Kerry J. Gilmore

Skeletal muscle cell proliferation and differentiation on polypyrrole substrates doped with extracellular matrix components

Conducting polymers have been developed as substrates for in vitro studies with a range of cell types including electrically-excitable cells such as nerve and smooth muscle. The goal of this study was to optimise and characterise a range of polypyrrole materials to act as substrates for electrical stimulation of differentiating skeletal myoblasts. Although all of the polymer materials provided suitable substrates for myoblast adhesion and proliferation, significant differences became apparent under the low-serum conditions used for differentiation of primary myoblasts. The significance of the work lies in the design and control of polymer materials to facilitate different stages of skeletal muscle cell proliferation and/or differentiation, opening up opportunities for engineering of this tissue. This paper therefore constitutes not just a biocompatibility assessment but a comprehensive study of how synthesis conditions affect the final outcome in terms of cell response.

Bio-ink for on-demand printing of living cells

Drop-on-demand bioprinting allows the controlled placement of living cells, and will benefit research in the fields of tissue engineering, drug screening and toxicology. We show that a bio-ink based on a novel microgel suspension in a surfactant-containing tissue culture medium can be used to reproducibly print several different cell types, from two different commercially available drop-on-demand printing systems, over long printing periods. The bio-ink maintains a stable cell suspension, preventing the settling and aggregation of cells that usually impedes cell printing, whilst meeting the stringent fluid property requirements needed to enable printing even from many-nozzle commercial inkjet print heads. This innovation in printing technology may pave the way for the biofabrication of multi-cellular structures and functional tissue.

The use of chloromethyl-X-rosamine (Mitotracker red) to measure loss of mitochondrial membrane potential in apoptotic cells is incompatible with cell fixation.

BACKGROUND A recent report by Macho et al. (Cytometry 25: 333-340, 1996) described the use of chloromethyl-X-rosamine (CMX-Ros) as a fixable probe for detection of loss of mitochondrial membrane potential (psi(mit)), an early event in many models of apoptosis. However, this previous report lacked a description of any direct comparisons between pre- and post-fixation analyses of normal and apoptotic cells stained with CMX-Ros. METHODS Using a variety of cell types, we investigated the effect of paraformaldehyde fixation on cellular retention of CMX-Ros and the implications of this for the subsequent analysis of changes in psi(mit) in cells undergoing apoptosis. RESULTS We found that following fixation, the resolution between normal cells with polarized mitochondria and apoptotic cells with depolarized mitochondria is reduced to the extent that accurate discrimination between the cell types is no longer possible. CONCLUSIONS Overall, our results are consistent with CMX-Ros being a valid probe for psi(mit) in intact cells but only when the cells are stained and analyzed immediately. Thus, our results suggest that the proposed applications for CMX-Ros in multiple parameter analysis of fixed cells are inappropriate and will lead to spurious results.

Reactive supramolecular assemblies of mucopolysaccharide, polypyrrole and protein as controllable biocomposites for a new generation of ‘intelligent biomaterials’

Abstract A method for the simple synthesis of supramolecular composites of polypyrrole, complex mucopolysaccharides and protein is described. These materials have interesting hydrogel-like properties such as high water content and biocompatibility. In addition they are capable of trapping protein in their structure during synthesis and releasing this protein in response to electrical stimuli. The materials are also electroconductive and electroactive. The improved mechanical properties of polypyrrole films over hydrogels and the facile control of their properties by the application of small electrical potentials make them interesting candidates for the design and synthesis of a new generation of ‘smarter’ biomaterials.

Electrical stimulation promotes nerve cell differentiation on polypyrrole/poly(2-methoxy-5 aniline sulfonic acid) composites

The purpose of this work was to investigate for the first time the potential biomedical applications of novel polypyrrole (PPy) composites incorporating a large polyelectrolyte dopant, poly (2-methoxy-5 aniline sulfonic acid) (PMAS). The physical and electrochemical properties were characterized. The PPy/PMAS composites were found to be smooth and hydrophilic and have low electrical impedance. We demonstrate that PPy/PMAS supports nerve cell (PC12) differentiation, and that clinically relevant 250 Hz biphasic current pulses delivered via PPy/PMAS films significantly promote nerve cell differentiation in the presence of nerve growth factor (NGF). The capacity of PPy/PMAS composites to support and enhance nerve cell differentiation via electrical stimulation renders them valuable for medical implants for neurological applications.

Creating Conductive Structures for Cell Growth: Growth and alignment of Myogenic cell types on polythiophenes.

Conducting polymers provide suitable substrates for the in vitro study of excitable cells, including skeletal muscle cells, due to their inherent conductivity and electroactivity. The thiophene family of conducting polymers offers unique flexibility for tailoring of polymer properties as a result of the ease of functionalization of the parent monomer. This article describes the preparation of films and electrospun fibers from an ester-functionalized organic solvent-soluble polythiophene (poly-octanoic acid 2-thiophen-3-yl-ethyl ester) and details the changes in properties that result from post-polymerization hydrolysis of the ester linkage. The polymer films supported the proliferation and differentiation of both primary and transformed skeletal muscle myoblasts. In addition, aligned electrospun fibers formed from the polymers provided scaffolds for the guided differentiation of linearly aligned primary myotubes, suggesting their suitability as three-dimensional substrates for the in vitro engineering of skeletal muscle tissue.

Preparation of hydrogel/conducting polymer composites

Abstract The electropolymerisation of a conducting electroactive polymer (polypyrrole) within a hydrogel matrix has been investigated. Using appropriate conditions a conductive electroactive polymer can be formed and the composite structure retains the hydration/rehydration properties of the hydrogel.

Direct Lipid Profiling of Single Cells from Inkjet Printed Microarrays

The on-demand printing of living cells using inkjet technologies has recently been demonstrated and allows for the controlled deposition of cells in microarrays. Here, we show that such arrays can be interrogated directly by robot-controlled liquid microextraction coupled with chip-based nanoelectospray mass spectrometry. Such automated analyses generate a profile of abundant membrane lipids that are characteristic of cell type. Significantly, the spatial control in both deposition and extraction steps combined with the sensitivity of the mass spectrometric detection allows for robust molecular profiling of individual cells.

Electrochemical production of conducting polymer colloids

The development of a flow-through electrochemical system that enables production of conducting polymer colloids has been addressed. It has been shown that polyaniline colloids can be produced using this system. These colloids can be electrocoagulated by application of an appropriate negative potential, however, the coatings that are formed re-dissolve once the negative potential is removed. It has also been shown that it is possible to produce conducting electroactive polymers containing active protein.

Gellan gum doped polypyrrole neural prosthetic electrode coatings

Surface modification of neural prosthetic electrodes with polymeric materials, in particular, conducting polymers and hydrogels, has the potential to circumvent many problems associated with currently used electrode platforms. These problems include the disparity in mechanical properties between implanted electrodes and host neural tissue and the lack of biofunctionality at the electrode surface, both of which dissuade favourable reception of the implanted device. We have developed conducting polymer electrode coatings doped with the polysaccharide gellan gum, as a platform for improved functionality of neural prosthetic electrodes. Our electrode coatings, prepared by galvanostatic electropolymerisation, significantly reduced the impedance magnitude at frequencies relevant to neural cells, relative to uncoated gold Mylar electrodes (24 ± 3 Ω at 1 kHz). Cyclic voltammetry was used to explore the electrochemical stability of the coatings, which lose only 23 ± 2% charge carrying capacity when subjected to 400 redox cycles. The coatings show no change in impedance magnitude at 1 kHz when subject to 32 h of clinically relevant charge balanced current stimulation.