2D/3D buccal epithelial cell self-assembling as a tool for cell phenotype maintenance and fabrication of multilayered epithelial liningsin vitro

Abstract

Maintaining the epithelial status of cells in vitro and fabrication of a multilayered epithelial lining is one of the key problems in the therapy using cell technologies. When cultured in a monolayer, epithelial cells change their phenotype from epithelial to epithelial-mesenchymal or mesenchymal that makes it difficult to obtain a sufficient number of cells in a 2D culture and to use them in tissue engineering. Here, using buccal epithelial cells from the oral mucosa, we developed a novel approach to recover and maintain the stable cell phenotype and form a multilayered epithelial lining in vitro via the 2D/3D cell self-assembling. Transitioning the cells from the monolayer to non-adhesive 3D culture conditions led to formation of self-assembling spheroids, with restoration of their epithelial characteristics after epithelial-mesenchymal transition. In 7 days, the cells within spheroids restored the apical-basal polarity, and the formation of both tight (ZO1) and adherent (E-cadherin) intercellular junctions was shown. Thus, culturing buccal epithelial cells in a 3D system allowed us to recover and durably maintain the morphological and functional characteristics of epithelial cells. The multilayered epithelial lining formation was achieved after placing spheroids for 7 days onto a hybrid matrix, which consisted of collagen layers and reinforcing poly (lactide-co-glycolide) fibers and was proven promising for replacement of the urothelium. Thus, we offer an effective technique of forming multilayered epithelial linings on carrier-matrices using cell spheroids that was not previously described elsewhere and can find a wide range of applications in tissue engineering, replacement surgery, and regenerative medicine.

Cell spheroid fusion: beyond liquid drops model

“…The revelation of more distinct mechanisms of cell migration in different types of spheroid fusion as well as the structure of the mixed fusion products requires further studies. As long as spheroids from single cell type have proven themselves to be a useful tool for studying tissue morphogenesis in vitro 58 and for obtaining tissue-engineered constructions with high cell density 6,59 , the further studies in the field of fusion of spheroids from different cell types will provide a new data on how to optimize the procedures of obtaining in vitro more complex tissue-engineered constructions that would more precisely reproduce the native tissue structure and function.…”

“…The formation of intercellular junctions in 3D cultures is one of the main processes that determine cell behavior, allows recovery and sustainability for the epithelial cell phenotype and controls the tissue structural organization 59 . Tight junctions (ZO-1+, Fig.…”

Skin tissue regeneration for burn injury

“…This is not surprising because one of the main skin component is collagen types I and III, and therefore, the product design will be more similar to the native tissue than others [139]. However, collagen possesses poor mechanical properties, and most scientists and manufacturers try to improve them via cross-linking or reinforcing with synthetic materials such as polylactide, polycaprolactone, and their copolymers [182, 183]. For instance, in TranCyte, the collagen gel is fortified with nylon mesh and covered with a silicone film; the latter enables the maintenance of moist environment.…”

Engineering a Model to Study Viral Infections: Bioprinting, Microfluidics, and Organoids to Defeat Coronavirus Disease 2019 (COVID-19)

“…Extrusion-based bioprinting is the most widely used technique[ 103 ]; however, only LIFT bioprinter can enable precise deposition at high speed and resolution and is considered to be the best option to print minor cell populations within complex 3D tissue-like structures[ 101 ]. Regarding bioinks, the most promising cell components are spheroids or organoids establish intercellular junctions and newly synthetized ECM compared to a single cell suspension and maintain cell phenotype[ 22 , 104 - 106 ]; the biomaterial component – hydrogel system – is usually presented by natural and synthetic polymers, including their conjugates such as acellularized ECM, alginate, gelatin, fibrin, hyaluronic acid, cellulose, polyethylene glycol, and Pluronic-F127. [ 101 , 107 - 109 ].…”