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Lothar Koch, Ole Brandt, Andrea Deiwick, et al.

Table 1. Parameters of the applied lasers 2.5 Preparation of the Collector Slide

Laser Nd:YAG 1 Yb:YAG 2
As collector slides, 1-mm thick glass slides (26×26 mm²)
Wavelength [nm] 355 532 1064 1064 were cleaned in an ethanol bath and with acetone us-
Pulse duration [ns] 0.5 0.52 0.75 8, 14, 20, 30, 50, ing lens cleaning tissue and sterilized by irradiating
100, 200
Max. pulse energy [µJ] 8 17.5 85 200 with UV-C light for 1 hour. For determination of the
survival rates of printed cells, the collector slides were
1 PULSELAS-P-355-100-HP, AlphalasGmbh; ² YLPM-1-A4-20-20, coated with 45 μL of 2 wt% alginate hydrogel. Pri-
IPG Photonics Corporation
marily, this hydrogel layer prevents the dying of print-
ed cells by drying-up, but it also cushions the impact
2.3 Cell Culture of the laser printing process. For analyzing printed
droplet sizes, uncoated glass slides were used.
For all cell experiments in this study, murine fibro-
blast cell line NIH-3T3 was used. Asa cell culture me- 2.6 Measuring Droplet Sizes
dium, Dulbecco’s Modified Eagle Medium/F12 sup- For measuring the sizes of printed droplets, the same
plemented with 10% fetal bovine serum and 1% peni- alginate was always printed on uncoated glass surfac-
cillin/streptomycin (all from PAN Biotech, Aidenbach, es. Therefore, there is a constant relation between the
Germany) was used and exchanged every third day. printed droplet diameter and volume. The volume

2.4 Preparation of the Donor Layer System can be calculated from the diameter by consideration
of the contact angle. The contact angle of the applied
For all presented printing experiments, 1-mm thick alginate on glass was measured by the sessile drop
glass slides (26×26 mm²) were cleaned with acetone method with contact angle measuring device OCA
and lens cleaning tissue. The slides were coated with a 40Micro (DataPhysics Instruments GmbH, Germany)
60-nm thick gold layer by sputter coating (208 HR, to be 31° ± 4°. The volume of a spherical segment is
Cressington Scientific Instruments Ltd., Watford, V droplet =/3a (sin)3(1cos) . (2+cos) with rad-
3
2
England, UK) with argon. Thickness variation on one ius a of the contact area and contact angle . With the
slide is low due to a planetary gear turning the glass contact angle of 31° ± 4°, the volume of the alginate
slides while they are coated. The thickness of the droplets on the glass surface is V droplet = (0.45±0.07)a .
3
coated layers was controlled with a thickness control-
ler (MTM-20, Cressington Scientific Instruments Ltd., 2.7 Characterization of Laser Pulses
Watford, England, UK). All experiments were con- The temporal pulse shapes were measured with a
ducted with several donor slides with the same layer photodiode (DET210, ThorlabsGmbh, Dachau, Ger-
system to avoid that results are affected by one donor many) with a rise time of one nanosecond and an os-
slide with potentially different layer thickness. cilloscope (WaveRunner 62Xi, Teledyne LeCroy
Onto this gold layer, a hydrogel layer, usually with GmbH, Heidelberg, Germany). 1000 pulses were ave-
embedded cells, was dispersed by blade coating. Here, raged for each measurement. The laser pulse energy
a blend of 1 part 4 wt% alginate (Sigma-Aldrich), was determined by measuring the laser power with a
dissolved in a 0.15 M NaCl solution and sterilized by laser power meter (Powermax PM10 + Fieldmax II
filtration with a 0.8-μm pore size filter, and 1 part TOP, Coherent Europe BV, Utrecht, The Netherlands)
EDTA blood plasma was applied. Cells were tryp- and divided by the laser pulse repetition rate. Spatial
sinized, resuspended in a certain volume of cell me- pulse shapes were recorded with a beam profiler
dium, and counted with a hemocytometer. They were (Beamstar FX, OphirSpiricon Europe GmbH, Darm-
centrifuged at 500g for 5 min and the supernatant stadt, Germany).
was removed. The pellet, containing 1.5 million cells, 2.8 Visualization of the Jetting and Measurement
was suspended in 45 μL of the alginate EDTA blood of Jet Velocity
plasma blend. This hydrogel suspension was pipetted
onto the gold-coated glass slide and dispersed on the The printing process and the material transfer by for-
gold surface with a blade coater to form a homogene- mation of a hydrogel jet for some hundred microsec-
ous layer of approximately 65-μm thickness. onds was visualized and surveyed with an microscopic
setup developed in-house with a digital SLR camera

International Journal of Bioprinting (2017)–Volume 3, Issue 1 45