Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible.

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Label-free cell separation and sorting in microfluidic systems

Biochemical sample mixtures are commonly separated in batch processes, such as filtration, centrifugation, chromatography or electrophoresis. In recent years, however, many research groups have demonstrated continuous flow separation methods in microfluidic devices. Such separation methods are characterised by continuous injection, real-time monitoring, as well as continuous collection, which makes them ideal for combination with upstream and downstream applications. Importantly, in continuous flow separation the sample components are deflected from the main direction of flow, either by means of a force field (electric, magnetic, acoustic, optical etc.), or by intelligent positioning of obstacles in combination with laminar flow profiles. Sample components susceptible to deflection can be spatially separated. A large variety of methods has been reported, some of these are miniaturised versions of larger scale methods, others are only possible in microfluidic regimes. Researchers now have a diverse toolbox to choose from and it is likely that continuous flow methods will play an important role in future point-of-care or in-the-field analysis devices.

Continuous flow separations in microfluidic devices

In the growth condition(s) of plants, numerous secondary metabolites (SMs) are produced by them to serve variety of cellular functions essential for physiological processes, and recent increasing evidences have implicated stress and defense response signaling in their production. The type and concentration(s) of secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage and environmental factors during growth. This suggests the physiological adaptive responses employed by various plant taxonomic groups in coping with the stress and defensive stimuli. The past recent decades had witnessed renewed interest to study abiotic factors that influence secondary metabolism during in vitro and in vivo growth of plants. Application of molecular biology tools and techniques are facilitating understanding the signaling processes and pathways involved in the SMs production at subcellular, cellular, organ and whole plant systems during in vivo and in vitro growth, with application in metabolic engineering of biosynthetic pathways intermediates.

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Stress and defense responses in plant secondary metabolites production

This paper describes a simple microfluidic sorting system that can perform size profiling and continuous mass-dependent separation of particles through combined use of gravity (1 g) and hydrodynamic flows capable of rapidly amplifying sedimentation-based separation between particles. Operation of the device relies on two microfluidic transport processes:  (i) initial hydrodynamic focusing of particles in a microchannel oriented parallel to gravity and (ii) subsequent sample separation where positional difference between particles with different mass generated by sedimentation is further amplified by hydrodynamic flows whose streamlines gradually widen out due to the geometry of a widening microchannel oriented perpendicular to gravity. The microfluidic sorting device was fabricated in poly(dimethylsiloxane), and hydrodynamic flows in microchannels were driven by gravity without using external pumps. We conducted theoretical and experimental studies on fluid dynamic characteristics of laminar flows in wide...

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Gravity-Driven Microfluidic Particle Sorting Device with Hydrodynamic Separation Amplification

Energy biotechnology with cyanobacteria.

Monitoring of live cells is important in the field of medical science, diagnostics, biology, and the pharmaceutical industry. In this study, live and dead CHO-K1 (Chinese Hamster Ovary) cells were fractionated by continuous-flow extraction in a microfluidic device using immiscible aqueous two-phase extraction technique. The polymer solutions offered stable two-phase flows in microchannel without diffusive mixing. The fundamentals of aqueous two-phase extraction can support stable and reproducible recovery and separation of biomolecules in microfluidic devices. Polyethylene glycol 8000 (PEG 8000, 4%) and dextran T500 (5%) were selected as model polymer solutions. The appropriate flow rates of polymer and cell solutions were suggested. The fractionation efficiency of live and dead CHO K-1 cells from the culture broth was compared in normal macroscale system and microfluidic device. The optimum pH for the fractionation was 6.6 in both the normal and micro-scale systems. The loss of target live cells by sedimentation was circumvented in microfluidic device because of the negligible effect of gravity on the sedimentation. Most live cells were distributed to PEG-rich phase, while dead cells were found at the interface of two polymer solutions in microchannel. In this case, the recovery and fractionation efficiency of live cells in the PDMS-based microfluidic device was 100% and 97.0%, respectively.

Continuous-flow fractionation of animal cells in microfluidic device using aqueous two-phase extraction.

Addition of an ethylene precursor, ethephon, was studied in plant-cell-suspension cultures of Coffea arabica and Thalictrum rugosum as an enhancer of secondary metabolite production. In both cases the compound was able to stimulate alkaloid production, even though the biochemical pathways are widely different. This suggests that ethephon, or more directly ethylene, may be valuable in increasing secondary metabolite levels in a variety of cell-suspension culture systems.

Ethephon Enhancement of Secondary Metabolite Synthesis in Plant Cell Cultures

Periosteum-derived progenitor cells (PDPCs) were isolated by characteristic surface markers. Reproducibility of immunophenotypes of the PDPCs was characterized by flow cytometric analysis using fluorescence-activated cell sorter (FACS). SH2+, SH3+, SH4+, CD9+, CD90+ and CD105+ were important eternal characteristic cell surface markers for the PDPCs. The characterized PDPCs maintained their chondrogenic potential in pellet cultures until the 15th passage from primary cell culture.

Isolation of human periosteum-derived progenitor cells using immunophenotypes for chondrogenesis.

A hybrid bioreactor was developed for the production of secondary metabolites from high density cultivation of plant cell suspensions. Some of the advantages of both air-lift and cell-lift by agitation were combined. The addition of a decanting column also made it possible to run a perfusion system for high density culture or to run a two-stage culture efficiently. Cell growth and the production of berberine from Thalictrum rugosum in the hybrid bioreactor are reported in this paper. A cell density up to 31 g/l was obtained by perfusion without any problems in mixing or loss of cell viability and the specific berberine productivity was comparable to that in shake flasks. The maximum berberine concentration was 88 mg/l at 3 weeks of operation and declined thereafter.

A hybrid bioreactor for high density cultivation of plant cell suspensions.

A proper flashing light is expected to enhance microalgal biomass productivity and photosynthetic efficiency. The effect of flashing light on high-density Chlorella kessleri (UTEX 398) cultures was studied using light-emitting diodes. A frequency modulator was designed to flash LEDs, and the device successfully provided wide range of frequencies and various duty cycles of flashing. A relatively high frequencies of 10, 20 and 50 kHz were used in this study. These frequencies have very short flashing time (2- 50 μs), which corresponded to the time constant of the light reaction of photosynthesis. The specific oxygen production rates of photosynthesis under flashing light were compared with those under an equivalent continuous light in specially designed illumination cuvette. The specific oxygen production rates under flashing light were 5- 25% higher than those under the continuous light. A range of cell concentration was discovered, where the benefit of flashing light was maximized. The photosynthetic efficiency was also higher under flashing light with frequencies of over 1 kHz, which was a clear indication of flashing light effect and the degree of mutual shading could be overcome by flashing lights, particularly at high-density algal cultures.

Dong-Il Kim

Papers

Continuous-flow fractionation of animal cells in microfluidic device using aqueous two-phase extraction.

Ethephon Enhancement of Secondary Metabolite Synthesis in Plant Cell Cultures

Isolation of human periosteum-derived progenitor cells using immunophenotypes for chondrogenesis.

A hybrid bioreactor for high density cultivation of plant cell suspensions.

Effect of Flashing Light on Oxygen Production Rates in High-Density Algal Cultures