Three-Dimensional Magnetic Levitation Culture System Simulating White Adipose Tissue

Abstract

White adipose tissue (WAT) has attracted interest for tissue engineering and cell-based therapies as an abundant source of adipose stem/stromal cells (ASC). However, technical challenges in WAT cell culture have limited its applications in regenerative medicine. Traditional two-dimensional (2D) cell culture models, which are essentially monolayers of cells on glass or plastic substrates, inadequately represent tissue architecture, biochemical concentration gradients, substrate stiffness, and most importantly for WAT research, cell phenotypic heterogeneity. Physiological cell culture platforms for WAT modeling must recapitulate the native diversity of cell types and their coordination within the organ. For this purpose, we developed a three-dimensional (3D) model using magnetic levitation. Here, we describe our protocol that we successfully employed to build adipose tissue organoids (adipospheres) that preserve the heterogeneity of the constituent cell types in vitro. We demonstrate the capacity of assembling adipospheres from multiple cell types, including ASCs, endohtelial cells, and leukocytes that recreate tissue organization. These adipospheres mimicked WAT organogenesis in that they enabled the formation of vessel-like endothelial structures with lumens and differentiation of unilocular adipocytes. Altogether, magnetic levitation is a cell culture platform that recreates tissue structure, function, and heterogeneity in vitro, and serves as a foundation for high-throughput WAT tissue culture and analysis.

Human adipose stromal-vascular fraction self-organizes to form vascularized adipose tissue in 3D cultures

“…Other work attempted to obtain 3-D-cultured adipose tissue models. These protocols used murine cells or organotypic-like cultures (i.e., culture of small AT explants), achieving AT-like structures with or without vascularization 27,40–44 . However, the use of exogenous ECs to form vessels inside the spheres could generate some artefacts.…”

Human Adipose-Derived Stromal/Stem Cell Culture and Analysis Methods for Adipose Tissue Modeling In Vitro: A Systematic Review

“…Spheroids exhibited improved viability and proliferation compared to non-levitated spheroids and 2D culture. Additionally, hASC experienced increased adipogenesis when cultured in spheroids compared to 2D; however, magnetic field levitation was associated with a smaller increase in adipogenesis compared to non-levitated spheroids [20].…”

“…Finally, none of the screened articles exclusively studied 2D culture; therefore, all articles included in this analysis report 3D culture methods. Data and analysis hereafter exclusively refer to the 27 articles [4,5,[8][9][10][11][12][13][15][16][17][19][20][21][22][23][24][25][26]28,30,31,33,38,44,45,48] identified to be most relevant to the central goal of this systematic review. Out of the 27 relevant articles, 26 articles used adipogenic medium to differentiate hASC into adipocytes in vitro.…”

The role of physical cues in the development of stem cell-derived organoids

“…Bone morphogenetic protein (BMP); epidermal growth factor (EGF); fibroblast growth factors (FGF); hepatocyte growth factor (HGF); insulin-like growth factor (IGF); microtubule associated protein kinase (MAPK); RHO-associated protein kinase (ROCK); transforming growth factor (TGF); vascular endothelial growth factor (VEGF); wingless-related integration site (Wnt) biological molecules (Sato et al 2009;Kurmann et al 2015;Workman et al 2017;McCracken et al 2017;Hohwieler et al 2017;Shah and Singh 2017;Chen et al 2017;Camp et al 2017;Yan et al 2018). Alternatively, organoids can be generated with the (i) suspension culture procedure accompanied by the use of spinner flasks or rotating bioreactors, which can be described as rotating cell culture systems (Nakano et al 2012;Qian et al 2018;Hoarau-Véchot et al 2018;Przepiorski et al 2018;Capowski et al 2019;Velasco et al 2020;Sander et al 2020); (ii) Air-liquid interface (ALI), where stem cells are exposed to culture medium on one side and to air on the other for maximizing the oxygen and nutrient supply (Takasato et al 2015;Neal et al 2018;Choi et al 2020;Lo et al 2020;Esser et al 2020;Gunti et al 2021); (iii) Magnetic levitation, which poses its bases in tagging cells with magnetic nanoparticles and then exposing them to a magnetic field that levitates them to the liquid-air interface where they aggregate and generate ECM components (Desai et al 2017;Tseng et al 2018;Ferreira et al 2019;Velasco et al 2020); (iv) 3D bioprinting, which could allow controlling the spatial positioning of cells and other biological components such as growth factors and ECM structural components (Fig. 3) (Duelen et al 2019;Reid et al 2019;Sun et al 2020;…”