Tridimensional cell culture of dermal fibroblasts promotes exosome-mediated secretion of extracellular matrix proteins

Abstract

Extracellular matrix (ECM) secretion, deposition and assembly are part of a whole complex biological process influencing the microenvironment and other cellular behaviors. Emerging evidence is attributing a significant role to extracellular vesicles (EVs) and exosomes in a plethora of ECM-associated functions, but the role of dermal fibroblast-derived EVs in paracrine signalling is yet unclear. Herein, we investigated the effect of exosomes isolated from stimulated human dermal fibroblasts. We report that tridimensional (3D) cell culture of dermal fibroblasts promotes secretion of exosomes carrying a large quantity of proteins involved in the formation, organisation and remodelling of the ECM. In our 3D model, gene expression was highly modulated and linked to ECM, cellular migration and proliferation, as well as inflammatory response. Mass spectrometry analysis of exosomal proteins, isolated from 3D cultured fibroblast-conditioned media, revealed ECM protein enrichment, of which many were associated with the matrisome. We also show that the cytokine interleukin 6 (IL-6) is predicted to be central to the signalling pathways related to ECM formation and contributing to cell migration and proliferation. Overall, our data suggest that dermal fibroblast-derived EVs participate in many steps of the establishment of dermis’s ECM.

Biomaterial-enabled 3D cell culture technologies for extracellular vesicle manufacturing

“…104 For example, Clément et al cultured dermal fibroblasts under ascorbic acid rich media for 28 days to produce a free-standing Fibroblast sheet composed of neosynthesized ECM protein meshwork. 138 Using this 3D culture they found significantly increased levels of 7 different MMP proteins and 3 different TIMP proteins in EVs derived from dermal epithelial cells in the cell media. 138 Overall, 3D cultures with biomimetic structures and microenvironment promote the cell cultures in vitro to produce EVs with enhanced expression of various cell adhesion regulating proteins that closely mimic the natural complex tissue in vivo .…”

“…EVs play crucial roles in various biological processes, including EV biogenesis, 135–137 cell adhesion, 93,104,138 cell proliferation and migration, 89,103,138 and immunomodulation (Fig. 4).…”

“…4). 103,138 These processes are facilitated by the complex mixture of protein and nucleic acid cargo present in EVs. For instance, cargo proteins like Alix and TSG101 promote EV biogenesis, 136,137 while transmembrane proteins like CD9, CD63, and CD81 on EV surfaces promote vesicle trafficking.…”

The Skin Microbiome: Current Landscape and Future Opportunities

“…Three-dimensional human skin equivalents provide a more dynamic way to explore cutaneous physiology, comprising epidermal and dermal structures produced by skin cells. They more closely model natural cellular behaviours by providing a more native ECM and enabling true paracrine signalling between cells [ 281 ]. As stratification is an important component of skin physiology and host response, a major aim of developing 3D constructs is to create an epidermal barrier that closely resembles native skin, with differentiated layers and a similar composition of epidermal lipids [ 282 ].…”

In Vitro 3D Modeling of Neurodegenerative Diseases

“…In addition, 2D culture also modifies cell division and triggers some phenotypic loss, a polarity change which can enhance apoptosis, and/or increase proliferation [51][52][53]. Another drawback of 2D culture is that monolayer culture may alter gene and protein expression levels, as well as extracellular vesicle release, when compared to a 3D environment [52,[54][55][56]. Furthermore, since the culture medium is homogeneously distributed, cells initially have unrestricted access to nutrients, oxygen, and growth factors, but over time, the levels decrease in amounts until the next culture medium renewal.…”