The goals of this proposal are to develop in vitro models of adipose tissue that allow a superior hypertrophic growth of adipocytes and facilitate investigation of metabolic stresses and signaling mechanisms during pathological culturing conditions mimicking those of progressing obesity. Existing in vitro adipocyte culture models are not optimal: 2-D monolayer culture does not represent the 3-D adipose morphology, 3-D cell encapsulation models (e.g., hydrogels) restrict the volume of differentiating adipocytes due to compressive stress and limited porosity, and 3-D ?scaffold-free? models (e.g., hanging drop, non-adherent coatings) do not support long term culture due to spheroid loss during media changes. Consequently, the existing in vitro models result in functionally impaired adipocytes that do not reach their full growth potential, seriously limiting the study of how a full range of intracellular triglyceride (fat) deposition affects adipocyte function in the development of obesity. This proposal directly addresses this technical limitation using a novel tissue engineering approach.
Specific Aims to prepare physiologically relevant in vitro models of adiposity are to: (1) Create the stable, surface-tethered 3-D spheroid model of adipocyte culture by using an array of copolymers of biocompatible elastin-like polypeptide (ELP) and charged polyelectrolytes (PE) as coating substrates. Here the positively-charged PEs encourage spheroid formation and ELP encourages stable surface-tethering of spheroids. We will systematically investigate the effect of charge content and chemistry on 3-D spheroid organization. Our overarching hypothesis is that we will achieve superior adipocyte maturation and functionality by this surface modification method that achieves 3-D culture without using a cell-size restrictive encapsulation scaffold and achieves long term culture through surface-tethering of spheroids. (2) Define the mechanism of adipogenesis in 3-D spheroid culture and determine the functionally superior model by comparing against 2-D monolayer and 3-D hydrogel cultures. We seek to define the mechanism of enhanced adipogenesis in the context of morphological cues (cell shape through the mmp14 pathway) regulating PPAR-?, a key effector of adipogenesis. (3) Determine the effects of multiple metabolic stresses (fatty acids and TNF-?) on adipocyte phenotype, viability, and function. The stability of 3-D culture atop our ELP-PE coatings allows a substantially longer culture period. This innovation allows us to expose the optimally developing 3-D spheroid cultures to nutritionally relevant fatty acids at physiological levels. Finally, by comparing the functional and genome-wide responses of the metabolically stressed 3-D spheroids to those of primary adipocytes from obese animal (mice) and human donors, we will recapitulate the effects of metabolic stresses predominant in progressing obesity. We expect the functionally superior 3-D spheroid model to be a clinically relevant in vitro adipocyte model with the potential to invent novel therapeutics by examining drug and nutrient treatments on an in vivo-like mature cell population.
Obesity has now affected more that two-thirds of the American population and may affect many more in future. Current treatment mode prescribes low-fat diet and exercise, but developing models of adipose tissue will help understand the altered cellular biology during obesity. We will follow a novel tissue engineering approach to simulate obesity development allowing scientists to test various hypotheses, study altered metabolic pathways due to the disease state, and therefore, facilitate invention of new therapeutics.
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|Turner, Paul A; Garrett, Michael R; Didion, Sean P et al. (2018) Spheroid Culture System Confers Differentiated Transcriptome Profile and Functional Advantage to 3T3-L1 Adipocytes. Ann Biomed Eng 46:772-787|
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