Paraffin Embedding and Histological Analyses of Sw71-Spheroids as Human Blastocyst-like Surrogates
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Although the topic of the paper is relevant and the described methodology is detailed, after a thorough review, I have observed that the topic addressed in the manuscript overlaps significantly with prior publications, such as [https://doi.org/10.1016/j.placenta.2023.01.005]. Consequently, the level of novelty may not meet the journal's threshold for publication. However, I believe this manuscript provides valuable methodological details and could be of interest to readers focused on experimental protocols. For this reason, I suggest considering submission to a journal specializing in protocols or experimental techniques (e.g. Methods and Protocols), where the work may be more suitable.
Author Response
Although the topic of the paper is relevant and the described methodology is detailed, after a thorough review, I have observed that the topic addressed in the manuscript overlaps significantly with prior publications, such as [https://doi.org/10.1016/j.placenta.2023.01.005]. Consequently, the level of novelty may not meet the journal's threshold for publication. However, I believe this manuscript provides valuable methodological details and could be of interest to readers focused on experimental protocols. For this reason, I suggest considering submission to a journal specializing in protocols or experimental techniques (e.g. Methods and Protocols), where the work may be more suitable.
We thank the Reviewer for his/her evaluation and notes. We are not agreed with the statement that our study address the same topic as that of the pointed out study [https://doi.org/10.1016/j.placenta.2023.01.005]. The latter describes the generation of spheroids from three trophoblastic cell lines incl. Sw71, whereas our study describes the conservation of already generated Sw71 spheroids by embedding in paraffin which preserves not only structure but also antigenicity of the model. Should be noted that the Sw71 spheroid as a model of implanting human embyo has been generated, in vivo validated and published by our group before Sandra Haider’s group – the study mentioned above. Please see
https://doi.org/10.1111/j.1600-0897.2011.01095.x
https://doi.org/10.1111/aji.13076
https://pubmed.ncbi.nlm.nih.gov/35715452/
https://doi.org/10.1111/aji.13800
In these studies, we have already proved Sw71 spheroids as human blastocyst-like surrogates for assessing the features and functionality (attachment, migration and invasion) of the trophoblast during human implantation. The novelty of the current study is paraffin-embedding of Sw71 spheroids in way to preserve both the structure and EVT-related antigens. Most important, while the authors of the study mentioned by the Reviewer report inability to create matrix embedded Sw71 organoids, we succeeded to embed them in paraffin wax using pre-embedding.
Reviewer 2 Report
Comments and Suggestions for Authors
Remarks to authors
In this study, Alexandrova and colleagues developed and embedded trophoblast Sw71-spheroids in paraffin for permanent histological preparations. The authors utilized human placenta cells to generate Sw71-spheroids, which serve as implanting human blastocyst-like surrogates (BLS). They established a reliable protocol for embedding Sw71-BLS spheroids in paraffin for permanent histological analysis, providing an invaluable tool for organoid and implantation studies. Using this optimized protocol, the authors observed the expression of EpCAM, HLA-C, and HLA-G in Sw71 trophoblasts. However, the data presented are still preliminary, and several key issues need to be addressed. My specific comments are outlined below.
- Sw71-BLS spheroid characterization: The Sw71-BLS spheroids require more detailed characterization, including the detection of key marker expressions, cell proliferation, apoptosis, and other relevant features to confirm their biological relevance and functionality.
- Lines 244: Figure references contain errors.
- Agarose pad pre-embedding method: More details on the Agarose Pad Pre-embedding method should be provided. Specifically, how is the agarose solidified, and what are the advantages of using this method over other embedding techniques, such as those that employ higher melting-point agarose or gelatin-based supports?
- Sample size clarification: The sample number in Figures 3 and 4 need to be clarified to ensure proper representation and reproducibility of the data.
- The quantification in Figure 4: Additional data should be included to provide more precise quantification of the digital signals, thereby improving the robustness of the results.
Author Response
In this study, Alexandrova and colleagues developed and embedded trophoblast Sw71-spheroids in paraffin for permanent histological preparations. The authors utilized human placenta cells to generate Sw71-spheroids, which serve as implanting human blastocyst-like surrogates (BLS). They established a reliable protocol for embedding Sw71-BLS spheroids in paraffin for permanent histological analysis, providing an invaluable tool for organoid and implantation studies. Using this optimized protocol, the authors observed the expression of EpCAM, HLA-C, and HLA-G in Sw71 trophoblasts. However, the data presented are still preliminary, and several key issues need to be addressed. My specific comments are outlined below.
We thank the Reviewer for his/her evaluation. Below we address his/her comment point-by-point.
Sw71-BLS spheroid characterization: The Sw71-BLS spheroids require more detailed characterization, including the detection of key marker expressions, cell proliferation, apoptosis, and other relevant features to confirm their biological relevance and functionality.
Since Sw71-BLS spheroid model has been well characterized already in the Introduction section we described the model citing most of the studies exploring this model and showing its key markers and specific functionality (lines 44-66). All the relevant studies were cited in the Discussion section as well to highlight the on-going work with this model (lines 320-324, 328-337). In this study we emphasized the restricted vitality of Sw71 BLS model and the need to be generated de novo for the functional and other experiments (line 66,67) which motivated us to search a possibility for its preservation in permanent histological preparations.
It is important to note that after de novo generation of Sw71 spheroids for whatever experiment we test them for migration and invasion to prove their functionality. We added now such an information in MandM section – line 97-99.
Lines 244: Figure references contain errors.
We did a correction – line 252 and also of the word “threshold” – line 249, 250.
Agarose pad pre-embedding method: More details on the Agarose Pad Pre-embedding method should be provided. Specifically, how is the agarose solidified, and what are the advantages of using this method over other embedding techniques, such as those that employ higher melting-point agarose or gelatin-based supports?
We thank the Reviewer for these notes. In part: 3.2. Protocol for FFPE of Sw71-BLS where we describe the protocol in detail, we added the sentence “The agarose was left to solidify at room temperature” – line 182. The advantages of using this method over other embedding techniques is the degree of preservation of the cell morphology, shape, and intact periphery of the embedded Sw71-spheroids as well as getting good slides as compared to directly embedding in paraffin. We mentioned that fact in the Discussion section – lines 299-302, 306-309. We also had better paraffin infiltration than with the agar support and higher melting-point agarose and mentioned that in Result section – line 224-226 and in the Discussion section – lines 292-294. We did not test a gelatin-based support.
Sample size clarification: The sample number in Figures 3 and 4 need to be clarified to ensure proper representation and reproducibility of the data.
We clarified the number of the samples (lines 182, 299) and experiments for Figure 3 – shown in the legend of the figure (line 231) and also in MandM section (lines 103-105). We clarified the number of the samples and experiments for Figure 4 – shown in the legend of the figure (line 264-265) and in MandM section – lines 120, 122.
The quantification in Figure 4: Additional data should be included to provide more precise quantification of the digital signals, thereby improving the robustness of the results.
To support the immunohistochemical findings, we have used published protocols using ImageJ software for digitalization of the signal (lines 141-144). The IHC analyses are well established plug-in of the software. We specified in the text that the threshold was set up using the negative control digital signal (J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D.J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, A. Cardona, Fiji: an open-source platform for biological-image analysis., Nat. Methods 9 (2012) 676–682. https://doi.org/10.1038/nmeth.2019.). The calculated signal represents the positive trophoblasts as area from the selected view (signal-threshold, %) (lines 248-252) [C. Shang, Analyze Immunohistochemistry images in ImageJ, (2018).
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
Thank you for the clarification of the study aim and background of this research idea.The paper is well written, but need some changes:
Line 30 please rephrase a term “is a hidden process” as it is not clear what do you mean.
Line 40 rephrase “moral dilemmas” to “ethical constraints”
Line 44 rephrase “We and others construct Sw71-spheroids ” to “In the previous research we and other others described the generation of Sw71-spheroids”
In the Introduction, line from 57 to 66 should be placed in discussion. Instead, describe here the challenges of using paraffin-embedded spheroid cultures for protein expression research such as: Preservation and fixation issues (e.g. Paraffin embedding requires fixation, often with formalin, which can alter protein structure and affect antigen recognition. This makes immunohistochemistry or Western blot analysis more difficult);Limited protein extraction efficiency ( Paraffin embedding creates a dense matrix that makes protein extraction inefficient. Harsh deparaffinization methods can lead to protein degradation or loss, impacting downstream applications); Loss of spatial context ( Spheroid cultures mimic in vivo conditions, providing a 3D cellular environment. However, paraffin embedding and subsequent sectioning can disrupt the original architecture, making it harder to study protein distribution and expression patterns); Antigen retrieval complexity(Many proteins require specific antigen retrieval methods post-embedding. Determining optimal conditions can be time-consuming and may not be universally applicable across different proteins); and finally, at the end of Introduction add comment how your model could adress these challenges.
In the Methodology section, the authors state the morphology and size of spheroids were assessed by the mean diameter and the spheroid surface on day 0, to day 2. Describe here the assessment of viability of spheroids, and at what day does viability decrease significantly? Also, were they tested for apoptosis and necrosis prior to sectioning? How old were the spheroids at embedding in paraffin?
Line 181 “liquefied low-gelling temperature agarose” add percentage of agarose used
Line 192 “Few spheroids were procesed without pre-embedding in low-gelling agarose or with pre-embedding in 2% agar pad.” Based on what criteria did you alter the protocol for these selected spheroids?
Line 310 rephrase “In our hands” to “In our experiment”
Line 314 change “permit” to “allow”
Author Response
Reviewer 1:
Thank you for the clarification of the study aim and background of this research idea. The paper is well written, but need some changes:
We thank the Reviewer for his/her efforts and time to evaluate our paper. Bellow we addressed point-by-point his/her suggestions and notes. We redacted the manuscript accordingly and our corrections are highlighted in yellow.
Line 30 please rephrase a term “is a hidden process” as it is not clear what do you mean.
We rephrased “is a hidden process” to “is a process difficult to observe directly” – line 29.
Line 40 rephrase “moral dilemmas” to “ethical constraints”
We rephrased “moral dilemmas” to “ethical constraints” – line 40.
Line 44 rephrase “We and others construct Sw71 spheroids” to “In the previous research we and other others described the generation of Sw71-spheroids”
We rephrased “We and others construct Sw71 spheroids” to “In the previous research we and others described the generation of Sw71-spheroids” – line 45.
In the Introduction, line from 57 to 66 should be placed in discussion. Instead, describe here the challenges of using paraffin-embedded spheroid cultures for protein expression research such as: Preservation and fixation issues (e.g. Paraffin embedding requires fixation, often with formalin, which can alter protein structure and affect antigen recognition. This makes immunohistochemistry or Western blot analysis more difficult); Limited protein extraction efficiency ( Paraffin embedding creates a dense matrix that makes protein extraction inefficient. Harsh deparaffinization methods can lead to protein degradation or loss, impacting downstream applications); Loss of spatial context (Spheroid cultures mimic in vivo conditions, providing a 3D cellular environment. However, paraffin embedding and subsequent sectioning can disrupt the original architecture, making it harder to study protein distribution and expression patterns); Antigen retrieval complexity (Many proteins require specific antigen retrieval methods post-embedding. Determining optimal conditions can be time-consuming and may not be universally applicable across different proteins); and finally, at the end of Introduction add comment how your model could adress these challenges.
We accept the suggestion of the Reviewer and accordingly moved the mentioned text from the Introduction (lines 59-68) to the Discussion section (lines 278-286). We added a new text in the Introduction section to describe the challenges of using paraffin-embedded spheroid cultures – Lines 70-73. In the Results and Discussion sections we thoroughly described how the steps in our protocol addressed all these challenges – we found that the mild fixation with 4% PFA for ON/4⁰C instead of 30 min/RT leads to better preserved/stabilized Sw71-BLS (line 231, 303-304), clearing with both cedarwood oil for 3 days and xylene for 3 x 3 h provides the easiest cutting and flattening of the section (line 228), the pre-embedding in low-gelling temperature agarose before embedding in paraffin preserve the structures better and allows easy move all the spheroids at once during the processing for paraffin embedding (line 292, 305-308, 322-323).
In the Methodology section, the authors state the morphology and size of spheroids were assessed by the mean diameter and the spheroid surface on day 0, to day 2. Describe here the assessment of viability of spheroids, and at what day does viability decrease significantly? Also, were they tested for apoptosis and necrosis prior to sectioning? How old were the spheroids at embedding in paraffin?
As we stated (lines 100- 101) some of the selected Sw71 spheroids were tested for migration and invasion and proved as functional. This is also a proof for their viability. In our experiments we usually use freshly generated spheroids. Thus, for embedding in paraffin we fixed the spheroids immediately after their differentiation (generation) and mentioned this in the text now – line 180-181. In the Discussion part we pointed out that the Sw71 spheroids remain viable with preserved morphology up to 2 weeks, shown in our previous research – line 287-288. We did not test the spheroids for apoptosis and necrosis prior to sectioning.
Line 181 “liquefied low-gelling temperature agarose” add percentage of agarose used
We added 2% of liquefied low-gelling temperature agarose – line 185, 196.
Line 192 “Few spheroids were procesed without pre-embedding in low-gelling agarose or with pre-embedding in 2% agar pad.” Based on what criteria did you alter the protocol for these selected spheroids?
We altered the protocol for these selected spheroids to see the effect of the pre-embedding on the preservation of the Sw71 spheroids having in mind their fragility. As we described in the Discussion (lines 324-325), the direct paraffin embedding of Sw71 spheroids impairs their peripheral zone.
Line 310 rephrase “In our hands” to “In our experiment”
We rephrased “In our hands” to “In our experiment” - Line 306, 324.
Line 314 change “permit” to “allow”
We changed “permit” to “allow” – line 328.
Reviewer 2 Report
Comments and Suggestions for Authors
The authors have addressed my concerns.
Author Response
Reviewer 2:
The authors have addressed my concerns.
We thank the Reviewer once again for his/her efforts and time to evaluate our manuscript.
Round 3
Reviewer 1 Report
Comments and Suggestions for Authors
The authors have adressed all issues and the paper could be accepted for publication.
In line 100 add the statement" We did not test the spheroids for apoptosis and necrosis prior to sectioning."
Author Response
Comments and Suggestions for Authors
The authors have adressed all issues and the paper could be accepted for publication.
In line 100 add the statement" We did not test the spheroids for apoptosis and necrosis prior to sectioning."
We thank the Reviewer for his/her positive evaluation.
In line 103 we added the statement " We did not test the spheroids for apoptosis and necrosis prior to sectioning".
