Functional evaluation of novel molecules that promote stem cell mediated endogenous repair often require multiplexed in vivo transplant studies that are low throughput and hinder the rate of discovery. Here, we optimized and miniaturized a previously developed muscle endogenous repair (MEndR) in vitro assay that captures significant events of in vivo muscle endogenous repair to offer greater throughput for functional validation studies. The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), prospectively isolated from mouse skeletal muscle tissue, are engrafted onto the engineered template. A regenerative milieu is then introduced by injuring the muscle tissue with a myotoxin. We evaluated two different commercially available human primary myoblast lines and were able to successfully generate miniaturized 3D muscle templates, as well as recapitulate the in vivo outcomes of a known modulator of MuSC mediated repair but only in the presence of both the stem cells and the regenerative milieu. Thus, the mini-MEndR culture assay captures the ability of different molecular treatments to modulate donor MuSC skeletal muscle production and niche repopulation. The miniaturized predictive assay offers a simple, scaled platform with which to investigate MuSC endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.

Obesity prevalence has reached pandemic proportions, leaving individuals at high risk for the development of diseases such as cancer and type 2 diabetes. In obesity, to accommodate excess lipid storage, adipocytes become hypertrophic, which is associated with an increased pro-inflammatory cytokine secretion and dysfunction of metabolic processes such as insulin signaling and lipolysis. Targeting adipocyte dysfunction is an important strategy to prevent the development of obesity-associated disease. However, it is unclear how accurately animal models reflect human biology, and the long-term culture of human hypertrophic adipocytes in an in vitro 2D monolayer is challenging due to the buoyant nature of adipocytes. Here we describe the development of a human 3D in vitro disease model that recapitulates hallmarks of obese adipocyte dysfunction. First, primary human adipose-derived mesenchymal stromal cells are embedded in hydrogel, and infiltrated into a thin cellulose scaffold. The thin microtissue profile allows for efficient assembly and image-based analysis. After adipocyte differentiation, the scaffold is stimulated with oleic or palmitic acid to mimic caloric overload. Using functional assays, we demonstrated that this treatment induced important obese adipocyte characteristics such as a larger lipid droplet size, increased basal lipolysis, insulin resistance and a change in macrophage gene expression through adipocyte-conditioned media. This 3D disease model mimics physiologically relevant hallmarks of obese adipocytes, to enable investigations into the mechanisms by which dysfunctional adipocytes contribute to disease.

Obesity prevalence has reached pandemic proportions, leaving individuals at high risk for the development of diseases such as cancer and type 2 diabetes. In obesity, to accommodate excess lipid storage, adipocytes become hypertrophic, which is associated with an increased pro-inflammatory cytokine secretion and dysfunction of metabolic processes such as insulin signaling and lipolysis. Targeting adipocyte dysfunction is an important strategy to prevent the development of obesity-associated disease. However, it is unclear how accurately animal models reflect human biology, and the long-term culture of human hypertrophic adipocytes in an in vitro 2D monolayer is challenging due to the buoyant nature of adipocytes. Here we describe the development of a human 3D in vitro disease model that recapitulates hallmarks of obese adipocyte dysfunction. First, human primary adipose-derived stromal cells are embedded in hydrogel, and infiltrated into a thin cellulose scaffold. The thin microtissue profile allows for efficient assembly and image-based analysis. After adipocyte differentiation, the scaffold is stimulated with oleic or palmitic acid to mimic caloric overload. Using functional assays, we demonstrated that this treatment induced important obese adipocyte characteristics such as a larger lipid droplet size, increased basal lipolysis, insulin resistance and activation of macrophages through adipocyte-conditioned media. This 3D disease model mimics physiologically relevant hallmarks of obese adipocytes, to enable investigations into the mechanisms by which dysfunctional adipocytes contribute to disease.