Adipose Tissue Engineering in Three-Dimensional Levitation Tissue Culture System Based on Magnetic Nanoparticles

Abstract

White adipose tissue (WAT) is becoming widely used in regenerative medicine/cell therapy applications, and its physiological and pathological importance is increasingly appreciated. WAT is a complex organ composed of differentiated adipocytes, stromal mesenchymal progenitors known as adipose stromal cells (ASC), as well as endothelial vascular cells and infiltrating leukocytes. Two-dimensional (2D) culture that has been typically used for studying adipose cells does not adequately recapitulate WAT complexity. Improved methods for reconstruction of functional WAT ex vivo are instrumental for understanding of physiological interactions between the composing cell populations. Here, we used a three-dimensional (3D) levitation tissue culture system based on magnetic nanoparticle assembly to model WAT development and growth in organoids termed adipospheres. We show that 3T3-L1 preadipocytes remain viable in spheroids for a long period of time, while in 2D culture, they lose adherence and die after reaching confluence. Upon adipogenesis induction in 3T3-L1 adipospheres, cells efficiently formed large lipid droplets typical of white adipocytes in vivo, while only smaller lipid droplet formation is achievable in 2D. Adiposphere-based coculture of 3T3-L1 preadipocytes with murine endothelial bEND.3 cells led to a vascular-like network assembly concomitantly with lipogenesis in perivascular cells. Adipocyte-depleted stromal vascular fraction (SVF) of mouse WAT cultured in 3D underwent assembly into organoids with vascular-like structures containing luminal endothelial and perivascular stromal cell layers. Adipospheres made from primary WAT cells displayed robust proliferation and complex hierarchical organization reflected by a matricellular gradient incorporating ASC, endothelial cells, and leukocytes, while ASC quickly outgrew other cell types in adherent culture. Upon adipogenesis induction, adipospheres derived from the SVF displayed more efficient lipid droplet accumulation than 2D cultures. This indicates that 3D intercellular signaling better recapitulates WAT organogenesis. Combined, our studies show that adipospheres are appropriate for WAT modeling ex vivo and provide a new platform for functional screens to identify molecules bioactive toward individual adipose cell populations. This 3D methodology could be adopted for WAT transplantation applications and aid approaches to WAT-based cell therapy.

Three-dimensional cell culturing by magnetic levitation

“…The MLM generally uses the magnetic nanoparticle assembly to promote delivery of the magnetic nanoparticles, making the MLM broadly applicable to most cell types 13,17,18 . Indeed, the MLM has been successfully used to make 3D cultures with all cell types tested to date, including cell lines, stem cells and primary cells 13,[17][18][19][20][21][22] (Table 1).…”

“…Indeed, the MLM has been successfully used to make 3D cultures with all cell types tested to date, including cell lines, stem cells and primary cells 13,[17][18][19][20][21][22] (Table 1). The most basic application of the MLM is to culture 3D cell cultures under different biochemical or environmental conditions, and then analyze them using common biological research techniques, such as IHC 17,18 and western blotting 19 . The ability to magnetically manipulate 3D cultures also allows for fine spatial control and more complex environments.…”

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting

“…Another successful example is the magnetic nanobeads-mediated spheroid formation [41][42][43][44][45][46][47][48][49] . This method (also known as 'magnetic levitation' when the cells are collected on top of the fluid in a tissue culture well rather than on its bottom 41 ), is extremely versatile.…”

“…This method (also known as 'magnetic levitation' when the cells are collected on top of the fluid in a tissue culture well rather than on its bottom 41 ), is extremely versatile. It has been used in several high-profile studies, being applied for 'bioprinting' of complex structures, such as valves 48 , bronchioles 45 , or adipose tissue 46 . Similar methods are being actively developed in other settings as well 50,51 .…”

A better brew

“…Magnetic levitation has been a good way to model adipose tissue, Kolonin adds, and to culture stem cells while retaining their ability to differentiate 7 .…”