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. 2022 Aug 29;23(17):9823.
doi: 10.3390/ijms23179823.

Theoretical and Experimental Assay of Shock Experienced by Yeast Cells during Laser Bioprinting

Erika V Grosfeld  1   2 , Vyacheslav S Zhigarkov  3 , Alexander I Alexandrov  1 , Nikita V Minaev  3 , Vladimir I Yusupov  3
Affiliations

Theoretical and Experimental Assay of Shock Experienced by Yeast Cells during Laser Bioprinting

Erika V Grosfeld et al. Int J Mol Sci. .

Abstract

Laser-induced forward transfer (LIFT) is a useful technique for bioprinting using gel-embedded cells. However, little is known about the stresses experienced by cells during LIFT. This paper theoretically and experimentally explores the levels of laser pulse irradiation and pulsed heating experienced by yeast cells during LIFT. It has been found that only 5% of the cells in the gel layer adjacent to the absorbing Ti film should be significantly heated for fractions of microseconds, which was confirmed by the fact that a corresponding population of cells died during LIFT. This was accompanied by the near-complete dimming of intracellular green fluorescent protein, also observed in response to heat shock. It is shown that microorganisms in the gel layer experience laser irradiation with an energy density of ~0.1-6 J/cm2. This level of irradiation had no effect on yeast on its own. We conclude that in a wide range of laser fluences, bioprinting kills only a minority of the cell population. Importantly, we detected a previously unobserved change in membrane permeability in viable cells. Our data provide a wider perspective on the effects of LIFT-based bioprinting on living organisms and might provide new uses for the procedure based on its effects on cell permeability.

Keywords: cell death; laser bioprinting; laser-induced forward transfer (LIFT); membrane perturbation; yeast.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic of the bioprinting setup. (a)—Sketch of experimental setup for laser bioprinting. 1—pulsed laser (1064 nm, 8 ns), 2—beam shaper, 3—galvanoscanner, 4—objective, 5—donor substrate with gel layer, 6—bioplate, 7—high-speed camera. (b)—schematic representation of a part of a donor plate with a formed jet and a separated microdroplet of gel with yeast cells. The shape of the laser pulse and the distribution of the laser intensity on the surface of the absorbing Ti film are shown.
Figure 2
Figure 2
Dependence of gel jet velocity on laser pulse energy. The inset shows an example frame from a high-speed video of a gel jet formed 100 μs after laser pulse absorption.
Figure 3
Figure 3
Effect of laser pulses on an absorbing Ti film of a donor substrate. (a)—Dependence of the square of the hole diameter D in the Ti film of the donor slide on the laser fluence in the absence of a gel layer on the surface of the Ti film. The inserts show the corresponding optical images of the holes. Linear trend shown. (b)—SEM image of a hole in the Ti film with melted edges when the gel layer was previously deposited on the surface of the Ti film. The yellow dotted line shows the area of the laser spot. The effective diameter of the formed hole D is shown. (F = 1 J/cm2). (c)—Green line shows the distribution of the maximum temperature in the Ti film under laser exposure in the presence of melting and vaporization of Ti. The dashed red Gaussian curve corresponds to the temperature distribution in the absence of phase transitions. The dotted red curve in the inner part of the hole corresponds to the temperature distribution in this area if vaporization of Ti does not occur. This curve is shifted down relative to the dashed curve by ΔT due to energy expended during melting. Melting (Tm), boiling (Tb), and critical (Tc) temperatures of Ti are shown.
Figure 4
Figure 4
Main stages of gel jet formation during laser pulsed heating of the Ti film of the donor slide after an 8 ns laser pulse. By the end of the pulse with τ = 8 ns, a thin (total ~135 nm) layer of glass and gel is heated simultaneously with the Ti film up to the temperature ≥ Tb/2 ≈ 1650 °C. By t = 300 ns, a gas-vapor bubble is formed near the Ti film and begins to expand. Ti nanoparticles are visible in the region of the laser spot, inside the bubble, and in the thin gel layer on the surface of the bubble. A shock wave leaves the area of laser impact (not to scale). By t = 10 µs, a gel jet begins to form. The inset shows the temperature jump ΔT distribution in the gel in the paraxial region at different times. The distance from the bottom wall of the bubble is indicated. By t = 20 µs, the jet elongates, and a counter jet is formed. The heated liquid layer is mainly concentrated on the surface adjacent to the bubble (the area is highlighted by the red dotted curve). The blue dotted curve 1 marks the region of the jet from which a microdrop is subsequently formed, which transfers to the acceptor surface.
Figure 5
Figure 5
Dependences of the transmitted laser energy (a) and transmittance of donor slide (b) in the cases of a pure Ti film without gel layer (Ti) and a gel layer on a Ti film (Ti + gel).
Figure 6
Figure 6
Distributions of laser intensity and fluence. (a)—Distribution of the laser intensity in the focusing region in the absence of a donor plate. (b)—Distribution of laser fluence during laser pulse action on a donor plate with Ti film and gel layer. For distributions in glass (above the Ti film) and in the gel layer their color palettes are shown.
Figure 7
Figure 7
Emergence of weak cytosolic PI-staining after LIFT. (a) Flow cytometric analysis of PI-fluorescence (b) Fluorescent and DIC microscopy of cells.
Figure 8
Figure 8
LIFT causes weak PI staining in most cells while resulting in minor, yet significant amounts of cell death not associated with strong PI staining. Laser irradiation alone does not have these effects. (a)—Share of cells with weak PI staining in a population of LIFTed cells vs. the laser pulse intensity (b)—Share of cells with strong PI staining in a population of LIFTed cells vs. the laser pulse intensity (c)—Dependence of % of dead cells in a population of LIFTed cells (see Materials and methods) vs. the laser intensity (p ≤ 0.1 (*), p ≤ 0.05 (**), p ≤ 0.01 (***)), Student’s t-test) (d) Dependence of % of dead cells in direct laser irradiation vs. the laser intensity.
Figure 9
Figure 9
LIFT of Ssa1-GFP cells shows no rise of GFP signal but GFP dimming in a small population of cells, which is also observed in a larger fraction of cells during short heat shock at 70 °C. (a) Relation between level of median GFP fluorescence and the laser pulse intensity (in PI-negative cells with noticeable GFP fluorescence) (b) Relation between share of cells with diminished GFP signal and the laser pulse intensity (p ≤ 0.05 (**), p ≤ 0.01 (***), Student’s t-test) (c,d) Cytometric analysis of PI (c) and GFP (d) fluorescence of yeast cells producing Ssa1-GFP after 1 min treatment with the indicated temperature.
Figure 10
Figure 10
Schematic of the experimental setup. Created with BioRender.com.
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Grants and funding

This work was supported by the Russian Science Foundation (Grant No. 20-14-00286) in part of improving the technology of microbial systems engineering, and partly supported by the Ministry of Science and Higher Education (within the State assignment FSRC «Crystallography and Photonics» RAS in part of «development of laser technologies») as well in the base funding of the work of AIA (within the State assignment of the FRC of Biotechnology of the RAS).

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