Improving 2D and 3D Skin <em>In Vitro</em> Models Using Macromolecular Crowding

Abstract

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls. The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as "bio-scaffolds" for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications. In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Cultivation of 3D Dermal Tissue by Application of Autologous Matrix

“…There is clear need to conduct further experiments on cultivation of fibrin matrix with existing and additional donor cell lines to obtain a sufficient data set related to patient specific factors that can influence gene and growth factor expression during cell cultivation (Pillet et al, 2017;Chung et al, 2016). Further studies should be addressed to evaluate advantages and disadvantages of cell culture, cell culturing in other types of matrix (biodegradable synthetic polymers and collagen matrix), and other growth factor gene expression profiles, such as VEGF, which is an important factor in angiogenesis and essential for the production of autologous skin transplant functionality (Benny et al, 2016).…”

Controlled and tuneable drug release from electrospun fibers and a non-invasive approach for cytotoxicity testing

“…In this way, we believe that the here described model could represent an interesting way to control the diffusion of encapsulated bio-active molecules and test them using a marker-independent, non-invasive approach. In the next future we aim to test the here presented model with other drugs and more adequate in vitro cell systems 85,86 . Especially 3D models 86–88 would better mimic the in vivo behaviour than monolayer cell cultures.…”

Human skin models: From healthy to disease‐mimetic systems; characteristics and applications

“…This system was further compared with a full-thickness skin mimetic model to evaluate the performance in animal tests for skin irritants, and both models revealed capacity to correctly classify the tested compounds according to their corrosive nature (Catarino et al, 2018). Other models comprises sucrose co-polymers and fibroblasts, leading to the formation of a macromolecular assembly, which potentiates collagen deposition (Benny, Badowski, Lane, & Raghunath, 2016). A 3D human skin model containing vitrified collagen that supported the culture of dendritic cells, keratinocytes, and fibroblasts (Uchino, Takezawa, & Ikarashi, 2009) Skin-engineered substitutes may be used not only as alternatives to ex vivo and in vitro non-cell-based models for testing pharmaceutical or cosmetic ingredients, in both healthy or pathological conditions, but they can also be applied in patients for regeneration of damaged skin, especially in the treatment of burn injuries and skin wounds (reviewed in Sarkiri et al, 2019;Yu et al, 2019).…”