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Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 μm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a global health threat that affects 10 million people worldwide. Human Immunodeficiency Virus (HIV) remains one of the major contributors to the reactivation of asymptomatic latent tuberculosis (LTBI). Over the recent years, there has been a significant focus in developing in-vitro 3D models mimicking early events of Mycobacterium tuberculosis (Mtb) pathogenesis, especially formation of the granuloma. However, these models are low throughput and require extracellular matrix. In this article, we report the generation of a matrix-free 3D model, using THP-1 human monocyte/macrophage cells and mCherryexpressing Mycobacterium bovis BCG (Bacilli Camille Guérin), henceforth referred as 3D spheroids, to study the host cell-bacterial interactions. Using mCherry-intensitybased tracking, we monitored the kinetics of BCG growth in the 3D spheroids. We also demonstrate the application of the 3D spheroids for testing anti-TB compounds such as isoniazid (INH), rifampicin (RIF), as well as a host-directed drug, everolimus (EVR) as single and combinational treatments. We further established a dual infection 3D spheroid model by coinfecting THP-1 macrophages with BCG mCherry and pseudotype HIV. In this HIV-TB co-infection model, we found an increase in BCG mCherry growth within the 3D spheroids infected with HIV pseudotype. The degree of disruption of the granuloma was proportional to the virus titers used for co-infection. In summary, this 3D spheroid assay is an useful tool to screen anti-TB response of potential candidate drugs and can be adopted to model HIV-TB interactions. [ABSTRACT FROM AUTHOR]

Myoblast differentiation is a key step in myogenesis and has long been considered to be controlled mainly by biochemical cues such as growth factors. However, the tissue engineering approaches based on biochemical cues demonstrate low reproducibility as a precise spatial control over their bioactivity is challenging. Recently, substrate micro/nano-structure and electro-responsive properties are recognized for their important roles in myoblast differentiation. In this study, we hypothesized that engineering biophysical features such as nano/micro-fibrous structure and conductive properties into a single biomaterial scaffold will instruct the myoblasts to differentiate into multinucleated myotubes even in the absence of differentiation media. We fabricated nanocomposite scaffolds composed of conductive graphene nanosheets and polycaprolactone (PCL), a widely used biocompatible material. The resulting graphene-PCL scaffolds possess excellent conductivity due to graphene nanosheets and great processability, biodegradability and elastic mechanical properties conferred by PCL. Additionally, physicochemical and mechanical properties of nanocomposite scaffolds can be tuned by varying graphene concentration. Further, graphene-PCL nanocomposites and their 8-week degradation products exhibited remarkable cytocompatibility and promoted adhesion and proliferation of C2C12 mouse myoblast cells. Importantly, these nanocomposite scaffolds induced graphene concentration-dependent differentiation of C2C12 cells into multinucleated myotubes even in normal growth media suggesting their cell-instructive potential. Thus, graphene-PCL nanocomposite scaffolds can serve as a strategy to promote skeletal muscle regeneration without biochemical cues. [ABSTRACT FROM AUTHOR]

Despite significant investments in cancer research and drug discovery/development, the rate of new cancer drug approval is ≤5% and most cases of metastatic cancer remain incurable. Ninety-five percent of new cancer drugs fail in clinical development because of a lack of therapeutic efficacy and/or unacceptable toxicity. One of the major factors responsible for the low success rate of anticancer drug development is the failure of preclinical models to adequately recapitulate the complexity and heterogeneity of human cancer. For throughput and capacity reasons, high-throughput screening growth inhibition assays almost exclusively use two-dimensional (2D) monolayers of tumor cell lines cultured on tissue culture-treated plastic/glass surfaces in serum-containing medium. However, these 2D tumor cell line cultures fail to recapitulate the three-dimensional (3D) context of cells in solid tumors even though the tumor microenvironment has been shown to have a profound effect on anticancer drug responses. Tumor spheroids remain the best characterized and most widely used 3D models; however, spheroid sizes tend to be nonuniform, making them unsuitable for high-throughput drug testing. To circumvent this challenge, we have developed defined size microwell arrays using nonadhesive hydrogels that are applicable to a wide variety of cancer cell lines to fabricate size-controlled 3D microtumors. We demonstrate that the hydrogel microwell array platform can be applied successfully to generate hundreds of uniform microtumors within 3-6 days from many cervical and breast, as well as head and neck squamous cell carcinoma (HNSCC) cells. Moreover, controlling size of the microwells in the hydrogel array allows precise control over the size of the microtumors. Finally, we demonstrate the application of this platform technology to probe activation as well as inhibition of epidermal growth factor receptor (EGFR) signaling in 3D HNSCC microtumors in response to EGF and cetuximab treatments, respectively. We believe that the ability to generate large numbers of HNSCC microtumors of uniform size and 3D morphology using hydrogel arrays will provide more physiological in vitro 3D tumor models to investigate how tumor size influences signaling pathway activation and cancer drug efficacy. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a public health concern that impacts 10 million people around the world. Current in vitro models are low throughput and/or lack caseation, which impairs drug effectiveness in humans. Here, we report the generation of THP-1 human monocyte/macrophage spheroids housing mycobacteria (TB spheroids). These TB spheroids have a central core of dead cells co-localized with mycobacteria and are hypoxic. TB spheroids exhibit higher levels of pro-inflammatory factor TNFα and growth factors G-CSF and VEGF when compared to non-infected control. TB spheroids show high levels of lipid deposition, characterized by MALDI mass spectrometry imaging. TB spheroids infected with strains of differential virulence, Mycobacterium tuberculosis (Mtb) HN878 and CDC1551 vary in response to Isoniazid and Rifampicin. Finally, we adapt the spheroid model to form peripheral blood mononuclear cells (PBMCs) and lung fibroblasts (NHLF) 3D co-cultures. These results pave the way for the development of new strategies for disease modeling and therapeutic discovery. [ABSTRACT FROM AUTHOR]