error of the mean.

Fig. 3

CD4+ cell capture specificity for control and release microchannels. (A) Bright field image of all the captured cells from blood (buffy coat) in control channels. (B) CD4 antibody stained cells in control channels. (C) All the captured cells in release channels. (D) CD4 antibody stained cells in release channels. (E) Specificity of captured cells at 37 °C was 83% (mean ± 2.5%) for control, and 91% (mean ± 1.3%) for thermoresponsive release channels. The brackets indicate statistically significant difference (n = 4 channels, 10 images per channel, t-test, p < 0.05). Error bars represent the standard error of the mean.

Fig. 4

Distribution of captured and released cells along the microfluidic channels. (A) In control channels, captured cells were concentrated closer to the inlet with a decreasing distribution along the channel. After cooling down and rinsing, the number of released cells were significantly lower along the channels. (B) In release channels, more cells were captured closer to the inlet and the number gradually decreased along the channels. After release steps, a comparable number of cells were released along the channels. The difference between the number of captured and unreleased cells (i.e., the number of released cells subtracted from the number of captured cells) along the channel was statistically significant (n = 6 channels, 10 images per channel, * indicates p < 0.05, paired t-test). Error bars represent the standard error of the mean.

Fig. 5

Effect of flow rate on release efficiency of captured CD4+ cells in microchannels. (A) The flow rates in the range of 50 μL min−1 to 500 μL min−1 were applied through a programmable syringe pump, whereas the manual flow rate was applied with standard pipettors as described in the methods section. A statistically significant effect on the release efficiency of captured CD4+ cells was not observed (P>0.05 for all comparisons). (B) Manual pipetting flow rate for phosphate buffered saline (PBS) and blood in microchannels. (C) The shear stress rate in channels when flowing PBS and blood. Error bars represent the standard error of the mean.

Fig. 6

Post-release cultivation of CD4+ cells. (A–C) CD4+ cells were collected after release and cultured for up to 8 days. Microscopic imaging was performed at day 1, 5 and 8 to assess the cellular morphologies. (D) Cell densities in culture (per millimetre square) at day-5 and day-8 displayed a statistically significant increase compared to day-1 (p < 0.05). The brackets indicate statistically significant difference. Error bars represent the standard error of the mean.

Fig. 7

Validation of the microfluidic capture/release system with CD34+ stem cells from whole blood. (A) CD34+ stem cells were successfully captured from whole blood. (B) CD34 fluorescent labeling of captured cells in microchannels indicated capture specificity greater than 90%. (C–D) The released CD34+ cells displayed greater than 90% viability as indicated by live/dead assay. (E) Post-release cultivation of CD34+ cells was possible with healthy cellular morphology after a day of culture.