Open-Source Culture Platform for Multi-Cell Type Study with Integrated Pneumatic Stimulation

Round 1

Reviewer 1 Report

The aim of Authors was to design and manufacture a small device which will modify the hydrostatic pressure in the standard cell culture conditions. They also assumed that this device should work with a commonly used multiwell plates placed in the standard cell incubators to keep the other environmental conditions like temperature and CO2 content stable. It looks like the set goals were fulfilled.  The paper presents the final result of the authors work in the form of the culture chamber, made from PMMA, equipped with diaphragm pump and dedicated electronic control system, based on ARDUINO Nano 3.0 board with ATmega328 microcontroller. This system can modify the pressure inside the cell chamber in preprogrammed time pattern. Authors performed all expected test like airtightness of the chamber and pressure and temperature stability. All those test were successful, demonstrating that the chamber is working properly. Then, this system was employed in the experiment, where proliferation tests were performed on two cell lines – human epidermal keratinocytes (HaCaT line) and human foreskin fibroblasts (HFF-1 line) – where one sample was grown in standard conditions serving as a control and the other sample was exposed to the cycling changing differential pressure of 12 kPa for 5 sec. duration time. The results have shown that there is no difference between control and treated cells. However, the device performed according to its specifications.

However, as one can notice, authors cite another paper (pos.9 in the Reference section) which describes the design and application of exactly the same device, even most of the  figures are the same. I do not see any benefit to public by publishing again the same design, fabrication procedure and performed tests. In contrary, I would not recommend publishing anything which artificially inflates the scientific achievements of authors. In my opinion, the paper is not ready to be accepted for publication in the present form. If Authors would like to proceed further with the material included in the reviewed manuscript, they should focus only on the description of electronics and software designed for this device and refer to the publication [9] for the other details.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Good production of teh device. The connection to cancer development is unclear and must be explained in more detail. Here significant literature must be given and discussed. Tests with fibroblasts are far away from the in vivo situation.

3D printing is increasingly important for the rapid prototyping and of advanced and tailor-made cell culture devices. Today, 3D printing is implemented in medical and biotechnological industry and will profoundly revolutionize these areas. Within the manuscript, the authors present a versatile Arduino based pneumatic system for the stimulation of a cell culture performed in a standard multi-well plate, designed to work inside a CO2 incubator. This system is capable to modify the hydrostatic pressure inside a cell culture plate thus providing a mechanical hydrostatic stimulus to a cell culture growing inside it.

Hydrostatic pressure regulates different cell behaviors like proliferation, differentiation, migration and apoptosis. Different tissue systems respond to hydrostatic pressure: tissues of the brain, eye, vasculature and bladder as well as articular cartilage. Abnormal hydrostatic pressure is associated with pathologies including glaucoma and hypertensive fibrotic remodeling. There is less information published that hydrostatic pressure causes cancer but the ability to quantify and alter hydrostatic pressure related volume regulation of cells may someday hold promise for killing cancer cells. The authors give a good overview of the produced pneumatic system, thereby discussing the advantages and the limitations. The design and the fabrication of the developed pneumatic device is quite good. The article provides a comprehensive description of the experiments. The list of materials and equipment needed looks complete. Most of the steps described are clearly explained. The construction/manufacturing part within this project is ok. In contrast, the cell biological characterization is by no means of best quality since the authors perform all analyses with cell lines. Especially fibroblasts are unsuited. The used cell lines are far away from the in-vivo situation.

 

Although this was hard work, the authors should improve their analyses and discussion concerning the following points:

-       Mechanical stimuli could be compression, tension, hydrostatic pressure, or fluid shear forces. The authors reduce this diversity to hydrostatic pressure.

-       The authors should strongly distinguish between cell culture effects (in –vitro experiments) and human cancer.

-       The authors have to present a sound biological interpretation of cancer development caused by hydrostatic pressure.

-       At least all experiments were performed without any biological variance. Therefore, the significance of the results is unclear.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have assembled a detailed method of hydrostatic conditions for cell culture. The method is presented clearly and applies it to cell culture with a reasonable O.D. readout for this initial manuscript. This paper is deserving of publication in this journal without further modification.

Author Response

Dear reviewer,

we sincerely appreciated your comment, and we hope to see our work published in the MDPI Electronics Journal.

Thank you for your kind consideration of our work.

 

Kind regards,

The submitting author

Nicolò Cacocciola

Round 2

Reviewer 1 Report

The manuscript still has parts literaly taken from the work [9]. This is not acknowledged in any way, nor I can see any agreement with the editors of "Micro and Nano Engineering" that authors can use already published figures. I still insist that authors remove any content which is already published.

Author Response

Dear reviewer, 

thank you very much for your suggestion. We followed your instructions deleting all the contents already published in our previous work, thus focusing the paper only on the electronics and software development, as you kindly suggested. We really hope that the revised version of our manuscript is now appropriate for publication in the MPDI Electronics Journal 

Best regards
Nicolò Cacocciola

Reviewer 2 Report

Thank you for the answers. sounds clear.

Author Response

Dear reviewer, 

thank you very much for your answer. We hope to see our work published on the MDPI Electronics journal

Best regards

The submitting author 

Nicolò Cacocciola

Round 3

Reviewer 1 Report

Thank you very much for introducing requested changes.