# Laser-induced forward transfer in picosecond regime for cell bioprinting

Lucas Duvert, Claris Murru, Ahme Al-Kattan, Anne-Patric Alloncle, Frederique Magdinier, Stefano Testa, Adrien Casanova

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Lucas Duvert, Claris Murru, Ahme Al-Kattan, Anne-Patric Alloncle, Frederique Magdinier, et al. Laser-induced forward transfer in picosecond regime for cell bioprinting. International Journal of Bioprinting, 2025, 10.36922/ijb.7788. hal-05016640

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RESEARCH ARTICLE

# Laser-induced forward transfer in picosecond regime for cell bioprinting

Lucas Duvert<fnref n="1" />,<fnref n="2" />✉, Clarissa Murru<fnref n="1" />✉, Ahmed Al-Kattan<fnref n="1" />✉, Anne-Patricia Alloncle<fnref n="1" />✉, Frederique Magdinier<fnref n="2" />✉, Stefano Testa<fnref n="2" />*✉, and Adrien Casanova<fnref n="1" />*✉

## Abstract

Based on interdisciplinary approaches, bioprinting methods aim to create and design highly organized 2D and 3D cultures. In this context, it has been more than a decade since laser-induced forward transfer (LIFT) was studied on a lab scale for its ability to transfer biomaterials, specifically bioink loaded with living cells, onto a substrate. Extreme physical and mechanical phenomena contribute to the jetting dynamic of the targeted bioink, raising a spontaneous biological question: does this process negatively affect the survival rate of transferred cells? This study demonstrates that laser pulse durations in the range of picoseconds to nanoseconds do not directly affect cell viability, indicating that LIFT is a valuable bioprinting method for transferring living cells. Moreover, we highlight the necessity of using hydrogel coatings on the surface of the receiver substrate to guarantee optimal post-printing viability of the cells. We demonstrate that the nature of the hydrogel also contributes to the resolution of the printed pattern. Among the tested materials, Matrigel demonstrated all the qualities required to ensure successful printing and should therefore be considered for future work. Overall, the results show the suitability of our LIFT setup for printing living cells in the picosecond regime with a high survival rate, paving the way for a wide range of biological applications.

**Keywords:** Cell viability; Laser-assisted bioprinting; LIFT bioprinting; Living cells printing; Regenerative medicine; Skeletal muscle; Tissue engineering

## 1. Introduction

Bioprinting techniques began to develop in the 2000s and have since proven to be an ideal tool for tissue engineering,<fnref n="1" />⁻⁶ representing a novel strategy to create complex biological constructs inspired by organs and tissues. Thanks to a multidisciplinary approach, bioprinting allows the accurate arrangement of cells in space, providing a well-structured 3D environment fundamental for cell survival, organization, and differentiation. Nowadays, 3D bioprinting can be divided into extrusion, inkjet, and laser-based bioprinting. In extrusion-based techniques, the bioink (the mixture of cells and biomaterials⁷) is deposited as a continuous filament by mechanical, pneumatic, or solenoid-based dispensing systems. On the other hand, inkjet systems make use of

**\*Corresponding authors:**

Stefano Testa
(stefano.TESTA@univ-amu.fr)

Adrien Casanova
(adrien.casanova@univ-amu.fr)

**Citation:** Duvert L, Murru C, Al-Kattan A, et al. Laser-induced forward transfer in picosecond regime for cell bioprinting.

*Int J Bioprint.*
doi: 10.36922/ijb.7788

Received: December 17, 2024

1st revised: January 14, 2025

2nd revised: January 23, 2025

Accepted: January 24, 2025

Published Online: January 24, 2025

**Copyright:** © 2025 Author(s).

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, permitting distribution, and reproduction in any medium, provided the original work is properly cited.

**Publisher's Note:** AccScience Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<footnote n="1">¹Laser, Plasmas et Procédés Photoniques (LP3), Aix-Marseille University, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, Marseille, France</footnote>

<footnote n="2">²Marseille Medical genetics (MMG), Aix-Marseille University, Institut national de la santé et de la recherche médicale (INSERM), Marseille, France</footnote>

<footnote>*(This article belongs to the *Special Issue: Intelligent 3D Bioprinting Strategies for Future Regenerative Medicine*)</footnote>
thermal, acoustic, or electrical stimulation to deposit small droplets of bioink. The laser-based bioprinting techniques exploit the laser-matter interaction to create organized biological systems.⁸ Among them, laser-induced forward transfer (LIFT), a printing technique first developed for electronic purposes,⁹⁻¹³ was recently applied for transferring biomolecules¹⁴⁻¹⁶ or even living cells.¹⁷⁻²⁶ This technique uses a short laser pulse to transfer tiny amounts of material from a thin donor film to a receptor substrate. Under appropriate conditions, the laser pulse induces the formation of a highly directive jet that propagates perpendicularly to the donor substrate. The bioink, previously spread as a thin film (a few tens of microns thick) on the donor substrate, is subsequently deposited as droplets onto the collector slide.

The donor substrate is composed of a thin glass slide with the potential addition of a metallic dynamic release layer (DRL), which the laser pulse will vaporize to induce the creation of the jetting.²⁷⁻²⁹ The DRL is employed when the ink is susceptible to damage from direct laser interaction, such as bioinks containing living cells or biological material, or when the ink is transparent to the wavelength of the laser; adding a thin metal layer allows controlled deposition of laser energy and promotes laser-matter interaction. The cells in direct proximity to the vapor bubble created by the vaporization of the DRL will be inevitably sacrificed due to the sudden elevation of temperature and immediate mechanical stress.¹⁷,³⁰ Nevertheless, as previously simulated,³¹ the use of an ultrashort pulse laser (with a duration of nanoseconds or less) in combination with a DRL makes any further thermal transfer to the bulk of the ink negligible, thus preventing cells from potential thermal damages. Considering these observations and the high-cell concentration within the targeted bioinks, the thermal effect and vaporization of the DRL are not critical parameters influencing post-printing cell survival. In this context, we extended the range of studied laser pulse duration typically found in the literature (nanosecond regime)³² to investigate the impact on “pre-transfer” cell survival over several orders of magnitude of pulse width, from nanoseconds to picoseconds, using the same optical setup and experimental protocols.

Despite these observations, it is essential to note that pulse duration is not only associated with thermal diffusion properties but also with jet ejection speed and shockwave formation.³³ The creation of the jet induced by the interaction of the short laser pulse with the DRL also causes high mechanical stress on the cells contained in the bioink. The jet acceleration reaches several meters per second in a few microseconds²⁸,³⁴ after the laser pulse interaction, which can potentially damage cell

membranes and reduce the overall cell survival rate. Adding to this initial shear stress, the propagation of the jet and its landing on the receiver substrate further increases the eventual damage to the cells. The necessity of a polymer layer to mitigate the impact on cells remains a topic of debate, with some studies supporting its use,³⁵⁻³⁸ while others suggest it may not be required.²,³⁹ Moreover, the nature and thickness of the receiver coating are also subject to variation and can lead to a wide range of observable cell survival rates.

This work aims to identify the key factors affecting cell survival during LIFT bioprinting. As mentioned before, the printing process can be divided into two distinct steps: ejection of the targeted material from the donor slide and landing of the created jet on the receiver substrate. Both events are accompanied by high mechanical stress that might heavily affect cell survival rates. Most studies in the literature are focused on the first step, investigating the effects of different laser wavelengths (UV or visible),⁴⁰ different bioink formulations,⁴¹ and energy levels on the jet formation and propagation, often reporting high cellular viability values.²⁵

In this study, we separately evaluated the respective contribution and significance of the ejection and landing processes to define optimized and successful cell transfer. We first studied the impact of picosecond and nanosecond laser pulses at different energy levels on the direct survival of the cells. The landing's mechanical strain was evaluated by studying the effects, benefits, and challenges of adding or omitting a thin protective layer to the receiver substrate. These results are finally used to print living cells with an extremely high survival rate, a high spatial resolution, and good reproducibility.

## 2. Material and methods

### 2.1. Cell culture and bioink preparation

Murine myoblasts C2C12 (ATCC, USA) were cultured following previously published protocols⁴² in T-75 Flasks (Sarstedt, Germany) at 37 °C with 5% CO₂ in DMEM GlutaMAX (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, EuroClone, Italy), penicillin (100 IU/mL, Gibco, USA), and streptomycin (100 mg/mL, Gibco, USA).

For bioink formulation, cells were harvested using a trypsin/EDTA 1x solution (Gibco, USA) and centrifuged in phosphate-buffered saline (PBS, Gibco, USA) at 115 × g for 10 min. PBS was carefully removed and replaced by an adequate volume of a 2% gelatin (Sigma-Aldrich, Germany) solution in DMEM (final concentration 5 × 10⁷ cells/mL).
## 2.2. LIFT printing setup

The bioink (5 µL) was spread on a titanium wafer (LAAS France) used as a donor substrate. This titanium layer acted as DRL and was chosen for its biologically inert behavior.⁴³ Several layers of polyimide tape were applied onto the titanium coating before being laser-cut to create individual wells on the substrate. The well was about 50 mm² for 80 µm of height (Figure 1B). The bioink was spread homogeneously in the confined area to guarantee a uniform layer of 80 µm. This method was inspired by Rapp et al.¹³

A motorized stage (X, Y, Z) controlled by a Newport Motion controller (Newport Corporation, USA) allows the translation of the donor substrate along the vertical direction (Z) of the incident laser beam and the perpendicular directions (X and Y). The receiver substrate is also motorized along the X and Y axes, allowing precise

and independent positioning of the samples. After the deposition of the bioink, the donor substrate is positioned with the ink facing downward (Figure 1A) and secured using a suction system. It is then lowered toward the receiver, with the gap between the two substrates precisely controlled using a camera.

The following two laser sources were used for these experiments: (i) a Light Conversion Carbide CB-1 Yb: KGW laser (Light Conversion, Lithuania) with a fundamental wavelength of 1030 nm (IR), a repetition rate of 60–1000 kHz, and a pulse duration of 10 ps; (ii) a Quantel Brio Nd: YAG laser (Lumibird, France) with a wavelength of 1064 nm (IR), a repetition rate of 20 Hz, and a pulse duration of 4 ns. Both lasers were set up on the same optical path with a removable mirror, allowing the switch between the two sources. A polarizer and a half-wave plate were positioned to reduce and tune the energy of the laser

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Scheme of the LIFT bioprinting process. (A) LIFT optical setup for cell-printing. (B) Schematic of titanium donor substrate preparation with individual wells. The bioink containing the cells is deposited inside the well, allowing the control of the layer thickness. (C) Experimental protocol for isolating laser-induced damages and evaluating post-transfer cell survival rates. Abbreviations: DRL, dynamic release layer; LIFT, laser-induced forward transfer. Created in BioRender. Magdinier, F. (2025) https://BioRender.com/a90p035</figcaption></figure>
pulses. An optical scan system (intelliSCAN 14, Scanlab, Germany) coupled with a 70 mm f-theta lens allowed us to direct and control the tuned laser beams on an area of 25 cm² to scan the entire donor substrate. The scanning system is motorized in the Z-axis direction allowing us to account for the donor-receiver gap, while maintaining accurate focus on the donor substrate.

Pulse energies for each laser were calibrated using an energy meter, with four increasing energy values (9-23-42-70 µJ) tested for both laser sources. The spot size and determination of the average fluence per pulse were based on the ablation size of the titanium layer on the donor substrate after the printing. The target surface was illuminated from above (through the glass of the wafer) with a 450-point matrix of single pulses. The large number of points allowed us to transfer enough bioink (and consequently cells) for statistical relevance. The illuminated areas were separated by 0.25 mm, guaranteeing the independence of the jetting mechanism and an intact titanium layer for each shot.

## 2.3. Cell survival: impact of laser pulses

In order to measure only the specific effect of the laser on cell survival, we used a homemade receiver substrate designed to isolate this factor. This substrate consisted of a 3D-printed well made of polylactic acid (PLA, Bambu Lab, China), with a height of 4 mm, attached to a 0.17-mm-thick glass slide, and filled with DMEM. The gap between the surface of the well and the donor slide was adjusted depending on the experiment, between 0.2 and 2 mm, and monitored through a Charge coupled device (CCD) camera. The important thickness of the culture medium (4 mm) was intended to minimize the mechanical impact with the receiver glass slide during cell deposition, allowing us to isolate and evaluate the sole effect of laser pulses on cell viability. After the transfer, particular attention was given to the presence of cells attached to the glass slide, indicating that the jetting reached the hard surface. In such cases, the gap was adjusted, and the experiment was repeated.

Immediately after printing, the well content (printed cells and medium) was retrieved and stained with 0.5% trypan blue before manual counting to determine cell viability (Figure 1C). Experiments were done in triplicates for each tested energy and both pulse durations (Carbide 10ps and Brio 4ns).

## 2.4. Cell survival: impact of the landing

A second set of experiments aimed at determining the effects of the jet's landing on cell survival. A glass slide with a gap of 0.5 mm was first used as a receiver, and the same matrix previously described was used as a printing pattern. The same process was repeated with thin layers

of Matrigel (Corning, USA) or 1.5% w/w sodium alginate (ThermoFisher Scientific, USA) in PBS as coatings on the receiver substrate before the printing (50 µL for 19 mm diameter glass slide). The gap between the donor and the surface of the coating was maintained at 0.5 mm.

For all conditions, glass slides were placed immediately after printing in a sterile culture dish, and 150 µL of LIVE-DEAD assay (ThermoFisher) supplemented with Hoechst solution (ThermoFisher) was added. This assay allows the simultaneous display of total cells (nuclear blue signal, Hoechst), live cells (green cytoplasmatic signal, calcein-AM), and dead cells (red nuclear signal, iodure BOBO-3). After 30 min of incubation at 37 °C, samples were observed by fluorescence microscopy (Evos M5000, Invitrogen, ThermoFisher, America), followed by culture medium addition and storage of the samples in an incubator for long-term analysis.

## 2.5. Colocalization analysis

To quantify the effect of the cell impact with the receiver substrate on cell survival, in the presence or absence of a coating layer, live/dead images were analyzed using the Fiji software⁴⁴ (https://fiji.sc;10.1038/nmeth.2019), using the “Colocalization Threshold” plugin. The “thresholded Mander’s split colocalization coefficients” were considered to quantify the ratio of dead cells (iodure BOBO-3, nuclei of dead cells) to the total cells (Hoechst signal, total nuclei), a coefficient ranging from zero (no colocalization) to one (perfect colocalization). The result can be considered as an index of cell mortality where “one” represents 100% of dead cells and “zero” 100% of live cells.

## 2.6. Statistical analysis

All experiments were performed in biological triplicates. Data were analyzed using GraphPad Prism 8, and values were expressed as means ± standard error. Statistical significance was tested using a one-way ANOVA test with the appropriate multiple comparison tests. A probability of less than 5% (*p* < 0.05) was considered as statistically significant.

# 3. Results

## 3.1. Impact of laser energy on cell survival

To study the effect of the direct laser impact on cell viability, four increasing energy values (9-23-42-70 µJ) were chosen for both tested lasers (with picosecond or nanosecond pulse durations). The energy range was chosen to widely cover usable energy for LIFT bioprinting and for the creation of the jetting mechanism. The experiment was first performed with a 10 ps pulse duration. To guarantee a sufficient number of transferred cells (minimum 1000 cells per replicate) and given the reduced size of the jets at
low energy, the distance between the donor and the top of the receiver well was adjusted to its minimum of 0.2 mm. For this experimental set, trypan blue staining showed 93-95% cell survival for all energy values considered, and no statistically significant differences were found compared to the control (survival rate of 96%) (Figure 2A).

Next, the optical setup was adjusted as previously described to accommodate nanosecond pulses. A longer pulse duration of 4 ns drastically changes the printing mechanism with a wider and more explosive jetting dynamic. For this reason, the gap was adjusted to 2 mm to avoid direct contact between the donor and the receiver as well as avoiding cell redeposition on the donor substrate. The staining showed a cell survival rate of about 90% with statistically significant differences compared to the control (Figure 2A). For this experimental set, we

observed a decrease in the cell survival rate that correlates with the increase in laser pulse energy. Even if statistically significant, this impact is limited, with a survival rate of about 89% for the highest energy considered, compared to 96% for the control.

The measurement of the associated fluences (energy per laser spot area) showed that the spatial repartition of the energy on the donor slide further influences cell survival (Figure 2B). The significant reduction in cell viability observed for the energies above 23 µJ, in the case of nanosecond pulses, can be explained by the important increase in laser fluence. At the maximum laser energy considered (70 µJ), the laser fluence is almost three times more important for the nanosecond pulses (Figure 2B). This point corresponds to the lowest observed cell viability of 89%.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Impact of laser on cell survival. (A) Post-printing statistical analysis of cell survival determined after Trypan Blue staining. For each pulse duration considered, the X-axis displays the laser pulse energy tested and the Y-axis, the percentage of viable (dark grey) or dead (light grey) cells. (B) The previously described cell survival rates (Y-axis) are displayed in terms of energy per surface area on the X-axis (fluence).</figcaption></figure>
**3.2. Impact of the landing on cell survival**

Given the previously described results, only the 10 ps pulse
duration was chosen for the subsequent experiments,
which focused on evaluating the impact of the receiving
surface during cell landing on survival (Figure 3). The same
range of energies was used. Printing on a dry receiver (glass
substrate) showed an extremely poor survival rate (3%) at
the lowest energy considered. Nevertheless, the survival
rate increased drastically for 23 µJ (around 71%) before
decreasing back to 51% for 70 µJ (Figure 3B). Interestingly,

the presence of a coating layer on the receiver for both
alginate and Matrigel attenuated the negative impact on
cell viability with a high rate of cell survival for each energy
tested and no statistically significant difference compared
to the control (Figure 3B).

For all the conditions tested, the fluorescent markers
(Live/Dead and Hoechst) were added directly after
printing to prevent drying of the cell suspension that
might negatively impact the viability (especially without
coating), and the samples were incubated for 30 min. The

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Cell survival in the presence of coating. (A) Representative immunofluorescence images of live/dead cell staining for each experimental condition (live cells in green, dead cells in red, nuclei in blue). (B) The graph shows the nuclear colocalization coefficient of dead cell nuclei over the total number of cell nuclei for each experimental condition. Created in BioRender. Magdinier, F. (2025) https://BioRender.com/l57q296. (C) Schematic representation of the effect of impact on the receiver surface during cell landing.</figcaption></figure>
immediate addition of a solution (fluorescent markers or culture medium) onto the receiving substrate caused a loss of printing resolution on uncoated or alginate-coated samples (Figure 3A) as the cells did not have sufficient time to adhere to the substrate. Furthermore, alginate is soluble in aqueous solutions, and attempts to add a calcium chloride solution immediately after printing to promote its crosslinking resulted in the formation of a floating hydrogel layer, as it could not adhere to the glass. In this context, Matrigel showed better adhesion to the glass substrate, and its fast gelation preserved the printing resolution even before the adhesion of the cells to the substrate (Figure 3A).

### 3.3. LIFT bioprinting resolution

Once conditions for optimal cell survival were successfully set up and characterized, picosecond pulses, and a Matrigel-coated receiver (50 µL on a 19-mm-diameter glass slide) were chosen to design the precise deposition of C2C12 cells in matrix (Figure 4A, top) and line patterns (Figure 4A, bottom). The laser energy used was tuned around the previously determined value of 23 µJ. Indeed, the amount of energy per pulse required for the creation of reproducible and stable jets is extremely sensitive to cell concentration, which modifies the rheological properties of the bioink, as well as to minor variations in the ink composition. To ensure well-defined and highly

concentrated cell deposition, empirical testing must be performed beforehand for each experimental session to slightly adjust the energy value. Cell survival just after printing was characterized by Live/Dead cell staining to confirm the safety of this printing system (Figure 4B). Cell adhesion and elongation were noticeable 24 h after printing (Figure 4C).

A thicker layer of Matrigel on the receiver substrates (70 µL for 19-mm-diameter glass slide) directly affected the proliferation of the printed cells. The cells deposited in a matrix pattern showed very different morphological arrangements, with clear signs of adhesion 1 day after printing for some of the matrix spots (Figure 5A, left, red circle) and a spheroidal organization for others (Figure 5A, left, blue circles). Live/Dead assay showed a very high survival rate for both configurations (adherent and spheroidal spots). Three days after printing, the printed cells that aggregated in spheroids reached the glass surface. Cell elongation was visible at the periphery of some spheroids with varying rates depending on the area considered (Figure 5A, right).

Interestingly, we could observe that some spheroids moved and merged due to the remodeling of the surface coating combined with cell migration. On the other hand, cells that reached the glass surface during the transfer

<figure><img src="image_5.png" /><figcaption>**Figure 4.** High resolution printing of living cells by LIFT bioprinting. (A) C2C12 cells printed by LIFT on a Matrigel coating in matrix patterns (top, scale bar = 200 µm) and line patterns (bottom, scale bar = 200 µm). (B) Post-printing Live (green)/Dead (red) cell assay showing excellent cell survival in both matrix (top, scale bar = 250 µm) and line (bottom, scale bar = 200 µm) deposition patterns. (C) C2C12 cells observed 24 h after the printing in matrices (top, scale bar = 200 µm) and lines (bottom, scale bar = 250 µm), showing elongated adherent cells. Abbreviation: LIFT, laser-induced forward transfer.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 5.** Variability in cell deposition during LIFT bioprinting. (A) C2C12 cells deposited by LIFT on Matrigel coatings 24 and 72 h after printing. Cells deposited close to the glass surface, leading to their fast adhesion and expansion, are circled in red. Spheroids and their apparent motility are highlighted in blue circles. Live cells still rounded are stained in green. Created in BioRender. Magdinier, F. (2025) https://BioRender.com/n99y226. (B) Schematic representation of cell deposition in three dimensions directly within the bulk of Matrigel by LIFT. The stiffening of Matrigel over time leads to jets depositing cells closer to the surface of the gel. The same effect could be obtained with a stable gel stiffness and a precisely controlled jet length. Abbreviation: LIFT, laser-induced forward transfer.</figcaption></figure>

exhibited cell adhesion and elongation precisely at their printing location (Figure 5A, right).

The distribution of the two configurations did not follow any explicit pattern on the receiver substrate, being randomly located on the coating of the receiver surface. We concluded that these differences in cell behavior were thus directly linked to the variability of the jetting mechanism, the local properties of the Matrigel, and its progressive stiffening over time. Longer jets directed the cells to the bottom of the coating layer, allowing them to adhere to the substrate, while shorter jets left the cells suspended in the middle of the Matrigel layer, generating spheroids (Figure 5B). In both cases, the quantity of gel allowed

the gentle deposition of cells onto the receiver substrate, ensuring their survival.

## 4. Discussion

Currently, the majority of studies investigating LIFT suitability for bioprinting living cells focus on the study and characterization of parameters that could affect cell viability during the initial phase of the process,41,45,46 with fewer studies addressing the implication of the landing phase.37,47 Based on our results, the latter constitutes a critical parameter that should be taken into account when defining an experimental LIFT bioprinting setup, in order to replicate results published in the field. For this reason,
we decided to study LIFT bioprinting as a whole, focusing on both the ejection and landing phases, to establish experimentally validated procedures that ensure high cell viability.

To deeply investigate the effect of LIFT on living cells, we conducted a systematic study to characterize the specific implication of the two main phenomena occurring during the printing process, the interaction of the laser with living matter (ejection phase) and the subsequent mechanical transfer of cells from a donor to a receiver substrate (landing phase). In this regard, a bioink composed of gelatin was used to facilitate the transfer of cells,⁴⁸,⁴⁹ while guaranteeing good compatibility with muscle cell culture.⁵⁰ The mechanical effect of the laser pulse on cells was first evaluated through bioprinting on a liquid container. Using a receiver specially designed to protect cells from potential landing damage, a high survival rate (about 90%) was observed across all tested energy conditions, and both laser pulse durations (10 ps and 4 ns). It is important to highlight that some of the energy values considered in this study are significantly higher than those typically reported in the literature for bioprinting applications,³⁵,⁵¹,⁵² further emphasizing the safety of the LIFT printing strategy. The only significant decrease in survival rate was noted for the highest energy considered with nanosecond pulses. This decrease can be directly linked to the significant increase in laser fluence associated with nanosecond pulses, combined with the characteristics of the jetting mechanism in the nanoseconds regime.⁴¹

Vogel et al. investigated the shockwave generation from pulsed lasers focused on water (an infinite medium) for 30 ps and 6 ns pulse durations.³³ Beyond a certain threshold, medium breakdown occurs, causing the laser pulse energy to be deposited in the medium, which creates a gas bubble (plasma) initially at high pressure and temperature, accompanied by a shockwave. This process can be compared to the mechanisms induced during the LIFT process. Vogel et al. demonstrated that, for equivalent laser pulse energy, longer pulse durations yielded significantly higher shockwave amplitude, higher initial bubble pressure, faster initial bubble wall velocities, and greater associated mechanical energy generation. This higher mechanical energy generation in the nanosecond regime is consistent with the lower cell survival rate observed in our study for energies higher than 23 µJ. Guven similar energy per pulse, nanosecond transfer is thus significantly more violent than its picosecond counterpart. Under those conditions, the mechanical impact of the cells on the liquid interface must be considered. The acceleration of the cells in the jet induced by the expansion of the vapor bubble is also a potential cause of cell death. Therefore, the impact of

the cells on the liquid receiver, along with the high ejection speed and associated shear stress, could explain the higher cell death rate observed.

Secondly, the landing of the cells on the receiver substrates was studied and evaluated, with and without a coating layer. The unusual survival trend observed when printing directly on glass could be explained by the explosive nature of the jetting dynamic at high energies. As mentioned in this work, higher pulse energy leads to a wider and more explosive jetting regime; more material is thus transferred from the donor to the receiver substrate in a more extended period. Similarly, to the cells printed at low energy, the first cells to land on the glass substrate will perish due to the mechanical impact. This early deposition of soft matter, however, creates a layer on the receiver that serves as a coating for the remaining cells contained in the jet (Figure 3C). Based on these observations and in the absence of a coating on the receiver substrate, an optimum energy of 23 µJ can be identified, where the mechanical strain created by the jet impact is balanced by the quantity of material transferred and the number of cells contained in the bioink, leading to a cell survival rate of about 71%. This hypothesis and explanation are in line with observations made in experiments conducted in the presence of a coating layer. Indeed, the presence of a coating applied to the substrate before the printing eliminates the previously mentioned mechanical forces and allows a smooth transfer of the cells onto the receiver substrate for all energy values considered. Therefore, a coating is essential for the printing process, allowing a high post-printing cell survival rate (with viability rates comparable to those of the control when using both alginate and Matrigel coatings, Figure 3B), while also preventing the drying out of the printed cells. The coating also plays a key role in maintaining the resolution of the printed droplets without waiting for them to adhere to the substrate, thus significantly speeding up the printing process and improving the overall versatility of the LIFT process. Once again, given the results presented, a 23 µJ pulse seemed to present the best compromise between the number of cells printed, droplet resolution on Matrigel, and cell survival with a 10 ps pulse.

Finally, the results and observations depicted in Figure 5 suggest the possibility of directly depositing cells in 3D within the bulk of the Matrigel. As shown in Figure 5B, the typical progressive stiffening of the Matrigel at room temperature can give rise to a gradual deposition of cells along the Z direction. Extending this concept, by adjusting and maintaining the stiffness of the receiver's polymer layer, controlling the viscosity of the bioink, and fine-tuning the energy deposited on the donor substrate, cells could be precisely deposited not only along the X and Y axes but also
along the Z-axis within the gel layer. This property could
enable the creation of organized 3D structures quickly
and efficiently within the gel's bulk, potentially replicating
tissues and organ architectures through LIFT bioprinting.
However, the motility of cells in suspension, as observed
in Figure 5A, remains an issue that must be addressed to
preserve LIFT's spatial resolution and its ability to deposit
cells at precise locations over time.

**5. Conclusion**

In this study, we demonstrate the conditions under which the LIFT printing process can ensure highly satisfying cell survival rates. The effect of the laser pulse deposition on cell viability was evaluated and we showed that the energy required to induce jetting does not jeopardize cell survival. This conclusion is supported by results across a wide range of laser pulse energies and durations. Mechanical strain and stress are, however, the primary cause of cell death during the bioprinting process. The presence of a coating on the receiver substrate is an ideal solution to achieve high survival rates with LIFT. Ideally, this coating should be carefully chosen to maintain the resolution of the printed droplets. Cross-linkable and polymerizable hydrogels are thus preferred to maximize the potential of this technology.

Nevertheless, we demonstrated that this coating is not
necessarily required, and in a worst-case scenario, moderate
cell survival (~71%) can be reached with an adapted laser
pulse energy and controlled jetting dynamic. These results
further emphasize the versatility of the LIFT process and
its ability to arrange cells across a wide range of substrates,
maintaining a moderate to extremely high survival rate. In
this work, both the ejection and the landing phases of the
bioink transfer are independently studied, highlighting
the key factors affecting cell survival and providing deeper
insights into the LIFT of living cells. The data reported in this
study are valuable for establishing frameworks and protocols
tailored to specific experimental needs, while contributing
to the growing knowledge in this emerging field.

Acknowledgments

Not applicable.

Funding

The work was funded by the French National Research
Agency (ANR Medilibs and Diagem projects) together
with the French Defense Innovation Agency (ANR-
DGA/AID-ICELARE Project ID: ANR-20-ASTR-
0004). The PhD thesis of Lucas Duvert was co-funded by
Aix-Marseille University, the French Defense Innovation
Agency and the MarMaRa funding scheme. This work
received support from the French government under

the France 2030 investment plan, as part of the Initiative
d'Excellence d'Aix-Marseille Université – A*MIDEX
"AMX-23-CPJ-05". The project leading to this publication
has received funding from the Excellence Initiative of Aix-
Marseille University-A*Midex, a French "investissement
d'avenir programme" AMX-19-IET-007, through the
MarMaRa funding scheme. This work was conducted
using LaMP facilities at LP3. The authors also acknowledge
the "Region SUD" for their financial support.

Conflict of interest

The authors declare that they have no competing interests.

Author contributions

Conceptualization: Anne-Patricia Alloncle, Frederique Magdinier, Adrien Casanova, Stefano Testa

Formal analysis: Lucas Duvert, Stefano Testa

Investigation: Lucas Duvert, Stefano Testa, Adrien Casanova, Anne-Patricia Alloncle

Methodology: Lucas Duvert, Stefano Testa, Ahmed Al-Kattan, Anne-Patricia Alloncle, Frederique Magdinier, Adrien Casanova

Writing – original draft: Lucas Duvert, Stefano Testa

Writing – review & editing: Lucas Duvert, Clarissa Murru, Ahmed Al-Kattan, Anne-Patricia Alloncle, Frederique Magdinier, Stefano Testa, Adrien Casanova

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data

Data are available from the corresponding author upon
reasonable request.

References

1. Tarassoli SP, Jessop ZM, Al-Sabah A, et al. Skin tissue engineering using 3D bioprinting: an evolving research field. *J Plast Reconstr Aesthet Surg.* 2018;71(5):615-623.
doi: 10.1016/j.bjps.2017.12.006

2. Guillemot F, Souquet A, Catros S, et al. High-throughput laser printing of cells and biomaterials for tissue engineering. *Acta Biomater.* 2010;6(7):2494-2500.
doi: 10.1016/j.actbio.2009.09.029

3. Zhu W, Ma X, Gou M, Mei D, Zhang K, Chen S. 3D printing of functional biomaterials for tissue engineering. *Curr Opin Biotechnol.* 2016;40:103-112.
doi: 10.1016/j.copbio.2016.03.014
4. Kačarević Ž, Rider P, Alkildani S, et al. An introduction to 3D bioprinting: possibilities, challenges and future aspects. *Materials*. 2018;11(11):2199.
doi: 10.3390/ma11112199

5. Li J, Chen M, Fan X, Zhou H. Recent advances in bioprinting techniques: approaches, applications and future prospects. *J Transl Med*. 2016;14(1):271.
doi: 10.1186/s12967-016-1028-0

6. Vinson BT, Sklare SC, Chrisey DB. Laser-based cell printing techniques for additive biomanufacturing. *Curr Opin Biomed Eng*. 2017;2:14-21.
doi: 10.1016/j.cobme.2017.05.005

7. Raees S, Ullah F, Javed F, et al. Classification, processing, and applications of bioink and 3D bioprinting: a detailed review. *Int J Biol Macromol*. 2023;232:123476.
doi: 10.1016/j.ijbiomac.2023.123476

8. Dey M, Ozbolat IT. 3D bioprinting of cells, tissues and organs. *Sci Rep*. 2020;10(1):14023.
doi: 10.1038/s41598-020-70086-y

9. Bohandy J, Kim BF, Adrian FJ. Metal deposition from a supported metal film using an excimer laser. *J Appl Phys*. 1986;60(4):1538-1539.
doi: 10.1063/1.337287

10. Bohandy J, Kim BF, Adrian FJ, Jette AN. Metal deposition at 532 nm using a laser transfer technique. *J Appl Phys*. 1988;63(4):1158-1162.
doi: 10.1063/1.340023

11. Rapp L, Cibert C, Nénon S, et al. Improvement in semiconductor laser printing using a sacrificial protecting layer for organic thin-film transistors fabrication. *Appl Surf Sci*. 2011;257(12):5245-5249.
doi: 10.1016/j.apsusc.2010.10.147

12. Rapp L, Constantinescu C, Delaporte P, Alloncle AP. Laser-induced forward transfer of polythiophene-based derivatives for fully polymeric thin film transistors. *Org Electron*. 2014;15(8):1868-1875.
doi: 10.1016/j.orgel.2014.04.029

13. Rapp L, Cibert C, Alloncle AP, et al. LIFT of organic materials for application in plastic micro-electronics. In: *3eme Russian-French Laser Physics Workshop for Young Scientist*; 2008. Accessed December 3, 2024.
https://hal.science/hal-00446484

14. Colina M, Serra P, Fernández-Pradas JM, Sevilla L, Morenza JL. DNA deposition through laser induced forward transfer. *Biosens Bioelectron*. 2005;20(8):1638-1642.
doi: 10.1016/j.bios.2004.08.047

15. Serra P, Colina M, Fernández-Pradas JM, Sevilla L, Morenza JL. Preparation of functional DNA microarrays through laser-induced forward transfer. *Appl Phys Lett*. 2004;85(9):1639-1641.
doi: 10.1063/1.1787614

16. Fernández-Pradas JM, Colina M, Serra P, Domínguez J,

Morenza JL. Laser-induced forward transfer of biomolecules. *Thin Solid Films*. 2004;453-454:27-30.
doi: 10.1016/j.tsf.2003.11.154

17. Barron JA, Spargo BJ, Ringeisen BR. Biological laser printing of three dimensional cellular structures. *Appl Phys A*. 2004;79(4-6):1027-1030.
doi: 10.1007/s00339-004-2620-3

18. Ringeisen BR, Kim H, Barron JA, et al. Laser printing of pluripotent embryonal carcinoma cells. *Tissue Eng*. 2004;10(3-4):483-491.
doi: 10.1089/107632704323061843

19. Guillotin B, Guillemot F. Cell patterning technologies for organotypic tissue fabrication. *Trends Biotechnol*. 2011;29(4):183-190.
doi: 10.1016/j.tibtech.2010.12.008

20. Koch L, Deiwick A, Chichkov B. Capillary-like formations of endothelial cells in defined patterns generated by laser bioprinting. *Micromachines (Basel)*. 2021;12(12):1538.
doi: 10.3390/mi12121538

21. Koch L, Deiwick A, Schlie S, et al. Skin tissue generation by laser cell printing. *Biotechnol Bioeng*. 2012;109(7):1855-1863.
doi: 10.1002/bit.24455

22. Bosmans C, Rodriguez NG, Karperien M, et al. Towards single-cell bioprinting: micropatterning tools for organ-on-chip development. *Trends Biotechnol*. 2024;42(6):739-759.
doi: 10.1016/j.tibtech.2023.11.014

23. Hakobyan D, Médina C, Dusserre N, et al. Laser-assisted 3D bioprinting of exocrine pancreas spheroid models for cancer initiation study. *Biofabrication*. 2020;12(3):035001.
doi: 10.1088/1758-5090/ab7cb8

24. Sorkio A, Koch L, Koivusalo L, et al. Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks. *Biomaterials*. 2018;171:57-71.
doi: 10.1016/j.biomaterials.2018.04.034

25. Chang J, Sun X. Laser-induced forward transfer based laser bioprinting in biomedical applications. *Front Bioeng Biotechnol*. 2023;11:1255782.
doi: 10.3389/fbioe.2023.1255782

26. Curley JL, Sklare SC, Bowser DA, Saksena J, Moore MJ, Chrisey DB. Isolated node engineering of neuronal systems using laser direct write. *Biofabrication*. 2016;8(1):015013.
doi: 10.1088/1758-5090/8/1/015013

27. Colina M, Duocastella M, Fernández-Pradas JM, Serra P, Morenza JL. Laser-induced forward transfer of liquids: study of the droplet ejection process. *J Appl Phys*. 2006;99(8):084909.
doi: 10.1063/1.2191569

28. Fernández-Pradas JM, Florian C, Caballero-Lucas F, Sopeña P, Morenza JL, Serra P. Laser-induced forward transfer: propelling liquids with light. *Appl Surf Sci*. 2017;418:559-564.
doi: 10.1016/j.apsusc.2016.10.197
29. Gruene M, Unger C, Koch L, Deiwick A, Chichkov B. Dispensing pico to nanolitre of a natural hydrogel by laser-assisted bioprinting. *BioMed Eng OnLine*. 2011;10(1):19.
doi: 10.1186/1475-925X-10-19

30. Barron JA, Krizman DB, Ringeisen BR. Laser printing of single cells: statistical analysis, cell viability, and stress. *Ann Biomed Eng.* 2005;33(2):121-130.
doi: 10.1007/s10439-005-8971-x

31. Mézel C, Souquet A, Hallo L, Guillemot F. Bioprinting by laser-induced forward transfer for tissue engineering applications: jet formation modeling. *Biofabrication*. 2010;2(1):014103.
doi: 10.1088/1758-5082/2/1/014103

32. Koch L, Brandt O, Deiwick A, Chichkov B. Laser-assisted bioprinting at different wavelengths and pulse durations with a metal dynamic release layer: A parametric study. *Int J Bioprint*. 2017;3(1):001.
doi: 10.18063/IJB.2017.01.001

33. Vogel A, Busch S, Parlitz U. Shock wave emission and cavitation bubble generation by picosecond and nanosecond optical breakdown in water. *J Acoust Soc Amer.* 1996;100(1):148-165.
doi: 10.1121/1.415878

34. Duocastella M, Fernández-Pradas JM, Morenza JL, Serra P. Time-resolved imaging of the laser forward transfer of liquids. *J Appl Phys*. 2009;106(8):084907.
doi: 10.1063/1.3248304

35. Deng Y, Renaud P, Guo Z, Huang Z, Chen Y. Single cell isolation process with laser induced forward transfer. *J Biol Eng.* 2017;11(1):2.
doi: 10.1186/s13036-016-0045-0

36. Koch L, Kuhn S, Sorg H, et al. Laser printing of skin cells and human stem cells. *Tissue Eng Part C Methods*. 2010;16(5):847-854.
doi: 10.1089/ten.tec.2009.0397

37. Hopp B, Smausz T, Kresz N, et al. Survival and proliferative ability of various living cell types after laser-induced forward transfer. *Tissue Eng*. 2005;11(11-12):1817-1823.
doi: 10.1089/ten.2005.11.1817

38. Schiele NR, Chrisey DB, Corr DT. Gelatin-based laser direct-write technique for the precise spatial patterning of cells. *Tissue Eng Part C Methods*. 2011;17(3):289-298.
doi: 10.1089/ten.tec.2010.0442

39. Guillotin B, Souquet A, Catros S, et al. Laser assisted bioprinting of engineered tissue with high cell density and microscale organization. *Biomaterials*. 2010;31(28):7250-7256.
doi: 10.1016/j.biomaterials.2010.05.055

40. Karakaidos P, Kryou C, Simigdala N, Klinakis A, Zergioti I. Laser bioprinting of cells using UV and visible wavelengths: a comparative DNA damage study. *Bioengineering*. 2022;9(8):378.

doi: 10.3390/bioengineering9080378

41. Kryou C, Theodorakos I, Karakaidos P, Klinakis A, Hatziapostolou A, Zergioti I. Parametric study of Jet/Droplet formation process during LIFT printing of living Cell-Laden bioink. *Micromachines*. 2021;12(11):1408.
doi: 10.3390/mi12111408

42. Murru C, Duvert L, Magdinier F, et al. Assessment of laser-synthesized Si nanoparticle effects on myoblast motility, proliferation and differentiation: towards potential tissue engineering applications. *Nanoscale Adv.* 2024;6(8):2104-2112.
doi: 10.1039/D3NA01020A

43. Hanawa T. Biocompatibility of titanium from the viewpoint of its surface. *Sci Technol Adv Mater.* 2022;23(1):457-472.
doi: 10.1080/14686996.2022.2106156

44. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. *Nat Methods*. 2012;9(7):676-682.
doi: 10.1038/nmeth.2019

45. Zhang Z, Xu C, Xiong R, Chrisey DB, Huang Y. Effects of living cells on the bioink printability during laser printing. *Biomicrofluidics*. 2017;11(3):034120.
doi: 10.1063/1.4985652

46. Xiong R, Zhang Z, Chai W, Chrisey DB, Huang Y. Study of gelatin as an effective energy absorbing layer for laser bioprinting. *Biofabrication*. 2017;9(2):024103.
doi: 10.1088/1758-5090/aa74f2

47. Catros S, Guillotin B, Bačáková M, Fricain JC, Guillemot F. Effect of laser energy, substrate film thickness and bioink viscosity on viability of endothelial cells printed by laser-assisted bioprinting. *Appl Surf Sci.* 2011;257(12):5142-5147.
doi: 10.1016/j.apsusc.2010.11.049

48. Riester D. High speed photography of laser induced forward transfer (LIFT) of single and double-layered transfer layers for single cell transfer. *JLMN*. 2016;11(2):199-203.
doi: 10.2961/jlmn.2016.02.0010

49. Antoshin AA, Churbanov SN, Minaev NV, et al. LIFT-bioprinting, is it worth it? *Bioprinting*. 2019;15:e00052.
doi: 10.1016/j.bprint.2019.e00052

50. Liu X, Zu E, Chang X, et al. Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration. *Dis Model Mech.* 2021;14(12):dmm049290.
doi: 10.1242/dmm.049290

51. Minaev NV, Yusupov VI, Zhigarkov VS, et al. Laser Printing of Gel Microdrops with Living Cells and Microorganisms. *KnE Energy*. 2018;3(3):23-31.
doi: 10.18502/ken.v3i3.2010

52. Ali M, Pages E, Ducom A, Fontaine A, Guillemot F. Controlling laser-induced jet formation for bioprinting mesenchymal stem cells with high viability and high resolution. *Biofabrication*. 2014;6(4):045001.
doi: 10.1088/1758-5082/6/4/045001