Gravitational field-flow fractionation of human hemopoietic stem cells

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.     

Magnetic field-flow fractionation using capillary tubing

Summary Separation of paramagnetic substances under the field of magnetic gradient by using capillary tubing was attempted. Teflon tubing of 0.5 mm i.d. and 270 cm in length was placed on the outside of one pole of an electric magnet which had 9 K gauss at 30 V. The Samples were bovine serum albumin, egg albumin, and EDTA, and their complexes with Ni ion. The mobile phase was water at a flow rate of 20 μl/min. Samples without Ni ion had the same retention volume irrespective of the existence of magnetic gradient. Samples which formed complexes with Ni ion showed retardation under the field of magnetic gradient, although the difference of retention volumes obtained with and without the magnetic field was not large.     

Separation of erythrocytes and latex beads by dielectrophoretic levitation and hyperlayer field-flow fractionation.

A model system consisting of a mixture of latex beads and erythrocytes has been investigated to demonstrate the practical feasibility of particle separation by means of the combined application of negative dielectrophoresis and hyperlayer field-flow fractionation. The dielectrophoretic levitation of latex beads is demonstrated by energizing interdigitated electrodes, of widths and separation ranging from 5 to 40 μm, with AC signals of 0–10 V (rms) in the frequency range 1 kHz–10 MHz. Maximum levitation was attained at 1 MHz, at which frequency levitation is relatively independent of the suspending medium conductivity. Levitation was also independent of particle size, but dependent on particle density and dielectric properties. At 1 MHz the erythrocytes were attracted to the electrodes by positive dielectrophoresis, and so could be separated from the latex beads by fluid flow. The electric field and field gradient above the electrodes were also computer modelled, and this information was used to design the electrode and chamber geometries for optimum DEP-field-flow fractionation.     

Hyperlayer hollow-fiber flow field-flow fractionation of cells

Interest in low-cost, analytical-scale, highly efficient and sensitive separation methods for cells, among which bacteria, is increasing. Particle separation in hollow-fiber flow field-flow fractionation (HF FlFFF) has been recently improved by the optimization of the HF FIFFF channel design. The intrinsic simplicity and low cost of this HF FlFFF channel allows for its disposable usage. which is particularly appealing for analytical bio-applications. Here, for the first time, we present a feasibility study on high-performance, hyperlayer HF FIFFF of micrometer-sized bacteria (Escherichia coli) and of different types of cells (human red blood cells, wine-making yeast from Saccharomyces cerevisiae). Fractionation performance is shown to be at least comparable to that obtained with conventional, flat-channel hyperlayer FIFFF of cells, at superior size-based selectivity and reduced analysis time.     

Subcellular fractionation of brown adipose tissue

The present study proposes a technique, using Metrizamide, which permits the preparation of brown adipose tissue plasma membranes from the crude mitochondria as well as from the crude microsome fraction. These plasma membranes have high relative specific activities of their marker enzyme, 5'-nucleotidase (15 +/- 3 and 14 +/- 2 respectively) and, particularly those originating in the crude microsomes, are relatively free of mitochondria contamination. This study also shows the influence of the mode of cell disruption on microsome integrity. When cell disruption was achieved by grinding in liquid nitrogen the purified microsome NADPH cytochrome c reductase specific activity was found to be 3.5 times greater than that of microsomes obtained after homogenization of the tissue.     

Characterization of oat proteins and aggregates using asymmetric flow field-flow fractionation

The soluble proteins and protein aggregates in Belinda oats were characterized using asymmetric flow field-flow fractionation (AF4) coupled with online UV–vis spectroscopy and multiangle light-scattering detection (MALS). Fractions from the AF4 separation were collected and further characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The AF4 fractogram of the oat extracts revealed three peaks which were determined to be monomeric forms of soluble proteins, globulin aggregates, and β-glucan, respectively. The early eluting monomeric proteins ranged in molar mass (MM) between 5 and 90 kg/mol and in hydrodynamic diameter (D _h) from 1.6 to 13 nm. The MM at peak maximum of the globulin aggregate peak was found to be ～300 kg/mol and the D _h was measured to be ～20 nm. SDS-PAGE of the collected fraction across this peak revealed two bands with MM of 37 and 27 kg/mol which correspond to the α and β subunits of globulin indicating the elution of globulin aggregates. A third peak at long retention time was determined to be β-glucan through treatment of the oat extract with β-glucanase and by injection of β-glucan standards. The amount of soluble protein was measured to be 83.1?±?2.3 wt.%, and the amount of albumin proteins was measured to be 17.6?±?5.7 wt.% of the total protein in the oats. The results for Belinda oat extracts show that the AF4-MALS/UV platform is capable of characterizing the physicochemical properties such as MM and hydrodynamic size distribution of proteins and protein aggregates within a complicated food matrix environment and without the need to generate protein isolates.

Separation of living red blood cells by gravitational field-flow fractionation.

The field-flow fractionation technique, using the earth's gravitational field, has been applied to peripheral blood cell populations. A more or less symmetrical, gaussian-like, elution peak is generally observed for the red cell population. The bimodal cell population obtained after a massive transfusion is shown to result in a shoulder on the red blood cell elution profile. In one case where a similar shouldering peak was obtained from a non-transfused donor, the existence of an immunological double population has been demonstrated. This suggests that field-flow fractionation has some potential for complementary biomedical diagnosis.     

Hybrid gravitational field-flow fractionation using immunofunctionalized walls for integrated bioanalytical devices

In this work, the biospecific recognition antigen–antibody reaction was implemented in gravitational field-flow fractionation (GrFFF), a flow-assisted separation technique for micron-sized particles, in order to realize a hybrid immunomodulated GrFFF system in which two different principles are combined to achieve highly versatile fractionation. Micron-sized polystyrene beads coated with horseradish peroxidase (HRP) were used as a model sample, and anti-HRP antibodies were immobilized on the accumulation wall of the GrFFF channel. Ultrasensitive chemiluminescence imaging was employed to visualize the beads during elution and to optimize experimental conditions. The same principle was then applied to real biological samples composed by Yersinia enterocolitica and Escherichia coli cells. Results show the possibility to modify the elution of selected sample components and even to retain them into the channel. The hybrid immunomodulated GrFFF system is a step towards the development of a module that could be integrated in a lab-on-a-chip-based point-of-care testing device which includes sample pre-analytical cleanup and analysis.     

Temporal variability of colloidal material in agricultural storm runoff from managed grassland using flow field-flow fractionation

This paper reports the use of flow field-flow fractionation (FlFFF) to determine the temporal variability of colloidal (<1 μm) particle size distributions in agricultural runoff waters in a small managed catchment in SW England during storm events. Three storm events of varying intensity were captured and the colloidal material in the runoff analysed by FlFFF. The technique had sufficient sensitivity to determine directly the changing colloidal profile over the 0.08–1.0 μm size range in the runoff waters during these storm events. Rainfall, total phosphorus and suspended solids in the bulk runoff samples were also determined throughout one storm and showed significant correlation (P < 0.01) with the amount of colloidal material. Whilst there are some uncertainties in the resolution and absolute calibration of the FlFFF profiles, the technique has considerable potential for the quantification of colloidal material in storm runoff waters.     

Composition and molecular weight analysis of styrene-acrylic copolymers using thermal field-flow fractionation

Thermal field-flow fractionation coupled with online multiangle light scattering, differential refractive index and quasielastic light scattering (ThFFF-MALS/dRI/QELS) was used to simultaneously determine the molecular weight (MW) and composition of polystyrene-poly(n-butyl acrylate) (PS-PBA) and polystyrene-poly(methyl acrylate) (PS-PMA) copolymers. The online measurement of the normal diffusion coefficient (D) by QELS allowed calculation of the copolymer thermal diffusion coefficient (D(T)) of sample components as they eluted from the ThFFF channel. DT was found to be independent of MW for copolymers with similar compositions and dependent on composition for copolymers with similar MW in a non-selective solvent. By using a solvent that is non-selective to both blocks of the copolymer, it was possible to establish a universal calibration plot of DT versus mole fraction of one of the monomer chemistries comprising the copolymer. PS-PBA and PS-PMA linear diblock polymers were determined to vary in composition from 100/0 to 20/80 wt% PS/acrylate and ranged in MWs between 30 and 360 kDa. The analysis of a PS-PBA miktoarm star copolymer revealed a polydisperse material with a weight percent PBA of 50-75% and MW ranging from 100 to 900 kDa. The presented ThFFF-MALS/dRI/QELS method allowed rapid characterization of polymers with MW and chemical distributions in a single analysis.     

Investigating the relationship between cell cycle stage and diosgenin-induced megakaryocytic differentiation of HEL cells using sedimentation field-flow fractionation

Differentiation therapy could be one strategy for stopping cancer cell proliferation. A plant steroid, diosgenin, is known to induce megakaryocytic differentiation in human erythroleukemia (HEL) cells. In recent studies, the use of sedimentation field-flow fractionation (SdFFF) allowed the preparation of subpopulations that may differ in regard to sensitivity to differentiation induction. The specific goal of this study was to determine the relationship between cell cycle stage and sensitivity to megakaryocytic differentiation induction of HEL cells. After first confirming the capacity of diosgenin to specifically select targets, hyperlayer SdFFF cell sorting was used to prepare fractions according to cell cycle position from crude HEL cells. The sensitivities of these fractions to diosgenin-induced differentiation were then tested. The coupling of SdFFF cell separation to imaging flow cytometry showed that G1-phase cells were more sensitive to differentiation induction than S/G2M-phase cells, confirming the relationship between cell status at the start of induction, the extent of the biological event, and the potential of SdFFF in cancer research.     

Investigation of red blood cell fractionation by gravitational field-flow fractionation.

Gravitational field-flow fractionation is used for the separation of particles according to their sizes in the range 1-100 microns: larger particles elute before smaller ones. This phenomenon can be explained as a result of the steric exclusion of the particles from the vicinity of the channel walls, and/or hydrodynamic effects supposedly associated with the inertia of the liquid. The method was used for the investigation of red blood cells. The dependence of the retention ratio on the flow-rate, sample volume, concentration of blood and relaxation time was studied. Analysis of fifteen individual fractions by Coulter counter and reinjection of three other fractions were studied in order to verify fractionation of red blood cells.     

Analysis of complex polymers by multidetector field-flow fractionation

Field-flow fractionation (FFF) is a powerful alternative to column-based polymer fractionation methods such as size-exclusion chromatography (SEC) or interaction chromatography (IC). The most common polymer fractionation method, SEC, has its limitations when polymers with very high molar masses or complex structures must be analysed. Another limitation of all column-based methods is that the samples must be filtered before analysis and shear degradation of large macromolecules may be caused by the stationary phase and/or the column frits. Finally, the separation of very polar polymers may be a challenge because such polymers interact very strongly with the stationary phase, causing irreversible adsorption or other negative effects. This article reviews the latest developments in field-flow fractionation of complex polymers. It is demonstrated that some of the limitations of column-based chromatography can be overcome by FFF. When appropriate, results from column-based fractionations are compared with those from FFF fractionations to highlight the specific merits and challenges of each method. In addition to the fractionations themselves, various detector setups are discussed to show that different polymer distributions require different experimental procedures. Examples are given of the analysis of molar mass distribution, chemical composition, and microstructure. Advanced detector combinations are discussed, most prominently the very recently developed coupling to ¹H NMR. Finally, analysis of polymer nanocomposites by asymmetric flow field-flow fractionation (AF4)–FTIR is presented.

Characterization of humic materials by flow field-flow fractionation

Several humic materials are characterized by flow field-flow fractionation, including humic acids, a fulvic acid, and aqueous leachates from compost. Hydrophilic and hydrophobic fractions of a compost leachate were also examined. After characterizing molecular weight distributions, the effect of pH and salt concentration on hydrodynamic size is studied. In general, the hydrodynamic size decreases as the pH is lowered. However, humic acids form large aggregates below pH 5. Small amounts of sodium chloride have little effect on the size distributions. In contrast, a little calcium chloride reduces the hydrodynamic size of individual molecules while inducing the formation of oligomers, although severe aggregation is absent. With further additions of calcium chloride, the decrease in hydrodynamic size continues but oligomer formation subsides. Precise characterization of the unaggregated material is hindered by sample penetration through the channel membrane.     

Hollow-fiber flow field-flow fractionation of whole blood serum

Hollow-fiber flow field-flow fractionation is here applied to untreated, whole human blood serum. Matrix-assisted, laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) of serum fractions shows mass signals in the <30,000 M(r) range where low-abundance, serum protein components are known to be present, though a membrane of nominal 30,000 Da cutoff was employed for the fractionation device. Using diluted sera spiked with low amounts (0.06-0.1%, w/w) of an artificial mixture constituted the human adrenocorticotropic hormone fragments 18-39 (M(r)=2465.7) and 7-38 (M(r)=3659.2), and of bovine insulin (M(r)=5734), horse cytochrome c (M(r)=12384) and chicken lysozyme (M(r)=14388), a hybrid fractionation/microfiltration mechanism shows to govern the separation of the low-M(r) components.     

Separation and characterization of particles in natural water and soil using flow field-flow fractionation

Flow field-flow fractionation (FlFFF) is used to characterize particles in natural water (ground and surface water) and soil. The opposed flow sample concentration (OFSC) mode of FlFFF (OFSC-FlFFF) is employed, where the colloidal sample is continuously fed into the channel so that the particles are focused into a narrow band near the inlet of the FlFFF channel before the separation is initiated. There is no need for stopping the flow for the sample relaxation, which is usually required in conventional FlFFF operations. First, the OFSC-FlFFF is tested with mixtures of polystyrene latex spheres. Then the OFSC-FlFFF procedure is optimized for the analysis of particles in natural water and soil by varying various experimental parameters including the flow rates. Ground water of up to 100 mL has been successfully loaded, concentrated, and characterized by OFSC-FlFFF. Results show that the OFSC-FlFFF provides a simplified alternative to existing off-line concentration procedures, and it shows high potential for application to analysis of dilute colloidal particles in environmental water. The composition of the samples was analyzed using atomic absorption spectrometry.     

Size characterization of drug-loaded polymeric core/shell nanoparticles using asymmetrical flow field-flow fractionation

Asymmetrical flow field-flow fractionation (AsFlFFF) was used to determine the size distribution of drug-loaded core/shell nanoparticles which have a lipid core of lecithin and a polymeric shell of a Pluronic. AsFlFFF provided separation of the drug-loaded core/shell nanoparticles from smaller coreless polymeric micelles, thus allowing accurate size analysis of the drug-loaded nanoparticles without interference by the coreless micelles. It was found from AsFlFFF that the drug-loaded nanoparticles have broad size distributions ranging from 100 to 600 nm in diameter. It was also found that, after the nanoparticles had been stored for 70 days, they disappeared as a result of self-degradation. Being a separation technique, AsFlFFF seems to be more useful than transmission electron microscopy or dynamic light scattering for size analysis of core/shell nanoparticles, which have broad and bimodal size distributions. Figure Separation by AsFlFFF     

Coupling gravitational and flow field-flow fractionation, and size-distribution analysis of whole yeast cells

This work continues the project on field-flow fractionation characterisation of whole wine-making yeast cells reported in previous papers. When yeast cells are fractionated by gravitational field-flow fractionation and cell sizing of the collected fractions is achieved by the electrosensing zone technique (Coulter counter), it is shown that yeast cell retention depends on differences between physical indexes of yeast cells other than size. Scanning electron microscopy on collected fractions actually shows co-elution of yeast cells of different size and shape. Otherwise, the observed agreement between the particle size distribution analysis obtained by means of the Coulter counter and by flow field-flow fractionation, which employs a second mobile phase flow as applied field instead of Earths gravity, indicates that yeast cell density can play a major role in the gravitational field-flow fractionation retention mechanism of yeast cells, in which flow field-flow fractionation retention is independent of particle density. Flow field-flow fractionation is then coupled off-line to gravitational field-flow fractionation for more accurate characterisation of the doubly-fractionated cells. Coupling gravitational and flow field-flow fractionation eventually furnishes more information on the multipolydispersity indexes of yeast cells, in particular on their shape and density polydispersity.     

Study of continuous two-dimensional thermal field-flow fractionation of polymers

Two-dimensional thermal field-flow fractionation (2D-ThFFF) is a new instrumental technique devised for continuous fractionation of soluble macromolecules and particles. The sample mixture is introduced into a disc-shaped channel and the separated sample components are collected continuously from the channel outlets. The method is based on a two-dimensional fractionation mechanism with radial and tangential flow components in the channel. The effects of flow components and thermal gradient on the fractionation were studied in the separation of polystyrene samples of different molecular masses using cyclohexane or a binary solvent consisting of 25% ethylbenzene and 75% cyclohexane as carrier. The continuous separation of polystyrene samples was improved with increasing thermal gradient and with the use of slow radial and tangential flow rates. The technique can be applied to preparative continuous separation of macromolecules.