Magnetic nanomanipulations inside living cells compared with passive tracking of nanoprobes to get consensus for intracellular mechanics.

During the last decade, the development of nanomaterials to penetrate inside living cells has been the focus of a large number of studies, with applications for the biomedical field. However, the further dynamics of these nanomaterials inside the cells is dictated by the intracellular environment and in particular its mechanical properties. The mechanical characteristics of the cell interior can be probed with either active or passive microrheological approaches. However, active intracellular microrheology is still in its infancy, owing to the difficulty of inserting probes that can be manipulated by external forces. Here we review recent active microrheology studies using magnetic nanoprobes inserted into endosomes or phagosomes as useful approaches for measuring frequency-dependent viscoelasticity, for mapping the viscoelastic landscape, as well as for identifying the contribution of individual cytoskeleton components and the influence of cell motility. The results of such direct measurements challenge the validity of more typical passive approaches in which the spontaneous displacement of embedded nanoprobes is measured. Here we discuss that one must distinguish probes suitable for use in conditions of thermal equilibrium, whose movements reflect the mechanical environment from probes that interact actively with the cytoplasm and cytoskeleton, in a state of nonequilibrium for which fluctuation-dissipation theorem no longer holds. However, when data on these probes' viscoelastic microenvironment is available, such passive probe movements can yield useful information on the forces responsible for intracellular activity.

Cell sorting by endocytotic capacity in a microfluidic magnetophoresis device.

Magnetically labelled cells are finding a wealth of applications for in vitro analysis as well as in vivo treatments. Sorting of cells into subpopulations based on their magnetite loading is an important step in such procedures. Here, we study the sorting of monocytes and macrophages which internalise nanoparticles to different extents based on their endocytotic capacity. Macrophages featured a high endocytotic activity and were found to internalise between 4 and 60 pg of iron per cell. They were successfully sorted into five subpopulations of narrow iron loading distributions via on-chip free-flow magnetophoresis, thus demonstrating the potential of sorting of relatively similarly loaded cells. Monocytes featured a low endocytotic capacity and took on 1 to 4 pg of iron per cell. Mixtures of monocytes and macrophages were successfully sorted within the free-flow magnetophoresis chip and good purity (>88%), efficacy (>60%) and throughput (from 10 to 100 cells s(-1)) could be achieved. The introduced method constitutes a viable tool for studies of endocytotic capacity and sorting/selection of cells based on this functionality.

In vivo determination of fluctuating forces during endosome trafficking using a combination of active and passive microrheology.

BACKGROUND: Regulation of intracellular trafficking is a central issue in cell biology. The forces acting on intracellular vesicles (endosomes) can be assessed in living cells by using a combination of active and passive microrheology. METHODOLOGY/PRINCIPAL FINDINGS: This dual approach is based on endosome labeling with magnetic nanoparticles. The resulting magnetic endosomes act both as probes that can be manipulated with external magnetic fields to infer the viscoelastic modulus of their surrounding microenvironment, and as biological vehicles that are trafficked along the microtubule network by means of forces generated by molecular motors. The intracellular viscoelastic modulus exhibits power law dependence with frequency, which is microtubule and actin-dependent. The mean square displacements of endosomes do not follow the predictions of the fluctuation-dissipation theorem, which offers evidence for active force generation. Microtubule disruption brings the intracellular medium closer to thermal equilibrium: active forces acting on the endosomes depend on microtubule-associated motors. The power spectra of these active forces, deduced through the use of a generalized Langevin equation, show a power law decrease with frequency and reveal an actin-dependent persistence of the force with time. Experimental spectra have been reproduced by a simple model consisting in a series of force steps power-law distributed in time. This model enlightens the role of the cytoskeleton dependent force exerted on endosomes to perform intracellular trafficking. CONCLUSIONS/SIGNIFICANCE: In this work, the influence of cytoskeleton components and molecular motors on intracellular viscoelasticity and transport is addressed. The use of an original probe, the magnetic endosome, allows retrieving the power spectrum of active forces on these organelles thanks to interrelated active and passive measures. Finally a computational model gives estimates of the force itself and hence of the number of the motors pulling on endosomes.

Magnetic micro-manipulations to probe the local physical properties of porous scaffolds and to confine stem cells.

The in vitro generation of engineered tissue constructs involves the seeding of cells into porous scaffolds. Ongoing challenges are to design scaffolds to meet biochemical and mechanical requirements and to optimize cell seeding in the constructs. In this context, we have developed a simple method based on a magnetic tweezer set-up to manipulate, probe, and position magnetic objects inside a porous scaffold. The magnetic force acting on magnetic objects of various sizes serves as a control parameter to retrieve the local viscosity of the scaffolds internal channels as well as the stiffness of the scaffolds pores. Labeling of human stem cells with iron oxide magnetic nanoparticles makes it possible to perform the same type of measurement with cells as probes and evaluate their own microenvironment. For 18 microm diameter magnetic beads or magnetically labeled stem cells of similar diameter, the viscosity was equivalently equal to 20 mPa s in average. This apparent viscosity was then found to increase with the magnetic probes sizes. The stiffness probed with 100 microm magnetic beads was found in the 50 Pa range, and was lowered by a factor 5 when probed with cells aggregates. The magnetic forces were also successfully applied to the stem cells to enhance the cell seeding process and impose a well defined spatial organization into the scaffold.