Mauricio Hoyos

Magnetophoretic Cell Sorting Is a Function of Antibody Binding Capacity

Antibody binding capacity (ABC) is a term representing a cellapos;s ability to bind antibodies, correlating with the number of specific cellular antigens expressed on that cell. ABC allows magnetically conjugated antibodies to bind to the targeted cells, imparting a magnetophoretic mobility on each targeted cell. This enables sorting based on differences in the cell magnetophoretic mobility and, potentially, a magnetic separation based on the differences in the cell ABC values. A cellapos;s ABC value is a particularly important factor in continuous magnetic cell separation. This work investigates the relationship between ABC and magnetic cell separation efficiency by injection of a suspension of immunomagnetically labeled quantum simply cellular calibration microbeads of known ABC values into fluid flowing through a quadrupole magnetic sorter. The elution profiles of the outlet streams were evaluated using UV detectors. Optimal separation flow rate was shown to correlate with the ABC of these microbeads. Comparing experimental and theoretical results, the theory correctly predicted maximum separation flow rates but overestimated the separation fractional recoveries.

Velocity profiles in thermal field-flow fractionation

Exact velocity profiles in thermal field-flow fractionation (FFF) were numerically computed for twelve solvents and forty different combinations of the temperature drop ΔT across the channel and of the cold wall temperature, Tc. An expression with six coefficients relating the ν parameter of a third-degree polynomial velocity profile which approximates the exact profile with ΔT and Tc. was derived for each solvent. Under typical experimental conditions, it provides a nearly two orders of magnitude improvement in the accuracy of the prediction of the retention over the equation based on the classical parabolic profile. A procedure is suggested for using this ν vs. ΔT and Tc expression for extracting the basic FFF parameter λ from retention data.

Thermal field-flow fractionation of colloidal materials: Methylmethacrylate-styrene linear di-block copolymers

SummaryModel methylmethacrylate-styrene, linear di-block, copolymers were used to investigate the respective influnnces of temperature, of molar mass and of chemical composition on their Soret coefficient, sT, by means of thermal field-flow fractionation (thermal FFF) in toluene and in THF. A recently developed thermal FFF retention model, which takes into account the variation of the basic FFF parameter λ with temperature, is applied to investigate the dependence of the Soret coefficient on temperature. It is found that the coefficient decreases approximately linearly with increasing temperature. At constant chemical composition and temperature, sT exhibits a power law dependence on molar mass with an exponent É ã 0.55. At constant molar mass and temperature, sT decreases monotonously with increasing weight percent styrene in the copolymer composition. At 300 K, sT values are slightly larger in THF than in toluene.

Controlled cell aggregation in a pulsed acoustic field.

Cell aggregation in ultrasonic resonators can be obtained in a few seconds. Hundreds even thousands of cells can be levitated in suspension and generate 2D or 3D aggregates. Nevertheless, the aggregation rate and the 2D or 3D configurations of the resultant aggregates are very difficult to control. This work reports on a novel way of generating and controlling particle and cell aggregates using pulsed ultrasound. This technique specifically explores (in addition to the ultrasound wave, frequency and amplitude) the time of ultrasound application, i.e. the number of pulses as well as the pulse repetition frequency. We demonstrate that with pulsed ultrasound, particles and/or cells levitate in suspension, as with continuous ultrasound, and the aggregation rate can be modified in a controlled manner. By carefully tuning the number of pulses and the repetition frequency, the 3-D and 2-D configurations of the aggregates can be selectively generated. In addition, pulsed ultrasound limits transducer heating, thus allowing for higher acoustic energies than those currently employed with continuous ultrasound.

Retention in Field-Flow Fractionation with a Moderate Nonuniformity in the Field Force

In some field-flow fractionation (FFF) techniques, the basic analyte-field interaction parameter, λ, is not constant but varies within the channel cross section as a result of the nonuniformity of the force exerted by the field on the analyte. This is the case, for instance, in thermal FFF, because of the temperature dependence of the relevant physicochemical transport parameters. To account for this effect, a new FFF retention model is developed, allowing a linear variation of λ from the accumulation to the depletion wall, which is assumed to describe correctly moderate nonuniformity in λ in the vicinity of the accumulation wall. A methodology for sample characterization on the basis of this model is proposed. It associates λ(app), the apparent λ value derived from the retention ratio by means of the classical retention model, with a specific distance from the accumulation wall. An empirical relationship between that distance and λ(app) is derived. In the high retention limit, it is found that this specific distance is not equal, as sometimes intuitively believed, to the mean distance of the molecule or particle cloud to the accumulation wall but is approximately equal to twice this mean distance. The validity of the proposed approach is checked.

Controlling the acoustic streaming by pulsed ultrasounds.

We propose a technique based on pulsed ultrasounds for controlling, reducing to a minimum observable value the acoustic streaming in closed ultrasonic standing wave fluidic resonators. By modifying the number of pulses and the repetition time it is possible to reduce the velocity of the acoustic streaming with respect to the velocity generated by the continuous ultrasound mode of operation. The acoustic streaming is observed at the nodal plane where a suspension of 800nm latex particles was focused by primary radiation force. A mixture of 800nm and 15μm latex particles has been also used for showing that the acoustic streaming is hardly reduced while primary and secondary forces continue to operate. The parameter we call pulse mode factor i.e. the time of applied ultrasound divided by the duty cycle, is found to be the adequate parameter that controls the acoustic streaming. We demonstrate that pulsed ultrasound is more efficient for controlling the acoustic streaming than the variation of the amplitude of the standing waves.

Acute hydrodynamic damage induced by SPLITT fractionation and centrifugation in red blood cells.

Though blood bank processing traditionally employs centrifugation, new separation techniques may be appealing for large scale processes. Split-flow fractionation (SPLITT) is a family of techniques that separates in absence of labelling and uses very low flow rates and force fields, and is therefore expected to minimize cell damage. However, the hydrodynamic stress and possible consequent damaging effects of SPLITT fractionation have not been yet examined. The aim of this study was to investigate the hydrodynamic damage of SPLITT fractionation to human red blood cells, and to compare these effects with those induced by centrifugation. Peripheral whole blood samples were collected from healthy volunteers. Samples were diluted in a buffered saline solution, and were exposed to SPLITT fractionation (flow rates 1-10 ml/min) or centrifugation (100-1500 g) for 10 min. Cell viability, shape, diameter, mean corpuscular hemoglobin, and membrane potential were measured. Under the operating conditions employed, both SPLITT and centrifugation maintained cell viability above 98%, but resulted in significant sublethal damage, including echinocyte formation, decreased cell diameter, decreased mean corpuscular hemoglobin, and membrane hyperpolarization which was inhibited by EGTA. Wall shear stress and maximum energy dissipation rate showed significant correlation with lethal and sublethal damage. Our data do not support the assumption that SPLITT fractionation induces very low shear stress and is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Measurement of membrane potential and cell diameter could provide a new, reliable and convenient basis for evaluation of hydrodynamic effects on different cell models, allowing identification of optimal operating conditions on different scales.

On void time determination in thermal field-flow fractionation.

Because of the temperature dependence of the carrier liquid density, the mass of carrier which is contained in a thermal field-flow fractionation channel depends on the cold wall temperature and on the temperature difference across the channel thickness. It is observed that the void time of the solvent peak decreases when increasing the average temperature in the channel. The void time is found to be directly proportional to the average carrier density in the channel. The determination of the void time from the knowledge of the channel geometrical volume and the measurement of the volumetric flow-rate leads to significant errors if the thermal expansion of the carrier between the temperature of the measurement and the average channel temperature is not taken into account. Recommendations are given for proper void time determinations in thermal FFF.

Separation of Leishmania-infected macrophages by step-SPLITT fractionation

After a primary infection protocol of macrophages with Leishmania amazonensis, the percentage of infection drops as infection progresses and the uninfected population of macrophages mask the effects of infection for electrophysiological studies. In order to increase or maintain the infection percentage, we introduce an enrichment process after primary infection, which increases the possibility of following the infection longer times than any known process. A membraneless separation technique, step-SPLITT fractionation, implying flow and transverse gravity field in a ribbon-like channel, was used for enriching samples of macrophages infected with particles and with L. amazonensis. We demonstrate the capability of the s-SPLITT of generating, from a mixture resulting from a primary infection, an enriched and a depleted fraction with infected cells, without using any selective labeling pre-processing. It is also shown that a continuous sorting is possible without damaging cells and the losses of matter into the separation chamber is minimal.