Development of a 3D cell culture system based on a microfluidic platform
-
1
Forschungszentrum Jülich, Institute of Complex Systems 8 (Bioelectronics), Germany
Motivation
Due to a rapidly aging population, more people are affected by neurodegenerative diseases like Alzheimer’s and Parkinson’s disease [1]. The current knowledge about biological processes has been partially uncovered by two dimensional (2D) culture systems. However, studies based on monolayer cultures lack cell-cell/cell-matrix contacts and interactions. Those are essential not only for physiological morphology but also for intracellular signaling and gene expression. Neurons cultured on 2D surfaces receive information from just the ventral plane, whereas neurons in the human body process signals from all three dimensions (3D) [2]. Consequently, observations analyzed from these culture systems might offer deviating results compared to a physiological environment.
Hence, there is an urgent need to develop in vitro models which better mimic the physiological environment of the brain. The project focuses on the development of a 3D hydrogel based system combined with microfluidics which potentially offers higher degrees of organization and advanced physiological characteristics, for instance mechanical properties as found in tissues and organs. This new culture system provides a valuable in vitro tool to investigate neural cell-cell and cell-substrate interactions in a 3D environment and signal processing that will be of higher biological relevance.
Materials and Methods
The human brain’s extracellular matrix (ECM) is generally composed of water, ions, glycoproteins, proteoglycans, hyaluronic acid and fibrous proteins [3]. Commonly used scaffolds to mimic the ECM are hydrogels. Naturally derived hydrogels are able to imitate the components of the native ECM and enhance the cell viability due to larger cell-surface contact area.
In this study, the hydrogels Alginate and Fibrin are used. Dissociated rat embryonic neurons (E18) are cultured in these hydrogels for two to four weeks. Different fibrin and alginate polymer concentrations are evaluated regarding viability and maturation. Therefore neuronal networks were analyzed with immunofluorescence stainings of neurons and astrocytes, as well as Live/Dead stainings were performed. The network activity will be evaluated using calcium imaging technique.
Embedded cells will be cultivated in a microfluidic platform, which offer new insights to biological processes on cellular and molecular levels. These structures are produced using 3D printing (stereolithography; ‘MiiCraft 100’). The 3D models for this process are created with ‘Autodesk Inventor 2018’. Especially for neurodegenerative research, microfluidic devices are of high interest, because they provide directed cell growth and signal propagation, as well as the possibility to create a neuronal network involving different cell types [4][5]. Thus, specific brain regions and their communication can be modeled for controlled measurements [4]. Common methods like optogenetics do not resolve 3D cell systems.
Results
We could show that unmodified alginate hydrogels promote higher degree of organization and advanced physiological characteristics. The cell viability showed to be negatively influenced by increasing alginate and calcium chloride concentration. Hence, complex network formation could be observed in 0.25% and 0.5% alginate hydrogels which showed spontaneous activity up to DIV (day in vitro) 28.
Similar cell behavior could be observed in fibrin hydrogels. Interconnected network maturation could be observed in low fibrin concentrations (1.25 mg/mL) as opposed to high concentrations where glial cells were present more strongly and maturation was weaker.
Discussion and conclusion
In this study 3D neural networks were developed, which offer higher degrees of organization and advanced physiological characteristics. As expected, cultured neurons build randomly connected networks. For better understanding of network functionality, a controlled growth of cells would be beneficial. This could be achieved using bio-printing technologies. However, in the first instance preliminary tests of the neurons and hydrogels printability have to be performed. Another approach inducing controlled network polarity are microfluidics. Before combining developed microfluidics using 3D printing, the materials biocompatibility has to be evaluated.
Acknowledgements
Many thanks to Prof. Dr. Jürgen Groll and Dipl-Ing. Tomasz Jüngst for cooperation and support and Bettina Breuer for neuron preparations.
References
[1] JPND research. Eu joint programme - neurodegenerative disease research, 2016
[2] Caliari, S. R., Burdick JA1. (2016). A practical guide to hydrogels for cell culture. Nature Publishing Group, 13(5), 405-14. doi:10.1038/nmeth.3839
[3] Maeda, N. (2015), Proteoglycans and neuronal migration in the cerebral cortex during development and disease. Frontiers in Neuroscience, 9, 98. doi:10.3389/fnins.2015.00098
[4] Peyrin, J.-M., Deleglise B, Saias L, Vignes M, Gougis P, Magnifico S, Betuing S, Pietri M, Caboche J, Vanhoutte P, Viovy JL, Brugg B. (2011). Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers. Lab on a Chip, 11(21), 3663-73. doi:10.1039/c1lc20014c
[5] Park, J. W., Vahidi B, Taylor AM, Rhee SW, Jeon NL. (2006). Microfluidic culture platform for neuroscience research. Nature Protocols 1, 2128 – 2136. doi:10.1038/nprot.2006.316
Keywords:
Microfluidics,
Alginate,
Fibrin,
Neurons,
3D culture,
Hydrogels,
3D printing
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Neural Networks
Citation:
Meeßen
C,
Graeve
A and
Offenhäusser
A
(2019). Development of a 3D cell culture system based on a microfluidic platform.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00050
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Mrs. Corinna Meeßen, Forschungszentrum Jülich, Institute of Complex Systems 8 (Bioelectronics), Jülich, Germany, c.meessen@fz-juelich.de