Biofilm architecture.

Microbial biofilms are complex self-organized communities of microbial cells that provide protective environments for the cells that inhabit the biofilm, enabling them to respond efficiently to challenges. The enhanced resistance and altered metabolism of the cells in the biofilm makes biofilms potentially very useful in chemical production processes, including the production of pharmaceuticals and biofuels. Synthetic biofilms in which the composition and architecture of the biofilm is controlled by the designer could help in harnessing this potential. In this chapter we discuss biofilm architecture, how it can be created by natural or artificial means, and how it affects biofilm function.

Formation of embryoid bodies using dielectrophoresis.

Embryoid body (EB) formation forms an important step in embryonic stem cell differentiation invivo. In murine embryonic stem cell (mESC) cultures EB formation is inhibited by the inclusion of leukaemic inhibitory factor (LIF) in the medium. Assembly of mESCs into aggregates by positive dielectrophoresis (DEP) in high field regions between interdigitated oppositely castellated electrodes was found to initiate EB formation. Embryoid body formation in aggregates formed with DEP occurred at a more rapid rate-in fact faster compared to conventional methods-in medium without LIF. However, EB formation also occurred in medium in which LIF was present when the cells were aggregated with DEP. The optimum characteristic size for the electrodes for EB formation with DEP was found to be 75-100 microns; aggregates smaller than this tended to merge, whilst aggregates larger than this tended to split to form multiple EBs. Experiments with ESCs in which green fluorescent protein (GFP) production was targeted to the mesodermal gene brachyury indicated that differentiation within embryoid bodies of this size may preferentially occur along the mesoderm lineage. As hematopoietic lineages during normal development derive from mesoderm, the finding points to a possible application of DEP formed EBs in the production of blood-based products from ESCs.

Construction by dielectrophoresis of microbial aggregates for the study of bacterial cell dormancy.

A study of the effect of aggregate size on the resuscitation of dormant M. smegmatis was conducted by constructing cell aggregates with defined sizes and shapes using dielectrophoresis and monitoring the resuscitation process under controlled laboratorial conditions in a long-term cell feeding system. Differently sized cell aggregates were created on the surface of indium tin oxide coated microelectrodes, their heights and shapes controlled by the strength of the induced electric field and the shape of the microelectrodes. Both two-dimensional (ring-patterned) and three-dimensional cell aggregates were produced. The cell aggregates were maintained under sterile conditions at 37 degrees C for up to 14 days by continuously flushing Sauton's medium through the chamber. Resuscitation of dormant M. smegmatis was evaluated by the production of the fluorescent dye 5-cyano-2,3-ditolyltetrazolium chloride. The results confirm that the resuscitation of dormant M. smegmatis is triggered by the accumulation of a resuscitation promoting factor inside the aggregates by diffusion limitation.

Separation by dielectrophoresis of dormant and nondormant bacterial cells of Mycobacterium smegmatis.

The dielectrophoretic behavior of active, dead, and dormant Mycobacterium smegmatis bacterial cells was studied. It was found that the 72-h-old dormant cells had a much higher effective particle conductivity (812+/-10 muS cm(-1)), almost double that of active cells (560+/-20 muS cm(-1)), while that of dead (autoclaved) M. smegmatis cells was the highest (950+/-15 muS cm(-1)) overall. It was also found that at 80 kHz, 900 muS cm(-1) dead cells were attracted at the edges of interdigitated castellated electrodes by positive dielectrophoresis, but dormant cells were not. Similarly, at 120 kHz, 2 muS cm(-1) active cells were attracted and dormant cells were not. Using these findings a dielectrophoresis-based microfluidic separation system was developed in which dead and active cells were collected from a given cell suspension, while dormant cells were eluted.

Creation of arrays of cell aggregates in defined patterns for developmental biology studies using dielectrophoresis.

It is shown that dielectrophoresis--the movement of particles in non-uniform electric fields--can be used to create engineered skin with artificial placodes of different sizes and shapes, in different spatial patterns. Modeling of the electric field distribution and image analysis of the cell aggregates produced showed that the aggregation is highly predictable. The cells in the aggregates remain viable, and reorganization and compaction of the cells in the aggregates occurs when the artificial skin is subsequently cultured. The system developed could be of considerable use for the in vitro study of developmental processes where local variations in cell density and direct cell-cell contacts are important.

Recreating the hematon: microfabrication of artificial haematopoietic stem cell microniches in vitro using dielectrophoresis.

The hematon is a three-dimensional aggregate of cells which is able to produce all blood types. To be able to do this, it must be able to create within the cell aggregate a microenvironment which enables haematopoietic stem cell maintenance, renewal and differentiation. A first step was taken towards the creation of artificial hematopoietic stem cell microniches in vitro by the creation with dielectrophoresis of hemispherical cell aggregates of a height of 50-100 mum with a defined internal architecture similar to that of a putative hematon. It is shown that, after their dielectrophoretic manipulation, the cells remain viable and active. Cells within the aggregate are in direct contact with each other, potentially allowing direct cell-cell communication within the cell construct. Some cell immobilisation methods are explored for further stabilising the 3-D organisation of the cell aggregate after its formation. The introduction of traceable individual cells into the artificial microniche is demonstrated.

The use of electric fields in tissue engineering: A review.

The use of electric fields for measuring cell and tissue properties has a long history. However, the exploration of the use of electric fields in tissue engineering is only very recent. A review is given of the various methods by which electric fields may be used in tissue engineering, concentrating on the assembly of artificial tissues from its component cells using electrokinetics. A comparison is made of electrokinetic techniques with other physical cell manipulation techniques which can be used in the construction of artificial tissues.

Tissue engineering with electric fields: investigation of the shape of mammalian cell aggregates formed at interdigitated oppositely castellated electrodes.

The shape of aggregates of cells formed by positive dielectrophoresis (DEP) at interdigitated oppositely castellated electrodes under different conditions was investigated and compared with calculations of the electric field gradient |nablaE(2)|, and the electric field E, and E(2). The results confirm that at low field strength the cells predominantly accumulate above the tips of the electrodes, but at higher electric field strengths the cells predominantly accumulate in the middle of the aggregate. For a given electrode size, a higher applied voltage significantly increases the aggregate footprint. Higher flow rates distort this pattern, with more cells accumulating at the electrodes that are upstream. Calculation of the electric field strength E, E(2) and the electric field strength gradient |nablaE(2)| in the interdigitated oppositely castellated electrode array shows that, at low flow rates, there is a strong correlation between the aggregate shape and the distribution of the electric field E and E(2), but not so between the aggregate shape and |nablaE(2)|. The results indicate that interparticle forces such as pearlchain formation strongly affect the aggregation process, but that, when positive DEP is used to make the aggregates, the distribution of the electric field E, or better E(2), can be used as a useful guide to the final aggregate shape.

Novel electrode structures for large scale dielectrophoretic separations based on textile technology.

The use of dielectrophoresis (DEP) to date has mainly been limited to processing small volumes due to difficulties in the fabrication of microelectrodes over large surface areas. To overcome this problem a novel approach to the construction of micro-electrode arrays has been developed based on weaving. A plain weave cloth was made from 100 microm diameter stainless steel wires and 75 decitex polyester yarns. The stainless steel wires formed the weft, and were kept parallel and apart by a warp of flexible polyester yarns, with a gap of around 150 microm between the metal wires. The metal wires were alternately connected to earth and signal of an AC power source, and it was shown that it was possible to collect yeast cells suspended in deionised water at the metal wire surfaces by dielectrophoresis. The polyester yarn was also found to distort the electric field, creating further areas of electric field non-uniformity around the polyester yarns, further enhancing the capability of the system to attract cells. A 14 ml separation chamber was built from the cloth by alternately sandwiching perspex slabs and cloth together. The DEP chamber was able to effectively collect life yeast from a flow of suspended cells through the cloth using an applied field of 1 MHz at flow rates up to 5 ml min-1. However, some loss occurred due to sedimentation. Also, the chamber was able to separate dead and live yeast cells at 30 Vpk-pk, 2 MHz, with some cell loss due to sedimentation.

Tissue engineering with electric fields: immobilization of mammalian cells in multilayer aggregates using dielectrophoresis.

Positive dielectrophoresis can be used to create aggregates of animal cells with 3D architectures. It is shown that the cells, when pulled together into an aggregate by positive dielectrophoresis in a low-conductivity iso-osmotic solution, adhere to each other. The adherence of the cells to each other is non-specific and increases in time, and after 10-15 min becomes strong enough to immobilize the cells in the aggregate, enabling the ac electric field to be released, and the iso-osmotic buffer to be replaced by growth or other media. Cell viability is maintained. The new method of immobilization significantly simplifies the construction of aggregates of animal cells by dielectrophoresis, and increases the utility of dielectrophoresis in tissue engineering and related areas.

Large scale dielectrophoretic construction of biofilms using textile technology.

Arrays of microelectrodes for AC electrokinetic experiments were fabricated by weaving together stainless steel wires (weft) and flexible polyester yarn (warp) in a plain weave pattern. The cloth produced can be used to collect cells in low conductivity media by dielectrophoresis (DEP). The construction of model biofilms consisting of a yeast layer on top of a layer of M. luteus is demonstrated, using polyethylenimine (PEI) as the flocculating agent. This technique offers an alternative to the formation of biofilms at microelectrodes made by photolithography, and would allow the construction of biofilms with defined internal architectures by DEP at much larger scales than was possible previously. Furthermore, the flexibility of the cloth would also allow it to be distorted or folded into various shapes.

Construction of artificially structured microbial consortia (ASMC) using dielectrophoresis: examining bacterial interactions via metabolic intermediates within environmental biofilms.

The construction of artificial biofilms with defined internal architectures is described. Bacterial cells are suspended in a low conductivity medium, guided to specific areas in a microelectrode array by dielectrophoresis (DEP), and then immobilised using the flocculating agent poly(ethylenimine). Multispecies biofilms can be constructed by introducing different species at different times. The rapid construction of such biofilms with defined internal architectures provides, when combined with visual reporters of gene activity, a powerful new method for the investigation of the effects of the spatial organisation on interactions between bacterial species in biofilms. To demonstrate the utility of the technique as a method for investigating metabolic interactions in biofilms, aggregates were constructed from Acinetobacter sp. C6 and Pseudomonas putida::gfp. The Acinetobacter degrades benzyl alcohol, overproducing benzoate, which in turn is consumed by the Pseudomonas strain. The P. putida has a chromosomally expressed cassette encoding a gfp downstream of the promoter which controls degradation of benzoate, making the interaction between the two strains in the metabolism of benzyl alcohol visible by the production of green fluorescent protein (GFP). Microscopic observation of the biofilms, including the use of confocal laser scanning microscopy (CLSM), confirmed that metabolic exchange occurred. In addition, it was observed that the bacteria appear to have a preferred biofilm architecture, with P. putida in the bottom layer, and Acinetobacter at the top.

Colonial architecture in mixed species assemblages affects AHL mediated gene expression.

Many bacterial species produce metabolites that accumulate in the extracellular environment and induce specific transcriptional responses in producing cells. This phenomenon, most often referred to as quorum sensing, is thought to constitute a self-cell-density sensing mechanism allowing bacterial populations to alter gene expression in response to increases in their own density. Quorum sensing systems involving N-acyl-L-homoserine lactone (AHL) production and response are the most intensively investigated example. In this study we have employed a novel technique, known as dielectrophoresis, to investigate the impact of colonial architecture on the induction of AHL mediated gene expression. Using dielectrophoresis, we constructed artificial mixed species microcolonies with specific architectures. In this way, we were able to show that approximately 1000 Escherichia coli cells layered over an immobilised cluster of approximately 500 AHL responsive cells alters the response of this cluster to AHLs supplied either exogenously or endogenously. These findings lend credence to the hypothesis that the accumulation of extracellular metabolites signifies generic crowding in mixed species assemblages.

Towards microbial tissue engineering?

Tissue engineering involves the creation of multicellular tissues from individual cells. It was previously perceived that tissues were only formed by higher organisms such as plants and animals. However, it is now known that multicellular systems of microorganisms, such as microbial colonies, biofilms, flocs and aggregates, can also show extensive spatial organization. Here, we discuss methods that can be used to spatially organize microorganisms--bacteria, in particular--into tissue-like materials with defined internal architectures. Some potential uses of such "microbial tissues" are covered.

Construction of biofilms with defined internal architecture using dielectrophoresis and flocculation.

A novel approach was developed for the construction of biofilms with defined internal architecture using AC electrokinetics and flocculation. Artificial structured microbial consortia (ASMC) consisting of localized layered microcolonies of different cell types were formed by sequentially attracting different cell types to high field regions near microelectrodes using dielectrophoresis. Stabilization of the microbial consortia on the electrode surface was achieved by crosslinking the cells using the flocculant polyethyleneimine (PEI). Consortia of Escherichia coli, Micrococcus luteus, and Saccharomyces cerevisiae were made as model systems. Also, more natural consortia were made of the bacteria Pseudomonas putida, Clavibacter michiganense, and Methylobacterium mesophilum, which are found together in consortia during biodegradation of metal-cutting waste fluids.

A simple method for the measurement of bacterial particle conductivities.

A method was developed for the measurement of the bacterial particle conductivity, based on the measurement of the conductivity of a bacterial cell suspension sigma(s) and the suspending medium sigma(m). A line plotted through sigma(s) - sigma(m) versus sigma(m) crosses the x-axis at sigma(m) = sigma(p), independent of the bacterial cell concentration. The method does not require anything more complex than a centrifuge and a conductivity meter. Knowledge of the bacterial particle conductivity is of importance in, for example, the dielectrophoretic separation, manipulation and trapping of bacterial cells, as well as the study of their physiological state.

Electro-orientation of Schizosaccharomyces pombe in high conductivity media.

The orientation of microbial cells may be important in cell-cell interactions within microbial consortia. As part of our research programme aimed at the construction of Artificial Structured Microbial Consortia (ASMC), we have investigated the electro-orientation of Schizosaccharomyces pombe in AC electric fields, and studied the effects of the applied frequency, voltage, and distance between the electrodes, at different medium conductivities. It is shown that the electro-orientation of S. pombe in media with conductivities similar to that of growth media is feasible using microelectrodes. Oriented growth of S. pombe can be obtained when continuously exposed to AC electric fields in growth medium over extended periods.