Spontaneous Epileptic Recordings from hiPSC-Derived Cortical Neurons Cultured with a Human Epileptic Brain Biopsy on a Multi Electrode Array

Round 1

Reviewer 1 Report

May I get such version of the manuscipt where the figures are readable, please? Now they are all too compressed and impossible to read. More comments after that.

Author Response

Reviewer 1:

 

Comment:

May I get such version of the manuscipt where the figures are readable, please? Now they are all too compressed and impossible to read. More comments after that.

Answer: There seemed to have been an issue with the pdf conversion as the Word version had higher definition figures. To accommodate the Reviewer, we used higher DPI standard (500DPI) and made the figures bigger and hopefully easier to assess. We look forward to further comments.

Reviewer 2 Report

Authors should read their text before submitting it. Text in lines 94-102 and 482-485 are part of the article instructions.

Otherwise, they provide interesting information. They should focus on epilepsy, not discuss other diseases (e.g. ALS), and critically evaluate their work (limitations).

Author Response

Comment:

Authors should read their text before submitting it. Text in lines 94-102 and 482-485 are part of the article instructions.

Otherwise, they provide interesting information. They should focus on epilepsy, not discuss other diseases (e.g. ALS), and critically evaluate their work (limitations).

Answer: We apologize for the mistakes that were left while formatting the original Word document into the specific journal template, prior to submission. Foremost, we removed the two sentences mentioned by the Reviewer that were indeed part of the article instructions. Following the suggestion of the Reviewer we went over the text and removed typo’s.  

Regarding to the specific comment on extrapolating to other diseases, in this case ALS, we can ensure that we now focus only on Epilepsy and removed the ALS sentence.

In addition, we want to mention that we already in the original version of the manuscript had a section on limitations of our study. As the Reviewer did not give specific instructions what to change we left it as is (lines 456-479). For the convenience of the Reviewer we added the particular paragraph below.

“One challenge is that the biopsy tissue also contains healthy material that surrounds diseased tissue when the latter is surgically removed. This means that some brain biopsy plugs may not contain diseased material or just very little. Therefore, we aimed to retrieve the biopsy plugs from the centre of the surgically removed biopsy. Human brain biopsies are hard to obtain and typically, brain slices generated from them, can only be measured for 6 to 12 hours [38]. Recording long-term network activity via multiple electrodes from a 3D biopsy or tissue construct has shown to be even more challenging [39]. In this respect, it is remarkable that we were able to record electrical activity from our hybrid model for 6 consecutive days within a relatively simple to use set-up and minimal culturing efforts. Four major advances of our new 3D model are, (1) extended time of measuring electrical activity from an intact brain biopsy tissue architecture ex vivo including all extracellular matrix compounds and the full plethora of cells to be found in human brain tissues, (2) good network functionality in a practical approach to assemble such biopsies on MEA that allows extracting of disease relevant electrophysiological fingerprints by means of a stable and reproducible hiPSC-derived neuron layer for taking reliable readouts, and (3) sufficient tissue volume to test various (non-) pharmacological interventions (e.g., neuromodulation, radiotherapy) and (4) capable of testing biopsy plugs punched along a gradient from healthy to diseased tissue form the edge of the biopsy tissue towards the centre, if sufficient material was retrieved during surgery, which has no further use in the conventional investigations of the pathologist’s laboratory. These advantages of our model offer a unique opportunity to study functional aspects of epilepsy in far greater detail than before. Finally, our approach to construct a hybrid ex vivo model may also be combined with 3D MEA technology, for example, a spike electrode array [40], to obtain information from measuring within the brain biopsy.”

Reviewer 3 Report

The topic is interesting:

The research described in the article focuses on developing a model of the brain that can be used to study epilepsy. Epilepsy can be difficult to study because it involves the interactions between different types of brain cells and the activity of those cells as a network. Animal models have often been used in epilepsy research, but there is growing awareness and concern about the use of animals in research, leading scientists to look for alternative methods. In this study, the researchers developed a model of the brain using a combination of human cells: one type of cell was derived from an epileptic brain tissue biopsy, and the other type was derived from induced pluripotent stem cells (hiPSCs). These two types of cells were grown together on multi-ectrode arrays which allowed to record the activity of the cells as a network. This model resembled the activity of an epileptic brain and may be a useful alternative to animal models for studying epilepsy and testing new therapies.

 

The topic text itself is easy to follow, but some elements suggests, that the manuscript has not been thoroughuly revised. Starting on page 1, line 94 there is a paragraph is undeleted text from a manuscript guidelines. This omnission is quite surprising for a manuscript that was supposedly read by 9 co-authors. The corresponding author should make sure that all co-authors read the final version before the submission. Similar situation is on the line 482

 

The main problem is with the presentation of the data in draft. I should be, as the reviewer able to asses the validity of the data. But I am not able to do it in some cases due to very low quality of graphs in the version I have received. 

 

Overall, the corresponding author should make sure all involved people has indeed read the manuscript, delete irrelevant text (the remains of template) and provide plots in sufficient quality.

Author Response

Comments:

The topic is interesting:

The research described in the article focuses on developing a model of the brain that can be used to study epilepsy. Epilepsy can be difficult to study because it involves the interactions between different types of brain cells and the activity of those cells as a network. Animal models have often been used in epilepsy research, but there is growing awareness and concern about the use of animals in research, leading scientists to look for alternative methods. In this study, the researchers developed a model of the brain using a combination of human cells: one type of cell was derived from an epileptic brain tissue biopsy, and the other type was derived from induced pluripotent stem cells (hiPSCs). These two types of cells were grown together on multi-ectrode arrays which allowed to record the activity of the cells as a network. This model resembled the activity of an epileptic brain and may be a useful alternative to animal models for studying epilepsy and testing new therapies.

 

The topic text itself is easy to follow, but some elements suggests, that the manuscript has not been thoroughuly revised. Starting on page 1, line 94 there is a paragraph is undeleted text from a manuscript guidelines. This omnission is quite surprising for a manuscript that was supposedly read by 9 co-authors. The corresponding author should make sure that all co-authors read the final version before the submission. Similar situation is on the line 482

 

Answer: We apologize for the oversight. Indeed text of the journal format was left while formatting the original Word document into the specific journal template, which we did just before submission. Foremost, we removed the two sentences mentioned by the Reviewer that were indeed part of the article instructions. Following the suggestion of the Reviewer we went over the text and removed typo’s. We can assure that all authors were much involved in the research and writing of the paper. In hindsight what has happened is that all editing was done in a Word file (because authors are used to that format) and only just prior to the last journal formatting step we moved the text to the specific journal template. Although the authors received that reformatted file just before submission they had not spotted the two sentences, concluded that the text was already finalized.

The main problem is with the presentation of the data in draft. I should be, as the reviewer able to asses the validity of the data. But I am not able to do it in some cases due to very low quality of graphs in the version I have received. 

 

Answer: There seemed to have been an issue with the pdf conversion as the Word version had higher definition figures. To accommodate the Reviewers we used a higher DPI standard (500DPI) and made the figures a bit bigger to make them hopefully easier to assess. 

Overall, the corresponding author should make sure all involved people has indeed read the manuscript, delete irrelevant text (the remains of template) and provide plots in sufficient quality.

Answer: Again we apologize for the oversight and hopefully have reassured the Reviewer that all authors were deeply involved in the research and manuscript writing. We contacted the Editorial Office how to deal with the lower quality of the figures and hope that the issue is now resolved.

Round 2

Reviewer 1 Report

The manuscript presents a concept to study epileptic brain biopsy slices on cortical neuron culture as a hybrid model where the culture acts as the "connector" of biopsy signal to the electrodes.

In general the manuscript is well-written and and the experiment is well designed. Now also the images are readable.

Something to improve anyway:
The introduction fails to explain what benefit you are going to achieve with the hybrid model (obviously transferring the signal from the biopsy plug to the electrodes, but you don't say it).

It remains unclear whether the improved signalling of the cell culture originates really from amplifying the biopsy signal and how much is just a reaction of bringing something "interfering" in the vicinity of the cell culture. If no suitable non-diseased human biopsy is available, maybe you could verify the concept with some animal cells and biopsies? Or at least with whatever add-on on top of the cell culture. Or put more effort on trying to get measurable signal from biopsy-only samples.

Please see also comments in the attachment.

Comments for author File: Comments.pdf

Author Response

We would like to thank the reviewer for his comments and suggestions. We provide a point by point answer in a separate document as well as the manuscript with track changes.

Author Response File: Author Response.docx