**3. Results**

For these experiments, we used the pressure pump aligned vertically in a homemade holder attached to a stand with a heavy base at a maximum force of 20 lbs (see **Figure 1**).

After preliminary testing, we realized the hypotonic alginate solution was causing the decrease in viability, so we compared alginate prepared in three different solutions: H<sup>2</sup> O, PBS, and complete culture media (as described in Section 2). **Figure 2** shows a very significant drop in viability compared to the control (95 ± 1%) when the cells were placed in alginate prepared in H2 O (Sham, 75 ± 11.6%, p = 0.048; 28G no coat, 21.8 ± 13.5%, p = 0.0056; 28G coat, 14 ± 1.3%, p = 6.3 × 10−8), while the cells placed in alginate prepared in either PBS (Sham, 94.3 ± 0.26, p = 0.16; 28G no coat, 92.9 ± 0.6%, p = 0.025; 28G coat, 87.8 ± 2.1%, p = 0.0062) or the complete culture media (Sham, 96.5 ± 0.4, p = 0.063; 28G no coat, 86.3 ± 2.8%, p = 0.0075; 28G coat, 85.5 ± 1.6, p = 0.00045) although sometimes significantly lower did not cause more than a 10% drop in cell viability using the same conditions: 3 mL syringe, 20 lbs of force giving 220 psi, and 28G nozzles of 1″ length. We show the data for 28G nozzles of 1″ length in **Figure 2**, but note that the ½ and 2″ lengths also did not cause a decrease of more than 10% in cell viability if the cells were placed in 3% (w/v) alginate prepared in PBS or complete culture media. Note that coating the nozzles did not improve the cell viability compared to those that were uncoated. The results for T = 0 are shown because the later time points did not yield more than a 10%

**2.7. Flow cytometry**

190 Tissue Regeneration

mL of 4–8 × 105

**2.8. Statistical analysis**

p < 0.05 to be significant.

**3. Results**

in H2

allowed to slowly drip out as small droplets.

ther analysis in the PerCP-Cy5-5-A fluorescent channel.

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

(unpublished results).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

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 fur-

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

For these experiments, we used the pressure pump aligned vertically in a homemade holder

After preliminary testing, we realized the hypotonic alginate solution was causing the

and complete culture media (as described in Section 2). **Figure 2** shows a very significant drop in viability compared to the control (95 ± 1%) when the cells were placed in alginate prepared

O (Sham, 75 ± 11.6%, p = 0.048; 28G no coat, 21.8 ± 13.5%, p = 0.0056; 28G coat, 14 ± 1.3%, p = 6.3 × 10−8), while the cells placed in alginate prepared in either PBS (Sham, 94.3 ± 0.26, p = 0.16; 28G no coat, 92.9 ± 0.6%, p = 0.025; 28G coat, 87.8 ± 2.1%, p = 0.0062) or the complete culture media (Sham, 96.5 ± 0.4, p = 0.063; 28G no coat, 86.3 ± 2.8%, p = 0.0075; 28G coat, 85.5 ± 1.6, p = 0.00045) although sometimes significantly lower did not cause more than a 10% drop in cell viability using the same conditions: 3 mL syringe, 20 lbs of force giving 220 psi, and 28G nozzles of 1″ length. We show the data for 28G nozzles of 1″ length in **Figure 2**, but note that the ½ and 2″ lengths also did not cause a decrease of more than 10% in cell viability if the cells were placed in 3% (w/v) alginate prepared in PBS or complete culture media. Note that coating the nozzles did not improve the cell viability compared to those that were uncoated. The results for T = 0 are shown because the later time points did not yield more than a 10%

O, PBS,

attached to a stand with a heavy base at a maximum force of 20 lbs (see **Figure 1**).

decrease in viability, so we compared alginate prepared in three different solutions: H<sup>2</sup>

cells/mL exposed overnight to a final concentration of 300 μM silver nitrate

**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 solutions: H2 O, PBS, and complete culture media with 1 mM EDTA. The "no coat" refers to the untreated nozzles and the "coat" refers to the nozzles coated with silicone as described in Section 2.The asterisks indicate significant differences from the controls.

decrease in viability, in agreement with what others found [10], and after 24 h, some of the surviving cells divided increasing the apparent viability.

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 described previously: H2 O, PBS, and complete culture media with 1 mM EDTA. Using the 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 in the alginate prepared in H2 O (14.1 ± 2.4%; p = 6.9 × 10−6), PBS (86 ± 10%; p = 0.21), or media

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

(78.3 ± 5.2%; p = 0.016) than after passing them through the 28G nozzle of the same length (**Figure 3**). Note that these differences in cell viability are not statistically significant from those obtained using the 28G nozzles (H2 O p = 0.22; PBS, p = 0.26; media, p = 0.051).

solution [10, 11], we decided to try and improve the cell viability by coating the nozzles with silicone in order to prevent membrane damage from nozzle imperfections and high pressures, as the latter was the proposed reason for the decrease in viability [14]. However, we discovered that neither the high pressures nor the membrane damage caused by nozzle imperfections was the reasons for the low cell viability; we found the low cell viability was

solution that causes cell lysis and bloating (swelling). Some studies that reported low cell viability using microextrusion and alginate solutions did not state what solvent the hydrogel was dissolved in, but the fact that low cell viability was observed after printing using increasing pressures suggests a hypotonic solution was the culprit. This appears to be a recurring problem in this field because numerous scientists cite these findings and reproduce them

During our investigation, we used a variety of syringes (3, 5, 10, and 60 mL) and nozzles (blunt-end needles; 28G with ½″, 1″, and 2″ lengths, and 30G with ½ and 1″ lengths) with only PBS or complete culture media containing H9 cells, but there was no effect on cell viability using the highest force (20 lbs) and 3 mL syringe to yield the smallest area (0.09 inches2

the highest pressure of ~220 psi, which is over five times the pressure (~40 psi) that is usually used and is over twice the pressure (~100 psi) most printers can accurately print. Only with

effect when the alginate was prepared in either PBS or complete culture media (**Figures 2** and **3**) or if the cells were placed in PBS or complete culture media without alginate (**Figure 4**). The water created a hypotonic solution because the counter cationic ion, alginate cannot enter the cell like chlorine ions (and sodium anions) can because it is too big (MW 216.12 g/mole), so about 25% of the cells initially lysed to create an isotonic solution and the rest of the cells survived but became "bloated" (swollen) during the process. However, our results suggest the bloating made the remaining cells more sensitive to mechanical pressure and caused the observed pressure-dependent decrease in cell viability. By preparing the alginate solutions in PBS or other isotonic solutions like complete culture media, we demonstrate significantly higher cell viability. We also put the cells in PBS and complete culture media without the potential protection of the alginate to see if they would be killed by the pressure or shear force alone, but we did not see any cell lysis or death using the same system (3 mL syringe, 20 lbs, 220 psi) and 28G nozzles of 1″ length (**Figure 4**). In addition, we did not see any significant decrease in cell viability with ½″ or 2″ long nozzles (results not shown). Furthermore, the 1″ long 30G nozzle also did not cause any appreciable decrease in cell viability (~7% decrease

The results presented here show the low cell viability found during some mircoextrusion 3D bioprinting studies using alginate was due to placing the cells in a hypotonic solution causing cell lysis and bloating that makes the cells more sensitive to mechanical pressure during printing, which has been modeled [14]. Here we show this problem can be easily resolved by using isotonic solutions like PBS or complete culture media (0.9% saline is also suitable). Furthermore, the so-called recovery or increase in cell viability after 24 h [10] can be attributed to the division of the living cells rather than the recovery of membrane or other cellular damage, as noted by the increase in total cell number. The reason there is a decline in viability with increasing pressure or decreasing nozzle diameter can be attributed to the increasing shear forces causing

O did we see an adverse effect on cell viability; there was no

O, which is a hypotonic

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

) for

193

really caused by preparing the hydrogel (alginate) solution in H2

using the same procedure.

the addition of alginate in H2

compared to 28G, **Figure 3**).

Finally, we wanted to see if we could decrease the cell viability by removing the alginate, as it might be affording protection by coating the cellular membranes. We show the results for 3-mL syringes with 28G nozzles of 1″ length (either coated or with no coat) but found no significant effect on cell viability even at the highest force of 20 lbs resulting in a pressure of 220 psi when in PBS or complete media (**Figure 4**). We also tried 30G nozzles of 1″ length and other syringe sizes, but they only slightly affected cell viability unless the cells were placed in a hypotonic alginate solution.

**Figure 4.** The viability of H9 cells was determined after control, sham, or passing the cells through the 3-mL syringe equipped with a 28G nozzle of 1″ length under 220 psi in either PBS or media in the absence of alginate. The cells were washed with either PBS or complete culture media three times prior to simulated microextrusion printing. The control and shams are as described in Section 2.

## **4. Discussion**

3D bioprinting using microextrusion is the most common and affordable way to print living cells. Microextrusion is a method of direct cell writing that can 3D bioprint using different substances like hydrogels, cell spheroids, and biocompatible polymers facilitating the deposition of multiple cell types with high resolution to accurately fabricate complex structures, like an ear, using computer-aided design software [13]. Among the multitudes of hydrogels, alginate is popular because it is inexpensive and is crosslinked using calcium to give it a solid structural form which can later be reversed using citrate or EDTA. One major advantage to microextrusion printing is the ability to print cells at high densities that are close to physiological conditions, which is needed to construct tissue-engineered organs, maintaining high cell viability using high pressures, and small nozzle sizes are required for fast printing speeds with high resolution. Because microextrusion 3D bioprinting was reported to result in a significant decrease in cell viability yielding between only 40 and 80% live cells in alginate solution [10, 11], we decided to try and improve the cell viability by coating the nozzles with silicone in order to prevent membrane damage from nozzle imperfections and high pressures, as the latter was the proposed reason for the decrease in viability [14]. However, we discovered that neither the high pressures nor the membrane damage caused by nozzle imperfections was the reasons for the low cell viability; we found the low cell viability was really caused by preparing the hydrogel (alginate) solution in H2 O, which is a hypotonic solution that causes cell lysis and bloating (swelling). Some studies that reported low cell viability using microextrusion and alginate solutions did not state what solvent the hydrogel was dissolved in, but the fact that low cell viability was observed after printing using increasing pressures suggests a hypotonic solution was the culprit. This appears to be a recurring problem in this field because numerous scientists cite these findings and reproduce them using the same procedure.

(78.3 ± 5.2%; p = 0.016) than after passing them through the 28G nozzle of the same length (**Figure 3**). Note that these differences in cell viability are not statistically significant from

Finally, we wanted to see if we could decrease the cell viability by removing the alginate, as it might be affording protection by coating the cellular membranes. We show the results for 3-mL syringes with 28G nozzles of 1″ length (either coated or with no coat) but found no significant effect on cell viability even at the highest force of 20 lbs resulting in a pressure of 220 psi when in PBS or complete media (**Figure 4**). We also tried 30G nozzles of 1″ length and other syringe sizes, but they only slightly affected cell viability unless the cells were placed in

3D bioprinting using microextrusion is the most common and affordable way to print living cells. Microextrusion is a method of direct cell writing that can 3D bioprint using different substances like hydrogels, cell spheroids, and biocompatible polymers facilitating the deposition of multiple cell types with high resolution to accurately fabricate complex structures, like an ear, using computer-aided design software [13]. Among the multitudes of hydrogels, alginate is popular because it is inexpensive and is crosslinked using calcium to give it a solid structural form which can later be reversed using citrate or EDTA. One major advantage to microextrusion printing is the ability to print cells at high densities that are close to physiological conditions, which is needed to construct tissue-engineered organs, maintaining high cell viability using high pressures, and small nozzle sizes are required for fast printing speeds with high resolution. Because microextrusion 3D bioprinting was reported to result in a significant decrease in cell viability yielding between only 40 and 80% live cells in alginate

**Figure 4.** The viability of H9 cells was determined after control, sham, or passing the cells through the 3-mL syringe equipped with a 28G nozzle of 1″ length under 220 psi in either PBS or media in the absence of alginate. The cells were washed with either PBS or complete culture media three times prior to simulated microextrusion printing. The control

O p = 0.22; PBS, p = 0.26; media, p = 0.051).

those obtained using the 28G nozzles (H2

a hypotonic alginate solution.

192 Tissue Regeneration

**4. Discussion**

and shams are as described in Section 2.

During our investigation, we used a variety of syringes (3, 5, 10, and 60 mL) and nozzles (blunt-end needles; 28G with ½″, 1″, and 2″ lengths, and 30G with ½ and 1″ lengths) with only PBS or complete culture media containing H9 cells, but there was no effect on cell viability using the highest force (20 lbs) and 3 mL syringe to yield the smallest area (0.09 inches2 ) for the highest pressure of ~220 psi, which is over five times the pressure (~40 psi) that is usually used and is over twice the pressure (~100 psi) most printers can accurately print. Only with the addition of alginate in H2 O did we see an adverse effect on cell viability; there was no effect when the alginate was prepared in either PBS or complete culture media (**Figures 2** and **3**) or if the cells were placed in PBS or complete culture media without alginate (**Figure 4**). The water created a hypotonic solution because the counter cationic ion, alginate cannot enter the cell like chlorine ions (and sodium anions) can because it is too big (MW 216.12 g/mole), so about 25% of the cells initially lysed to create an isotonic solution and the rest of the cells survived but became "bloated" (swollen) during the process. However, our results suggest the bloating made the remaining cells more sensitive to mechanical pressure and caused the observed pressure-dependent decrease in cell viability. By preparing the alginate solutions in PBS or other isotonic solutions like complete culture media, we demonstrate significantly higher cell viability. We also put the cells in PBS and complete culture media without the potential protection of the alginate to see if they would be killed by the pressure or shear force alone, but we did not see any cell lysis or death using the same system (3 mL syringe, 20 lbs, 220 psi) and 28G nozzles of 1″ length (**Figure 4**). In addition, we did not see any significant decrease in cell viability with ½″ or 2″ long nozzles (results not shown). Furthermore, the 1″ long 30G nozzle also did not cause any appreciable decrease in cell viability (~7% decrease compared to 28G, **Figure 3**).

The results presented here show the low cell viability found during some mircoextrusion 3D bioprinting studies using alginate was due to placing the cells in a hypotonic solution causing cell lysis and bloating that makes the cells more sensitive to mechanical pressure during printing, which has been modeled [14]. Here we show this problem can be easily resolved by using isotonic solutions like PBS or complete culture media (0.9% saline is also suitable). Furthermore, the so-called recovery or increase in cell viability after 24 h [10] can be attributed to the division of the living cells rather than the recovery of membrane or other cellular damage, as noted by the increase in total cell number. The reason there is a decline in viability with increasing pressure or decreasing nozzle diameter can be attributed to the increasing shear forces causing increasing amounts of cellular damage resulting in increasing cell death via apoptosis [11] (and our unpublished observations) but only when the cells are placed in hypotonic solutions.

**Abbreviations**

**Author details**

Spring, MD, USA

**References**

Dianne Eyvonn Godar

2014;**32**:773-785

Review

EDTA ethylenediaminetetraacetic acid

Address all correspondence to: dianne.godar@fda.hhs.gov

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US Food and Drug Administration, Center for Devices and Radiological Health, Silver

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PBS phosphate buffered saline

psi pounds per square inch

PI propidium iodide

SD standard deviation
