Development and Optimization of a Lactate Dehydrogenase Assay Adapted to 3D Cell Cultures

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The manuscript “Development and Optimization of a Lactate Dehydrogenase 2 Assay adapted to 3D cell cultures” prepared by Castiglione H. et al., shows how easily the LDH assay protocol used in classic 2D cell cultures can be adapted to 3D models. This is undoubtedly a very interesting and important aspect, especially at a time when research on organoids is increasingly replacing one-dimensional cultures. However, despite the fact that I find the work very interesting and useful, I find several elements that the authors need to improve/clarify.

1.       Line 81. “the LDH activity value is usually normalized by the number of 81 seeded cells” –this is not entirely true because it is often also normalized by protein concentration or LDH activity in cell lysates.

2.       Figure 1A – as well as its description in the text should be in methods not in results description.

3.       Figure 1B - description of the horizontal axis – LDH activity is not the same as absorbance.

4.       The literature is in the wrong format, not adapted to the journal requirements .

5.       No conclusion, no summary showing innovation or usefulness.

 

Author Response

Dear reviewer,

Thank you for your valuable feedback and suggestions for improvement. We have carefully considered your comments and have made the necessary revisions to enhance the clarity and quality of our work. We hope the changes meet your expectations. Please find below our detailed, point-by-point responses.

1. Line 81. “the LDH activity value is usually normalized by the number of 81 seeded cells” –this is not entirely true because it is often also normalized by protein concentration or LDH activity in cell lysates.

We agree with your observation. Initially, we did not mention these normalizations because the number of seeded cells was, to us, the most used value. However, to address your point, we have now included these normalization possibilities, line 70, along with a brief comment about their use in 3D culture, line 80. Additionally, we have updated the relevant sections in the discussion to reflect these changes, specifically on lines 291 and 296.

2. Figure 1A – as well as its description in the text should be in methods not in results description.

We have divided Figure 1 into two separate figures, now labeled as Figures 1 and 2. The first part has been moved into the methods section, line 183. Consequently, all figures have been renumbered, and the captions for Figures 1 and 2 have been updated. Additionally, we have relocated the description of the initial Figure 1A to the methods section, lines 156 and 172.

3. Figure 1B - description of the horizontal axis – LDH activity is not the same as absorbance.

We believe the absorbance measured is directly related to LDH activity in this context. However, we acknowledge that LDH abundance also influences this value. To enhance clarity, we have modified the horizontal axis title to “LDH detection – Absorbance at 490 nm” line 214.

4. The literature is in the wrong format, not adapted to the journal requirements.

We apologize for this mistake. We have corrected the reference format to align with the journal’s guidelines. Additionally, we have reordered the reference list according to the order of appearance rather than alphabetical order. For clarity, we have not highlighted these changes.

5. No conclusion, no summary showing innovation or usefulness.

Initially, we omitted a conclusion section based on the journal guidelines, which suggest including it only if the discussion is lengthy and intricated. However, we have now incorporated a brief conclusion to succinctly summarize our findings and underscore their significance, located at line 391. 

Reviewer 2 Report

Comments and Suggestions for Authors

Recommendation: Major revisions necessary

Comments:

The study introduced the LDH assay from 2D cell culture to 3D cell culture, and the authors aim to provide a standardized framework. This is very important for the evaluation of 3D cell cultures. The authors have provided comprehensive investigations and detailed protocols. However, to enhance the paper's advantages, understandability, and logical flow, a major revision is necessary.

1.     The author conducted experiments to investigate the BSA buffer effect on the storage. They claimed that the buffer could help to maintain the LDH activity. Please explain the mechanism behind this. In addition, please explain why the total protein concentration in buffer and medium/buffer started to increase at day 120 in Fig. 1C.

2.     In the experiments of Fig. 1B and C, there lacked the control experiment, such as pure PBS.

3.     In the optimization of dilution experiments, the authors offered a number of dilution factors. However, there was no definition of the dilution factors. To make it clear, it is better to provide relative descriptive information.

4.     In the validation experiments, the description of cerebral organoids was missing, such as the size, cultured time, derivation (human, or mouse), etc.

5.    In line 45, when it comes to organoid on chip, the authors should cite the following papers.

‘Guocheng Fang, et al., Enabling peristalsis of human colon tumor organoids on microfluidic chips, Biofabrication, 2021, 14, 1: 015006 ’

‘Chak Ming Leung, et al., A guide to the organ-on-a-chip, Nat Rev Methods Primers 2, 33 (2022).’

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear reviewer,

We sincerely appreciate your feedback and suggestions aimed at improving the clarity of our paper. We have carefully considered them and made the necessary adjustments to enhance its comprehensibility to the best of our ability. We hope these revisions meet your expectations. Please find below our detailed point-by-point responses.

1. The author conducted experiments to investigate the BSA buffer effect on the storage. They claimed that the buffer could help to maintain the LDH activity. Please explain the mechanism behind this. In addition, please explain why the total protein concentration in buffer and medium/buffer started to increase at day 120 in Fig. 1C.

Initially, our decision to use BSA to preserve LDH activity at -20°C was influenced by the numerous beneficial effects of BSA described in the literature, regarding protein integrity and activity maintenance. While the exact mechanism in our context remains unclear, such uncertainty is not uncommon in the literature, despite various attempts to decipher it. To further demonstrate these beneficial effects and explore potential underlying mechanisms, we have added a discussion in the relevant section, line 304.

In Fig. 1C, now named 2B, it may appear that the total protein concentration in the buffer and medium/buffer increases at day 120. However, we believe this observation is the result of an experimental artifact rather than a true increase. Absorbance assays are highly sensitive to incubation time, and slight variations may occur. To confirm the non-significance of this apparent increase, we conducted a statistical analysis using a mixed-effects analysis to assess repeated measures over time, followed by a Tukey’s post-hoc test. Our analysis revealed no statistical differences between the values at day 90 and day 120. We have included these statistical results in the figure’s caption, line 222.

2. In the experiments of Fig. 1B and C, there lacked the control experiment, such as pure PBS.

We acknowledge the absence of this control in our study. Our primary objective was to demonstrate that LDH integrity cannot be maintained at -20°C and that adding a preservation buffer could ensure prolonged integrity. We appreciate your suggestion and agree that including a vehicle control would have been beneficial to confirm that the observed effect is solely due to BSA and not to the vehicle solution or a dilution effect. However, it would be impractical for us to repeat the experiment, due to the duration of our study spanning four months, and we believe our findings sufficiently support our conclusion of prolonged integrity for one-month.

3. In the optimization of dilution experiments, the authors offered a number of dilution factors. However, there was no definition of the dilution factors. To make it clear, it is better to provide relative descriptive information.

Thank you for bringing this issue to our attention. We had not anticipated that these values might be ambiguous. To address this concern, we have included relative descriptive information in Table E of Figure 3 line 244. Additionally, we have specified these details in the methods section, line 163.

4. In the validation experiments, the description of cerebral organoids was missing, such as the size, cultured time, derivation (human, or mouse), etc.

We apologize about these missing elements. To enhance accessibility, we have updated Figure 4 by incorporating a timeline of the experiment and the growth profiles of the organoids. Consequently, we have adjusted the caption of the figure, lines 273 and 282, and the corresponding description in the results section, lines 255 and 265. Additionally, we have included a brief description of the cerebral organoid growth evolution measurements in the methods section, line 151. However, the derivation (human) was already mentioned in the methods section, as indicated in line 104.

5. In line 45, when it comes to organoid on chip, the authors should cite the following papers.

‘Guocheng Fang, et al., Enabling peristalsis of human colon tumor organoids on microfluidic chips, Biofabrication, 2021, 14, 1: 015006 ’

‘Chak Ming Leung, et al., A guide to the organ-on-a-chip, Nat Rev Methods Primers 2, 33 (2022).’

We sincerely thank you for these additional references, which align well with our point. We have included them line 43.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all my concerns. This paper can be published in present form.