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PONE-D-21-23497Engineering solutions for biological studies of flow-exposed endothelial cells on orbital shakersPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #2: Yes

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Reviewer #1: The manuscript is clear, well-structured and contains the required protocol information in the supplementary materials file.

Reviewer #2: The paper “Engineering solutions for biological studies of flow-exposed endothelial cells

on orbital shakers” by Fernandes et al., describes two technical/engineering improvements on methods/devices for studying (endothelial) cells exposed to diverse flow patterns in an orbital shaker platform using circular cell culture chambers (wells of a 6-well plate).

General criticism:

1. The idea to avoid interference from heterogenous flow patterns (disturbed, oscillatory flow /recirculating eddies in the center vs, unidirectional, laminar flow) which elicit distinct physiological responses, is good, but not innovative. This idea has been promoted in an elegant 2018 paper by Ghim et al. The relatively small engineering innovation here is in the specific way, how the central zone of the culture dish is rendered non-adherent. A critical question, however, that has not been addressed here, is whether this really matters, i.e., whether signals originating from the cells in the disturbed central zone impact the cells elsewhere in the culture dish? The Ghim paper is quite ambivalent about this (“depends on what parameter you are looking at”). Comparisons of the physiological responses of their cells (e.g. expression / upregulation of EC activation molecules) in the laminar flow zones in the presence or the absence of cells in the center of circular culture dishes, would increase the confidence in this methodology .

2. The proposed approach for generating a multi-staining platform is technically elegant, but, in principle, not novel: A similar idea has been published by Ghim et al., 2018. This new paper seems to build on and extend the Ghim paper. Moreover, the engineering approaches for creating this multi-staining platform, appear to be technically involved and fairly cumbersome. Previous work by others (e.g., Brooks et al., Endothelium, 11:45–57, 2004) has employed a much simpler, yet effective approach, placing small circular coverslips in various locals of a circular culture dish.

3. The authors should validate their system by computational fluid dynamics (CFD) since their experimental conditions are similar to, but no identical to those used by Ghim et al.

Specific comments

1. The authors’ description of the flow patterns in the center of the dish as “turbulent’ (3 occurrences in the text) is incorrect. Rather, it is characterized as disturbed/oscillatory. For a more quantitative, engineering approach, that authors should characterize their system by CFD.

2. Immunostaining should not just be limited to visualizing VE-cadherin for EC identification, but should be extended to includee functional analysis assessing the expression/upregulation of activation molecules, such as VCAM-1 or E-Selectin

3. Figure 3 b is not publication-quality, the distinct staining patterns (immune) are not discernible.

4. Figure 4 is instructive, but in its complexity indicative for the highly complicated engineering approach. Much simpler solutions have been described in the past (see, e.g., Brooks et al., 2004)

5. Figure 5 , Panels a and b are not publication-quality

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Reviewer #1: Yes: Patricia Diaz-Rodriguez

Reviewer #2: Yes: Peter I Lelkes

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Engineering solutions for biological studies of flow-exposed endothelial cells on orbital shakers

PONE-D-21-23497R1

Dear Dr. Lovchik,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Panayiotis Maghsoudlou

Academic Editor

PLOS ONE