Malardalen Univerity is characterised by its close collaboration with companies and with the public sector in the region. A main strategic directions of the university is to develop co-production w ...

Rethinking effectiveness : Addressing managerial paradoxes by using a process perspective on effectiveness

We present a computer-controlled scanning electroporation method. Adherent cells are electroporated using an electrolyte-filled capillary in contact with an electrode. The capillary can be scanned over a cell culture and locally deliver both an electric field and an electroporation agent to the target area without affecting surrounding cells. The instantaneous size of the targeted area is determined by the dimensions of the capillary. The size and shape of the total electroporated area are defined by these dimensions in combination with the scanning pattern. For example, striped and serpentine patterns of electroporated cells in confluent cultures can be formed. As it is easy to switch between different electroporation agents, the method is suitable for design of cell cultures with complex composition. Finite element method simulations were used to study the spatial distributions of the electric field and the concentration of an electroporation agent, as well as the fluid dynamics related to scanning and flow of electroporation agent from the capillary. The method was validated for transfection by introduction of a 9-base-pair-long randomized oligonucleotide into PC12 cells and a pmaxGFP plasmid coding for green fluorescent protein into CHO and WSS cells.

We here report on a concept for creating well-defined electric field gradients between the boundaries of capillary electrode (a capillary of a nonconducting material equipped with an interior metal electrode) outlets, and dielectric surfaces. By keeping a capillary electrode opening close to a boundary between a conducting solution and a nonconducting medium, a high electric field can be created close to the interface by field focusing effects. By varying the inner and outer diameters of the capillary, the span of electric field strengths and the field gradient obtained can be controlled, and by varying the slit height between the capillary rim and the surface, or the applied current, the average field strength and gradient can be varied. Field focusing effects and generation of electric field patterns were analyzed using finite element method simulations. We experimentally verified the method by electroporation of a fluorescent dye (fluorescein diphosphate) into adherent, monolayered cells (PC-12 and WSS-1) and obtained a pattern of fluorescent cells corresponding to the focused electric field.

higher surface tension, which in turn causes a shear-driven movement of the contained fluid.Besides allowing membrane material and small particles to be transported between individual containers, charged materials can also be transported by electrophoresis in addressed nanotubes by application of an electric potential over two nanotube-connected liposomes (results not shown).In addition to applications relating to chemical and physical processes in cell systems as well as chemical computation, large and complex fluid membrane networks can be used as templates for designing solidstate devices with potential applications in microfluidics and microelectronics 6,10 .

We have constructed complex two-dimensional microscopic networks of phospholipid bilayer nanotubes and containers in which we are able to control the connectivity, container size, nanotube length, and angle between the nanotube extensions. Containers within these networks can be chemically differentiated and materials successfully routed between two containers connected by a common nanotube. These networks will enable model systems to be devised for studying confined biochemical reactions1,2,3, intracellular transport phenomena4 and chemical computations5.

We describe the fabrication of nanoengineered holding pipets with concave seating surfaces and fine pressure control. These pipets were shown to exhibit exceptional stability in capturing, transporting, and releasing single cells and liposomes 1-12 microm in diameter, which opens previously inaccessible avenues of research. Three specific examples demonstrated the utility and versatility of this manipulation system. In the first, carboxyrhodamine was selectively incorporated into individual cells by electroporation, after which nearly all the medium (hundreds of microliters) surrounding the docked and tagged cells was rapidly exchanged (in seconds) and the cells were subsequently probed by laser-induced fluorescence (LIF). In the second study, a single liposome containing carboxyrhodamine was transported to a dye-free solution using a transfer pipet, docked to a holding pipet, and held firmly during physical agitation and interrogation by LIF. In the third study, pairs of liposomes were positioned between two microelectrodes, held in contact, and selectively electrofused and the resulting liposomes undocked intact.

As organisms, we are amazingly complex living laboratories. As we move, breathe, think, and eat, seemingly endless chemical reactions and interactions occur inside us. The test tubes, beakers, and flasks used to separate and selectively mix the myriad of reactants involved are cells, vesicles, and organelles. Taking the analogy further, whereas chemists typically mix chemicals milliliters or more in volume, biological systems carry out their biochemistry in containers that are femtoliters or less in volume. As researchers we assume, with good reason, that the material surfaces of our laboratory test tubes do not substantially affect the kinetics we measure. This assumption might not hold were we to shrink our containers to the femtoliter scale. At such a small scale, collision rates between reactants and their container walls become significant [1], and the inner surface, particularly in biological containers, is chemically complex. The bilayers of cells and organelles are composed of a variety of lipids. These varieties assemble into domains [2] in a process partly controlled by the transmembrane proteins in them [3]. Cellular and organellar control of chemical reactions may thus come, in part, from alterations in the composition and arrangement of the molecular species making up the bilayer membrane [4]. How do systematic alterations to the bilayer composition of a liposome alter the kinetics of reactions within the liposome interior? We may find that the potential physiological significance of lipid domains within bilayers to the kinetics of in-plane reactions [5] (i.e., for proteins and other molecules moving within the bilayer) has applications to molecules within liposomes that interact with the inner bilayer surface.

This paper describes the use of focused electric fields and focused optical fields for the high-resolution manipulation of single cells. A focused electric field, obtained with the use of ultramicroelectrodes (tip diameter approximately 5 μm), is used to electroporate and electrofuse individual cells selectively and with high spatial resolution. A focused optical field, in the form of an optical tweezer, is used to isolate single organelles from a cell as well as to position liposomes incorporated with receptors and transporters along the cell for the high-resolution sampling and probing of the cellular microenvironment.

We describe an electrofusion-based technique for combinatorial synthesis of individual liposomes. A prototype device with containers for liposomes of different compositions and a fusion container was constructed. The sample containers had fluid contact with the fusion container through microchannels. Optical trapping was used to transport individual liposomes and cells through the microchannels into the fusion container. In the fusion container, selected pairs of liposomes were fused together using microelectrodes. A large number of combinatorially synthesized liposomes with complex compositions and reaction systems can be obtained from small sets of precursor liposomes. The order of different reaction steps can be specified and defined by the fusion sequence. This device could also facilitate single cell-cell electrofusions (hybridoma production). This is exemplified by fusion of transported red blood cells.

A method for cell-cell and cell-liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell-cell or cell-liposome pair was achieved by the application of a highly focused electric field through a pair of 5-micrometer o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell-liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (gamma-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.

A combined electroporation and pressure-driven microinjection method for efficient loading of biopolymers and colloidal particles into single-cell-sized unilamellar liposomes was developed. Single liposomes were positioned between a approximately 2-microm tip diameter solute-filled glass micropipet, equipped with a Pt electrode, and a 5-microm-diameter carbon fiber electrode. A transient, 1-10 ms, rectangular waveform dc voltage pulse (10-40 V/cm) was applied between the electrodes, thus focusing the electric field over the liposome. Dielectric membrane breakdown induced by the applied voltage pulse caused the micropipet tip to enter the liposome and a small volume (typically 50-500 x 10(-15) L) of fluorescein, YOYO-intercalated T7-phage DNA, 100-nm-diameter unilamellar liposomes, or fluorescent latex spheres could be injected into the intraliposomal compartment. We also demonstrate initiation of a chemical intercalation reaction between T2-phage DNA and YOYO-1 by dual injection into a single giant unilamellar liposome. The method was also successfully applied for loading of single cultured cells.

We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.