**3D Bioprinting: Surviving under Pressure**

**3D Bioprinting: Surviving under Pressure**

#### Dianne Eyvonn Godar Dianne Eyvonn Godar Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73137

#### **Abstract**

Because 3D bioprinting using microextrusion was reported to yield cells with low viability (~40%) after pneumatic pressure (40 psi) printing through stainless steel nozzles, or blunt-end needles, with about 150 μm diameters (28 and 30G), we set out to improve the viability by coating the interior of the nozzles with silicone. For these studies, H9 human lymphoma cells were used to simulate human stem cells in suspension, and cell viability was measured using propidium iodide dye exclusion and flow cytometry. We tried to improve the viability by coating the inside of the 28 and 30G nozzles (1″ length) with silicone to protect the cell membranes from being damaged by the imperfections in the stainless steel nozzle. However, we discovered silicone coating had little effect on viability because imperfections in the nozzle were not the problem. Instead, the cells being placed in hypotonic 3% (w/v) alginate prepared in water prior to printing caused significant cell death (~25%) and considerably more (≥50%) after simulated printing under pressure. By preparing the alginate in isotonic solutions of either phosphate buffered saline or complete culture media, we could use pressures over five times (>220 psi) what most printing procedures use and obtain ~80% viability.

DOI: 10.5772/intechopen.73137

**Keywords:** force, hydrogel, hypotonic, isotonic, microextrusion, viability

## **1. Introduction**

3D bioprinting is the wave of the future for constructing viable, functional, and biocompatible human organs that will be created from the patient's own stem cells so that antirejection drugs will not be needed after transplantation. Currently, there are three main 3D bioprinting methods: inkjet or "drop on demand," laser-assisted, and microextrusion [1].

Inkjet printing uses thermal or acoustic (piezoelectric) forces to create and eject droplets. Thermal inkjet bioprinting yields good cell viability (>85%), and although localized heating of 200–300°C occurs and the temperature at the head only rises 4–10°C for short durations

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

(~2 μs), cells can be heat shocked. The heat-shock protein chaperones can protect cells from dying [2] so that they have the potential of passing along DNA mutations that could ultimately result in a cancerous cell. Only a couple of degrees rise in temperature ≥2°C (~39°C) is needed to induce some heat-shock proteins [3]. Acoustic inkjet bioprinting applies voltage across polycrystalline piezoelectric ceramics to induce a rapid change in their shape that creates the pressure to eject droplets. However, the 15–25 kHz frequencies shock the cells: causing membrane damage and cellular lysis [4] and allowing molecules up to 40,000 Daltons (90 Å) in size to enter or exit the cell [5].

media (see below) with the addition of 1 mM ethylene diamine tetra acetic acid (EDTA; Sigma Aldrich, St. Louis, MO). The 1 mM EDTA was added to crosslink the 2 mM calcium present in the media so the alginate would not solidify. Note that this solution of alginate in complete culture media with EDTA did not solidify for over a year. We sterilized these solutions using either a 0.45 μm syringe filtration system (Nalge Nunc International Corporation, Rochester, NY) with the cell dispensing device described below (**Figure 1**) at the highest force (20 lbs) overnight or a 0.45 μm filtration unit with vacuum suction overnight. Additionally, we tested the viability after passing cells through the syringe and blunt-end needles using this force (20 lbs) in complete culture media or PBS (after washing the cells three times to remove any bound proteins that might afford membrane

3D Bioprinting: Surviving under Pressure http://dx.doi.org/10.5772/intechopen.73137 187

**Figure 1.** The KD scientific model 100 series screw-driven pressure pump with maximum force of 20 lbs is shown housed in a homemade holder attached to a vertical stand with a heavy base equipped with a 3-mL syringe and a 28G nozzle of 1″ in length that was used for the experiments shown in **Figures 2** and **4** (**Figure 3** has the 30G nozzle but same device).

protection).

Laser-assisted bioprinting yields high cell viability (>95%) [6] and is nozzle-free so that the problem of clogging with materials or cells that other printing methods have is circumvented. In addition, it has microscale resolution of a single cell per drop. The drawbacks to laser-assisted bioprinting are low flow rates due to the high resolution requiring rapid gelation kinetics [7], time-consuming preparation of the ribbons used for printing, metal residues from vaporization of the metallic laser-absorbing layer during printing (nanoparticles), the complexity of making ribbons to print multiple cell types, and the high cost; more germane, it is not clear if this technology can be scaled up for larger tissue sizes other than skin [8], let alone organs.

Finally, microextrusion is the most common and affordable 3D bioprinting method that uses either pneumatic (air) or mechanical (screw or piston) forces to create pressurized dispensing systems [9]. However, this 3D bioprinting method is reported to yield the lowest cell viability of all three methods (40–80%) [10]. In previous studies, this low cell viability was completely attributed to the biofabrication mechanical forces or high pressures applied to the cells [11] rather than to structural imperfections inside the stainless steel nozzles creating membrane damage.

We reasoned if some of this membrane damage was actually due to the imperfections inside the stainless steel nozzle, then we might be able to improve the viability by coating their interiors with silicone. By minimizing these interior structural flaws, we would increase the viability during printing under pressure. However, in the course of our investigation, we found that neither the biofabrication of mechanical forces nor the structural flaws inside the nozzle were causing the reduction in viability but rather it was the hypotonic solution the cells were placed in when the alginate was prepared in water; the cells were placed directly into that hypotonic solution without first adjusting it with salt to be isotonic. This lysed many cells (~25%) until evidently the solution became isotonic from the released intracellular salts leaving the remaining cell population "bloated" or swollen and very sensitive to mechanical forces.
