Laser Bioprinting of Cells Using UV and Visible Wavelengths: A Comparative DNA Damage Study

Abstract

Laser-based techniques for printing cells onto different substrates with high precision and resolution present unique opportunities for contributing to a wide range of biomedical applications, including tissue engineering. In this study, laser-induced forward transfer (LIFT) printing was employed to rapidly and accurately deposit patterns of cancer cells in a non-contact manner, using two different wavelengths, 532 and 355 nm. To evaluate the effect of LIFT on the printed cells, their growth and DNA damage profiles were assessed and evaluated quantitatively over several days. The damaging effect of LIFT-printing was thoroughly investigated, for the first time at a single cell level, by counting individual double strand breaks (DSB). Overall, we found that LIFT was able to safely print patterns of breast cancer cells with high viability with little or no heat or shear damage to the cells, as indicated by unperturbed growth and negligible gross DNA damage.

Keywords: DNA damage; double strand breaks; laser fluence; laser-induced forward transfer (LIFT); wavelength.

Figure 1

Schematic of LIFT setup.

Figure 2

Visualization of the jet dynamics induced by laser pulses with different wavelengths (a) at 532 nm and 0.6 ns pulse duration, and (b) at 355 nm and 10 ns pulse duration at different laser fluencies at the indicated timeframes. Representative images from each condition are depicted. All experiments were conducted in triplicates, n = 5 Scale bar 100 μm.

Figure 3

(a) LIFT-printed droplet diameter as a function of laser fluence for both wavelengths. (b) Droplet volume as a function of laser fluence for both wavelengths. Error bars depict SEM. n ≥ 5 droplets per condition, in at least three independent experiments.

Figure 4

The dependence of the cell suspension jet front position vs. time. (a) at 532 nm and (b) at 355 nm for various laser fluences.

Figure 5

The dependence of produced velocities as a function of laser fluence for both wavelengths. (a) at 532 nm and (b) at 355 nm for various laser fluences.

Figure 6

Number of LIFT-printed MDA-MB-468 cells per droplet (a) at 532 nm and (b) at 355 nm for the indicated laser fluencies. The experiments were conducted at least in triplicates and the number of counted droplets was a minimum of 5 droplets per condition. Error bars indicate SEM.

Figure 7

Bright-field (BF) optical microscopy images of LIFT-printed MDA-MB-468-H2B-GFP at 0 h, 4- and 6-days post-printing, at 360 ((a1–c1) at 532 nm) and 330 mJ/cm² ((a2–c2) at 355 nm), respectively. Scale bar 200 μm.

Figure 8

Presence of γH2AX foci on LIFT-printed breast cancer cells at 532 nm (MDA-MB-468 on the left and MDA-MB-231 on the right). (Top) Representative IF images of γH2AX foci on control or printed cells at the indicated timepoints. Cell nuclei were counterstained with DAPI, scale bar = 20 μm. (Bottom) The percentage of high- and low-DSB-bearing populations at control or given time points after LIFT-printing, fpn-foci per nucleus. The measurements obtained from at least 3 independent experiments and the total cells analyzed were: for MDA-MB-468 control = 1221, 0 h = 1705, 6 h = 1584, 24 h = 1456 and for MDA-MB-231 control = 1609, 0 h = 1896, 6 h = 1769, 24 h = 1746.

Figure 9

Live-dead assay on printed MDA-MB-468 cells. Representative images of printed cells at 0 h (left) and 24 h (right). All cells were counterstained with DAPI (blue), while dead cells were also positive for PI (red). The insets on top right are enlargements of the squares in the center of each image with shifted overlay of the red channel (~10 μm) to the right to visualize the DAPI-stained nuclei underneath. Scale bars: 100 μm.

Figure 10

(Top) Representative IF images of γH2AX foci of control or printed MDA-MB-468 cells at 355 nm. Cell nuclei were counterstained with DAPI, scale bar = 20 μm. (Bottom) The percentage of high- and low-DSB-bearing populations at control or given timepoints after LIFT-printing of MDA-MB-468 at 355 nm, fpn-foci per nucleus. The measurements were obtained from at least 3 independent experiments and the total cells analyzed were: control = 1221, 0 h = 521, 6 h = 1177, 24 h = 1084.

Figure 11

The distribution of high (>5)-DSB-bearing MDA-MB-468 cells in terms of γH2AX foci per nucleus.