Design of Electroporation Process in Irregularly Shaped Multicellular Systems

Round  1

Reviewer 1 Report

In this paper, the authors studied the eletroporation phenomenon in the multicellular systems. As a neuroscientist, I am quite interested in this work since eletroporation has been widely used in the neuroscience research. I am not an expert in electrical model. So I will give my comments about this work as a neuroscientist. In general, this work is interesting and well written. However, I think it might be better if the authors could introduce more features in their test model to show the potential application of this model in physiological condition.

1. The authors used their model to study two 7-cell system. Is it possible to study the cells with different orientation of long axis? That would be more physiological since the cells will not line up in the medium.

2.  It is good that the authors took the shape of cells into the model. Is it possible to introduce mulishape cells in the 7-cell system? In most cases, we will have multishape cells in the experiment especially in the primary cultured cells.

3. It has been a struggle for most neuroscience lab using eletroporation because different types of cells such as different type of neurons and glia cells always have different efficiency in transporting molecules through the membranes. We need to adjust frequency or power to increase the efficiency. Could the authors introduce different types of cells into the 7-cell system to show whether there is any differences among types of cells? For example, neurons and glia cell may have different feature of membrane and shape. Such analysis may make this work more interesting.

4. As I mentioned before, we have to adjust frequency or power to get higher efficiency. Could the authors adjust their parameters to study the effect of different frequency or power?

Author Response

See attached file

Author Response File: Author Response.pdf

Reviewer 2 Report

In the paper, the authors make a model of a cell and calculate electroporation (i.e. pore formation) during 10 ns pulse on dense and sparse multicellular arrangement. They take into account dispersive properties of cell membranes and other compartments. They also model inclusion of a nucleus. After performing the inverse Fourier transform, the dielectric properties, described with Debye relation, can be included in the time domain calculation of pore formation.
The paper is nicely written and well structured. However, methodology regarding the spatial dimension (2D vs 3D) is not clear and needs better explanation. A comparison of their study with already existing studies is missing. Section ‘Results and Discussion’ seems more as only ‘Results’ section and the presented work is not put into the frame of existing works.
Major comments
- You claim that the built model is in 3D, although all equations are later only dependent on x and y coordinate. It is not clear what geometry you used as you keep writing about 3D, although the images (figures 1, 2, 4, 6, 7, 8) show only 2D cells. It is very confusing. Please explain.
- Line 51: what is realistic about 2D cell with sharp edges and one circular organelle inside?
- The superformula enables construction of numerous different cell shapes. Why did you decide for the chosen shape. Please comment.
- Paragraph after line 59: ‘…a medium of regular shape without any corner…’. It seems to me that there was a corner in the cells you modelled in this paper.
- Paragraph after line 59: ‘… in consists of Cn cells with an inner membrane system…’. It seems only circular nucleus was modeled. Is this means as a generalization of you model? If yes, I think it should go under discussion and possible upgrades of you model, not under M&M section, as you do not model several differently shaped organelles. For example, in a (conference) paper Denzi et al., doi: 10.1109/SMACD.2017.7981606, reticulum has been more realistically modeled (although still in 2D). Please comment.
- Eq. 5, 6: What is the sign φ? Please, explain all symbols used in equations.
- Eq. 6: Is the equation missing the permittivity from eq. 5?
- Line 78 and the text afterwards: Why did you model the dispersion of the plasma membrane in a different way than the membrane of the intracellular organelles (first order vs second order Debye expression)? Please comment in the discussion section.
- Eq. 7: Electric field E is missing from the expression
- Eq. 11 in the same as eq. 12. Is this intentional?
- Plasma membrane is the external membrane of the cell and the expression cannot be used for membranes of the intracellular structures. Please, correct it through the text. The wording for the plasma membrane and membranes of the intracellular structures in confusing. Also the symbols referring to them are confusing – why is ‘Pc’ plasma of the intracellular organelles and ‘Mc’ cell membranes? Then later you state that Pc,i1 (line 79) refers to the cytoplasm. What is correct?
- Line 82 and the text afterwards: ‘…affect TMV til the end of the pulse’. If my understanding is correct, the TMV is influenced by the increased membrane conductivity until the membrane conductivity is increased. And membrane conductivity is increased as long as pores exist in the membrane, which depends on membrane resealing and is in the pore model usually in the range of 20 seconds?
- Line 100: could you show on one cell where is this 75° angle? Why did you choose this angle?
- From figs. 4 and 6 is difficult to know what was the TMV on the cells. Consider adding a
figure where you show the TMV on each cell as a function of angle at a fixed time point. Line
114: the asymmetric distribution is impossible to see from this figure.
- Line 110: my understanding is, that TMV decreases very fast after the end of the pulse while
N stays increased for the duration of membrane resealing. Thus, I do not understand why at
t=20 ns (after the pulse ended) TMV is still increased (fig. 4)
- Could you please add numbers of cells also to figs 4, 6, 7, 8 to facilitate understanding
- Fig. 5: adapt the y-axis of figs (b) and (d) to be the same size
- Add spatial scale to all figures with shown cells
- The conclusion section is not really a conclusion as you only state the results.
- Comparison of you model with models not including the dispersion properties is lacking.
- I don’t understand, if dispersion due to intracellular organelles is already included in the
Debye relation, why do you also model the geometry of the organelles? Isn’t it redundant?
Minor comments
- Line 20: Electroporation is usually regarded electric phenomena not electromagnetic.
- Line 31: based on which assumptions? Not clear what you are referring to.
- Line 32: imaging of TMV is limited not only by the membrane thinness, also by the time range
of the phenomena which is in the range of pulse duration and is difficult to acquire with
existing imaging system.
- Line 50: The computation did not take into account the magnetic field, as you neglected the
time variation of magnetic fields.
- Paragraph after line 59: did you model the schematic cell in Fig. 1 with the superformula? Or
is this just ay drawing? It is not clear.
- Through the text, you grammatically incorrectly use ‘because’ although you should write
‘because of’.

Author Response

See attached file

Author Response File: Author Response.pdf

Round  2

Reviewer 1 Report

Instead of simply stating their model has the ability to test several conditions, I hope the authors could give examples of adjusting the parameters in the paper to show the application of their model in different physiological conditions. The 7-cell sample case is too simple. 

Author Response

1) Instead of simply stating their model has the ability to test several conditions, I hope the authors could give examples of adjusting the parameters in the paper to show the application of their model in different physiological conditions. The 7-cell sample case is too simple. 

Reply

Following the reviewer suggestion, an irregularly shaped multicellular arrangement having more complex physiological conditions has been considered and the following sentence has been added in section 3 “Finally, with the aim to highlight the broad capability of the developed numerical model to manage more complex cellular configurations in Fig. 14 has been reported an example having cells with different geometric shapes, orientation and nucleus position.”

Reviewer 2 Report

All my comments have been adequately addressed. I have only three minor comments which need addressing.

1)Paragraph after line 88: you write about improvement of membrane conductivity. Improvement implies something is getting better, you express your opinion. However, in the case of the membrane, the conductivity is increased. The same comment goes to line 174, where the EPRL of the nuclear membranes slightly ‘improve’. Again, please use expression increase and not improve.

2)Fig. 12 and fig. 13: unit for the color scale is missing.

3) Line 17, sentence ‘It is clear, the most important …’. It is not clear to what you are referring to and what is the most important interference phenomenon. Please, specify. 

Author Response

1) Paragraph after line 88: you write about improvement of membrane conductivity. Improvement implies something is getting better, you express your opinion. However, in the case of the membrane, the conductivity is increased. The same comment goes to line 174, where the EPRL of the nuclear membranes slightly ‘improve’. Again, please use expression increase and not improve.

Reply

Following the reviewer suggestion, the expressions “improvement” and “improves” have been replaced with “increase” and “increases”, respectively.

2) Fig. 12 and fig. 13: unit for the color scale is missing.

Reply

Following the reviewer highlight, unit for the color scale to Fig. 12 and Fig.13 has been added.

3) Line 17, sentence ‘It is clear, the most important …’. It is not clear to what you are referring to and what is the most important interference phenomenon. Please, specify. 

Reply

In order to provide a clear feedback to the reviewer suggestion in section 3 the sentence “It is clear, the most important interference phenomenon characterizing the packed system.” has been replaced with the following sentence: “The obtained numerical results highlight in the packed system the electric field and current flow are strongly affected by the neighboring cells.”