Reproducible Spheroids Research Articles

DESIGN AND DEVELOPMENT OF A CUSTOM PLATFORM TO GROW AND CHARACTERIZE OSTEOSARCOMA SPHEROIDS

Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research.Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms.To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode.

Effect of Polyplex Size on Penetration into Tumor Spheroids.

Ovarian cancer is one of the most lethal gynecological cancers in the world. In recent years, nucleic acid (NA)-based formulations have been shown to be promising treatments for ovarian cancer, including tumor nodules. However, gene therapy is not that far advanced in clinical reality due to unfavorable physicochemical properties of the NAs, such as high molecular weight, poor cellular uptake, rapid degradation by nucleases, etc. One of the strategies used to overcome these drawbacks is the complexation of anionic NAs via electrostatic interactions with cationic polymers, resulting in the formation of so-called polyplexes. In this work, the role of the size of pDNA and siRNA polyplexes on their penetration into ovarian-cancer-based tumor spheroids was investigated. For this, a methoxypoly(ethylene glycol) poly(2-(dimethylamino)ethyl methacrylate) (mPEG-pDMAEMA) diblock copolymer was synthesized as a polymeric carrier for NA binding and condensation with either plasmid DNA (pDNA) or short interfering RNA (siRNA). When prepared in HEPES buffer (10 mM, pH 7.4) at a nitrogen/phosphate (N/P) charge ratio of 5 and pDNA polyplexes were formed with a size of 162 ± 11 nm, while siRNA-based polyplexes displayed a size of 25 ± 2 nm. The polyplexes had a slightly positive zeta potential of +7-8 mV in the same buffer. SiRNA and pDNA polyplexes were tracked in vitro into tumor spheroids, resembling in vivo avascular ovarian tumor nodules. For this purpose, reproducible spheroids were obtained by coculturing ovarian carcinoma cells with primary mouse embryonic fibroblasts in different ratios (5:2, 1:1, and 2:5). Penetration studies revealed that after 24 h of incubation, siRNA polyplexes were able to penetrate deeper into the homospheroids (composed of only cancer cells) and heterospheroids (cancer cells cocultured with fibroblasts) compared to pDNA polyplexes which were mainly located in the rim. The penetration of the polyplexes was slowed when increasing the fraction of fibroblasts present in the spheroids. Furthermore, in the presence of serum siRNA polyplexes encoding for luciferase showed a high cellular uptake in 2D cells resulting in ∼50% silencing of luciferase expression. Taken together, these findings show that self-assembled small siRNA polyplexes have good potential as a platform to test ovarian tumor nodulus penetration..

Abstract 4266: Reproducible spheroid formation using functionalized hyaluronan 3D scaffolds

Abstract Extracellular matrix (ECM) characteristics, including stiffness, porosity, composition and spatial interaction with the surrounding cells and soluble factors are key components for cell growth in a tissue microenvironment. However, poor performance of 2D in vitro systems and animal models demands physiologically relevant well controlled 3D platforms for mechanistic assays, drug resistant phenotypes, new drug efficacy, toxicity assessment. Technical limitations in the current use of multicellular spheroids prevent their widespread use in cancer research and drug development. Existing systems for spheroid formation require lengthy processing times, and make simple tasks like media exchange, cell retrieval and microscopy analysis challenging. An ideal 3D cell culture system would form spheroids in an in vivo like microenvironment, while being easy to handle and compatible with all analytical methods. To address this unmet needs, we use a controlled hyaluronic acid-based scaffolds for spheroid formation. We have enriched 2 hyaluronan scaffolds with other components of the ECM such as collagen I, collagen IV, collagen VI, RGDs motif or galactosamine. Functionalised scaffolds overpass the 2D flat culture limitations by recreating cell/cell interactions and cell/matrix interaction to recreate a more physiologically authentic 3D architecture. The 2 functionalized scaffolds recapitulate the microenvironment for Hepatocyte and Adipocyte growth. Once formed, the spheroids can be cultured long-term, the scaffold is transparent allowing reproducible High Content Screening, the spheroids and the cells can be retrieve, avoiding the technical issues of other 3D systems to retain samples. Moreover, the scaffold is compatible with fluorescence/luminescent kit and immunofluorescent microscopy. We demonstrated this technology using cell lines, primary cells (adipocytes, hepatocytes). We assayed the spheroids over time using various endpoint spheroid morphology, growth and viability, resistance to anti-cancer drug, relevant cell organization formation and toxicity endpoint. Thus, this study introduces functionalized HA scaffold for the use of in vitro culture model as that represent native cell environments is ready to ready to use and compatible with HTS and all analytical methods for drug development and compound screening. Citation Format: Pauline Pannetier, Fiona Louis, Zied Souguir, Agathe Devaux, Didier Le Cerf, Jean-Pierre Vannier, Elise Demange, Guillaume Vidal. Reproducible spheroid formation using functionalized hyaluronan 3D scaffolds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4266.