Cellular manipulations in microvortices
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- Chiu, D.T. Anal Bioanal Chem (2007) 387: 17. doi:10.1007/s00216-006-0611-2
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The ability to tailor the size of microfluidic systems to match the length scale of single biological cells has led to a plethora of new microfluidic techniques geared towards the manipulation, culturing, and analysis of single cells [1–11]. Examples include the use of laminar flows for the rapid exchange of the solution environment around single cells  and the targeting of small molecules to localized regions of single cells . By miniaturizing the channels to cellular or subcellular dimensions [4, 5], single cells can be trapped in defined regions of the fluidic system for subsequent detailed studies. Miniaturized devices for the high-throughput manipulation and detection of streams of single cells have also been demonstrated, and include such widely used methods as flow cytometry and cell sorting . By combining electrical or optical methods with microfluidics such as laser trapping [7, 11], single cells and particles can be precisely positioned. In addition to single-cell manipulation, a range of integrated microfluidic platforms has been designed for the analysis of the contents of single cells [8, 9] or even that of single subcellular components [10, 11].
Some of these microfluidic methods originated from the miniaturization of their widely used macrofluidic counterparts. Many other new microfluidic techniques, however, are based on the unique fluidic behavior in microchannels; these latter techniques are especially interesting because they exploit the physics of low-Reynold’s number flow and offer new capabilities that are otherwise unattainable. Here we describe one unique microfluidic system that we have explored in the past years for exerting rotational control over single cells [12–16]. This system is based on the use of microvortices, which are tiny re-circulating flows that we controllably generate in microchannels. This system illustrates the intriguing possibilities when unique microfluidic behaviors are applied towards biological and cellular manipulations.
Controlled generation of microvortices
As radial acceleration scales quadratically with velocity and inversely with radius, the combination of high flow velocities in microchannels and small radii of microvortices enables the generation of high radial accelerations . Although many designs produce re-circulating flows in microchannels, only a few designs generate stable vortices whose center of rotation stays relatively spatially confined. Furthermore, we found the average rotational velocity as well as the formation and the shape of the microvortex to depend critically on the geometries of the microchamber, especially the angle of the chamber opening and the aspect ratio of the channel width to the width of the opening. Microvortices formed in secondary chambers (Fig. 1d), that is, chambers where the re-circulation is driven by another microvortex, often provide the stable conditions required for single-cell rotation.
Fluidic behavior of microvortices
To study the fluidic properties of microvortices and to verify its ability to generate high radial accelerations, we must be able to measure fast fluid flow in such small dimensions. Using single-molecule detection and spatially defined uncaging of caged fluorescein, we were able to map flows at speeds up to tens of meters per second within confines of micrometers . With this sensitive flow mapping method, we were able to measure experimentally rotational velocities as high as ∼12 m/s at 10 μm from the vortex core, which corresponds to a radial acceleration of ∼1×107 m/s2 or ∼1×106 g (Fig. 1g) .
This ultra-high rotational velocity was attained in a specially designed trapezoidal microchamber connected to an asymmetrically constricted microchannel (Fig. 1e). There were two effects that made this design attractive for generating high rotational velocities. The first effect was that, by reducing the cross-sectional area, the channel constriction increased the overall flow velocity at the opening of the trapezoid chamber, provided that sufficient pressure was applied to counter the pressure-drop associated with the constriction. The second effect arose from the asymmetric nature of the constricted channel, which skewed the velocity maxima towards the chamber opening and drove the microvortex more effectively.
Separation and fractionation
Rotation of single cells and molecules
In addition to using the microvortex as a method of separating or fractionating particles, it offers a new venue for exerting rotational control over single cells . To demonstrate controlled rotation and orientation, we optically trapped single cells and positioned them at the center of the microvortex. Because of the no-slip boundary condition at the fluid-particle interface, the shear stress from the re-circulation flow in the microvortex caused the rotation of the cell placed at the center of the vortex.
Figure 2d–f shows the fast rotation of a mouse B-lymphocyte, where the appearance of the cell is shown before (Fig. 2d), immediately after (Fig. 2e), and 1 min after (Fig. 2f) rotation at ∼200 Hz. The area defined by the nuclear membrane (arrow) measured ∼125 μm2 (∼70 % of the cell area) prior to spinning. Immediately after spinning (Fig. 2e), the area within the nuclear membrane was observed to expand to ∼150 μm2 (∼85% of the cell area). The expanded nucleus slowly relaxed back to its original size, and measured ∼125 μm2 after 1 min (Fig. 2f). Although we have achieved ultra-high rotational frequencies of 2×105 Hz with a corresponding radial acceleration of greater than 106 (Fig. 2g) in a microvortex, the rate at which we can spin a single cell is currently limited by our ability to use optical trapping to maintain the cell at the center of rotation. Because of the instability of the vortex center at high rotational rates, we can only retain a spinning cell with optical trapping up to an estimated 200 Hz (∼12,000 rpm). Nevertheless, this rate is still much faster than current electrical or optical methods of single-cell rotation.
In addition to the presence of centrifugal force, the steep velocity gradients present in microvortices can cause significant shear stress on a fast rotating cell. Figure 2g–i shows a mouse mast cell before (Fig. 2g), during (Fig. 2h), and after rotation at ∼60 Hz (Fig. 2i). The cell was rotated periodically over a period of 10 mins. The shear stress at the surface of the cell, which was produced by the steep velocity gradient present in the microchamber, caused a shear-induced detachment of the glycocalyx at the membrane-fluid interface. We estimated the local shear stress to be as high as ∼100 dyn/cm2 . Endothelial cells can respond to shear stress as low as 0.5 dyn/cm2; however typical in-vivo arterial shear stress level is ∼12 dyn/cm2.
Besides single cells, microvortices also can be applied towards the manipulation of subcellular structures, such as single DNA molecules. Figure 2j illustrates the rotation of a single λ-DNA molecule that has been compacted into a globular state . A free DNA molecule tracing the flow in the microvortex caused the circular streak in the image. Rotation of a single DNA is difficult to observe under fluorescence; thus, we trapped simultaneously three DNA molecules, which caused these DNAs to form an aggregate within the optical trap. Figure 2k–m shows the rotation of this DNA aggregate. One advantage of using the microvortex to rotate objects is the great dynamic range (less than one Hz to hundreds of Hz) available and the precision with which one can rotate single cells and microparticles.
Microvortices offer intriguing potential in exerting controlled and precise rotation and shear stress on single cells, both for mechanical control and for fundamental studies of cellular behavior. The high radial acceleration attainable in microvortices may be harnessed for the fractionation of single-cell contents. To achieve these goals, however, is not without challenges. Besides high radial acceleration, also present in microvortices are hydrodynamic forces and shear stress derived from the steep velocity gradient inherent in the vortices . To fully exploit the utility of this interesting microfluidic phenomenon will require both an in-depth understanding of its properties and behavior as well as clever microfluidic designs that can tailor this system with the desired cellular applications.
Support from the NSF and NIH is gratefully acknowledged.