Orane Guillaume-Gentil

Force-controlled manipulation of single cells: from AFM to FluidFM

The ability to perturb individual cells and to obtain information at the single-cell level is of central importance for addressing numerous biological questions. Atomic force microscopy (AFM) offers great potential for this prospering field. Traditionally used as an imaging tool, more recent developments have extended the variety of cell-manipulation protocols. Fluidic force microscopy (FluidFM) combines AFM with microfluidics via microchanneled cantilevers with nano-sized apertures. The crucial element of the technology is the connection of the hollow cantilevers to a pressure controller, allowing their operation in liquid as force-controlled nanopipettes under optical control. Proof-of-concept studies demonstrated a broad spectrum of single-cell applications including isolation, deposition, adhesion and injection in a range of biological systems.

pH-controlled recovery of placenta-derived mesenchymal stem cell sheets

Widely used in different biomedical applications, polyelectrolyte multilayers provide inter alia an attractive way for manufacturing of bio-functionalized, stimuli responsive surface coatings to control cellular behavior. In this study a novel polyelectrolyte-based platform for the engineering and controllable detachment of human mesenchymal stem cell (MSC) sheets is presented. Thin films obtained by layer-by-layer deposition of cationic poly(allylamine hydrochloride) (PAH) and anionic poly(styrene sulfonate) (PSS) polyelectrolytes on conductive indium tin oxide (ITO) electrodes allowed for the fast formation of viable sheets from human placenta-derived mesenchymal stem cells (PD-MSCs). Resulting stem cell sheets retained their phenotypical profile and mesodermal differentiation potency. Both electrochemically-induced local pH lowering and global decrease of the environmental pH allowed for a rapid detachment of intact stem cell sheets. The recovered stem cell sheets remained viable and maintained their capacity to differentiate toward the adipogenic and osteogenic lineages. This novel polyelectrolyte multilayer based platform represents a promising, novel approach for the engineering of human stem cell sheets desired for future clinical applications.

Rapid and Serial Quantification of Adhesion Forces of Yeast and Mammalian Cells

Cell adhesion to surfaces represents the basis for niche colonization and survival. Here we establish serial quantification of adhesion forces of different cell types using a single probe. The pace of single-cell force-spectroscopy was accelerated to up to 200 yeast and 20 mammalian cells per probe when replacing the conventional cell trapping cantilever chemistry of atomic force microscopy by underpressure immobilization with fluidic force microscopy (FluidFM). In consequence, statistically relevant data could be recorded in a rapid manner, the spectrum of examinable cells was enlarged, and the cell physiology preserved until approached for force spectroscopy. Adhesion forces of Candida albicans increased from below 4 up to 16 nN at 37°C on hydrophobic surfaces, whereas a Δhgc1-mutant showed forces consistently below 4 nN. Monitoring adhesion of mammalian cells revealed mean adhesion forces of 600 nN of HeLa cells on fibronectin and were one order of magnitude higher than those observed for HEK cells.

Ion-induced cell sheet detachment from standard cell culture surfaces coated with polyelectrolytes

Polyelectrolyte multilayers (PEMs), formed by alternating layer-by-layer deposition of polyanions and polycations, are an ideal substrate for controlling cellular adhesion and behavior. In the present study we propose a simple mechanism for the controlled detachment of C(2)C(12) myoblasts cell sheets from PEMs consisting of poly(l-lysine) and hyaluronic acid with a topmost layer of fibronectin. The multilayers were deposited on two standard cell culture surfaces: glass and polystyrene. Adding a low concentration of nontoxic ferrocyanide to the cell culture medium resulted in erosion of the polyelectrolyte multilayer and rapid detachment of viable cell sheets. Additional Quartz Crystal Microbalance and Atomic Force Microscopy measurements indicated that the detached cells retained their extracellular matrix and that no polyelectrolyte molecules remained bound to the cell sheets. The dissolution of polyelectrolyte multilayers by multivalent ions is a promising approach to cell sheet engineering that could potentially be used for regenerative medicine.

Electrochemically switchable platform for the micro-patterning and release of heterotypic cell sheets

This article describes a dynamic platform in which the biointerfacial properties of micro-patterned domains can be switched electrochemically through the spatio-temporally controlled dissolution and adsorption of polyelectrolyte coatings. Insulating SU-8 micro-patterns created on a transparent indium tin oxide electrode by photolithography allowed for the local control over the electrochemical dissolution of polyelectrolyte mono- and multilayers, with polyelectrolytes shielded from the electrochemical treatment by the underlying photoresist stencil. The platform allowed for the creation of micro-patterned cell co-cultures through the electrochemical removal of a non-fouling polyelectrolyte coating and the localized adsorption of a cell adhesive one after attachment of the first cell population. In addition, the use of weak adhesive polyelectrolyte coatings on the photoresist domains allowed for the detachment of a contiguous heterotypic cell sheet upon electrochemical trigger. Cells grown on the ITO domains peeled off upon electrochemical dissolution of the sacrificial polyelectrolyte substrate, whereas adjacent cell areas on the insulated weakly adhesive substrate easily detached through the contractile force generated by neighboring cells. This electrochemical strategy for the micro-patterning and detachment of heterotypic cell sheets combines simplicity, precision and versatility, and presents great prospects for the creation of cellular constructs which mimic the cellular complexity of native tissues.

Tunable Single-Cell Extraction for Molecular Analyses

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level.

The quantification of single cell adhesion on functionalized surfaces for cell sheet engineering.

The use of force spectroscopy to measure and quantify the forces involved in the adhesion of 3T3 fibroblasts to different chemically functionalized surfaces has been investigated. Cells were grown on glass surfaces as well as on surfaces used for cell sheet engineering: surfaces coated with polyelectrolyte multilayers (poly-L-lysine and hyaluronic acid) and thermally-responsive poly(N-isopropylacrylamide) (PNIPAM) brushes. Individual adherent cells were detached from their culture substrate using an AFM cantilever coated with fibronectin. The maximum forces of detachment of each cell were measured and taken as characteristic of the cellular adhesion. Large differences in cellular adhesion were observed on polyelectrolyte coatings depending on the number of polyelectrolyte layers. On PNIPAM-grafted surfaces, changes of more than an order of magnitude were observed in cell adhesion above and below the lower critical solution temperature. Glass surfaces patterned with periodic PNIPAM microdomains were also investigated, and it was shown that cellular adhesion could be reduced while keeping cellular morphology unchanged.

Global and local view on the electrochemically induced degradation of polyelectrolyte multilayers: from dissolution to delamination

The electrochemically tailored degradation of polyelectrolyte assemblies holds great promises for the design of inexpensive, easily prepared and precise controlled release systems. However, the conception of such electrochemically responsive platforms for drug or gene delivery requires a detailed understanding of the degradation process of the polyelectrolyte multilayer in which the active species to release are incorporated. To this end, we assess here the influence of an applied electric potential on different polyelectrolyte systems, combining global and local investigation techniques. In situ atomic force microscopy allows us to evidence morphological changes at the nano- and micro-scale, while the investigation at a larger scale by optical waveguide lightmode spectroscopy brings complementary information relating not only to material release into the bulk solution, but also to ion migration and swelling. Weak and highly hydrated poly(L-lysine)/hyaluronic acid assemblies with thicknesses up to several hundreds of nanometre continuously dissolve upon electrochemical trigger. However, stronger and more compact films made of poly(allylamine hydrochloride) and poly(styrene sulfonate) dissolve only if their thickness is of a few tens of nanometres, while thicker films delaminate from the electrode. Additional results obtained with composite films combining both polyelectrolyte systems allow us to present mechanisms based on the continuous formation of protons at the electrode surface due to water electrolysis which fully describe the dissolution and the delamination processes. In addition, the study also reveals a novel approach for the release of free-standing polyelectrolyte membranes, which is of great interest for the development of mechanical sensors, separation membranes, or micromechanical devices.

Single-Cell Mass Spectrometry of Metabolites Extracted from Live Cells by Fluidic Force Microscopy

Single-cell metabolite analysis provides valuable information on cellular function and response to external stimuli. While recent advances in mass spectrometry reached the sensitivity required to investigate metabolites in single cells, current methods commonly isolate and sacrifice cells, inflicting a perturbed state and preventing complementary analyses. Here, we propose a two-step approach that combines nondestructive and quantitative withdrawal of intracellular fluid with subpicoliter resolution using fluidic force microscopy, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed method enabled the detection and identification of 20 metabolites recovered from the cytoplasm of individual HeLa cells. The approach was further validated in 13C-glucose feeding experiments, which showed incorporation of labeled carbon atoms into different metabolites. Metabolite sampling, followed by mass spectrometry measurements, enabled the preservation of the physiological context and the viability of the analyzed cell, providing opportunities for complementary analyses of the cell before, during, and after metabolite analysis.

From nanodroplets to continuous films: how the morphology of polyelectrolyte multilayers depends on the dielectric permittivity and the surface charge of the supporting substrate

Using atomic force microscopy, we investigated how the morphology of layer-by-layer deposited polyelectrolyte multilayers is influenced by the physical properties of the supporting substrate. The surface coverage of the assembly and its topography were found to be dependent on the dielectric permittivity of the substrate and the strength of the electrostatic interactions between polyanions and polycations. For poly(allylamine hydrochloride)/poly(styrene sulfonate) (PAH/PSS), a strongly interacting polyelectrolyte couple, no dependency of the surface morphology on the physical properties of the underlying substrate was observed. In contrast, the weakly interacting pair poly(L-lysine)/hyaluronic acid (PLL/HA) formed rapidly continuous, flat layers on substrates of low dielectric permittivity and inhomogeneous droplet-films on substrates of high dielectric permittivity. Variations in the dielectric permittivity account for changes in the image charges that are induced in the substrate. These changes influence the balance between repulsive electrostatic forces (and image forces) and attractive van der Waals interactions, and thus cause the differences in surface morphology. Differences in surface charge did not influence the morphology of the polyelectrolyte multilayers, but higher surface charge resulted in more polymeric material adsorbed on the surface. A comparison between (PLL/HA) multilayers with and without an initial layer of poly(ethyleneimine) (PEI) supports this hypothesis.