Living Organisms Join The Jet Set

Suwan N. Jayasinghe is passionate about jet-based fabrication process technologies. In his small office at University College London (UCL), he talks enthusiastically about his research on the development of electrospraying, electrospinning, and related jet technologies to encapsulate a wide range of materials, including, most recently, living organisms.

Jayasinghe calls himself a biophysicist. "I work at the interface of engineering and biology," he tells C&EN. "My research is multidisciplinary. It cuts across the primary colors of academic science—chemistry, physics, and biology—and also brings in engineering and medicine."

Born in Colombo, Sri Lanka, Jayasinghe came to England in 1995. He graduated with bachelor's and master's degrees in engineering at Brunel University in 1999. Three years later, he completed his doctorate in materials science at Queen Mary, University of London. In January this year, he was appointed lecturer in bioengineering and biomaterials at UCL's department of mechanical engineering, where he has a laboratory, not much bigger than his office, in the basement of the building.

In a recent paper, Jayasinghe and coworkers showed that electrospraying—also known as electrohydrodynamic jet processing—can be used to process living cells (Small 2006, 2, 216). He carried out the research at Queen Mary in collaboration with research technician Amer N. Qureshi and lecturer in biology Peter A. M. Eagles at Kings College London.

"Jet-based techniques such as ink-jet printing and electrospraying are being extensively investigated for the safe processing of living organisms, biomolecules, and biocompatible micro- and nano-sized materials," Jayasinghe says. The use of such techniques for tissue engineering is potentially revolutionary, he explains. One of the goals of this work is to fabricate finely tuned 2- and 3-D biologically active structures by depositing living cells on one another. Such architectures, when constructed in vivo, could, in theory at least, be used to repair damaged tissues and might one day be used to replace organs.

Jet techniques rely on a pressure gradient, potential difference, or some other means to generate a forceful stream by driving a fluid from a reservoir through a nozzle or needle. "In electrospraying, the liquid acquires an electrical charge within a conducting needle," Jayasinghe explains. "When the charged liquid enters an external electric field set up between the charged needle and a grounded electrode below the needle, the liquid fragments into a spray of droplets that accelerate toward the grounded electrode."

Ink-jet printers, on the other hand, employ heating elements or piezoelectric crystals within needles to propel ink droplets onto paper. Ink-jet printing technology has been extensively investigated for processing biological materials such as suspensions of cells in cell media. The size and size distribution of these biosuspension droplets generated by ink-jet printing is limited, however.

The resolution of ink-jet printing is constrained by the diameter of the internal orifice of the jetting needle, according to Jayasinghe. The droplets spread to more than 100 µm in diameter, even when the needle diameter is around 60 µm, he says.

For some potential applications—for example, the repair of damaged nerves—smaller droplets having a narrow distribution of droplet sizes are required.

"Smaller droplets, when deposited on one another, can potentially fabricate tissues with fine or enhanced features," Jayasinghe says. "The concentrated suspensions of cells in cell media needed for tissue repair can also cause serious needle blockage with ink-jet needles.

"Electrospraying does not suffer from these disadvantages," he continues. "Droplets a few micrometers in diameter can be generated from concentrated suspensions of biomaterials by using needles with internal diameters of several hundred micrometers."

In the Small paper, Jayasinghe and coworkers showed that concentrated suspensions of Jurkat cells—an immortalized line of T lymphoblast cells—could be processed into a jet of micrometer-sized droplets by electrospraying. They also reported that a centrifuged fraction, the buffy coat, of human blood cells could be processed in the same way. In all cases, they found no evidence that the jetting technique, and particularly the high-intensity electric field used, affected the cells structurally or adversely affected their activity or rate of division. However, the size of the droplets varied widely.

Jayasinghe subsequently demonstrated, in work carried out with UCL lecturer in biochemistry Andrea Townsend-Nicholson, that droplet size could be controlled more precisely by using a coaxial needle system and a biodegradable polymer to encapsulate the electrosprayed droplets (Lab. Chip 2006, 6, 1080). The experimental setup involves an inner electrospray needle, which holds a highly concentrated suspension of living organisms, enclosed by an outer needle that accommodates the polymer (medical-grade polydimethylsiloxane, or PDMS).

Droplets of a nearly uniform size are generated that are an order of magnitude smaller than those generated by competing jet-based technologies, the two researchers note. The use of a PDMS liquid coating assists as a binding agent for the droplets.

To date, Jayasinghe and Townsend-Nicholson have used their jet-based techniques only on immortalized cell lines to show proof of concept. The long-term aim is to use these techniques to construct tissue, layer by layer, by using primary cells such as fibroblasts or stem cells taken from patients.

In a recent paper, the two researchers describe a jet-based technique known as electrospinning to fabricate continuous biocompatible polymer fibers encapsulating suspensions of living organisms (Biomacromolecules, DOI: 10.1021/bm060649h).

"Electrospinning is a versatile electric-field-driven polymeric fiber production process where a charged polymer is drawn out of a conducting needle toward a grounded or near-grounded collector," Jayasinghe explains. The process most commonly produces a nano- to micrometer-diameter continuous thread.

"We used coaxial simultaneous electrospinning of PDMS and the biosuspension to generate biologically active microthreads," Jayasinghe adds. "It demonstrates that living organisms encapsulated within a biopolymer could be spun directly onto an open wound as a biologically active scaffold. The biopolymer would then degrade, leaving the active organisms to assist in rapid healing of the wound. Cell electrospinning could also be used in regenerative and therapeutic medicine to fabricate tissues for repair and organs for repair or replacement."

Cells collected after electrospinning remain viable and show no evidence they have incurred cellular damage, Jayasinghe and Townsend-Nicholson note. "Even though the cells undergo mitosis and are viable, we still need to prove that cellular components, such as DNA, chromosomes, and RNA, are not damaged or altered in any way," Jayasinghe says.

Electrospinning is an old technology, comments chemistry professor Benjamin S. Hsiao at the State University of New York, Stony Brook. "Coaxial electrospinning is also not new, but its use to encapsulate and connect living cells is quite novel and offers some unique advantages over existing methods," he says.

But there are potential problems with this approach, Hsiao says. Though the electrospinning conditions used by the UCL team—for example, high electric field—did not appear to compromise the integrity of the chosen cell type, "this may not be true for many delicate cell types," he says.

Hsiao and coworkers also have been developing electrospinning techniques for tissue engineering. Last year, for example, the team showed that electrospinning can be used to fabricate biodegradable nonwoven poly(lactide)- and poly(glycolide)-based scaffolds for heart tissue engineering applications (Biomaterials 2005, 26, 5330).

Another electrospinning expert is Paul D. Dalton, a research fellow in the School of Biological Sciences at the University of Southampton, in England. Earlier this year, Dalton reported work carried out with coworkers at Aachen University of Technology (RWTH), in Germany, on the use of polymer melt electrospinning to deposit fibers onto cultured cells. The work paves the way to build up layer-on-layer tissue constructs of cells and electrospun polymers (Biomacromolecules 2006, 7, 686). The in vitro experiments were successful and did not cause cell death, according to the authors.

"The concept of encapsulating cells within electrospun fibers has been informally discussed by numerous electrospinning colleagues and scientists at conferences," Dalton tells C&EN. "Jayasinghe and coworkers have conceived a system where this is achievable, although the methods used will undoubtedly require further modification to improve the process. The significance of this work is difficult to measure, as the benefits may not be known for years. Even so, their experiments and observations push the limits of what many scientists may have deemed feasible."

Working with postdoc Sumathy Arumuganathar, Jayasinghe is developing another jet-based technique, known as aerodynamically assisted jetting, for encapsulation. The technology employs high pressure to drive fluids through an exit orifice and generate a jet stream of droplets or a continuous nano- or micrometer-diameter thread.

"Our idea is to use a coaxial needle configuration to prepare hollow or compound droplets and threads," Jayasinghe says. "We aim to use the inner needle to accommodate the flow of a gas or a concentrated suspension of a bio- or functional material, while the outer one accommodates the flow of another suspension."

The development of jet-based approaches such as aerodynamically assisted jetting, electrospraying, and electrospinning should lead to a host of precision "drop and place" biotechniques, according to Jayasinghe. When coupled with computer control on the x-, y-, and z-axes, it is possible to contemplate the fabrication of active 3-D biological architectures for use in a wide range of tissue engineering and other biological and medical applications, he concludes.