Field-flow fractionation and biotechnology

Field-flow fractionation and biotechnology

Review TRENDS in Biotechnology Vol.23 No.9 September 2005 Field-flow fractionation and biotechnology Pierluigi Reschiglian1, Andrea Zattoni1, Barba...

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Review

TRENDS in Biotechnology

Vol.23 No.9 September 2005

Field-flow fractionation and biotechnology Pierluigi Reschiglian1, Andrea Zattoni1, Barbara Roda1, Elisa Michelini2 and Aldo Roda2 1 2

Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, I-40126 Bologna, Italy Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy

The gentle separation mechanism has made field-flow fractionation particularly suited to samples of biotechnological interest, from proteins and nucleic acids to viruses, subcellular units and whole cells. Recent progress in field-flow fractionation technology, as well as the development of coupled techniques combining field-flow fractionation capabilities with the specificity and sensitivity of well-established analytical methods, opens up new biotechnological applications for fieldflow fractionation. The most recent appealing applications include: sorting and fingerprinting of bacteria for whole-cell vaccine production; noninvasive and tagless sorting of immature and stem cells; separation of intact proteins and enzymes in top-down proteomics; and the development of flow-assisted, multianalyte immunoassays using nano- and micron-sized particles with immobilized biomolecules.

Introduction The explosive growth of analytical methods demands separation techniques with a wide range of applications, good resolution and versatility with respect to the analysis of complex biological samples. Gel (GE) and capillary zone (CZE) electrophoresis have recently been improved for the separation of nucleic acids for genomic analysis, and for proteins and peptides to be further characterized in proteomics. Recent years have also seen the continued growth of different liquid chromatographic (LC) techniques applied to many samples of biological interest, and of flow cytometry (FC) for cell analysis. Parallel to the development of GE, CZE, LC and FC, although somewhat slower and less visible, has been the development of fieldflow fractionation (FFF). FFF, together with LC, is a flow-assisted separation technique for the separation of analytes in a 1015 molar mass range, from macromolecules such as proteins to micron-sized particles such as whole cells [1,2]. As in LC, FFF starts with the injection of a narrow sample band into a stream flowing through a thin, empty flow chamber, called a ‘channel’. A flow stream drives sample components along the channel, eventually flushing them out into a detector and/or collection device for further characterization. Unlike LC, FFF has no stationary Corresponding author: Roda, A. ([email protected]).

phase. Separation is structured by the interaction of sample components with an externally generated field, which is applied perpendicularly to the direction of the mobile phase flow. Table 1 lists the fields that are currently used effectively in FFF (in bold) and some hypothetical fields that could be used in future FFF. The fields implemented to date are listed according to the current, most frequent usage. The typical FFF system is described in Figure 1. The FFF separation mechanism is simple. Sample components (the analytes) differing in molar mass, size and/or other physical properties are driven by the applied field into different velocity regions within the parabolic flow profile of the mobile phase across the channel. In parabolic flow, the flow velocity at the channel walls is zero, and this increases towards the channel center, where it reaches the maximum value. The analytes are then carried downstream through the channel at different speeds, and exit the channel after different retention times. The relative distribution of the analytes over the parabolic flow profile thus determines the separation characteristics. Different types of distributions correspond to different operating modes. The two most frequently used operating modes are described in Box 1. Whatever the type of field and operating mode used, the key feature of FFF is the absence of a stationary phase. Thus, unwanted interactions of bioanalytes with the stationary phase are avoided, and FFF has significant advantages over other bioseparation techniques in terms of high biocompatibility, ‘soft’ fractionation mechanism, reduction of sample carry-over and simple sterility issues. Table 1. Practical (in bold) and hypothetical FFF fields, and the corresponding techniques Field type Crossflow (Fl) Sedimentation (Sd)

Thermal (temperature gradient) (Th) Electrical (El) Magnetic (Mg) Dielectric (Dl) Photophoretic (Ph) Concentration gradient (Cg) Acoustical (Ac) Shear (Sh)

www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.07.008

Technique Flow FFF (FlFFF) Hollow-fiber FlFFF Sedimentation FFF (SdFFF) Centrifugal SdFFF Gravitational (GrFFF) Thermal FFF (ThFFF) Electrical FFF (ElFFF) Magnetic FFF (MgFFF) Dielectric FFF (DlFFF)

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Detector signal

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Figure 1. The FFF system. The mobile phase flow inside the channel is usually delivered by an LC, a peristaltic or a syringe pump. Sample mixtures are injected into the channel inlet through an injection port. A detection system with a flow-through sample cell is connected downstream of the channel outlet for online recording of the signal generated by the eluted analyte (the fractogram). Most commonly used detection systems are the ultraviolet–visible spectrophotometric detectors for LC. Laser light scattering, refractive index, luminescence detectors and, most recently, soft-impact mass spectrometers have also been combined with ultraviolet–visible detection. A fraction collector can be positioned downstream of the detection system if the fractionated analytes need further characterization or to be reused. Different applied fields require different channel designs and configurations. In centrifugal SdFFF, the channel is spooled inside a centrifuge bowl, whereas GrFFF channels are rectangular. Flat channels are employed in FlFFF, except for HF FlFFF, in which the channel is made up of a porous, cylindrical hollow fiber (HF). In flat-channel FlFFF, at least one channel wall must be permeable to enable a crossflow of mobile phase to generate the field. In macro- or microfabricated ElFFF, the channel walls are made of electrically conductive materials. Some FFF systems are commercially available from Postnova Analytics (http://www.postnova.com), ConSenxus (http://www.consenxus.com) and Wyatt Technology (http://www.wyatt.com).

Open-channel configuration makes FFF advantageous also for continuous fractionation of bioanalytes on a preparative scale. Nonetheless, FFF has long been considered to be the ‘best-kept secret’ in the field of bioseparation (Box 2). Most recent FFF applications in the life sciences, however, indicate biotechnology to be one of the most suitable application niches for exposing this technique to a wider audience.

Emergence and evolution of FFF in life sciences and biotechnology Proteins The first application of FFF to proteins was reported in 1972 [3]. The authors used electrical FFF (ElFFF), a variant of FFF in which an electric field is used. ElFFF revealed several advantages with respect to protein electrophoresis, such as low required voltage, lack of adverse heating and support effects, and the existence of a mobile phase flow to amplify separation. Flow FFF (FlFFF), which instead uses a mobile phase crossflow as the applied field, has been the most widely applied FFF technique for protein fractionation. The ability of FlFFF to measure the diffusivity of intact proteins, within a mass range of w105 in a single run, was first shown in 1977 [4]. This technique was used to characterize intact proteins of www.sciencedirect.com

different origin, from wheat proteins [5] and enzyme mutants [6] to lipoproteins screened in patients with coronary artery disease [7]. Proteins can denature or dissociate into smaller subunits or they can associate to form large aggregates. Protein complexes can differ according to the degree of aggregation, size, charge, density, shape and biological activity. The study of protein complexes is important in proteomics because protein conformation, self-dissociation and dissociation are strongly related to the biological activity and to the interaction of proteins with other proteins, protein receptors, drugs or cell metabolites. FlFFF is particularly appealing for the separation of intact ultralarge proteins and protein complexes [8,9]. This is because the separation of such complexes using other methods, such as LC, is seriously limited, owing to the possible interaction between the proteins and the stationary phase and/or to the possible presence of organic modifiers in the mobile phase, which can cause degradation.

Nucleic acids Sedimentation field-flow fractionation (SdFFF), which uses a sedimentation field generated by a centrifuge, has been demonstrated to be sufficiently gentle to fractionate l

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Box 1. FFF operating modes Normal mode The normal FFF mode drives the elution of macromolecules and submicrometer particles. As the macromolecules or particles that constitute the sample are driven by the field toward the accumulation wall, their concentration increases with decreasing distance from the wall (Figure Ia). This creates a concentration gradient that causes sample diffusion away from the wall. When these two opposite transport processes balance, the sample cloud reaches a characteristic average elevation from the wall. The lower the molar mass or size of the sample component, the greater the component cloud elevation, the deeper the cloud penetration into the faster streamlines of the parabolic flow profile and the shorter the time required by the component to exit the channel. Retention time in normal FFF is therefore shorter for lower molar mass or size.

(a)

External field

Steric and hyperlayer mode

(b)

External field

(c)

External field

If the sample components are micron-sized particles, their diffusion away from the wall is negligible. Particles are in fact driven by the field directly to the accumulation wall. Particles of a given size form a thin layer of a given thickness, hugging the wall. Larger particles form thicker layers that penetrate into faster streamlines of the parabolic flow profile, and they are eluted more rapidly than smaller particles. This is just the opposite of normalmode elution: it is then referred to as a reversed mode. This elution mode is in fact governed by the physical (steric) barrier of the accumulation wall, so is called ‘steric’ (Figure Ib). Retention in steric FFF then depends only on particle size. During elution, however, the micron-sized particles make very little contact with the wall. Instead, their moves toward the wall are opposed by mobile phase flow-induced lift forces (Figure Ic, green arrows). When particles are driven from the wall by a distance that is greater than their diameter, the retention mode is called hyperlayer (Figure Ic). Retention in hyperlayer mode is still reversed with respect to particle size but it also depends on the various physical features of the particles, which will have a varying influence on the intensity of the flow-induced lift forces.

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Figure I. Most-frequently-used FFF operating modes. Different mechanisms of separation for particles of different size. (a) Normal, (b) steric and (c) hyperlayer mode.

DNA and smaller supercoiled plasmids, without altering the conformations during fractionation [10]. FlFFF was applied to the fractionation of plasmid fragments [11], to elucidate DNA conformation and measure the different diffusion coefficients of linear and circular DNA [12]. The ribosomal composition of recombinant proteins can be monitored by evaluating the cell translation capacity that produces the proteins. FlFFF was used to separate and quantify tRNA in recombinant Escherichia coli cells and, subsequently, to relate protein production levels to tRNA levels [13,14]. Another promising FFF application that has been partially explored concerns the separation of lipid–DNA complexes for structure–activity studies. Self-assembled cationic lipid–DNA complexes are widely used for cell transfection, although their efficiencies are not as high as those observed for viral vectors. This is probably owing to the presence of a heterogeneous population, in terms of size and net charge, which lowers the transfection efficiency. Standard chromatographic techniques are not www.sciencedirect.com

suitable for the fractionation of such large, insoluble species. FlFFF made it possible to separate cationic lipid– DNA complexes prepared at various lipid–DNA ratios, by distinguishing subtle changes in the physical properties of such vectors [15]. These results pave the way for new potential tools using FFF, for clinical applications such as gene therapy, in which high transfection efficiencies are required. Organelles SdFFF was the first FFF technique to be applied to the fractionation of a wide variety of subcellular particle populations. Eluted fractions containing mitochondria, microsomes, Golgi membranes and plasma membranes were examined, and the subcellular particles appeared to remain mostly intact [16]. FlFFF is also effective for the fractionation of bacterial ribosome subunits. E. coli ribosomal contents were accurately determined, with short analysis times (less than ten minutes), and the ribosome size was accurately determined from retention

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Box 2. FFF: the best-kept secret in bioanalysis articles published after the death of J.C. Giddings, the inventor of FFF. In April 1998, R. Stevenson (American Biotechnology Laboratory) focused on this particularly slow adoption of FFF in the separation science arena. He defined FFF as the ‘best-kept secret in separation science’, and concluded that what will probably be needed to increase FFF visibility, ‘. is more people using the technique to solve chemical problems . it would help immensely if the users would publish more reports of their success.’. Has his suggestion been acknowledged? If we look at the number of FFF publications on bioapplications over the total number of FFF publications, since 1998 (Figure Ic), the number of reports on bioapplications has shown a rapidly increasing trend.

FFF was invented almost 40 years ago. However, currently, we observe a difference of two orders of magnitude between the number of FFF and of LC publications (Figure Ia). LC, in fact, exploded in the 1980s, probably because of the increasing LC applications to biomolecules and pharmaceuticals. Although the number of LC publications reached a plateau in the 1990s, this technique has recently undergone a renaissance, mainly because of the introduction of techniques hyphenated with LC methods, particularly the use of MS for proteome studies. Unlike LC, if we observe the publication trend for FFF through the years (Figure Ib), it has been linear since its invention in 1966, with only one exception (in 1997), owing to the high number of memorial

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Figure I. The slow adoption of FFF in the bioseparation science arena (data source: Caplus). (a) The total number of literature references: FFF versus LC. (b) Comparison between the total number of references on FFF and the number of references on FFF bioapplications. (c) Percentage ratio of the number of references on FFF bioapplications over the total number of references on FFF.

times [17]. Through FlFFF, the protein production levels in recombinant E. coli cells were found to be associated with the ribosomal content [13,14,18,19]. The analysis time for isolating ribosomes through FlFFF was always substantially shorter than typical ultracentrifugation run times. A microfabricated ElFFF device has recently been used for sorting organelles, for separating mitochondria from whole cells and nuclei and for the separation of mitochondrial subpopulations [20]. The micro-ElFFF system provided fast separation in very small samples, while avoiding large voltages and heating effects. Viruses The main advantages of FFF over conventional LC and CZE techniques can be seen when separating very high molar-mass bioanalytes. The first example of the application of FFF to viruses dates back to 1975, when SdFFF was applied to the separation and molar mass determination of the T2 phage [21]. SdFFF fractionated oligomeric aggregates of rod-shaped viral particles of nuclear polyhedrosis virus from a complex mixture of enveloped aggregated forms and monomers [22]. SdFFF was shown to be sufficiently gentle that passage through the system had very little effect on the infectivity of the T4D virus [23]. Accurate and precise determination of the molar mass and density of viruses, such as the Paramecium www.sciencedirect.com

bursaria chlorella virus (PBCV), was also possible using SdFFF [24]. As in the case of lower molar-mass analytes discussed in previous sections, FlFFF is also effective and rapid for virus analysis. Determination of virus diffusivity from FlFFF retention times was reported, and a clean, quick separation of viruses was observed [11,25]. The use of a multiangle laser-scattering detector coupled with FlFFF was also explored to separate and elucidate the size of the tobacco mosaic virus [26]. Bacteria The first FFF of whole bacteria was reported in 1991, when a specifically designed variant was used to separate phenotypically different E. coli strains [27]. Among different FFF techniques for separating bacteria, SdFFF showed the highest resolution power [28]. SdFFF was applied to the separation of bacteria from sediments [29], and SdFFF-based procedures to isolate bacteria from circulating blood or mouse ascitic fluid were developed with high recovery and total maintenance of cell viability [30,31]. Isolation of microorganisms was also demonstrated through gravitational field-flow fractionation (GrFFF), the simplest FFF variant, which uses the gravitational field. In 1991, isolation of live and dead microfilariae from blood showed the potential of GrFFF for diagnostic screening of parasite infections for the first

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time [32]. The hybrid variant dielectrophoretic–GrFFF (DEP–GrFFF) was applied for the isolation of cell specimens for the sensitive diagnosis of malaria-infected cells [33]. The microcolumn variant of FlFFF, which makes use of a porous hollow-fiber (HF) channel (HF FlFFF), was used to fractionate different types of bacteria within a short analysis time [34,35]. Yeast cells The bioprocess of yeast cell growth can be monitored by FFF in terms of variations in cell number, size and shape. SdFFF was applied to fractionate Saccharomyces cerevisiae cell samples in less than four minutes, obtaining several subpopulations differing in their size, as well as in their number of buds [36]. The association of SdFFF with FC was then applied to the characterization of active dry winemaking yeast strains [37,38]. Winemaking yeast strains were also fractionated and characterized by GrFFF [39], and comparison with Coulter counter measurements confirmed the ability of GrFFF to reveal physical differences in yeast cells other than size [40]. This ability was further evaluated by two-dimensional FFF, in which two different separation stages were obtained by applying two different fields [41]. Offline coupling of GrFFF to FlFFF made it possible to obtain multidimensional information on the biophysical indexes of yeast cells [42]. This information, which strictly correlates with yeast viability, is crucial in several processes, ranging from the food industry (mainly regarding wine production, baking and beer brewing) to the production of therapeutic recombinant proteins. Mammalian cells The use of SdFFF for the size-based separation of human and animal cells was reported for the first time in 1984 [43]. SdFFF and GrFFF were then the most widely used FFF techniques for applications in whole mammalian cells. GrFFF was first applied to fractionate populations of human red blood cells (HRBCs) [44]. It was demonstrated through GrFFF of normal and glutaraldehyde-fixed HRBCs that cell density and membrane rigidity, in addition to cell size, significantly influence cell retention [45]. Efficient SdFFF methods, depending on different cell sorting objectives (i.e. analytical or semipreparative sorting), were further developed for living cell fractionation, using biocompatible materials and sterilization protocols. Cells collected after fractionation fully maintained their characteristics and viability [46–49]. SdFFF further confirmed its potential in living cell separation and purification based on differences in early and specific biophysical modifications. SdFFF made it possible to obtain a purified or enriched neuron cell culture from a cortical cell suspension [50], and to monitor the induction of cell apoptosis in a human osteosarcoma cell line [51]. Although SdFFF and GrFFF have been the most widely used FFF techniques for human cell fractionation, other FFF variants have also been successfully applied. FlFFF and, most recently, its microcolumn variant HF FlFFF have been applied to separate different types of cells, including RBCs [35,52]. Small sample loadings and possible disposable use of the HF FlFFF channel show www.sciencedirect.com

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interesting perspectives for living cell fractionation methods in which the requirements of small sample amounts and sterility, and strict avoidance of run-to-run contamination, constitute serious issues. Split-flow thin cells (SPLITT) are FFF-like systems for continuous, preparative-scale fractionation [53]. A SPLITT system using the centrifugal field was applied to continuous fractionation of human blood into proteins, platelets, RBCs and leukocytes [54]. A SPLITT system using the magnetic field generated by a quadrupole was applied to a cell model system of human peripheral T lymphocytes (CD4C, CD8C and CD45C cells) [55]. Hybrid FFF variants were also applied to mammalian cell selection. Separation of B and T lymphocytes was shown by FFF–adhesion chromatography using a surface-modified channel wall [56]. DEP–GrFFF was used to separate, in less than five minutes, a mixture of cultured human breast cancer cells from normal blood cells, based on the difference in dielectric and density properties between cell populations [57]. Principles of hybrid and two-dimensional FFF variants for possible application to continuous fractionation of cells were also described [58,59]. The role of FFF in modern biotechnology Beads, recombinant cells and vaccines Surface-modified nanobeads are widely used in biotechnology, from immunoassays to bioreactors in protein arrays, to cell sorting methodologies. The ability of SdFFF to characterize surface-modified nanobeads for biotechnological applications has been explored. An SdFFF-based method was described for the evaluation of surface concentrations of human immunoglobulin G adsorbed to polystyrene latex spheres of different sizes [60]. Unlike conventional techniques, the method enables direct evaluation of mass adsorbed per unit area, without the need for labeling reactions. In similar approaches, SdFFF was used to establish the relationship between the extent of antibody binding on different modified bead surfaces and the specific activity of the surface-bound antibody, and to characterize oligonucleotide, surfacehybridized nanobeads [61,62]. The ability of FFF to isolate a particular type of recombinant cell was also used for a cell separation based on transient physical properties, such as differential swelling kinetics [63]. The surface features of deactivated bacteria used for whole-vaccine production affect the immune response to bacteria-associated antigens. Sorting and quantification of the different bacterial cells are then related to the quality assessment of whole-cell vaccines. The low-cost GrFFF and the highly size-selective FlFFF techniques, including the microcolumn variant HF FlFFF, were used to distinguish and quantify different deactivated E. coli strains used for whole-cell vaccines, which differed only in the presence of fimbriae on the bacterial membrane [35,64]. HF FlFFF was also used to select, by their fractogram profiles, two serotypes of deactivated Vibrio cholerae strains used for whole-bacteria vaccine production, with the key advantages of short analysis times, high reproducibility and low limits of detection [34]. These

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Box 3. FFF applications in biotechnology seek orthogonality

results show the potential use of FFF to enhance the process of separation and selection of surface-engineered bacteria. Display of heterologous proteins on the microorganism surface, which has been made possible by recombinant DNA technology, represents an area of intense ongoing research, with current uses and future applications in vaccine technology. Stem cells The gentle fractionation mechanism of FFF makes possible the collection of viable cell fractions, which can be used for further culture and/or transplantation. FFF might therefore potentially be used as an alternative or complementary method to standard stem cell sorting www.sciencedirect.com

Field (a)

Field (b)

Void peak

CL signal

As FFF techniques have been applied to an increasing degree to life science, it appears that FFF might substantially increase the analytical information that can be derived if it is combined with orthogonal methods. Coupling FFF with highly specific and sensitive techniques which are broadly and routinely applied to biology and biochemistry, currently shows an interesting capacity to enhance the analytical information obtained when each individual method is applied as stand-alone technique. Combination of FFF with methods such as luminescence detection, flow cytometry (FC) and soft-impact mass spectrometry has been explored. Using these techniques in combination with FFF as the separation step shows promise for the development of cell sorting methods, for multianalyte, flow-assisted immunoassays in dispersed phase and in top-down and whole-cell proteomics. The association of FFF with the powerful characterization capacity of FC has been shown to be an effective tool for cell analysis. Characterization through FC of different cell subpopulations obtained from FFF-based cell sorting can also be used as a feedback check to evaluate the actual cell sorting capabilities of FFF. Some highly specific methods for cell detection at the single-cell level are also based on chemiluminescence (CL). Some cells show natural CL activity (bioluminescence, BL) or they can express CL when labeled with an appropriate CL tracer. BL from cells can also be obtained by means of genetic engineering, when cells are transfected with gene sequences that are able to codify the synthesis of a CL reactioncatalyzing enzyme when the gene transcription is activated by stimulation of the specific receptor. These features make interesting use of CL or BL detection for FFF-based cell sorting methods. CL detection is well established in immunoassays. However, much work is still required for the simultaneous detection of different analytes. When a competitive-type format is used, the most crucial step becomes the possibility to achieve an efficient separation of bound and free tracer, and to distinguish the specific signals originating from each CL tracer. A possible solution lies in the use of micrometer-sized particles as the supporting solid phase for the immobilized antibodies. Differently sized nano- or microbeads can be used to immobilize different analyte-specific antibodies, using FFF as the size-based separation method to sort the various beads coated with the different antibodies, as well as to isolate the beads from the free CL tracers in solution (Figure I). Coupling soft-impact mass spectrometry with separation techniques has become of great relevance for proteome analysis. However, separation techniques often show intrinsic limitations when applied to intact proteins, enzymes and protein complexes, which can undergo denaturation during separation. They often have a poor capacity for monitoring conformational and morphological changes to intact proteins as a result of the interaction between the proteins and other surrounding proteins or molecules. Because of the gentle separation mechanism, coupling FFF with soft-impact mass spectrometry shows promise for the characterization of intact proteins and protein complexes in multidimensional, top-down proteome analysis.

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Figure I. The concept of multianalyte, competitive enzyme immunoassay in the dispersed phase, based on FFF–CL. (a) Sample injection and incubation. (b) Separation of the free tracers from the bound tracers, and separation of the different tracers bound to beads of varying size.

methods, providing that stem cells differ sufficiently from other cells. FFF-based stem cell sorting would have the advantage, in principle, of not requiring cell labeling (Box 4). The first application of FFF to stem cell separation was reported in 1996 [65]. It employed GrFFF for the micropreparation of stem cells from mouse bone marrow. The effectiveness of SdFFF to provide selective, immature cell isolation without inducing cell differentiation was further shown by fast purification (in less than 15 minutes) of an immature neural cell fraction from a human neuroblastic cell line [66]. Among immature cells, embryonic stem cells (ESs) are an important biotechnological tool. ESs are used as a vehicle for transgenesis and

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Box 4. Could cell labeling be no longer necessary for stemcell sorting? It is known that very small numbers of different stem cells can be found in the human body. In some cases, only one stem cell in 100 000 cells is present in the circulating blood, and under the microscope it looks similar to other cells. Isolation and identification (sorting) of a number of stem cells from complex cell populations has therefore become a challenging task. A wide panel of methodologies are available for cell sorting. Most of these methodologies make use of immunological markers, and a combination of multiple markers is used to identify a particular stem cell type. When a marker is labeled with a magnetic bead or fluorescent tag, magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) techniques, respectively, are used to define the different types of cells as positive or negative for the given marker, and consequently to isolate the labeled cells. Fluorescent tags are also used for microscopic visualization of the appearance of stem cells in the tissues. Alternatively, a geneticengineering approach can be applied to stem cell sorting, by introducing a reporter gene, the expression of which can be made dependent on the pluripotency of the cell. All of these sorting techniques, however, present some limitations. Firstly, specific markers for pluripotent stem cells, which do not have clearly recognizable functions, are not as yet available. Moreover, the presence of surface markers is not evidence that stem cells are in their primitive state Secondly, any cell labeling might interfere with the differentiation process of stem cells or affect their in vivo expansion. Indeed, FFF is able to sort cells based on very small differences in biophysical properties (e.g. cell size, density, shape, flexibility, membrane roughness) between cells. FFF-based cell-sorting methods alone might be sufficient without the need for cell labeling, providing that stem cells differ from other cells in their biophysical characteristics. After sorting by FFF, a process that usually takes just a few minutes, unlabeled, viable cells can be collected and cultured for further use, and enrichment of a target subpopulation might be possible.

can be cultured in vitro onto a layer of embryonic fibroblasts, and collected by using time-consuming and difficult methods. A cell suspension (w106 cells/ml) of ESs at various stages of proliferation was selectively fractionated within a few minutes using SdFFF, and collected cell fractions with in vivo potential development were used to derive transgenic mice by the generation of chimeras [67]. The hybrid DEP–GrFFF variant was used for cancer cell purging from normal T lymphocytes and from CD34C hematopoietic stem cells, for the separation of the major leukocyte subpopulations, and for the enrichment of leukocytes [68]. FFF and luminescence detection Over the years, several methods for the evaluation of yeast viability in fermentation processes have been explored. Among these, the most widely used are based on FC, which is a rapid but expensive technique, or on the estimation of cell viability by determination of colony forming units, a laborious and time-consuming method. A GrFFF method using fluorescence detection was recently developed to determine the viability of several commercial winemaking yeast strains before the wine fermentation process [69]. Chemiluminescence (CL) and bioluminescence (BL) are potent and versatile detection tools for a wide range of biotechnological applications. GrFFF–CL was recently proposed for the development of flow-through www.sciencedirect.com

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immunoassays [70]. The CL signal was generated inside the GrFFF channel and detected in situ by use of a chargecoupled device (CCD)-based camera. Horseradish peroxidase, free or immobilized on polystyrene (PS) microbeads, was then visualized during separation, as shown in the animation supplied as supplementary material (see Online Supplementary Material; Adobe Reader 6.0 or higher required). Based on these results, development of the multianalyte, FFF-based immunoassay concept described in Box 3 was carried out by offline FlFFF–CL. Fractionation and selective detection of different enzymes linked to PS microbeads of different sizes was possible in a single, quick FlFFF run [71]. GrFFF–CL methods were also developed for ultrasensitive cell detection using HRBCs as a model sample [72]. Whole-cell and top-down proteomics: FFF–MS FFF can be coupled to mass spectrometry (MS) equipped with low-fragmentation ion sources such as matrixassisted, laser desorption–ionization (MALDI) and electrospray ionization (ESI) sources for whole-cell and topdown proteomics. MALDI–time-of-flight (TOF)–MS is a straightforward method for whole-cell protein analysis and identification. For example, species desorbed from whole bacterial cells by MALDI, and detected in TOF–MS spectra are intact proteins, which can be identified through proteomic database searches. FlFFF was used to fractionate whole bacterial cells for further analysis by MALDI–TOF–MS [73]. The FlFFF–MALDI–TOF–MS method was then improved using HF FlFFF [74]. A mixture of two bacteria (Bacillus subtilis and E. coli) was fractionated through HF FlFFF, and MALDI–TOF–MS analysis performed on each separated bacterial species. MALDI–TOF–MS characterization demonstrated that mixed bacteria were fully separated through HF FlFFF, because each fractionated population preserved the most characteristic ion signals from ribosomal proteins of the species, without the presence of characteristic signals from ribosomal proteins from the other species. Separation and characterization of intact proteins in their native conditions is a crucial process in biotechnology. Highly accurate measurement of the actual molar mass is a prime goal for identifying intact proteins and protein complexes. Using ESI–MS, accurate mass measures, and indications of the higher-order structure of proteins and noncovalent protein complexes can be obtained. However, direct ESI–TOF–MS shows limited success for complex protein mixtures. The spectra become very complex because of the presence of many proteins and/or contaminants such as nonvolatile salts. Rapid and efficient separation methods that purify the sample and affect neither the three-dimensional structure of proteins nor the noncovalent chemistry of protein complexes can significantly enhance the power of ESI–TOF–MS methods for top-down proteomics. HF FlFFF has been coupled online to ESI–TOF–MS for the analysis and characterization of intact proteins [75]. Protein samples maintained their native structure, and were desalted online during fractionation. Correlation between the molar mass values independently measured by HF FlFFF retention and ESI– TOF–MS provided information on the quaternary

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structure of the proteins. HF FlFFF has been shown to be effective as a separation and purification method which is complementary to LC when implemented in MS-based, comprehensive approaches to the characterization of proteins in their native form. Concluding remarks Recent technical developments for increasing biocompatibility, in addition to hyphenation with the powerful, wellcharacterized bioanalytical methods described here, have made FFF a promising and increasingly effective technique in the field of modern biotechnology, from the separation and purification of native proteins and lipid– DNA complexes to the separation and characterization of immuno- or surface-hybridized nanobeads, whole-cell vaccines and stem cells. However, FFF has not, as yet, enjoyed what can be considered as an explosive growth phase, as occurred with LC and GE, even though the basic principles of FFF were established as early as in the late 1960s. A combination of different factors might be responsible for this slow development. Most FFF users agree that one major hindrance to the widespread use of FFF as a routine tool is due, quite paradoxically, to its greatest asset of versatility. FFF is applicable to a broad range of different biosamples but there is no simple answer as to which FFF method should be best used for a given application. The development of disposable or microfabricated devices for different FFF techniques, which will become possible with further developments of the FFF concept, should, however, reduce the monetary investments necessary for a laboratory to acquire different FFF methods for different biotechnological applications. Very few FFF techniques are currently commercially available, and these are produced by very few companies. Low market competition imposes a limit on what can be offered by FFF systems, and might not sufficiently encourage new technical advances and applications. Another explanation might be that since early times, FFF has been considered and applied as if were an ‘absolute’ technique - in other words, as a family of methods to separate and to characterize analytes. Nonetheless, although FFF can be unique in terms of separation selectivity, particularly for very high molarmass and particulate samples, other well-established techniques have been shown to be superior in characterizing biosamples. The authors are confident that further development of comprehensive analytical approaches using FFF coupled with other techniques will enable FFF to have the more widespread role in future biotechnology that these techniques deserve. Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.tibtech.2005.07.008

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