Fractionation of bone-marrow cells by counter-current distribution in aqueous polymer two-phase systems. Relation between settling time and the efficiency of separation

The fractionation of heterogeneous populations of rat and human bone-marrow cells has been studied by counter-current distribution in a charged 5% dextran-4% poly(ethylene glycol) two-phase system. The subfractionation into two broad populations has been achieved at a low top/bottom phase volume ratio by increasing (up to 20 min) the settling time allowed for the phases to be separated after each mixing step. No effect of this parameter on a homogeneous population of erythrocytes has been observed. However, heterogeneous cell separations can be improved by exploiting different phase settling times.     

Erythrocyte partitioning in dextran-poly(ethylene glycol) aqueous phase systems. Events in phase and cell separation.

Early events in the partitioning process which involve characteristic kinetics of cell- and phase-specific interactions and phase separation have been described previously. This paper reports on red cell-phase droplet interactions pertaining at the time of usual phase sampling (i.e., the time at which a clear bulk interface is first apparent) and beyond in cell partitioning and countercurrent distribution experiments. In non-charge-sensitive phase systems close to the critical point, cells can be free or attached to phase droplets. Cells that are free are virtually completely in the top phase, whereas different cell populations that show essentially complete binding to droplets can nevertheless have different partition ratios and be separated, thus reflecting the effects of the difference in the cells' avidity for the phase droplets during the early, elapsed events in partitioning. At higher polymer concentrations (i.e., higher interfacial tensions), the cell populations, completely bound to phase droplets, partition completely to the interface, and consequently cannot be separated. When such systems are made charge-sensitive by the generation of a Donnan potential between the phases or made into affinity systems by the incorporation of PEG ligands (e.g., PEG-palmitate), there is a decrease in the avidity of the cells for phase droplets. The resulting increase in the ratio of free to droplet-bound red cells in the top phase at the time of sampling correlates with an increase in the partition ratio, P, observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modelling counter-current and dual counter-current chromatography using longitudinal mixing cell and eluting counter-current distribution models

Two well known approaches are considered to analyze the processes of counter-current and dual counter-current chromatography: the longitudinal mixing cell model and the Craig's counter-current distribution model. The cell model represents perfectly mixed, equally sized cells in series. The number of cells characterizes the rates of longitudinal mixing in the stationary and mobile phases. In the eluting counter-current distribution (CCD) model, the CCC process is considered as a continuous form of Craig's counter-current distribution. For a cascade of equilibrium stages theoretical elution profiles of the CCC process by using the CCD and cell model approaches have been compared. It is shown that in general, distribution functions of the CCD and cell models differ. It is established that the distribution of a solute between two solvent phases in the dual CCC process is determined by the extraction factor c, the total number of equilibrium stages n and the position of the sample inlet m by the equation Q(x)=(1-c(m))/(1-c(n+1)) with c=F(2)K(D)/F(1) (K(D), F(1), F(2) and Q(x) are the distribution constant, the phase flow-rates and the portion of solute eluted by the first phase, respectively).     

Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography. III. Effects of physical properties of the solvent systems and operating temperature on the distribution of two-phase solvent systems

Statistical studies were made to correlate the hydrodynamic behavior of two-phase solvent system in counter-current chromatography (CCC) to their physical properties including interfacial tension, viscosity, and the difference in density of the two phases. Settling time measured under unit gravity provided a reliable numerical index for the hydrodynamic behavior of the solvent systems in a centrifugal force field. Viscosity and settling time were strongly correlated (correlation coefficient, r = +0.88) while interfacial tension (r = -0.65) and phase density difference (r = -0.45) showed moderate and weak correlation, respectively. Studies of the effect of temperature on settling time as well as a preliminary apparatus operated at higher temperature show that raising the temperature will improve the performance of high-speed CCC.     

Partitioning behavior of erythrocytes in aqueous two-phase systems containing hydroxypropyl starch and polyethylene glycol

The partitioning behavior of erythrocytes in Reppal PES 200 (a hydroxypropyl starch produced by Reppe Glykos AB, V?xj?, Sweden)-polyethylene glycol (PEG) and in dextran (Dx)-PEG aqueous phase systems made isotonic with phosphate is similar in a number of ways: (i) There is a correlation between the relative electrophoretic mobilities and partition ratios, P, of red blood cells from different species; (ii) The cell P is reduced when, at constant polymer concentrations, phosphate is systematically replaced by sodium chloride (with the total concentration isotonic); (iii) The cell P is increased with reduced polymer concentrations (decreased interfacial tensions); (iv) Treatment of erythrocytes with neuraminidase results in a reduced P value; (v) Rat red cells of different ages can be fractionated by counter-current distribution; and (vi) Differences between red blood cells from genetically distinct rats or between humans can be detected. Aquaphase (a hydroxypropyl starch marketed by Perstorp AB, Lund, Sweden) has been tested as in ii-iv above with analogous results. The partitioning behavior of erythrocytes in PES-PEG and Dx-PEG aqueous phase systems containing sodium chloride differs in a number of ways: (vii) The correlation, apparent in Dx-PEG systems, between the P value of red blood cells from different species and the ratio of their membrane poly- to monounsaturated fatty acids is absent in PES-PEG systems. It is replaced by a correlation as in i; (viii) The increase in P value in Dx-PEG observed from red blood cells after treatment with neuraminidase is replaced by a decrease in P value in PES-PEG or Aquaphase-PEG systems. We conclude that PES (and Aquaphase) can be substitutes for dextran in cell partitioning studies when charge-sensitive phases are used (e.g., those containing phosphate) while separations based on properties reflected by Dx-PEG systems containing sodium chloride are not duplicated by PES-PEG (and probably not by Aquaphase-PEG). The hydroxypropyl starch-PEG systems containing sodium chloride, unlike the analogous Dx-PEG systems, have a significant electrostatic potential difference between the phases.     

Liquid-liquid extraction of membranes from calf brain using conventional and centrifugal counter-current distribution techniques

Neural membranes isolated from calf brain have been partitioned in aqueous two-phase systems containing dextran and polyethyleneglycol. When the partition was repeated several times, using counter-current distribution technique, the distribution of the membranes between the upper phase and the interface changed in a non-ideal manner and in favour of the interface. By using a centrifugal counter-current distribution device the time for the experiment could be reduced by a factor of 7-8 and the distribution was similar to what could be expected for ideally behaving membranes. The time-dependent change of the membranes is discussed in terms of aggregation and lateral membrane perturbations. Despite this effect a certain fractionation has been achieved as deduced from analysis of cholesterol content, opiate receptor activity and acetylcholinesterase activity along the counter-current distribution row of fractions. Compared to the starting material these activities were enriched some two-fold in certain fractions.     

Isolation of bacitracins A and F by high-speed counter-current chromatography

The major components of bacitracin were separated and purified using high-speed counter-current chromatography (HSCCC). A systematic search for optimum two-phase solvent systems resulted in two systems: chloroform-ethanol-methanol-water (5:3:3:4) and chloroform-ethanol-water (5:4:3). These were selected based on the determination of the partition coefficients of all the components and the settling time of the phases. HSCCC with these solvent systems separated two components, bacitracins A and F. Improvements in the flow-cell arrangement eliminated noise in detection, making in-line monitoring possible. A tandem mass spectrometric technique was used to characterize the isolated components.     

Partitioning of cells in dextran-poly(ethylene glycol) aqueous phase systems. A study of settling time, vessel geometry and sedimentation effects on the efficiency of separation.

The effect of prolonged settling times (up to 2 h), in high- and low-phase columns, on the cell partition ratios measured and on the separability of cell populations was examined. With closely related cell populations, modelled by rat erythrocytes in which subpopulations of red blood cells of distinct age were labeled isotopically, it was found that partitioning proceeds over the entire time period examined as evidenced by the continuous change in relative specific activity of cells in the top phase as the partition ratio falls. In control cell sedimentation experiments in top phase there was almost no change in the quantity of cells present when vertical settling (i.e., high-phase columns) was used and no separation of specific subpopulations was found. In the horizontal settling mode the initially higher cell partition ratio, as compared to vertical settling, decreased to a greater extent with longer time intervals; a given purity of cells only being obtained at a lower partition ratio than in the vertical settling mode. Cell sedimentation in top phase was appreciable with time in the horizontal settling mode but did not result in a separation of cell subpopulations. The effect of relative cell partition ratios and sizes in high- and low-phase columns on the efficiency of separation was examined by use of rat or sheep 51Cr-labeled red cells mixed with an excess of human unlabeled erythrocytes. Rat and sheep red cells are appreciably smaller than human erythrocytes. Rat red cells have higher, and sheep red cells lower partition ratios than human erythrocytes. With vertical settling, over a 2-h period, there is no appreciable contribution to the change in relative specific activities by cell sedimentation. However, the more rapid sedimentation of the larger human red cells has, with time, a measurable effect on the relative specific activities obtained during cell partitioning when run in the horizontal mode: enhancing the rat-human and diminishing the sheep-human cell separations. Partitioning cells in high-phase columns is of advantage with respect to increasing separation efficiency and virtually eliminating the influence of other physical parameters (e.g., cell size). Since the cell partitioning process continues for long periods of time, yielding ever-lower partition ratios with increasing proportions of cells with higher P values, a time may be selected which balances desired relative cell purity and yield.     

Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography. II. Phase distribution diagrams for helical and spiral columns

Extensive studies have been performed on hydrodynamic distribution of two immiscible solvent phases in helical and spiral columns by means of coil planet centrifugation. Under the Scheme IV [see Fig. 1 in Y. Ito, J. Chromatogr., 301 (1984) 377] planetary motion, the two phases display unilateral distribution in the coil, i.e., one phase travels toward the head and the other phase toward the tail. This hydrodynamic trend is sensitively affected by various factors such as physical properties of the solvent system, helical diameter of the column, revolutional radius, etc. Phase distribution diagrams obtained from a set of conventional two-phase solvent systems under various experimental conditions provide extremely useful information for both basic hydrodynamic studies and practical application in high-speed counter-current chromatography.     

Isolation of all-trans lycopene by high-speed counter-current chromatography using a temperature-controlled solvent system

The effect of solvent system, partition coefficient, retention of stationary phase, column, revolution speed, and flow rate of mobile phase are well known parameters to effect HSCCC (high-speed counter-current chromatography) separations. Temperature effects on chromatographic techniques like HPLC and GC are well studied, but the influence of temperature on CCC solvent systems is hardly investigated. This paper presents the influence of temperature on several key parameters (partition coefficient, settling time, volume ratios) in the hydrophobic HSCCC solvent system hexane:dichloromethane:acetonitrile (30:11:18, v/v/v) used for the isolation of lycopene from tomato paste at 10, 15, 20 and 25 degrees C.     

Microfluidic lithography of SAMs on gold to create dynamic surfaces for directed cell migration and contiguous cell cocultures

A straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics-microfluidic lithography-has been developed. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling the solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients with a single microfluidic chip. To demonstrate the utility of microfluidic lithography, multiple cell experiments were conducted. By patterning cell adhesive regions in an inert background, a combination of selective masking of the surface and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous cocultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous cocultures with multiple sites for heterogeneous cell-cell interactions.     

Scenarios of heterogeneous nucleation and growth studied by cell dynamics simulation

The dynamics of phase transformation due to homogeneous nucleation has long been analyzed using the classic Kolmogorov-Johnson-Mehl-Avrami (KJMA) theory. However, the dynamics of phase transformation due to heterogeneous nucleation has not been studied systematically even though it is vitally important technologically. In this report, the author studies the dynamics of heterogeneous nucleation theoretically and systematically using the phenomenological time-dependent Ginzburg-Landau (TDGL)-type model combined with the cell dynamics method. In this study the author focuses on the dynamics of phase transformation when the material is sandwiched by two supporting substrates. This model is supposed to simulate phase change storage media. Since both homogeneous and heterogeneous nucleations can occur simultaneously, the author predicts a few scenarios of phase transformation including homogeneous nucleation regime, heterogeneous nucleation regime, and the homogeneous-heterogeneous coexistence regime. These predictions are directly confirmed by numerical simulation using the TDGL model. The outcome of the study was that the KJMA formula has limited use when heterogeneous nucleation exists, but it could still give some information about the microscopic mechanism of phase transformation at various stages during phase transformation.     

Purification of astilbin and isoastilbin in the extract of smilax glabra rhizome by high-speed counter-current chromatography

Purification of astilbin from the rhizome extract of Smilax glabra was conducted using a high-speed counter-current chromatograph equipped with a 700 mL column. In a single operation, 1.5 g of crude sample was separated to yield 105 mg of component astilbin and 48 mg of isoastilbin while the upper phase of the two-phase solvent system composed of n-hexane-n-butanol-water (1:1:2, v/v/v) was used for stationary phase. The chemical structures of the two flavonoid glycosides were confirmed by electrospray ionization ion trap multiple mass spectrometry and nuclear magnetic resonance experiments.     

Partitioning behaviour in aqueous two-phase systems and fractionation by counter-current distribution of chick-embryo erythrocytes with numerical resolution of distribution curves

The partition of chick-embryo and young-chick erythrocytes in dextran-poly-(ethylene glycol) two-phase systems depends on the interfacial tension and electrical potential differences between the phases. Counter-current distribution with charged 5% dextran-poly(ethylene glycol) systems has proved to be an adequate method for the separation of primitive and definitive erythrocytes present in chick embryos when a phase settling time of 20 min is used. The computer-aided numerical resolution of experimental curves has shown the existence of subpopulations which could not have been detected by using conventional methods.     

Subfractions of membranes from calf brain synaptosomes obtained and studied by liquid-liquid partitioning.

Synaptosomes isolated from calf brain cortex were lysed and fragmented by Yeda press treatment. The obtained membranes have previously been fractionated in a counter-current distribution process using a liquid-liquid two-phase system consisting of water, dextran, Ficoll and poly(ethylene glycol) [J. Chromatogr., 358 (1986) 147]. Using the fact that there are discrete membrane populations, a rapid preparative method for isolation of the two main fractions is presented in the present work, as well as a subfractionation of one of them using liquid-liquid extraction with dextran-bound Procion yellow HE-3G. The content of several membrane constituents, i.e. protein, acetylcholinesterase, succinate dehydrogenase and ATPase, as well as opiate binding, were determined for the three fractions. Counter-current distribution of the fractions elucidates their heterogeneity and the effectiveness of the purification.     

Metabolomics studies of cell–cell interactions using single cell mass spectrometry combined with fluorescence microscopy

Cell–cell interactions are critical for transmitting signals among cells and maintaining their normal functions from the single-cell level to tissues. In cancer studies, interactions between drug-resistant and drug-sensitive cells play an important role in the development of chemotherapy resistance of tumors. As metabolites directly reflect the cell status, metabolomics studies provide insight into cell–cell communication. Mass spectrometry (MS) is a powerful tool for metabolomics studies, and single cell MS (SCMS) analysis can provide unique information for understanding interactions among heterogeneous cells. In the current study, we utilized a direct co-culture system (with cell–cell contact) to study metabolomics of single cells affected by cell–cell interactions in their living status. A fluorescence microscope was utilized to distinguish these two types of cells for SCMS metabolomics studies using the Single-probe SCMS technique under ambient conditions. Our results show that through interactions with drug-resistant cells, drug-sensitive cancer cells acquired significantly increased drug resistance and exhibited drastically altered metabolites. Further investigation found that the increased drug resistance was associated with multiple metabolism regulations in drug-sensitive cells through co-culture such as the upregulation of sphingomyelins lipids and lactic acid and the downregulation of TCA cycle intermediates. The method allows for direct MS metabolomics studies of individual cells labeled with fluorescent proteins or dyes among heterogeneous populations.

We combined single cell mass spectrometry and fluorescence microscopy techniques to study metabolites affected by interactions between different types of cells under ambient conditions.     

Partial purification of glucose 6-phosphate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems

Glucose 6-phosphate dehydrogenase shows a high partition coefficient in poly-(ethylene glycol)-dextran aqueous two-phase systems in comparison with those for 6-phosphogluconate dehydrogenase, phosphofructokinase and the bulk of proteins present in rat erythrocyte haemolysates. As a consequence, fractions highly enriched in glucose 6-phosphate dehydrogenase can be obtained after multiple partitions in the above systems with a counter-current distribution procedure. Phosphofructokinase shows a high affinity for Cibacron Blue and, as a result, the enzyme can be extracted in the top phase of poly(ethylene glycol)-dextran systems containing Cibacron Blue-poly(ethylene glycol) (affinity systems). The efficiency for the purification of the enzymes by partitioning is increased up to 10-fold when enzyme-rich fractions, obtained by precipitation with poly(ethylene glycol), are used instead of original haemolysate. The recovery of enzyme activities is near 100% in both instances.     

A study of DNA from chloroplasts separated by counter-current distribution.

Three types of chloroplasts (Peak I, Peak II, and Peak III chloroplasts) isolated by counter-current distribution in aqueous polymer two-phase systems have been studied with respect to DNA content. (The characterization was performed by studying the buoyant density, the melting profile and the amount of DNA of each type). The DNA was found to differ between the three types of chloroplasts.     

Observations of established dual flow in a spiral dual-flow counter-current chromatography coil

This paper describes the observations made in dual-flow counter-current chromatography. For the first time, the behaviour of the phases inside a spiral dual-flow coil has been studied using stroboscopic visualisation. During the study it was observed that the phase distribution and the linear flow rate in the tubing were not uniform throughout the coil, but behaved differently at each end of the coil with a transition area in between. The location of the transition area is dependent on the flow rate of both the upper and the lower phase. Understanding and then explaining such counter-current flow will significantly help in the prediction of elution behaviour in true moving bed chromatography.