Biomimetic Full-Thickness Skin-on-a-Chip Based on a Fibroblast-Derived Matrix

Round 1

Reviewer 1 Report

The paper report on the comparison of reconstructed skin cultured on a microfluidic chip or in static conditions. The paper is well written and shows interesting differences between the two culture conditions. 

There are only a few remarks:

1. Title: I would not write "an in vivo-like microenvironment". The in vivo microenvironment of the skin is much more complex.
2. Lines 30-31. is not only the elderly population that is affected by chronic skin diseases. Atopic dermatitis for example is found more often in children. Reference 1 is not appropriate to support this sentence.
3. Fig. 6 and 7. It would be useful to have an analysis of fluorescence intensity of several sections using imageJ.

 

Author Response

Reviewer 1

1.Title: I would not write "an in vivo-like microenvironment". The in vivo microenvironment of the skin is much more complex.

We agree with the reviewer. The sentence “in vivo-like microenvironment” could be misleading. Therefore, we altered the title of the manuscript to “Biomimetic full-thickness skin-on-a-chip based on a fibroblast-derived matrix”. Considering that the use of a fibroblast-derived matrix to recreate the dermal compartment is one of the main features of our skin-on-a-chip model, we think it is appropriate to mention it in the title.

2.Lines 30-31. is not only the elderly population that is affected by chronic skin diseases. Atopic dermatitis for example is found more often in children. Reference 1 is not appropriate to support this sentence.

Thank you for pointing out the problem with the reference. We agree that the reference is not appropriate to support the affirmation and we’ve changed it accordingly. Furthermore, we have removed the reference to the elderly population to make the sentence more comprehensible.

(Lines 30-31) The global topical drug delivery market is growing fast due to the increasing prevalence of chronic skin diseases [1].

[1] Seth, D.; Cheldize, K.; Brown, D.; Freeman, E.F. Global Burden of Skin Disease: Inequities and Innovations. Curr. Dermatol. Rep. 2017, 6, 204–210, doi:10.1007/s13671-017-0192-7

3.Fig. 6 and 7. It would be useful to have an analysis of fluorescence intensity of several sections using imageJ.

We agree with the reviewer. As suggested, we have added the analysis of the fluorescence intensity using software ImageJ for figures 4, 6 and 7.

Author Response File: Author Response.docx

Reviewer 2 Report

The article Biomimetic full-thickness skin-on-a-chip with an in vivo-like 2 microenvironment is an interesting article describing preparation skin on a chip. The article organization, as well as the choice of methods of characterization are scientifically sound and describe the potential of the prepared model.

Nevertheless, there are parts of the text that need either clarification of require correction, therefore I suggest minor revision of the article. If the authors are willing to address the following issues or comment on the below stated suggestions accordingly, I would reconsider the publication of this article in the Micro.

GENRAL REMARKS

1. You are writing about 3D full-thickness SoC, however your model does not include subcutis, which is a part of skin. So you have just dermis with epidermis and endothelial cells included.
2. Please define the difference of your skin on a chip regarding described in this article: Skin-on-a-chip model simulating inflammation, edema and drug-based treatment MaierdanjiangWufuer1,2,*, GeonHui Lee3,*, Woojune Hur1,2, Byoungjun Jeon1,2, Byung Jun Kim1, Tae Hyun Choi1 & SangHoon Lee3,4, 2016, Scientific Reports

 

CONCRETE REMARKS

Results

1. Figure 7 should be moved to the section where is mentioned (into 3.3.).

Discussion

1. You are mentioning the influence of mechanical forces on the physiology of tissue – it would make sense to show also the mechanical properties of your construct – the mechanical properties should be similar to skin, for be able to react similar to mechanical forces.
2. Page 15, line 525: Increased thickness… regarding what? The thickness should simulate the structure of the skin.

Author Response

Reviewer 2

 

General Remarks

1.You are writing about 3D full-thickness SoC, however your model does not include subcutis, which is a part of skin. So you have just dermis with epidermis and endothelial cells included.

In the area of tissue engineering, the term full-thickness skin is commonly used to describe models that include the epidermal and dermal compartments. This term probably originates from the field of skin transplantation, in which a full-thickness skin graft consists of a complete epidermis and dermis (Ramsey, 2018). In the context of 3D skin models, please see below multiple examples of this term described in the literature:

 

“More advanced models represent the full-thickness skin based on the fibroblast populated collagen matrices (dermis equivalent) and an epidermal overlay representing NHKs” (Semlin et al., 2011).

 

“Already well-established are full thickness (FT) models which consist of dermal compartment enabling cellular cross-talk between keratinocytes and fibroblasts.” (Mathes et al 2014)

 

“3D full thickness skin models which contain both epidermal and dermal layers have also been developed for a variety of applications and studies…” (Pupovac et al 2018)

 

“More complex full thickness skin models include both the dermal and epidermal components.” (Roger et al, 2019)

2.Please define the difference of your skin on a chip regarding described in this article: Skin-on-a-chip model simulating inflammation, edema and drug-based treatment Maierdanjiang Wufuer1,2,*, GeonHui Lee3,*, Woojune Hur1,2, Byoungjun Jeon1,2, Byung Jun Kim1, Tae Hyun Choi1 & SangHoon Lee3,4, 2016, Scientific Reports

In the introduction, we describe the state-of-the-art skin-on-a-chip models. We briefly describe the work by Wufuer et al:

 

(Lines 54 – 61) More recently, skin models generated on-chip have been reported. Wufuer et al developed a SoC consisting of 3 cell layers (keratinocytes, fibroblasts and endothelial cells) separated by two porous membranes to simulate inflammation and edema [21]. Ramadan et al further explored the potential of the SoC devices by including an immune component, describing the interaction between the HaCaT cell line and a monocytic cell line in a bi-channel microfluidic device [22]. Although these studies replicated some important features of healthy and diseased skin, they included monolayer systems that do not replicate the 3D environment of in vivo human skin.

The model described by Wufuer et al does not include a 3D fully-differentiated epidermal and dermal compartment. The group simulated the different skin layers by culturing monolayers of keratinocytes and fibroblasts in porous membranes. This model was successful in simulating some features of inflammation and edema. However, it does not represent the complexity and 3D architecture of the native human skin. Our work focuses on the recreation of the 3D skin architecture on-chip. We achieve this by integrating a 3D porous scaffold on the platform and by culturing the epidermal cells for approximately 2 weeks under air-liquid interface to achieve a fully-differentiated architecture. This strategy increased the physiological relevance of the developed skin models.

Concrete remarks

Results

1.Figure 7 should be moved to the section where is mentioned (into 3.3.).

Author response: We agree with the reviewer. As suggested, we have moved figure 7 to the section where it is mentioned.

Discussion

1.You are mentioning the influence of mechanical forces on the physiology of tissue – it would make sense to show also the mechanical properties of your construct – the mechanical properties should be similar to skin, for be able to react similar to mechanical forces.

Author response: This manuscript compares the skin constructs developed on-chip and off-chip (controls) using histological and immunofluorescence analysis as well as transepithelial resistance measurements and permeation assays. These are the most commonly used techniques to study the architecture and physiology of the skin. We agree with the reviewer that it would be interesting to perform a more in-depth analysis by studying the mechanical properties of the skin models such as the determination of the young’s modulus. However, in the context of the skin-on-a-chip model, the most relevant forces are fluid-induced forces such as shear stress, imparted by the fluid exerting a tangential force on the cells. This force mainly depends on the fluid’s properties such as fluid velocity and it has been simulated using finite element modelling (Figure A2). The interstitial flow (fluid flow through the 3D matrix) around the fibroblasts also provides a specific mechanical environment to the cells. Future experiments should be performed to determine interstitial flow velocity using Darcy’s law which relates velocity to the pressure gradient and the hydraulic conductivity of the dermal compartment (Lines 524-526).

2.Page 15, line 525: Increased thickness… regarding what? The thickness should simulate the structure of the skin.

Author response: Thank you for pointing out that the sentence was incomplete. We have produced skin models with increased thickness compared to static models (controls). The epidermal thickness in the SoC model better simulates the thickness of the native epidermis [83.7±17 μm (Sandby-Møller et al, 2003)]. We corrected the text:

(Lines 538 - 540) Using the developed OoC platform, we generated a mature, pluristratified and orthokeratinized epidermal construct with increased thickness and barrier function compared to the static models.

Author Response File: Author Response.docx