Conducting polymer hydrogels with electrically-tuneable mechanical properties as dynamic cell culture substrates.

Hydrogels are a popular substrate for cell culture due to their mechanical properties closely resembling natural tissue. Stimuli-responsive hydrogels are a good platform for studying cell response to dynamic stimuli. Poly(N-isopropylacrylamide) (pNIPAM) is a thermo-responsive polymer that undergoes a volume-phase transition when heated to 32 °C. Conducting polymers can be incorporated into hydrogels to introduce electrically responsive properties. The conducting polymer, polypyrrole (PPy), has been widely studied as electrochemical actuators due to its electrochemical stability, fast actuation and high strains. We determine the volume-phase transition temperature of pNIPAM hydrogels with PPy electropolymerised with different salts as a film within the hydrogel network. We also investigate the electro-mechanical properties at the transition temperature (32 °C) and physiological temperature (37 °C). We show statistically significant differences in the Young's modulus of the hybrid hydrogel at elevated temperatures upon electrochemical stimulation, with a 5 kPa difference at the transition temperature. Furthermore, we show a three-fold increase in actuation at transition temperature compared to room temperature and physiological temperature, attributed to the movement of ions in/out of the PPy film that induce the volume-phase transition of the pNIPAM hydrogel. Furthermore, cell adhesion to the hybrid hydrogel was demonstrated with mouse articular chondrocytes.

Stretchable microchannel-on-a-chip: A simple model for evaluating the effects of uniaxial strain on neuronal injury.

BACKGROUND: Axonal injury is a major component of traumatic spinal cord injury (SCI), associated with rapid deformation of spinal tissue and axonal projections. In vitro models enable us to examine these effects and screen potential therapies in a controlled, reproducible manner. NEW METHOD: A customized, stretchable microchannel system was developed using polydimethylsiloxane microchannels. Cortical and spinal embryonic rat neurons were cultured within the microchannel structures, allowing a uniaxial strain to be applied to isolated axonal processes. Global strains of up to 52% were applied to the stretchable microchannel-on-a-chip platform leading to local strains of up to 12% being experienced by axons isolated in the microchannels. RESULTS: Individual axons exposed to local strains between 3.2% and 8.7% developed beading within 30-minutes of injury. At higher local strains of 9.8% and 12% individual axons ruptured within 30-minutes of injury. Axon bundles, or fascicles, were more resistant to rupture at each strain level, compared to individual axons. At lower local strain of 3.2%, axon bundles inside microchannels and neuronal cells near entrances of them progressively swelled and degenerated over a period of 7 days after injury. COMPARISON WITH EXISTING METHOD(S): This method is simple, reliable and reproducible with good control and measurement of injury tolerance and morphological deformations using standard laboratory equipment. By measuring local strains, we observed that axonal injuries occur at a lower strain magnitude and a lower strain rate than previous methods reporting global strains, which may not accurately reflect the true axonal strain. CONCLUSIONS: We describe a novel stretchable microchannel-on-a-chip platform to study the effect of varying local strain on morphological characteristics of neuronal injury.

Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning.

Cell patterning is becoming increasingly popular in neuroscience because it allows for the control in the location and connectivity of cells. A recently developed cell patterning technology uses patterns of an organic polymer, parylene-C, on a background of SiO2. When cells are cultured on the parylene-C/SiO2 substrate they conform to the underlying parylene-C geometry. Parylene-C is, however, just one member of a family of parylene polymers that have varying chemical and physical properties. In this work, we investigate whether two commercially available mainstream parylene derivatives, parylene-D, parylene-N and a more recent parylene derivative, parylene-HT to determine if they enable higher fidelity hNT astrocyte cell patterning compared to parylene-C. We demonstrate that all parylene derivatives are compatible with the existing laser fabrication method. We then demonstrate that parylene-HT, parylene-D and parylene-N are suitable for use as an hNT astrocyte cell attractive substrate and result in an equal quality of patterning compared to parylene-C. This work supports the use of alternative parylene derivatives for applications where their different physical and chemical properties are more suitable.

Investigating parylene-HT as a substrate for human cell patterning.

We demonstrate, for the first time, how parylene-HT on SiO2 substrates can be used as a human cell patterning platform. We demonstrate this platform with hNT astrocytes, derived from the human NTera2.D1 cell line. We show how hNT astrocytes are attracted to Parylene-HT and repelled by the SiO2 and are shown to adopt a similar morphology as that attained on standard tissue culture polystyrene. Furthermore, parylene-HT was capable of patterning the astrocytes achieving a ratio of 8:1 for cells on parylene compared to SiO2. Thus, as parylene-HT has similar physical properties to parylene-C with the addition of UV and thermal resistance, parylene-HT represents a desirable alternative substrate for human cell patterning.

Investigation of the Ca2+ response of human hNT astrocytes to laser removal of cellular processes.

We demonstrate, for the first time, UV laser ablative microsurgery as a method for pruning astrocytic processes from live hNT astrocytic networks in vitro. Calcium fluorescence imaging was used to evaluate the cellular response to process ablation. The results showed that ablation of astrocyte processes induced an immediate increase in intracellular calcium level which propagated through the cells cytoplasm as a wave originating from the ablation site. The increased intracellular calcium dissipated from the body of the cell but remained high in the vicinity of the ablation site. Cell viability post ablation was confirmed by observing the integrity of the cell membrane. Ablation of astrocytic processes did not compromise cell viability whereas ablation of the cytoplasm using the same laser energy resulted in cell lysis.

Low cost, patterning of human hNT brain cells on parylene-C with UV & IR laser machining.

This paper describes the use of 800nm femtosecond infrared (IR) and 248nm nanosecond ultraviolet (UV) laser radiation in performing ablative micromachining of parylene-C on SiO2 substrates for the patterning of human hNT astrocytes. Results are presented that support the validity of using IR laser ablative micromachining for patterning human hNT astrocytes cells while UV laser radiation produces photo-oxidation of the parylene-C and destroys cell patterning. The findings demonstrate how IR laser ablative micromachining of parylene-C on SiO2 substrates can offer a low cost, accessible alternative for rapid prototyping, high yield cell patterning.