**2. Materials and methods**

#### **2.1. Chemical formulations**

The alginate solution was prepared using medium viscosity sodium alginate (Sigma, St. Louis, MO) dissolved in high-performance liquid chromatography grade water (Sigma Aldrich, St. Louis, MO) to make a 3% (w/v) solution as described previously [10, 11]. We also prepared 3% (w/v) alginate solutions in Dulbecco's phosphate buffered saline (PBS) without magnesium or calcium (GIBCO, Gaithersburg, MD) and in complete culture 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 protection).

(~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

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 remain-

The alginate solution was prepared using medium viscosity sodium alginate (Sigma, St. Louis, MO) dissolved in high-performance liquid chromatography grade water (Sigma Aldrich, St. Louis, MO) to make a 3% (w/v) solution as described previously [10, 11]. We also prepared 3% (w/v) alginate solutions in Dulbecco's phosphate buffered saline (PBS) without magnesium or calcium (GIBCO, Gaithersburg, MD) and in complete culture

ing cell population "bloated" or swollen and very sensitive to mechanical forces.

**2. Materials and methods**

**2.1. Chemical formulations**

Daltons (90 Å) in size to enter or exit the cell [5].

186 Tissue Regeneration

**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).

#### **2.2. Cell culture**

Human lymphoma H9 cells (ATTC, Manassas, VA) were cultured and maintained in the incubator at 37°C using complete culture medium: CO2 -independent media (GIBCO, Gaithersburg, MD) supplemented with 10% (v/v) heat inactivated, mycoplasma-tested, and endotoxin-free, fetal bovine serum (GIBCO, Gaithersburg, MD), 4 mM glutamine (GIBCO, Gaithersburg, MD), antibiotic (10,000 IU penicillin and 10,000 μg/mL streptomycin), and antimycotic (25 μg/mL amphotericin B) solution (Sigma, St. Louis, MO). We use CO2 independent media so the cells do not undergo pH shock while being manipulated during or after experiments. This enabled us to leave the cells in culture tubes at 37°C using a constant temperature controlled heating block, Hema-Bath Block Module Heater Type 12,200 Dribath (Baxter Scientific Products, Deerfield, IL), under sterile conditions in the biosafety cabinet until monitored on the first day (0 and 4 or 6 h postexposure). For the 24-h time point, we diluted the samples 1:1 with CO2 -independent complete culture media and maintained the cells in 5 mL sterile culture tubes at 37°C in the heating block or in the incubator (results not shown). These suspension cells were grown and maintained below 1 × 106 cells/mL in culture and were usually used between 4 and 8 × 105 cells/mL for experiments with viability ≥90% as determined by dye exclusion of propidium iodide (PI) using flow cytometry.

**2.4. Microextrusion cell dispensing system**

dles are referred to as nozzles.

**2.5. Chemical coating nozzles**

saline (isotonic).

**2.6. Cell viability**

ages using flow cytometry.

per square inch (psi) using Eq. (1):

P = \_\_F

We employed a mechanical device that uses a screw to create a force with subsequent pressure dependent on the radius of the syringe. The KD Scientific Model 100 series (Harvard Apparatus, Holliston, MA) screw-driven pressure pump with maximum force of 20 lbs was housed in a homemade holder attached to a vertical stand with a heavy base (see **Figure 1**). We used syringes of various sizes (3–60 mL) with Hamilton blunt-end 28G or 30G needles (Harvard Apparatus, Holliston, MA) of different lengths: ½″, 1″, and 2″. These blunt-end nee-

The experiments were performed using various pressures "P" that were calculated in pounds

where "F" is the applied force (maximum of 20 lbs was used) and "A" is the area (in inches squared) of the applied force. We performed various experiments using syringes of different sizes, 3, 10, 30, and 60 mL, having radii of 0.17 (8.59 mm), 0.285 (14.48 mm), 0.425 (21.59 mm),

220, 78, 35, and 23 psi, respectively. Pressure experiments were conducted at room tempera-

In order to help minimize membrane damage incurred during pressurized simulated microextrusion printing, presumably from imperfections in the stainless steel, we coated the interior of the 28 and 30G blunt-end needles for 5–15 min at room temperature using ~10% (w/v) high molecular weight (500,000 g/mole) polydimethylsiloxane, trimethylsiloxy terminated (Gelest, Inc., Morrisville, PA) in high-performance liquid chromatography grade hexane (Sigma Aldrich, St. Louis, MO). To sterilize the blunt-end needles, we luerlocked them on the syringe, submerged them in 70% ethanol, and then used three 0.5 mL interior washes of 70% ethanol followed by three 0.5 mL interior washes of sterile 0.9%

Cell viability was assessed by the dye exclusion method using PI (Molecular Probes, Eugene, OR) at a final concentration of 1 μg/mL using a slightly modified procedure [12]. Briefly, the PI was added directly to the samples so as not to lose any of the representative cell populations or to create false positives by centrifuging and disrupting the membranes of partially damaged cells. Live cells completely exclude PI, while dead cells allow it to almost instantly pass through their membranes. Cell viability was quantified as percent-

and 0.524 inches (26.6 mm), with areas of 0.09, 0.255, 0.568, 0.86 inches2

ture, and the cells were placed at 37°C after treatment until analyzed.

<sup>A</sup> (1)

3D Bioprinting: Surviving under Pressure http://dx.doi.org/10.5772/intechopen.73137 189

, yielding pressures of

Cells were centrifuged in 50 mL centrifuge tubes (Corning, Tewksbury, MA) at 300 × *g* for 7 min, and the media were aspirated to leave cell pellets that were loosened by quickly (2–3 s) vortexing at low speed. These cell pellets were very gently and briefly mixed in the viscous 3 mL of 3% (w/v) alginate solutions with a Pasteur pipet by swirling and slowly pipetting up and down three times to homogeneously disperse the cells as previously described [10]. The cell density was ~3 × 106 cells, as determined by hemocytometer readings, before mixing into 3 mL of alginate to give ~1 × 106 cells/mL.

#### **2.3. Procedure for simulated microextrusion pressure printing**

Cells were grown to 4–8 × 105 cells/mL in complete culture media whereby 100 mL was centrifuged at 300 × *g* for 7 min, media aspirated, and then the cell pellet was vortexed and suspended in one of the three, 3% (w/v) alginate solutions (H2 O, PBS, or complete culture media) or suspended in solutions of either PBS or complete culture media to a final density of ~1 × 106 cells/mL or 4–8 × 105 cells/mL, respectively. Different concentrations were used to know if high cell density afforded protection for the cells. For the PBS and complete culture media comparison studies, we washed the cells three times with either PBS or complete culture media, respectively, prior to microextrusion. For the T = 0 time point (actual time < 10 min), we simply collected the cells in a test tube containing 0.25–0.5 mL of complete culture media at room temperature. For the longer daily time points of 4 or 6 h, we put the test tubes at 37°C in a dry-block incubator under the biosafety cabinet. For overnight studies at 24 h, we further diluted the cells 1:1 with complete culture media and put them in tightly capped sterile 5 mL polypropylene culture tubes or T-25 flasks (Corning, Tewksbury, MA).

#### **2.4. Microextrusion cell dispensing system**

**2.2. Cell culture**

188 Tissue Regeneration

diluted the samples 1:1 with CO2

and were usually used between 4 and 8 × 105

described [10]. The cell density was ~3 × 106

Cells were grown to 4–8 × 105

T-25 flasks (Corning, Tewksbury, MA).

final density of ~1 × 106

ings, before mixing into 3 mL of alginate to give ~1 × 106

**2.3. Procedure for simulated microextrusion pressure printing**

and suspended in one of the three, 3% (w/v) alginate solutions (H2

cells/mL or 4–8 × 105

Human lymphoma H9 cells (ATTC, Manassas, VA) were cultured and maintained in the

Gaithersburg, MD) supplemented with 10% (v/v) heat inactivated, mycoplasma-tested, and endotoxin-free, fetal bovine serum (GIBCO, Gaithersburg, MD), 4 mM glutamine (GIBCO, Gaithersburg, MD), antibiotic (10,000 IU penicillin and 10,000 μg/mL streptomycin), and antimycotic (25 μg/mL amphotericin B) solution (Sigma, St. Louis, MO). We use CO2

independent media so the cells do not undergo pH shock while being manipulated during or after experiments. This enabled us to leave the cells in culture tubes at 37°C using a constant temperature controlled heating block, Hema-Bath Block Module Heater Type 12,200 Dribath (Baxter Scientific Products, Deerfield, IL), under sterile conditions in the biosafety cabinet until monitored on the first day (0 and 4 or 6 h postexposure). For the 24-h time point, we

cells in 5 mL sterile culture tubes at 37°C in the heating block or in the incubator (results not

Cells were centrifuged in 50 mL centrifuge tubes (Corning, Tewksbury, MA) at 300 × *g* for 7 min, and the media were aspirated to leave cell pellets that were loosened by quickly (2–3 s) vortexing at low speed. These cell pellets were very gently and briefly mixed in the viscous 3 mL of 3% (w/v) alginate solutions with a Pasteur pipet by swirling and slowly pipetting up and down three times to homogeneously disperse the cells as previously

centrifuged at 300 × *g* for 7 min, media aspirated, and then the cell pellet was vortexed

culture media) or suspended in solutions of either PBS or complete culture media to a

tions were used to know if high cell density afforded protection for the cells. For the PBS and complete culture media comparison studies, we washed the cells three times with either PBS or complete culture media, respectively, prior to microextrusion. For the T = 0 time point (actual time < 10 min), we simply collected the cells in a test tube containing 0.25–0.5 mL of complete culture media at room temperature. For the longer daily time points of 4 or 6 h, we put the test tubes at 37°C in a dry-block incubator under the biosafety cabinet. For overnight studies at 24 h, we further diluted the cells 1:1 with complete culture media and put them in tightly capped sterile 5 mL polypropylene culture tubes or

shown). These suspension cells were grown and maintained below 1 × 106

determined by dye exclusion of propidium iodide (PI) using flow cytometry.


cells/mL for experiments with viability ≥90% as

cells, as determined by hemocytometer read-

cells/mL, respectively. Different concentra-

cells/mL.

cells/mL in complete culture media whereby 100 mL was


cells/mL in culture

O, PBS, or complete


incubator at 37°C using complete culture medium: CO2

We employed a mechanical device that uses a screw to create a force with subsequent pressure dependent on the radius of the syringe. The KD Scientific Model 100 series (Harvard Apparatus, Holliston, MA) screw-driven pressure pump with maximum force of 20 lbs was housed in a homemade holder attached to a vertical stand with a heavy base (see **Figure 1**). We used syringes of various sizes (3–60 mL) with Hamilton blunt-end 28G or 30G needles (Harvard Apparatus, Holliston, MA) of different lengths: ½″, 1″, and 2″. These blunt-end needles are referred to as nozzles.

The experiments were performed using various pressures "P" that were calculated in pounds per square inch (psi) using Eq. (1):

$$\mathbf{P} = \frac{\mathbf{F}}{\mathbf{A}}\tag{1}$$

where "F" is the applied force (maximum of 20 lbs was used) and "A" is the area (in inches squared) of the applied force. We performed various experiments using syringes of different sizes, 3, 10, 30, and 60 mL, having radii of 0.17 (8.59 mm), 0.285 (14.48 mm), 0.425 (21.59 mm), and 0.524 inches (26.6 mm), with areas of 0.09, 0.255, 0.568, 0.86 inches2 , yielding pressures of 220, 78, 35, and 23 psi, respectively. Pressure experiments were conducted at room temperature, and the cells were placed at 37°C after treatment until analyzed.

#### **2.5. Chemical coating nozzles**

In order to help minimize membrane damage incurred during pressurized simulated microextrusion printing, presumably from imperfections in the stainless steel, we coated the interior of the 28 and 30G blunt-end needles for 5–15 min at room temperature using ~10% (w/v) high molecular weight (500,000 g/mole) polydimethylsiloxane, trimethylsiloxy terminated (Gelest, Inc., Morrisville, PA) in high-performance liquid chromatography grade hexane (Sigma Aldrich, St. Louis, MO). To sterilize the blunt-end needles, we luerlocked them on the syringe, submerged them in 70% ethanol, and then used three 0.5 mL interior washes of 70% ethanol followed by three 0.5 mL interior washes of sterile 0.9% saline (isotonic).

#### **2.6. Cell viability**

Cell viability was assessed by the dye exclusion method using PI (Molecular Probes, Eugene, OR) at a final concentration of 1 μg/mL using a slightly modified procedure [12]. Briefly, the PI was added directly to the samples so as not to lose any of the representative cell populations or to create false positives by centrifuging and disrupting the membranes of partially damaged cells. Live cells completely exclude PI, while dead cells allow it to almost instantly pass through their membranes. Cell viability was quantified as percentages using flow cytometry.

#### **2.7. Flow cytometry**

The viability of 10,000 cells from each sample was determined by a FACSCanto II (Becton Dickinson, CA, USA) triple beam flow cytometer at medium flow rate in the PerCP-Cy5-5-A (equivalent to FL-2) channel using PI dye exclusion [12]. We gated on the single-cell population. To set the marker for the live cell population, we used cultured cells from the incubator (≥90%) and created the marker for the dead cell population (with viability <10%) using 10 mL of 4–8 × 105 cells/mL exposed overnight to a final concentration of 300 μM silver nitrate (unpublished results). The sham-exposed cells underwent the same treatment as the exposed cells except they were not put under pressure through the syringes and nozzles; the cells were carefully mixed in alginate or other solutions and then gently placed into the syringe and allowed to slowly drip out as small droplets.

For each experiment, the live and dead cell controls were used to set the gates for the live and dead cell populations in the PerCP-Cy5-5-A fluorescent channel. We used forward scatter characteristics (FSC-W and FSC-H) to distinguish between single and multiple, or clumped, cell populations. The gate was set on the single-cell population to collect 10,000 cells for further analysis in the PerCP-Cy5-5-A fluorescent channel.

decrease in viability, in agreement with what others found [10], and after 24 h, some of the

the "coat" refers to the nozzles coated with silicone as described in Section 2.The asterisks indicate significant differences

**Figure 2.** The viability of H9 cells immediately (T = 0 h) following direct cell writing biofabrication using the homemade microextrusion device shown in **Figure 1** equipped with a 3-mL syringe and 28G nozzle of 1″ length using maximum force of 20 lbs to produce 220 psi. Concentrated H9 cells were mixed with 3 mL of 3% (w/v) alginate prepared in different

O, PBS, and complete culture media with 1 mM EDTA. The "no coat" refers to the untreated nozzles and

As shown in **Figure 2**, the nozzle surface chemistry did not affect cell viability, but the hypotonic solution did, so we did not compare coated with uncoated nozzles in **Figure 3**. We wanted to know how a lower gauge nozzle with a smaller diameter would affect cell viability, so we used a 30G nozzle of 1″ length and placed the cells in 3% (w/v) alginate solutions as

same conditions employed for the 28G nozzle of 1″ length (i.e., 3 mL syringe and maximum force of 20 lbs yielding 220 psi), we did not observe more than ~8% decrease in cell viability

**Figure 3.** The viability of H9 cells immediately (T = 0 h) following direct cell writing biofabrication using the homemade microextrusion device equipped with a 3-mL syringe and 30G nozzle of 1″ length using maximum force of 20 lbs to produce 220 psi. Concentrated H9 cells were mixed with 3 mL of 3% (w/v) alginate prepared in different solutions: H<sup>2</sup>

PBS, and complete culture media with 1 mM EDTA. The asterisks indicate significant differences from the controls.

O, PBS, and complete culture media with 1 mM EDTA. Using the

O (14.1 ± 2.4%; p = 6.9 × 10−6), PBS (86 ± 10%; p = 0.21), or media

3D Bioprinting: Surviving under Pressure http://dx.doi.org/10.5772/intechopen.73137 191

O,

surviving cells divided increasing the apparent viability.

described previously: H2

solutions: H2

from the controls.

in the alginate prepared in H2

#### **2.8. Statistical analysis**

The data in the text and figures are presented as the mean (n = 3) ± standard deviation (SD) computed using the Student's t-test for two samples assuming unequal variance and consider p < 0.05 to be significant.
