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 in 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 cells, soluble factors and extracellular matrix (ECM). The objectives of this PhD were i) to study the influence of these three microenvironment components 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).

First, the BIOMIMESYS® Brain technology has been developed. This acid hyaluronic based-matrix allows the simulation of the ECM and a 3D culture of cerebral cells in 96-well plates. The sensitivity of Luhmes cells, a dopaminergic neuronal cell line, to PD inducers has been studied: the cells displayed a lower sensitivity in BIOMIMESYS® Brain 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 lower neuronal maturity compared to cells cultured in 2D. 

The importance of the cellular microenvironment has been studied through a co-culture of Luhmes cells and primary human astrocytes in 2D. This co-culture has then been transposed in BIOMIMESYS® matrix, to form a complex model including both the glial and the matricial microenvironments.

In parallel, the influence of the molecular microenvironment has been studied on the SH-SY5Y cells, a cell line derived from a neuroblastoma, commonly used for neurotoxicity assessment. In this study, the 24 major differentiation media described in the literature to differentiate these cells into neurons have been screened. The 3 most differentiating conditions in terms of proliferation slowdown and neurite elongation have been selected: retinoic acid, staurosporine, and cyclic Adenosine Monophosphate (cAMP) combined to B21 supplement. The neuronal protein marker expression and the cell sensitivity to compounds of known-toxicity have been measured, in 2D and in 3D in BIOMIMESYS® Brain. Both maturity and sensitivity of these neurons varied according to the differentiation medium, and were higher in B21+cAMP. The 3D cell culture modified also the cell response, with a lower sensitivity of cells cultured in 2D.

This study highlighted that the microenvironment of neurons, including the ECM, the glial cells and the soluble factors, 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.

Overview and perspectives of the studied microenvironment components and their interactions. A. Visual abstract of the studied components. Both the soluble factors and the matrix influenced the neuronal maturity and sensitivity. Astrocytes also modified neuronal sensitivity and can secrete matrix molecules. B. Perspectives for improving the model. Addition of sulfated Glycosaminoglycans (GAG) in the matrix; Addition of microglia to implement the neuroinflammatory process; Addition of the Blood-Brain Barrier (BBB) to test the crossing of drugs; Study of neuronal secretions (matrix compounds, α-synuclein, etc.). Solid lines: observed effects, dotted lines: hypothetical effects. Pointed arrows: up-regulation ; Round arrows: down-regulation. BBB: Blood Brain Barrier. PG: Proteoglycans. Images were realized with Servier Medical Art.

This PhD work has been successfully defended by Véronique De Conto in December 21sd 2021. Feel free to contact us if you want more information about this work or about BIOMIMESYS® hydroscaffold for 3D cell culture !


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