Biomedical manufacturing

Cell seeding and impregnation is an indispensable step for research and development in tissue engineering, regenerative medicine, biosensor/bioactuator, and other different cell-associated biological applications. Homogeneous cells have been conventionally seeded using soft lithography, dipping, microlitre syringe dispatching, to name a few. As a result, seeded cell are usually uniform and mostly in a two-dimensional (2D) form, which may not be sufficient for some controlled gradient and/or three-dimensional (3D) cell applications.

As a promising alternative, cell direct writing has emerged as a revolutionary advance in tissue engineering with great potential for use in the manufacture of arbitrary cell patterning as well as heterogeneous two or three-dimensional (2D or 3D) living scaffolds [H1-H3]. Generally speaking, direct-writing technologies include any techniques or processes capable of depositing, dispensing, or processing different types of materials over various surfaces to create structures with controlled architecture and composition in a consequent manner based on any computer-aided design [H2, H4]. During a typical direct-write approach, patterns or layered structures are built directly without the use of masks, allowing rapid prototyping. Either the substrate or the direct-write head or the both can be designed to be movable with respect to each other in order to fabricate 2D or 3D patterns.

By utilizing fluid jetting phenomena, maskless jet-based (mainly laser- or ink-based) direct-write technologies as shown in Fig. H1, also known as jet-based technologies, have been most pioneered to precisely position both nonviable and viable biological patterns and constructs over different substrates [H1, H5, H6]. For example, successful inkjet printing endeavors include E. coli bacteria [H7] and viable mammalian cells [H8] deposition using a modified thermal inkjet printer. Laser-based technologies mainly include the use of laser light to form living cell clusters [H9], matrix-assisted pulsed-laser evaporation direct-write (MAPLE DW) to deposit 2D [H10] and 3D [H11] mammalian cellular structures, and laser-induced forward transfer [H12] to assist rat Schwann and astroglial cell deposition. Recently, electrohydrodynamic jetting (EHDJ) method has also been successfully demonstrated to print viable cells [H13] and even zebrafish embryos [H14].

Laser-based approach mainly includes laser guidance direct writing using laser optical force and laser-induced forward transfer (LIFT) and its modified forms using laser pulse energy, and the aforementioned MAPLE DW is a form of LIFT. Ink-based approach relies on the deposition of cell culture-based inks to create structures layer-by-layer using mechanical pressure and/or electric force, and it mainly includes inkjet direct writing and EHDJ. Using a bottom-up approach, different direct-write methods are envisioned to seed cells and biomaterials to resemble a multiculture, multimaterial, heterogeneous structure, which could be an enhanced environment for regenerative growth and other medical treatment purposes such as organ printing for donor shortage. The following sections introduce some common cell direct-write technologies from the manufacturing process development point of view.

Fig. H1. Classification of jet-based cell direct-write technologies

2.1 Laser-based direct writing

Laser guidance direct writing uses the optical force to direct write different particles, including cells and other biomaterials. Optical force was first demonstrated to levitate aerosol droplets and dielectric spheres in 1970 [H15]. Since that, various biological and inorganic materials have been successfully manipulated optically in an aqueous suspension [H16-H18], and viable cells were successfully patterned using a laser beam-based optical force [H9, H19].

The optical force utilized during cell direct writing comes from the scattering of laser beam energetic photons off the surface of a microscopic particle. If the refraction index of a particle is larger than that of the surrounding medium, the particle experiences both a radial force pulling the particle towards the center of laser beam and an axial force pushing the particle in the propagation direction of the light [H1, H16]. The larger the difference of the refraction indexes between the particle and the medium, the stronger the optical force. Depending on whether a hollow optical fiber is used or not to minimize the effect of the fluid motion from the suspension medium, laser guidance direct writing is usually implemented in two different ways as shown in Fig. H2. For most biomedical applications, since water absorbs less light energy from near-infrared wavelength lasers, lasers with a near-infrared wavelength have been favored to reduce possible the laser-induced thermal damage to cells [H18, H20].

Fig. H2. Laser guidance direct writing system: (a) simple setup and (b) system with a guidance fiber

During a laser guidance cell direct writing process, the cell droplet is generally formed using sonic agitation or nebulization. Once the particle droplets are formed, they are guided onto the substrate by either gas flow or optical force momentum effects. The resultant droplet velocity is usually as low as 10^-5—10^-4m/s [H1].

Laser has also been widely used to deposit different engineering materials using the high pressure generated during the laser-matter interaction process. LIFT and MAPLE were firstly demonstrated the capacity to deposit metals and dielectric materials over various substrates. During the LIFT process, the coated transparent thin film substrate (called the donor substrate) is paired with an uncoated substrate (the acceptor substrate to which the pattern is to be deposited). A laser is focused through the transparent donor substrate onto the thin film causing the formation of vapor at the film-substrate interface and the ejection of a portion of the film as a high speed jet. The ejected thin film material then strikes the acceptor substrate, bonding with it. MAPLE is a variation of the conventional pulsed-laser evaporation. It is a more gentle mechanism for transferring many different compounds that include small and large molecular weight species such as sugars and polymeric molecules, from the condensed phase into the vapor phase. When a substrate is positioned in the path of the plume, a continuous film is formed from the evaporated solute, while the lower molecular weight solvent is rapidly removed by continuous evacuation. It is conducted in a conventional vacuum chamber equipped with a window of high transmittance for the wavelength of the pulsed laser to be used. The laser interaction primarily occurs with the solvent, and the material of solute remains intact. The solute concentration is intentionally kept low so that the incident laser energy is mostly absorbed by the solvent molecules rather than by the solute molecules.

By combining the beauties of LIFT and MAPLE, modified LIFT has been pioneered to transfer living cells and other biomaterials [H1, H11, H21, H22]. As shown schematically in Fig. H3, in MAPLE DW focused ultraviolet (UV) laser pulses are directed perpendicularly through the backside of a UV laser transparent quartz support that is coated with a solution of materials to be transferred. The laser pulses are then absorbed by the matrix at the interface causing extremely localized heating and evaporation of a small portion of the coating to form a small vapor/plasma pocket. Finally, the sublimation releases the remaining beneath coating as a droplet from the interface by ejecting it away from the quartz support to a receiving substrate directly underneath (on the order of 100 um) [H23]. MAPLE DW is schematically similar to laser-induced forward transfer (LIFT), but the matrix coating and thus the novelty of the laser-material interaction are what makes MAPLE DW unique.

2.2 Inkjet-based direct writing

2.2.1 Inkjet direct writing

Inkjetting technology recently has been modified to successfully print E. coli bacteria [H7] and viable mammalian cells [H8] as a promising tissue engineering technology. A typical inkjet system consists of a printhead and an ink cartridge/reservoir, and the bio-ink is delivered through one of the two main mechanisms: thermal and piezoelectric. Regardless of the working mechanism, an inkjet direct-write process involves three stages: pressure generation, cell droplet formation and ejection through an orifice, and cell droplet landing on a receiving substrate. The cell droplet velocity in inkjet printing is usually around 10 m/s [H1].

As shown in Fig. H4, a thermal inkjet system can be implemented as roof-shooter or side-shooter using the following mechanism. First, a drive circuitry is used to produce a heating pulse that can raise the temperature of a heating element to several hundred degrees. Then a bubble is formed near the element surface, producing a bubble expansion-induced outwards pressure. This pressure forces a controlled amount bio-ink, which is in the pressure chamber, out of the orifice as an ink jet. Finally, the ejected bio-ink forms a cell droplet towards a receiving substrate. The collapse of bubble pulls bio-ink from the cell suspension inlet into to fill the pressure chamber, and the cell suspension inlet usually connects to a bio-ink reservoir. Since most biological materials such as living cells, proteins, peptides, nucleic acid, and various nucleotide bases are very sensitive to heat, excessive heat should be controlled to avoid any possible thermal damage to these biological materials.

As shown in Fig. H4, a piezoelectric inkjet system can be implemented in bend or push mode using the following mechanism. First, a controlled voltage pulse is applied to a piezoelectric element, and the deformation of diaphragm shape causes a contraction in the volume of the bio-ink pressure chamber, jetting a controlled amount of fluid from an orifice. Then the ejected bio-ink forms cell droplet and lands on a receiving substrate. The relaxation of the piezoelectric element causes the pressure chamber to return to its original geometry, refilling from a connected cell suspension inlet. While the process-induced temperature rise is negligible in a piezoelectric inkjet system, cells are exposed to deleterious electric fields [H26].

In inkjet-based cell direct writing, the thermal or piezoelectric printheads and ink cartridges should be charged with cell culture. Besides, biocompatibility requirement must be fulfilled before using an inkjet system to direct write biomaterials. When heterogeneous patterns of cells are incorporated into a tissue scaffold, another requirement is multifunctional direct writing, which means that several types of cells and cultures can be arranged in order to manufacture biological tissues [H26]. Some other inkjetting-evolved biodispensing technologies [H2] have also been successfully applied to deposit living cells in both continuous extrusion and drop-on-demand droplet modes [H27] as shown in Fig. H6.

Fig. H6. Biodispensing deposition modes: continuous extrusion and drop-on-demand droplet

During MAPLE DW, a matrix is selected as the laser energy absorbing material. For some other applications, a sacrificial coating layer, which functions as the laser energy absorbing vehicle is coated between the quartz support and the biological coating to be transferred, forming a sandwich structure [H10-H12, H24, H25] as illustrated in Fig. H3. While such a laser-assisted deposition approach is named as absorbing film-assisted LIFT (AFA LIFT) and biological laser printing (Bio LP) [H10-H12, H24, H25], it is usually classified as modified LIFT [H1].

2.2.2 Electro-hydrodynamic jetting

Electro-hydrodynamic jetting (EHDJ) has emerged as a new endeavor in cell direct writing. As shown in Fig. H7, during EHDJ the bio-ink is pumped through a needle-like nozzle, which is connected to a high voltage power supply, using a syringe pump. Jetting of the bio-ink is controlled by adjusting the strength of high voltage-induced electric field which is on the order of 1 kV/mm. At the nozzle outlet, the applied high voltage and Taylor instability make the ejected bio-ink into droplets under the effects of surface tension and electric field gradients.

The distance between the nozzle outlet and the target substrate is usually on the order of 10 mm, which may result in some deleterious evaporation or cooling effects to cells [H1]. The cell droplet velocity in EHDJ is about 0.1 m/s, which is far lower than that in inkjetting. The high applied voltages and strong electric field gradients during EHDJ can induce deleterious damage to cells in the bio-ink; however, some successes have been achieved in direct writing of various biological constructs including zebrafish embryos [H14].

This work of this web site was partially supported by the National Science Foundation (NSF), the National Textile Center (NTC), and the South Carolina Space Grant Consortium (SCSGC).

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Table of Contents

2.1 Laser-based direct writing

2.2 Inkjet-based direct writing

2.2.1 Inkjet direct writing

2.2.2 Electro-hydrodynamic jetting