Cameron J. Ferris

Biofabrication: an overview of the approaches used for printing of living cells

The development of cell printing is vital for establishing biofabrication approaches as clinically relevant tools. Achieving this requires bio-inks which must not only be easily printable, but also allow controllable and reproducible printing of cells. This review outlines the general principles and current progress and compares the advantages and challenges for the most widely used biofabrication techniques for printing cells: extrusion, laser, microvalve, inkjet and tissue fragment printing. It is expected that significant advances in cell printing will result from synergistic combinations of these techniques and lead to optimised resolution, throughput and the overall complexity of printed constructs.

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.

Conducting bio-materials based on gellan gum hydrogels

Hydrogels, a class of highly hydrated polymer materials, are emerging as a viable bio-material for tissue engineering applications due to their bio-degradability, process-ability, and similarity with the natural extra-cellular matrix. Here, we report on gellan gum hydrogels and demonstrate that the gelation temperature can be tailored to be physiologically relevant. Furthermore, we demonstrate the biocompatibility of these hydrogels and show that cell behaviour is influenced by gel modulus and the incorporation of surface topographical features. Carbon nanotubes were incorporated into hydrogels as conducting fillers to achieve an electrically conducting hydrogel for the future purpose of electrical cell stimulation. Percolation studies revealed that a carbon nanotube concentration of 1.3% by weight is required to achieve electrical conduction through 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.

Gel–carbon nanotube composites: the effect of carbon nanotubes on gelation and conductivity behaviour

The gelation and conductivity behaviour of gellan gum–carbon nanotube composite materials was investigated. It was shown that addition of carbon nanotubes allowed for more efficient cooling and heating of composite dispersions compared to gellan gum solutions. Free-standing films were prepared by an evaporative casting process. The resistance decreased with increasing MWNT mass fraction. Exposure to a humid atmosphere resulted in further reduction in the resistance. It was shown that cation mobility and polymer conformation play an important role in this water-sensitive behaviour. In particular, it was suggested that the current contains an electrical contribution from electron transport through the nanotubes and an ionic contribution due to cations. The ionic contribution was shown to increase with increasing nanotube mass fraction, which allowed us to suggest that the nanotube network consists of pathways dominated by inter-nanotube junctions and polymer tunnelling barriers. The observed water sensitivity was explained as resulting from the contribution to the current of the polymer-dominated pathways, which are enabled under humid conditions and disabled under ambient conditions.

Characterization of Gellan Gum by Capillary Electrophoresis

Gellan gums were characterised for the first time using free-solution capillary electrophoresis (CE) or CE under critical conditions (CE-CC). CE-CC is a fast method that separates the polysaccharide. Gellan gums are shown to be heterogeneous in terms of their electrophoretic mobility at 55°C revealing: oligomer peak(s), broad peaks of polymers with a random coil conformation with different degrees of acylation (composition), aggregates, and polymers with double-helix conformation. CE-CC is complementary with the rheological analysis also performed in this work. Sonication of gellan gums is shown to decrease the viscosity of gellan gum mainly by breaking up aggregates. The effect of sonication is stronger on the high-acyl gellan gum since the latter has a far higher tendency to aggregate.

Peptide modification of purified gellan gum

Gellan gum (GG) is an anionic polysaccharide with potential as a biopolymer for additive manufacturing (3D-bioprinting) and tissue engineering. Previous studies have shown GG to be highly cytocompatible, but lacking specific attachment sites required for anchorage-dependent cells. In this work, we modify purified-GG polymer with a short peptide containing the arginine-glycine-aspartic acid (RGD) sequence that is known to enhance integrin-mediated cell attachment. Radiolabelling of the peptide was used in optimisation of the conjugation procedure to achieve an overall efficiency of 40%. The purification of divalent cations from commercial GG samples was found to be critical for successful conjugation. Rheological studies revealed that the peptide coupling did not prevent gelation behaviour. C2C12 cells showed improved attachment on the surface of and encapsulated within RGD-GG hydrogels, differentiating to multinucleated myofibers after 5-7 days. PC12 cells showed minimal interactions with both GG and RGD-GG, with formation of cell clusters and impedance of terminal differentiation and neurite extension.

Diffusion of vitamin B 12 in gellan gum-carbon nanotube hydrogels

We report on the diffusion of vitamin B<inf>12</inf> through gellan gum-carbon nanotube hydrogels. The addition of carbon nanotubes to gellan gum reduced the vitamin B12 diffusion coefficient from 1.70 × 10⁻⁶ cm²/s to 0.70 × 10⁻⁶ cm²/s.

Printed hydrogel materials

An inkjet printer has been developed for printing hydrogel materials. The printer uses two peizo industrial printheads to print out combinations of cationic and anionic polymers, which then form gels on a substrate in a clear demonstration of reactive printing. The synthetic polymers poly-diallyl-dimethyl ammonium chloride and polystyrene sulfonate, and the naturally occurring polymers, alginate, chitosan and gellan gum all formed gels via inkjet deposition.