An In vitro Model for Bacterial Growth on Human Stratum Corneum

Abstract

The diversity and dynamics of the skin microbiome in health and disease have been studied recently, but adequate model systems to study skin microbiotas in vitro are largely lacking. We developed an in vitro system that mimics human stratum corneum, using human callus as substrate and nutrient source for bacterial growth. The growth of several commensal and pathogenic bacterial strains was measured for up to one week by counting colony-forming units or by quantitative PCR with strain-specific primers. Human skin pathogens were found to survive amidst a minimal microbiome consisting of 2 major skin commensals: Staphylococcus epidermidis and Propionibacterium acnes. In addition, complete microbiomes, taken from the backs of healthy volunteers, were inoculated and maintained using this system. This model may enable the modulation of skin microbiomes in vitro and allow testing of pathogens, biological agents and antibiotics in a medium-throughput format.

Microbially competent 3D skin: a test system that reveals insight into host–microbe interactions and their potential toxicological impact

“…This limits the choice of naturally available nutrients to peptides and lipids, restricting access to urea, ammonia, vitamins or sugars (Scharschmidt and Fischbach 2013). However, the latter will at least partially be compensated for by diffusion of glucose from the cell culture medium into the dermal and epidermal layers of the model (Khalil et al 2006;Ullah et al 2018). This provides some selectional advantage for the more invasive P. oleovorans as even a thin stratum corneum layer constitutes a considerable barrier for passive diffusion (Ullah et al 2018).…”

“…Systems used to study the pathophysiology of skin–microbe interactions (i.e., during infectious or inflammatory settings) comprise cell cultures with added bacterial endotoxins (Kampfer et al 2017 ; Lai et al 2009 ), two-dimensional cell cultures in the presence of live pathogens or the odd three-dimensional approach with bacteria added to the culture medium (Barrila et al 2017 ; Mason et al 1998 ; Mohiti-Asli et al 2014 ). Additionally, there is a stratum corneum model from van der Krieken et al ( 2016 ), where bacteria are applied to the apical surface of dead corneocytes or a model from de Breij et al ( 2012 ), where an epidermal skin equivalent is colonised by Acinetobacter species. While the latter models come close to the in vivo situation, at least structurally, they nevertheless lack the option to study microbial interaction with living keratinocytes and fibroblasts.…”

3D skin models for 3R research: The potential of 3D reconstructed skin models to study skin barrier function

“…The viability of relevant bacterial strains upon coculture with the HEEs should be determined, as the molecular approaches that are currently in use are unable to distinguish between microbial genomic DNA derived from living vs dead organisms. We recently successfully used the propidium monoazide (PMA) treatment of micro‐organisms, resulting in isolation and amplification of viable cell genomic DNA only . Finally, the window of opportunity for evaluating the effect of pre‐, pro‐ or antibiotic strategies in vitro skin microbiome models could be examined.…”

Skin microbiota and human 3D skin models

“…When working with skin‐derived microorganisms, one has to consider that the switch from the skin milieu to conventional culture media will affect the physiology of the bacteria. To study skin‐derived bacteria in vitro and to avoid physiological stress associated with changes of the environmental conditions, van der Krieken et al developed an in vitro model for bacterial growth using human stratum corneum. This relatively simple model is based on a 2% agar layer in a 24‐well plate with a 2% callus suspension, originating from the heel of healthy volunteers, on top.…”