Title: Improving 2D and 3D skin in vitro models, using macromolecular crowding
Authors : Benny, Paula
Badowski, Cedric
Lane, E. Birgitte
Raghunath, Michael
Published in : Journal of visualized experiments : video journal
Volume(Issue) : 2016
Issue : 114
Publisher / Ed. Institution : JoVE
Issue Date: 2016
License (according to publishing contract) : Licence according to publishing contract
Type of review: Peer review (Publication)
Language : English
Subject (DDC) : 571: Physiology and related subjects
610: Medicine and health
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.
Departement: Life Sciences and Facility Management
Organisational Unit: Institute of Chemistry and Biotechnology (ICBT)
Publication type: Article in scientific Journal
DOI : 10.3791/53642
ISSN: 1940-087X
URI: https://digitalcollection.zhaw.ch/handle/11475/12194
http://europepmc.org/articles/PMC5091943?pdf=render
Appears in Collections:Publikationen Life Sciences und Facility Management

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