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Poster presented during the SLAS Building Biology in 3D symposium event in Cambridge on April, 21th 2023.
Title: 3D cell culture taking into account the extracellular matrix for phenotypic screening in the frame of Parkinson’s disease
V. De Conto 1*, Cheung V.1, Alejandra Mogrovejo – Valdivia1, Maubon N.1, Vandenhaute E.1, Bérézowski V.2
Aim: About 90% of drug candidates fail in clinical trials, for efficacy- and toxicity-related reasons, which often involve the Central Nervous System (CNS). This high failure rate highlights a lack of relevance of experimental models used upstream, including human in vitro models. Indeed, they do not take into account the complexity of the CNS, in which neurons are organized in 3 dimensions (3D) and interact with their microenvironment, composed of other cell types like glial cells, and the extracellular matrix (ECM). The objectives of this work were i) to study the influence of the microenvironment on neuronal cells in cerebral in vitro models by automatized cellular imaging, and ii) to develop more relevant cerebral in vitro models for phenotypic screening, to assess neurotoxic or therapeutic effects, in the frame of Parkinson’s Disease (PD).
Methods: In this purpose, we developed BIOMIMESYS®, a hyaluronic acid-based matrixbio-functionalized with structural and adhesion molecules of the ECM, which forms a physiological microenvironment for in vitro 3 dimensional cell culture. This ECM-like hydroscaffold™ combines both the advantages of solid scaffold (porosity and structure maintain) and of hydrogels (cell-matrix interactions). Its composition, porosity and stiffness can be modified, in the aim to reproduce the organ-specificity of the native ECM, including the brain. This matrix is chemically defined, translucent and provided ready-to-used in 96 well plate format. It can be therefore used for High Content Screening. First, the sensitivity of Luhmes cells, a dopaminergic neuronal cell line, to PD inducers has been studied in this model. Then, we performed a co-culture of Luhmes cells and primary human astrocytes in this matrix, to form a complex model including both the glial and the matricial microenvironments. All the results were acquired by automated confocal fluorescent microscopy and quantitative image analysis.
Results: In the hydroscaffold, the neuronal cells were organized in clusters and exhibited neurites. They displayed a lower sensitivity in 3D compared to cells cultured in 2 dimensions (2D). This difference was explained by two phenomena: a partial retention of toxic molecules in the matrix, and a difference in neuronal protein expression compared to cells cultured in 2D. In the co-culture, we observed spheroids containing both neurons and astrocytes. The analysis of matrix component expression in the co-culture, in healthy and pathological conditions, is ongoing.
Conclusions: This work highlighted that the microenvironment of neurons can modify the neuronal response in vitro, and should thus be considered carefully in academic research and as early as possible in the drug discovery industrial process.
1 HCS pharma, 250 rue Salvador Allende, Biocentre Fleming Bâtiment A, 59120 Loos, France
2Lille Neuroscience & Cognition, Univ. Lille, Inserm, CHU-Lille, Lille, FranceTherapies, F-59000, Lille, France.