Active cell sorting methods

Active sorting and uses an external marker e.g., dielectric marker attached via immunological means or the use of a fluorescent marker e.g., green fluorescent protein (GFP) to differentiate between the cell types present. Examples of this type of immunological sorting at the macroscopic scale are based on commercial cell separation methods such as FACS and MACS (35).

FACS machines can detect and sort cells according to a large number of parameters, and readily sort cells at rates in the order of 105 cells per second (36). In a FACS device, cells contained in liquid droplets are discharged from an acoustic vibrating nozzle and streamed through a detection region. In the detection region, the light scattering and fluorescence properties of a given cell are recorded onto photodetectors as the cell transverses a laser beam. The cells may be tagged with appropriate fluorescent markers (fluorescently labeled monoclonal antibodies), allowing specific cells to be recognized. If the light scattered from a cell corresponds to the chosen fluorescence signal, an electrical charge is applied to the droplets containing the selected cells. One or more droplets are then separated from the main stream of droplets into a collection chamber. In the same fashion, droplets containing different cell types are directed toward separate collection vials by a static electrical field (37). In this process, the FACS machine can also record the cell size, volume or viscosity (granularity), DNA or RNA content as well as the presence of surface antigens or internal proteins. FACS has a variety of uses and has found application in the diagnosis of leukaemia, lymphoma and immunodeficiencies.

The MACS cell-sorting technique is mostly used in immunology (35), and offers a direct and rapid separation between two cell types. Prior to the magnetic separation process, the cells are incubated with paramagnetic micro beads that are coated with the appropriate antibodies. This permits them to preferentially attach to the cells that are expressing the specific surface antigens in the sample. Subsequently the cells of interest may be sorted by use of an externally applied magnetic field. This technique is reliant upon the need for suitable antigens on the cell surface allowing the paramagnetic beads to accurately bind to or “tag” cells of interest. Furthermore, the number of paramagnetic beads that can be used in parallel is more limited than for FACS.

Both the FACS and MACS cell sorting techniques possess high specificity and selectivity because they consist of extremely precise immunoreactions between the membrane marker proteins and labeling antibodies. Another advantage of FACS and MACS cell sorting schemes is the achievement of high-throughput cell separation. This fact not only means that there is a requirement of large numbers of cells for efficient separation, but

immunologically isolated cells may often experience damage during follow on processes such as the elution of cells from the capturing antibodies, and additionally overall these cell separation systems are bulky and expensive. New technologies for cell sorting are emerging; these can be used with rare or precious cell samples in nano-liter or micro-liter volumes. For such minute analyte volumes, innovative sorting methodologies that readily deal with small sample volumes and are easily adapted to microfluidic environments are necessary. So, in contrast to the macroscopic sorting schemes previously mentioned, the next portion of this sub-section therefore describes a recent active sorting technique performed in a micro sample chamber (microscopic active-sorting).

In this example active optical sorting exists, where the combination of optical forces typically with microfluidics aims to replicate more bulky FACS machines but in a more compact geometry. Specifically, in the study of Wang et al, 2005 (38), a microFACS (µFACS) system that used a microfluidic cartridge and a laser at 488 nm to excite fluorescence, and a subsequent 1064 nm laser to extract the sorted cells in a microfluidic flow was developed. This therefore produced a fluorescence-activated microfluidic cell sorter. The researchers assessed the performance of this device on live, stably transfected HeLa cells that expressed a fused histone-green fluorescent protein. Viability was measured by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress, and no detrimental effects on the cells from the optical sorting were observed. Another example is shown in figure 2.9 (39) below, where the principle of active cell sorting via optical forces in such µFACS devices is illustrated. Several groups have implemented microfluidic forms of FACS, as mentioned in the review by Andersson and Berg (40). The methods used for cell identification are similar to those of macroscopic FACS machines. Hydrodynamic focusing is used to generate a laminar cell flow into a detection area, and subsequent deflection into the appropriate extraction channel is performed by electrical or optical switches. At the microfluidic scale level, the requirement of separating cells from micro liter samples may lead to a reduced throughput compared to that obtained in macroscopic sorting apparatus.

Figure 2.9: Principle and illustration of µFACS based on optical forces. The hydrodynamically focused macrophage: (A) is detected by forward scattering; (B) enters the IR laser spot; (C) is deflected by optical gradient forces; (D) and finally is released in a different laminar flow stream (reprint by permission from

Analytical Chemistry (39)).