Addition of ROCK Inhibitors Alleviates Prostaglandin-Induced Inhibition of Adipogenesis in 3T3L-1 Spheroids

Abstract

To elucidate the additive effects of the ROCK inhibitors (ROCK-i), ripasudil (Rip) and Y27632 on bimatoprost acid (BIM-A), a prostaglandin analog (PG), on adipose tissue, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells, the most well characterized cells in the field of lipid research, were used. The cells were subjected to a variety of analyses including lipid staining, real-time cellular metabolic analysis, the mRNA expressions of genes related to adipogenesis and extracellular matrices (ECMs) as well as the sizes and physical properties of the 3D spheroids by a micro-squeezer. BIM-A induced strong inhibitory effects on most of the adipogenesis-related changes in the 2D and 3D cultured 3T3-L1 cells, including (1) the enlargement and softening of the 3D spheroids, (2) a dramatic enhancement in lipid staining and the expression of adipogenesis-related genes, and (3) a decrease in mitochondrial and glycolytic metabolic function. By adding ROCK-i to the BIM-A, most of these BIM-A-induced effects were cancelled. The collective findings reported herein suggest that ROCK-i eliminated the PG-induced suppression of adipogenesis in the 3T3-L1 cells, accompanied by the formation of enlarged 3D spheroids. Such effects of adding ROCK-i to a PG in preadipocytes on cellular properties appear to be associated with the suppression of PG-induced adverse effects, and provide additional insight into our understanding of lipid-related research.

Biofabrication of 3D adipose tissue via assembly of composite stem cell spheroids containing adipo-inductive dual-signal delivery nanofibers

“…According to the literature, at a distance greater than 100 µm from the spheroid surface, molecular signaling and glucose concentrations decrease to zero due to diffusion limitation, resulting in cell death [40,45]. In a previous study, a heterogeneous distribution of oil droplets was observed only in 3T3-L1 cells on the surfaces of spheroids with a diameter of 200-300 µm [46]. Our IHC results confirmed elevated levels of adipogenic markers characteristic of differentiated cells, consistent with previous findings in 3T3-L1 cells [47,48].…”

Unraveling White Adipose Tissue Heterogeneity and Obesity by Adipose Stem/Stromal Cell Biology and 3D Culture Models

“…On the other hand, organoids must be formed from stem cells derived from healthy or pathological tissues, having at least one physiological function corresponding to the desired organ and not necessarily showing a spheroidal 3D structure ( Table 1 ). However, most studies of adipogenic 3D models using spheroids still rely on the 3T3-L1 cell lineage (mouse preadipocytes) [ 95 , 96 , 97 , 98 ]. In fact, few articles explored the use of ASCs to form and maturate adipose tissue spheroids [ 99 ], and studies with organoid models are still recent [ 6 , 100 ].…”