Laser-Induced Forward Transfer in Organ-on-Chip Devices
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
This manuscript introduces the application of laser-induced forward transfer (LIFT) bioprinting in organ-on-chip devices. Through parameter analysis, a microfluidic device (designed using LCD 3D printing and biocompatible resin) was manufactured. The fluid conditions within the device were validated through computational fluid dynamics (CFD) simulations, and LLC cells were successfully printed in the device. This study confirms the feasibility and accuracy of LIFT bioprinting for integrating cells into microfluidic platforms, offering potential applications in organ chips and biomedical research. There are several issues:
- It is concluded that the optimal donor-receiver distance for LIFT bioprinting is 2.0 mm, based on experimental results using LLC cells and specific bio-ink concentrations. Does this threshold also apply to other types of cells (such as stem cells or epithelial cells) or bio-inks with different viscosities?
- In the high-speed imaging analysis, Python scripts are used to quantify droplet trajectory and velocity, though the script's function is only briefly mentioned. Could the authors provide more details on the core algorithms used in the script, such as the particle tracking methods, calibration process, and verification data?
- Cell viability experiments indicate that the survival rate of LLC cells exceeds 95% 24 hours after printing. However, functional indicators of the cells (e.g., migration ability, cytokine secretion levels) are not discussed.
- The CFD simulation of the microfluidic device shows a 24% deviation from the particle shadow velocimetry experimental results. Could the authors provide more specific characterization data, such as surface roughness parameters or the deviation distribution between measured and design values for key sizes?
The English could be improved to more clearly express the research.
Author Response
Reviewer 1
This manuscript introduces the application of laser-induced forward transfer (LIFT) bioprinting in organ-on-chip devices. Through parameter analysis, a microfluidic device (designed using LCD 3D printing and biocompatible resin was manufactured. The fluid conditions within the device were validated through computational fluid dynamics (CFD) simulations, and LLC cells were successfully printed in the device. This study confirms the feasibility and accuracy of LIFT bioprinting for integrating cells into microfluidic platforms, offering potential applications in organ chips and biomedical research. There are several issues:
Comments 1: It is concluded that the optimal donor-receiver distance for LIFT bioprinting is 2.0 mm, based on experimental results using LLC cells and specific bio-ink concentrations. Does this threshold also apply to other types of cells (such as stem cells or epithelial cells) or bio-inks with different viscosities?
Response 1: The optimal donor-receiver distance of 2.0 mm determined in our study specifically applies to LLC cells and the bioink concentration tested. This threshold is anticipated to be valid primarily for cells with similar diameters (15–20 µm) (average tumor cell is 10-20um, source: https://ccr.cancer.gov/pediatric-oncology-branch/gist-clinic/research/cell-line) and bioinks with comparable viscosities and concentrations. For bioinks with significantly different rheological properties or cell types, the optimal distance may vary due to altered droplet dynamics.
Comments 2: In the high-speed imaging analysis, Python scripts are used to quantify droplet trajectory and velocity, though the script's function is only briefly mentioned. Could the authors provide more details on the core algorithms used in the script, such as the particle tracking methods, calibration process, and verification data?
Response 2: We appreciate this suggestion and will include the Python scripts in the supplementary material. The algorithms employed tracking techniques, utilizing user-interactive annotations for frame-by-frame jet front trajectory tracking. Pixel-to-micrometer calibration is performed using predefined reference scales. Additionally, data extraction includes droplet displacement for each frame, in structured formats (CSV files) for analysis.
No algorithms were used for the particle shadow velocimetry.
Comments 3: Cell viability experiments indicate that the survival rate of LLC cells exceeds 95% 24 hours after printing. However, functional indicators of the cells (e.g., migration ability, cytokine secretion levels) are not discussed.
Response 3: Previous studies have demonstrated that LIFT bioprinting does not damage cellular DNA (Koch L, Deiwick A, Franke A, et al. Biofabrication. 2018;10(3):035005. doi: 10.1088/1758-5090/aab981 & Catros, Sylvain, et al. Biofabrication 3.2 (2011): 025001), preserving normal cellular functions post-printing. Therefore, significant alterations in the functional indicators of migration ability or cytokine secretion levels, due to the printing process, are not anticipated.
Comments 4: The CFD simulation of the microfluidic device shows a 24% deviation from the particle shadow velocimetry experimental results. Could the authors provide more specific characterization data, such as surface roughness parameters or the deviation distribution between measured and design values for key sizes?
Response 4: We appreciate the reviewer’s observation regarding the 24% deviation between CFD-predicted and experimentally measured flow velocities.
Initially, we hypothesized that internal surface roughness, introduced by the 3D printing process, might be responsible. However, upon characterization, we found the average surface roughness to be Rx=2.63 μm in the x axis and Ry=3.25 μm in the y axis (Veeco Dektak 150 Profilometer), which is negligible compared to the 500 μm channel height and has minimal effect on flow velocity in low-Reynolds-number (laminar, viscous-dominated) conditions. The difference of the roughness in the two axis is attributed to the 60º angle of the 3D printing procedure.
To investigate further, we 3D printed an open cross-section of a microchannel and analyzed the cross-sectional geometry using ImageJ, in order to quantify geometrical differences, due to the printing procedure. This allowed us to directly measure the printed geometry and compare it with the initial design. We found that the actual printed cross-sectional area was approximately 207,000 μm², significantly lower than the initial design of this channel which was of 250,000 μm² (based on 500 μm × 500 μm), corresponding to a 17.2% area difference. Moreover, the 3D printing process, as a layer-by-layer additive manufacturing technique, inherently introduces dimensional imperfections in cross sections of the microchannels, as well as geometric irregularities at channel junctions and corners. Such geometrical deviations, from the initial microfluidic design imported in the CFD calculations, have a notable impact on the hydraulic resistances of microchannels, which in turn affect the pressure and flow rate distributions within the multichannel microfluidic chip.
The manuscript has been revised accordingly, attributing the observed 24% deviation in flow velocities to the above-mentioned factors.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear autors,
I have read the manuscript titled “Laser Induced Forward Transfer in organ-on-chip devices” and offer the following observations:
1. The phrase "sub-nanosecond Nd:YAG laser system" is vague and requires clarification. An approximate pulse duration should be provided, as the term could refer to durations ranging from 1 picosecond to as short as 100 femtoseconds.
2. The number of laser pulses used per spot in the LIFT (Laser-Induced Forward Transfer) printing experiments should be clearly stated. If a single laser pulse was used, energy fluctuations become particularly critical and should be characterized. Additionally, beam pointing stability is another important parameter that can affect the precision and reproducibility of the printed arrays (e.g., spatial deviations), and should also be addressed in the text.
3.The rationale for selecting a Ti layer should be explained, especially in relation to the laser wavelength used for the material transfer.
4. It is unclear how the optimal focus position was determined during the experiments. This should be described in more detail.
5. Was the Ti film transferred simultaneously with the target material? Were any Ti debris observed on the donor slide surface? Clarifying this would provide insight into the transfer fidelity and potential contamination.
6. The scale numbers in Figure 8 are too small and should be enlarged for clarity. Additionally, the inlet and outlet in Figure 8a should be clearly labeled, with arrows indicating the direction of flow.
7. The Discussion section lacks a coherent structure and needs to be rewritten. Typically, new results are compared with previously published work to highlight advancements. In the current version, such references are missing, and the contribution relative to existing literature is not clearly articulated.
Author Response
Reviewer 2
Comments 1: The phrase "sub-nanosecond Nd:YAG laser system" is vague and requires clarification. An approximate pulse duration should be provided, as the term could refer to durations ranging from 1 picosecond to as short as 100 femtoseconds.
Response 1: We thank the reviewer for pointing out this oversight. The term "sub-nanosecond" was indeed an error; the correct laser pulse duration utilized in this study is 6 nanoseconds, and this correction will be made explicitly in the revised manuscript.
Comments 2: The number of laser pulses used per spot in the LIFT (Laser-Induced Forward Transfer) printing experiments should be clearly stated. If a single laser pulse was used, energy fluctuations become particularly critical and should be characterized. Additionally, beam pointing stability is another important parameter that can affect the precision and reproducibility of the printed arrays (e.g., spatial deviations), and should also be addressed in the text.
Response 2: Only one laser pulse per spot was used in our experiments. We acknowledge that energy fluctuations are a critical factor affecting reproducibility and precision, however this has not been observed in the laser employed in this experiment. To support this, we characterized 50 single pulses using a laser energy detector (QE8SP-B-MT-D0 - Pyroelectric detector for laser energy measuremens, Gentec-EO) in combination with a laser power monitor (11MAESTRO - Laser Power/Energy Monitor, standa). It was found the average value was 11.3μJ with RMS stability 0.34%. Beam pointing stability is negligible in our setup since we are using 60μm focal spot.
Comments 3: The rationale for selecting a Ti layer should be explained, especially in relation to the laser wavelength used for the material transfer.
Response 3: Titanium thin film was selected as the sacrificial absorption layer due to its absorption characteristics at the laser wavelength used (532 nm) (Hass, Georg, and Alan P. Bradford., Journal of the Optical Society of America 47.2 (1957): 125-129.), facilitating efficient energy transfer and controlled droplet ejection. This rationale will be clearly stated in the manuscript.
Comments 4: It is unclear how the optimal focus position was determined during the experiments. This should be described in more detail.
Response 4: Initial laser beam alignment and focus, on specific donor position, was achieved using a FLIR camera, in combination with a motorized x–y translation stage (Standa), enabling high-precision positioning of the donor slide loaded with bioink. We will include these methodological details in the revised text to clarify the procedure.
Comments 5: Was the Ti film transferred simultaneously with the target material? Were any Ti debris observed on the donor slide surface? Clarifying this would provide insight into the transfer fidelity and potential contamination.
Response 5: Thank you for this comment. Titanium debris was not observed, as the sacrificial Ti layer is specifically designed to vaporize.
Comments 6: The scale numbers in Figure 8 are too small and should be enlarged for clarity. Additionally, the inlet and outlet in Figure 8a should be clearly labeled, with arrows indicating the direction of flow.
Response 6: Figure 8 will be revised as suggested, enlarging the scale numbers and clearly labeling inlet and outlet with directional flow arrows for improved readability and clarity.
Comments 7: The Discussion section lacks a coherent structure and needs to be rewritten. Typically, new results are compared with previously published work to highlight advancements. In the current version, such references are missing, and the contribution relative to existing literature is not clearly articulated.
Response 7: We appreciate this important suggestion. The Discussion section will be thoroughly reorganized to explicitly compare our findings with relevant previously published studies, clearly highlighting the novelty, improvements, and contributions of our work relative to the existing literature.
Reviewer 3 Report
Comments and Suggestions for AuthorsThis study quantifies laser-induced forward transfer (LIFT) technology under limited conditions through detailed analysis of jet dynamics, printing resolution, fluid behavior, and cell viability. This study demonstrates clear biomedical relevance and systematic experimental design. The data strongly supports these conclusions. The innovation is also quite good. I suggest accepting after making modifications to the following points.
- In Figure 3, the spatial displacement is described in detail, but there seems to be no measurement of the geometric dimensions of the droplets. Will the size of the droplets affect the final cell culture and directional printing? Is there a certain correlation between the size of geometric dimensions and offset?
- In this work, it was noted that a laser with a frequency of 1kHz and a wavelength of 532nm was used, but there seems to be a lack of description of the power, which is the biggest factor affecting the energy density of the laser power. The size of the light spot affects the transfer and distribution of energy. The absorption capacity of different absorption layer materials for laser energy is also significantly different. Did the author fully consider these issues and optimize the parameters in the early experimental design.
- It is best for the author to carefully review the manuscript again to avoid low-level errors such as "2. Results" and "(Error! Reference source not foundation.)", which may raise doubts about the rigor of this work.
Author Response
Reviewer 3
This study quantifies laser-induced forward transfer (LIFT) technology under limited conditions through detailed analysis of jet dynamics, printing resolution, fluid behavior, and cell viability. This study demonstrates clear biomedical relevance and systematic experimental design. The data strongly supports these conclusions. The innovation is also quite good. I suggest accepting after making modifications to the following points.
Comments 1: In Figure 3, the spatial displacement is described in detail, but there seems to be no measurement of the geometric dimensions of the droplets. Will the size of the droplets affect the final cell culture and directional printing? Is there a certain correlation between the size of geometric dimensions and offset?
Response 1: We thank the reviewer for this comment. In our study, a fluence of 400 mJ/cm² and a fixed spot size of 60 μm was employed, resulting in a front droplet diameter of 145 ± 12 μm; this measurement was obtained from high-speed imaging frames at donor–receiver distances ranging from 1 mm to 3 mm and will be added to the manuscript. The laser fluence value was selected based on previous in-house experiments showing optimized results in terms of cell immobilization post-printing.
In our investigation, the main parameters examined were donor–receiver distance and jet trajectory stability. Fig.3 presents a centroid analysis, in order to determine the optimum distance for our experiments.
We can assume that if a hypothetic larger/smaller droplet diameter was selected, it could affect the cell number introduction hence cell proliferation and confluency in the final culture.
Comments 2: In this work, it was noted that a laser with a frequency of 1kHz and a wavelength of 532nm was used, but there seems to be a lack of description of the power, which is the biggest factor affecting the energy density of the laser power. The size of the light spot affects the transfer and distribution of energy. The absorption capacity of different absorption layer materials for laser energy is also significantly different. Did the author fully consider these issues and optimize the parameters in the early experimental design.
Response 2: Thank you for this comment. Laser fluence (power density) was precisely controlled and optimized at 400 mJ/cm², explicitly considering laser spot size (60 µm). Prior to the calculation of the energy fluence the laser power was measured with a laser energy detector (QE8SP-B-MT-D0 - Pyroelectric detector for laser energy measuremens, Gentec-EO) in combination with a laser power monitor (11MAESTRO - Laser Power/Energy Monitor, standa) at 11.3μJ (RMS stability 0.34% for 50 single pulses). These procedures will be thoroughly detailed in the revised manuscript to clarify the rationale behind the chosen parameters.
Comments 3: It is best for the author to carefully review the manuscript again to avoid low-level errors such as "2. Results" and "(Error! Reference source not foundation.)", which may raise doubts about the rigor of this work.
Response 3: Thank you for pointing out these errors. They will be carefully corrected in the revised manuscript, and we will perform additional proofreading to ensure manuscript rigor and clarity, thus avoiding any such errors.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI have no further comments.
Comments on the Quality of English LanguageThe English could be improved to more clearly express the research.
Author Response
We thank the reviewer for his/her acceptance.
