Aim: About 90% of drug-candidates failed in clinical trials, in particular in neurology, due to a lack of efficacy. That highlights a lack of relevance in preclinical models, including in vitro models, which do not take into account the microenvironment, composed by glial cells and the Extracellular Matrix (ECM). The objective was to study the influence of the microenvironment in cerebral in vitro models, in the frame of Parkinson’s Disease (PD). Methods: First, we analyzed the influence of astrocytes on Luhmes cell sensitivity, a dopaminergic neuronal cell line, in 2D culture. Then, we developed a hyaluronic acid-based hydroscaffold for 3D cell culture, which mimics the ECM, and study the sensitivity of Luhmes cells in this model. Thirdly, we performed a co-culture of Luhmes cells and astrocytes in this matrix, to form a complex model including both the glial and the matricial microenvironments. Results: We observed a protective effect of astrocytes in 2D culture. In the hydroscaffold, Luhmes cells displayed a lower sensitivity compared to 2D culture, that was explained by a partial retention of toxic molecules in the matrix, and differences in neuronal protein expression. In the co-culture, we observed spheroids containing both neurons and astrocytes. 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 in drug discovery. This model can be now used to study the microenvironment modifications in pathological conditions, and to develop innovative drugs targeting the microenvironment.

1. 1. Introduction
• Low success rate in drug discovery:
90% of failure in clinical trials (safety and efficacy)
• The Central Nervous System is highly concerned:
This highlights a lack of relevance in preclinical models used
upstream to select drug candidates:
• Animal: inter-species differences
• In vitro models: too simple, do not take into account the
microenvironment of neurons (glial cells and ECM).
Ex in Parkinson disease (PD), with the death of dopaminergic
neurons involving the microenvironment:
• Neuroinflammation mediated by glial cells
• ECM modifications
Importance of microenvironment in cerebral in vitro models for phenotypic screening
De Conto V.1*, Cheung V.1, Maubon G.1, Souguir Z.1, Maubon N.1, Vandenhaute E.1, Bérézowski V.2 .
1HCS Pharma, Loos, France ; 2Lille Neuroscience & Cognition, Univ. Lille, Inserm, CHU-Lille, Lille, France
2. What is the extracellular
matrix (ECM)?
The ECM has diverse
and crucial roles,
in health and disease
*GAG: Glycosaminoglycans
Ratio GAG*/Collagen
= 10/1
 Low stifness
Goal : To improve the predictivity of in vitro models
• Taking into account the 3D cell organization
• Including the cellular and matricial
microenvironments
3. What is BIOMIMESYS®
hydroscaffoldTM?
Hydroscaffold
Hydrogels:
Biohydric medium
Cells encapsulated
Pathological or
irrelevant ECM
Solid Scaffolds:
Porous, No ECM
Decellularized organs
Complete ECM
Difficult tissue
access,
not
reproducible
(Scanning electron microscopy)
Hyaluronic acid grafted with other ECM components:
Composition and physico-chemical properties tailored to mimic
the ECM of each tissue/organ
BIOMIMESYS® Brain: HA, RGDS, Collagen IV, cationic polymer
Elastic modulus 0.1 kPa.
Porous, physiological, reproducible matrix
4. Methods
5. Results
For more information, go to:
hcs-pharma.com or scan this QR-code.
* : veronique.deconto@hcs-pharma.com
Neurons and astocytes in mono and co-
culture, in 2D and in BIOMIMESYS® Brain
Scale bar = 200µm
GFAP TUBB3
Reference : Barnes, J.M., Przybyla, L., and Weaver, V.M. (2017). Tissue mechanics regulate brain development, homeostasis and disease. J Cell Sci 130, 71–82 ; Morgan, P., Brown, D.G., Lennard, S., Anderton, M.J., Barrett, J.C., Eriksson, U., Fidock, M., Hamrén, B., Johnson, A., March, R.E., et al. (2018). Impact of a five-dimensional
framework on R&D productivity at AstraZeneca. Nat Rev Drug Discov 17, 167–181 ; Lu, P., Weaver, V.M., and Werb, Z. (2012). The extracellular matrix: A dynamic niche in cancer progression. The Journal of Cell Biology 196, 395.
The cellular microenvironment
influences the neuronal sensitivity (2D)
The presence of astrocytes decreased the cell sensitivity to
PD inducers, probably linked to their secretion properties,
like antioxydative molecules.
The matricial microenvironment
influences neuronal sensitivity (3D)
In the hydroscaffold, Luhmes cells displayed a lower
sensitivity compared to 2D culture, which might explained
by a partial retention of toxic molecules in the matrix, and
differences in neuronal protein expression (data not
shown).
Source : CMR (2010-2017)
Morgan et al., 2018
Barnes et al., 2017
Adapted from Lu et al. 2021
Brain ECM:
20% of cerebral volume
6. Conclusion and perspectives
The cellular and matricial microenvironments
modify the neuronal response in vitro, and
should thus be considered carefully in academic
research and in drug discovery. This model can
now be used to study the microenvironment
modifications in pathological conditions, and to
develop innovative drugs targeting the
microenvironment.