Three-dimensional in vitro cell model of adipose pathophysiology.

摘要

The prevalence of overweight and obese individuals has increased steadily within the United States, affecting men and women of all age groups and all racial and ethnic groups. Obesity is a contributing factor to many systemic and metabolic diseases including type-2 diabetes, hypertension, coronary heart disease, osteoarthritis, and cancer. The common mode of treatment for obesity involves low-fat diet and exercise to control the patient's body weight and blood sugar levels. An optimal treatment should include intervention at the cellular level, but available in vitro cellular models of the adipose tissue do not address the current challenges. Therefore, the overall goal of this project involved the development of an in vitro model of adipose tissue displaying induced lipid accumulation and subsequently investigating the cellular phenotypes during pathological culturing conditions mimicking that of progressing obesity. Conjugates of elastin-like polypeptide (ELP) and polyethyleneimine (PEI) were engineered as substrates for inducing 3-D organization of adipose cells. Adipocyte response to metabolic stresses relevant to clinical pathologies - more specifically, elevated extracellular free fatty acids that induce oxidative stress, promoting inflammation and apoptosis - were investigated. H35 rat hepatoma and 3T3-L1 mouse preadipocytes were used initially to develop protocols and prove feasibility. Human adipose derived stem cells (hASCs) were also implemented in the model to explore the differences between mouse and human metabolism.;Using a carbodiimide coupling scheme, we optimized reaction conditions to achieve conjugation ratios approaching 30 mol% for ELP-PEI. Our investigation has yielded a system for effectively and efficiently producing spheroids using this biocompatible and thermal-transitioning substrate. Our ELP-PEI system indicated the potential for influencing spheroid dimensions, with spheroid size inversely related to polyelectrolyte conjugation. Overall, substrates of ELP-PEI concentrations of 5 mol% or greater were determined to be optimal for cell culture based on less variability in spheroid sizes and minimum incidence of overgrown aggregates.;Following studies using undifferentiated cell lines, we conducted experiments to prove the feasibility of using our in vitro model to evaluate differentiated 3T3-L1 and hASCs. Measurements taken from spheroid micrographs indicated spheroid size increased with differentiation and fatty acid accumulation, though neither fatty acid dosage nor class significantly affected spheroid dimensions compared to controls. The increased spheroid size correlated with increased triglyceride accumulation in 3T3-L1 model cultures. Our 3-D adipogenic culture system showed a sustained overall increase in intracellular triglyceride storage and fatty acid uptake by 3T3-L1 cells compared to those in a 2-D monolayer and indicated a differential advantage provided by the 3-D spheroid culture. As such, the triglyceride accumulation was less profound in hASCs, correlated with smaller average spheroids, suggesting a relatively less complete differentiation state. CD36 and CD40 proteins, markers for cell competency for consuming fatty acids and interacting with exogenous cytokines, were consistently found to be upregulated in 3T3-L1 and hASC 3-D cultures compared to 2-D monolayers. This outcome may prove advantageous for more rapidly promoting a differentiated phenotype in adipose cell cultures for investigating the influence of exogenous drugs and nutrient treatments on a mature cell population over a shorter culture period compared to conventional methods.;Next, our 3T3-L1 spheroid model was used to test the effects of multiple metabolic insults, namely maturation in an environment of elevated fatty acids followed by acute TNF-alpha exposure. We hypothesized this treatment should invoke an inflammatory response from the adipocytes including a reduction in cell viability and changes in protein expression, ultimately leading to lipolysis (triglyceride breakdown) resulting in the release of free glycerol and fatty acids. Cell viability was reduced across cultures exposed to TNF-alpha, increased by potentially pro-inflammatory LA treatment. However, analysis of cell lysates failed to indicate reduction of intracellular triglyceride following TNF-alpha treatment. Conversely, extracellular glycerol and fatty acids both increased after TNF-alpha treatment, particularly in cultures matured with elevated fatty acid concentrations. We have concluded that glyceroneogenesis or glycerol formation from glucose provided reactants for esterification of intracellular free fatty acids, which likely made up for the triglyceride reduction from lipolysis caused by fatty acid-fasting and TNF-alpha pro-inflammatory stimuli. Taken together, we believe our model presents a more physiologically-relevant model of differentiated adipocycte organization and function than 2-D analogues which, following further development and improvement, will help to elucidate key phenotypic differences that contribute on the cellular level to health problems associated with obesity and other pathologies.