52 SKIN MICROBIOME
Figure 3: Reconstructed human epidermis (EpiSkin™). Red circles highlight very early signs of microcolony formation.
2: Assessing bacterial colonisation in the 3-dimensional, reconstructed skin For the second challenge, we wanted to scale up to robustness of the cell model to introduce in vitro 3-dimensional (3D), skin models. We selected a commercially available human reconstructed epidermis model. This model, known as EpiSkin™, is a validated model for use in the assessment of skin irritation and skin corrosion for cosmetic products. What differed from that of the in vitro keratinocyte model, is not only the 3-dimensional structure and overall robustness, but the similarities of this model to the epidermal layer of the skin. This model sits within culture on an air-liquid interface, which means that the surface of the model, or ‘skin’, is exposed to air and in effect dry, similar to human skin. The intention of this experiment was to determine the attachment of the same strain of S. aureus on the surface of the skin. Bacterial attachment was assessed in a time and dose-dependent manner. The output of this assay was the microbiological quantification of attached bacteria to the skin and the imaging of bacterial attachment using scanning electron microscopy (SEM). The results of this study also showed positive S. aureus attachment between the contact times of 60 and 240 minutes. The degree of bacterial attachment to the skin model was also in a dose and time dependent manner, as seen with the keratinocyte model. In addition, we noted excellent reproducibility and good cell viability. The beauty of using such models in this way means that we can also assess the active soluble factors from the tissue in response to the addition of bacteria. Such downstream analysis means that we can also assess parameters such as collagen production and inflammation in the same model. Upon visualisation of the surface of the skin
we identified S. aureus attachment. What was incredibly interesting is that we saw very early signs of microcolony formation, indicative of biofilm formation. This highlights even further the natural propensity for microorganisms to form micro-communities to encourage bacterial cell communication, nutritional support and protection.
PERSONAL CARE January 2021
Figure 4: Biofilm formation detected.
3: Assessing bacterial colonisation in wounded skin (E.g. compromised wound barrier) The next step beyond the colonisation of 3D skin, was to assess bacterial attachment to skin whereby the epidermal barrier is compromised, not too dissimilar to that of certain skin conditions such as acne. To assess this, we scaled up the use of a 3D
epidermis model to a commercially available 3D full thickness skin model. A full thickness skin model not only contains the keratinocyte rich epidermal layer but it also contains a fibroblast- rich dermal layer (we use these models quite frequently to assess wound closure). We obtained this model pre-wounded and applied S. aureus for a much longer contact time, to simulate a worst-case scenario. From this, we imaged the bacterial attachment inside the wound bed. The bacterial attachment at 24 hours
was extremely significant, with indications of biofilm formation. We also noted that the presence of bacteria within the compromised skin resulted in the impediment of wound closure. This model presents as an excellent example of modelling skin pathologies. In the context of the skin microbiome
and products to support this area there are different ways we can use these models to show different modes of action. Firstly, we can apply a formulation before the addition of bacteria to assess whether the formulation can prevent or block microbial attachment. We can also use the same approach to determine whether the application of the product can increase or decrease the attachment and growth of microorganisms on the skin. It is also possible to see how a product could disrupt existing microbial populations, for example the use of cleansers or toners, in which case the effect of your product can be evaluated against microbial removal or a change in mixed species populations.
Conclusion To summarise, what we have presented are viable in vitro models that can be used at various stages of the personal care product development pipeline. For instance, high throughput hard
surface models are highly beneficial when at the proof-of-concept phases of product development and wishing to narrow down candidate formulations. From that point onwards, the level of complexity of each model increases to suit the evaluation of the selected formulations or even market-ready products. With increased complexity we move one step closer to ‘real-life’ scenarios but also increase the amount of information that we can obtain as an end point. The impact of the body’s microbiome is
increasingly being linked to health, and not just skin health, but gut health relating to pathologies such as inflammatory bowel diseases, obesity or research into the gut-brain axis. This article highlights the importance of
challenging skin microbiome products using robust, reproducible science to help define the capability of product. In doing so, there is emphasis on the importance of having valid and reproducible data, and that we also use models that focus on a real-world relevance. Whether that is using live skin models or testing whole skin care regimes. With this, we hope to set a standard across the board to support with the regulatory guidance for the skin microbiome products of the future.
Acknowledgment The data and images in Figures 1 to 4 were obtained by Perfectus Biomed during a study commissioned by Symrise. We would like to thank Symrise for their permission to publish.
References 1
https://hmpdacc.org/ 2 Prescott SL, Larcombe DL, Logan AC, et al. The skin microbiome: impact of modern environments on skin ecology, barrier integrity, and systemic immune
programming.World Allergy Organ J 2017; 10: 29.
3 Findley K, Grice EA. The Skin Microbiome: A Focus on Pathogens and Their Association with Skin Disease. PLoS Pathog 2014; 10: (11)
4 Kong HH, Oh J, Deming C, Conlan S, Grice EA, et al. Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res 2012; 22: 850–859
5
https://thesecretlifeofskin.com/2020/06/19/ pre-pro-postbiotics-back-to-basics/
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