Institute for Regenerative Medicine Sarthak Patnaik, Prafulla Chandra, Christina Ross, Yuanyuan Zhang, and Benjamin S. HarrisonWake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157

IntroductionWith recent advances in technology, researchers have devised methods for the development and implantation of engineered biomaterials to replace injured tissues and organs. This has become increasingly important as the supply of tissues and organs does not come close to meeting the demand worldwide. Through the general evolution toward microscale patterning, it has become possible to potentially offset this lack of supply through the development of piezoelectric ink jet processing of biomaterials and cells. This work aims to develop of living cells via small droplets and layering them within hydrogels in order to develop three-dimensional substitute tissue. There are several parameters on our bioprinter that we can change to affect the printing process. As this technology is a very recent development, we still do not know how to change these parameters to print cells in the most effective manner; this became the main problem for us to solve.

It is hypothesized that temperature of cell cartridges, along with stirring of cell suspensions within cartridges affect the general distribution and viability of cells. Our goal was to determine which specific settings would optimize the printing process.

Materials & Methods• All experiments were conducted on a multi-channel bioprinter (custom-made by

Microfab Technologies Inc., Plano, TX). • Mouse macrophages and 3T3 Fibroblasts were printed using piezoelectric inkjet

dispensing (Figure 1) onto microscope slides in 7 x 10 dot patterns. • To test for average cell distribution (number of cells printed per drop), the slides

were analyzed using Leica microscopes, and cells in samples of 30 drops per slide were counted.

• Effective working time for each cell type was determined by obtaining cell viability within the cartridges at various time points (0 min, 30 min, 60 min, 90 min, 2 hours, 4 hours, and 6 hours) . These procedures were conducted for all parameters tested, which are listed in Figure 2. Conclusions

From the optimization of bioprinting parameters using macrophages and fibroblasts, we can conclude that medium stir speeds resulted in the longest effective working time with least loss of cell viability, and also increases the number of drops which printed cells. This same effect is seen with cells containing cartridges maintained at 30° C temperatures.

References1. Jan Sumerel, John Lewis, Andy Doraiswamy, Leila F. Deravi, Sarah L. Sewell, Aren E. Gerdon, David W. Wright, and Roger J. Narayan. “Piezoelectric ink jet processing of materials for medical and biological applications.” Biotechnology Journal, 2006, 976-987.2. Kalyani Nair, Milind Gandhi, Saif Khalil, Karen Chang Yan, Michele Marcolongo, Kenneth Barbee, and Wei Sun. “Characterization of cell viability during bioprinting processes.” Biotechnology Journal, 2009, 1168-1177.

AcknowledgementsWe would like to thank Eben Adarkwa and Dr. Salil Desai for their training and assistance with the printing process. This work was supported in part by USAMRMC through contract #W81XWH-11-1-0658.

Optimization of Cell Bioprinting Parameters for Tissue Engineering

Figure 1: Diagram illustrating piezoelectric drop-on-demand bioprinting. This process utilizes a piezoelectric crystal as a transducer, which changes the size of the nozzle opening to affect droplet shape and diameter. This crystal uses any waveform to expand and constrict the nozzle opening at constant time intervals. The rate at which it does this is affected by voltage and wave attributes. The formed droplets are then printed onto the substrate in the specified pattern.

Optimization ExperimentParameter Type Parameter Settings Tested

Stir Speed No Stirring Medium Stirring(500 rpm)

Maximum Stirring (1000 rpm)

Temperature 23 ° C 30° C 37 ° C

Figure 2: Table listing the two parameters tested: stir speed and cartridge temperature. Experiments were done with three different settings for each parameter. Note: All slides printed for the stirring experiment were done so at 24.4° C, the temperature within the bioprinting apparatus..

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Figure 3: Effect of Stir speed and cartridge temperature on printing of macrophages.. a) Graph showing cell viability within the cartridge at various time points for stirring experiment. Unpaired student t test shows that there is no significant difference between the three stir settings up to 2 hours, after which the viability drops off. Also, viability changes remain insignificant for only 30 minutes at maximum stir speeds and 60 minutes for no stirring; however, cell viability remains relatively constant at medium stir speeds up to 2 hours, which suggests that medium stir speeds increase effective working time. b) Table listing percentage of drops that printed with cells, and average number of cells within those drops, for stirring experiment. Unpaired student t test shows that stir speed does not affect average number of cells per drop, but does have a significant effect on percentage of drops printed with cells. Medium stir speed produces more drops with cells in them, than do other settings. c) Graph showing effect of cartridge temperature on cell viability. Once again, it is observed that for all three temperatures, cell viability is very similar up to 2 hours, after which viability drops significantly for all three settings. However, t tests show that effective working time is only 60 minutes at 37° and 90 minutes at 23°, but is 2 hours for 30° C. d) Table listing effect of cartridge temperature on cell distribution within the printed drops. T test shows that temperature does not have a significant effect on average number of cells per drop, but at 30° C, significantly more printed drops contain cells compared to ones at 23° C or 37° C. Fibroblasts produced similar results.

Figure 4: Microscopic images of bioprinted droplets with cells. Top row are images of printed drops containing macrophages fluorescently labeled with CellTrackerTM Red CMPTX dye (Life Technologies Corp., Carlsbad, CA) (Black and White Images shown: Scale bar: 50 μm) Middle row are drops containing Fibroblasts and Macrophages labeled with CellTrackerTM Green CMFDA dye (Scale bar: 50 μm). Bottom row are bright field images of printed drops containing Macrophages. (Scale bar: 100 μm). Only drops imaged with fluorescence microscopy (top & middle row; average 17 μm in diameter) were included in the data presented in Figure 3. All images taken with Leica microscopes at 200x total magnification.

Results and Discussiona) b)

c) d)

0 60 120 180 240 300 3600

102030405060708090

100

Viability vs. Time (Stir Speed)

No StirringMed StirringMax Stirring

Time (min)

Viab

ility

(%)

0 60 120 180 240 300 3600

20

40

60

80

100

120

Viability vs. Time (Temperature)

23° C 30° C37° C

Time (min)

Viab

ility

(%)

Stirring None Med Max

% of Drops with Cells 27% 58% 37%

Average Cells per Drop 1.2 ± 0.7 1.3 ± 0.7 1.4 ± 0.6

Temperature 23°C 30°C 37°C

% of Drops with Cells 27% 38% 26%

Average Cells per Drop 1.3 ± 0.6 1.5 ± 0.7 1.2 ± 0.4