Abstract
Adipose tissue dysfunction is critical to the development of type II diabetes and other metabolic diseases. While monolayer cell culture has been useful for studying fat biology, 2D culture often does not reflect the complexity of fat tissue. Animal models are also problematic in that they are expensive, time consuming, and may not completely recapitulate human biology because of species variation. To address these problems, we have developed a scaffold-free method to generate 3D adipose spheroids from primary or immortal human or mouse pre-adipocytes. Pre-adipocytes self-organize into spheroids in hanging drops and upon transfer to low attachment plates, can be maintained in long-term cultures. Upon exposure to differentiation cues, the cells mature into adipocytes, accumulating large lipid droplets that expand with time. The 3D spheroids express and secrete higher levels of adiponectin compared to 2D culture and respond to stress, either culture-related or toxin-associated, by secreting pro-inflammatory adipokines. In addition, 3D spheroids derived from brown adipose tissue (BAT) retain expression of BAT markers better than 2D cultures derived from the same tissue. Thus, this model can be used to study both the maturation of pre-adipocytes or the function of mature adipocytes in a 3D culture environment.
Highlights
Adipose function and signalling play critical roles in the development of type II diabetes and other metabolic diseases
We have found that the dioxin-like polychlorinated biphenyl (PCB) 126 inhibits adipogenesis with concomitant downregulation of PPARγ, and this is associated with a strong pro-inflammatory phenotype[20]
normal pre-adipocyte (NPAD) pre-adipocyte droplets consisting of 1 million cells per milliliter of pre-adipocyte growth media were deposited on the underside of the lid of a cell culture plate using a multichannel pipette at 20 μl (20,000 cells) per droplet (Fig. 1A)
Summary
Adipose function and signalling play critical roles in the development of type II diabetes and other metabolic diseases. While animal model systems more closely resemble human conditions, they are expensive, time consuming, and due to species variation do not always faithfully predict human biology[1,2,3] To address these problems, we have developed a facile method to fabricate hundreds of 3D adipose spheroids from human and mouse pre-adipocytes that exhibit morphological and physiological behaviours that mimic in vivo adipose tissue. Because adipocytes need to be replaced, disruption of adipogenesis can lead to dysfunctional adipose tissue Certain drugs such as the thiazolinediones (the glitazones) increase adipogenesis through activation of the adipocyte-specific transcription factor PPARγ and have been used for treatment of type II diabetes because they are insulin sensitizers[7]. Because cells in 2D culture are under vastly different microenvironmental and physical conditions than in vivo tissue, cell morphology and function of 2D adipocyte cultures do not mirror their in vivo counterparts
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