Application Note: Co-culture of Astrocytes and Cortical Neurons

 

Andrew Mearns Spragg; Jellagen Limited, UK and David Wallbank; CENSO Biotechnologies Ltd, UK and Sophie Escott-Morgan; Jellagen Limited, UK.

Introduction

Understanding and deciphering the immeasurable number of parallel functions occurring within the brain remains an overwhelming challenge.  Numerous culturing methods and their use in planning and conducting toxicology experiments have become essential for modern toxicologists, in the drive to reduce or replace the need for living animals1.

In vitro human co-culture models allow the establishment of biologically relevant cell-cell interactions that recapitulate the tissue micro-environment and better mimic its physiology. However, the number of publications on this specific subject is limited2. The utilisation of precise co-culture methods using chemically defined culture media could provide an additional valuable model in toxicological studies3. The engineering of well-defined culture media for the development of functional neuronal micro-environments may also be able to offer reduced complexity of in vitro experiments and may yet better control experimental variables to provide significant value for fundamental research on how the nervous system develops and functions. These “bottom up” approaches may also offer the potential of developing low-medium throughput neuroscience methods into versatile tools for high-throughput pharmacological screening and development of new drug targets against neurodevelopmental and neurodegenerative disorders1-3. The intensified interest in functional micro-environments is leading towards new ways of designing cell culture systems that are able to increase physiological relevance in vitro.

Neuronal co-culture using human- and iPSC-derived astrocytes offer an important tool for investigating the fundamental questions in neurobiology for applied applications in cell therapy and drug discovery. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures have been shown to improve the quality of in vitro assays.  Astrocytes secrete factors that promote neuron survival, synapse formation, and plasticity4. Understanding how these factors perform these roles requires a robust in vitro system that can effectively assess the impact of individual glial factors on neuronal properties without being influenced by the initial cell culture matrix used in the assay. In addition, the potential of monitoring neuronal network function may be better modelled.

To date, neuromodulation has been studied with a focus on either neurons or astrocytes at the single-cell level or on neuronal activity in networks. Studies of neuron-astrocyte interactions have been mostly limited to the single synapse level6. How the influence that neuromodulators have over network level signalling between neurons and astrocytes has been generally overlooked. Recent reports have shown the effect of neuromodulation on neuron-astrocyte network communications and explored the role of astrocyte signalling in brain networks5.

In this Application Note we report that, for the first time, Jellagen® jellyfish collagen offers a chemically defined alternative to the co-culture of Astrocytes and cortical neurons which could lead to the development of better predictive assays of neuronal cell interaction modelling for disease and drug screening.


Materials & Methods

Materials

  • Jellagen Biomaterial (JL100ML 3)
  • Cell Grade Culture Water
  • Astrocyte cell line (BIONi010-C, EBISC)
  • Primitive cortical neuron cell line (BIONi010-C, EBISC)
  • Astrocyte Maturation Media
  • Cortical Maturation Media
  • Uridine (U6381-5G, Sigma)
  • 5-Fluro-2-deoxyuridine (F0503-100MG, Sigma)
  • 4% Paraformaldehyde (PFA) (43368, Alfa Aesar)
  • Staining Antibodies: GFAP (Astrocyte) (AB7260, Abcam), TUJ-1 (Neuronal) (MAB1195, RND Systems), Hoechst 33342 (Nucleic Acid) (H1399, ThermoFisher)
  • 15ml Falcon Tubes
  • TPP 96 well culture plate (Z707910-162EA, Sigma)
  • Incubator 37OC in 5% CO2
  • Sterile microbiological safety cabinet
  • Water bath 37OC
  • Centrifuge
  • Microscope

Methods

Plate Coating

  1. Jellagen was diluted to a concentration of 32μg/mL by adding 85μL of 3mg/mL stock Jellagen to 7.915mL Cell Grade Culture water.
  2. 100μL of Jellagen was added to each well of a TPP 96 well culture plate and incubated for 24 hours at 4˚C.
  3. After incubation, all Jellagen was removed and the plate allowed to air dry in a sterile microbiological safety cabinet overnight.
  4. Stored at 4˚C until use.
  5. Prior to seeding wells were rinsed with cell culture media and then cells were added to the plate at desired density.

Cell Culture – Astrocytes Thaw and Culture

  1. Astrocytes were thawed in a 37oC water bath and transferred to a 15mL falcon.
  2. 10mL of Astrocyte Maturation media was added drop-by-drop to the Astrocytes.
  3. Cells were centrifuged at 300 xg for 5 min and the supernatant was discarded.
  4. Cells were re-suspended in 1mL of Astrocyte Maturation media and a cell count performed.
  5. Cells were re-suspended at 3.3×106/mL (for 100k/cm2) or 1.65×106/mL (for 50k/cm2) and 100μL was plated onto Jellagen coated 96 well plates.
  6. The astrocytes were cultured for 7 days before addition of neurons. The Astrocyte Maturation media was changed every other day. On the second day of culture, the wells were treated with Astrocyte mitomix (Astrocyte Maturation media + 10uM Uridine + 10uM 5-Fluro-2-deoxyuridine).

Cortical Neuron Thaw and Culture

  1. Neurons were thawed in a 37oC water bath and transferred into a 15mL falcon.
  2. 10mL of Neuronal Maturation media was added drop-by-drop to the Neurons.
  3. The solution was centrifuged at 300xg for 3 mins and the supernatant was discarded.
  4. Cells were re-suspended in 1mL of Cortical Maturation media and a cell count performed.
  5. Cells were then re-suspended at a density of 6.6×106/mL and 100μL was plated onto the wells of the 7 day cultured astrocytes to achieve 200k/cm2.
  6. The cells were cultured for a further 7 days, with media changes every other day before fixing with 4% PFA.

Immunohistochemistry

  1. Standard IHC protocol was used to stain for astrocytes (GFAP), Neuronal cells (TUJ-1) and with a nuclear marker (Hoechst 33342).
  2. Cells were the visualised and the following results obtained.

Results and Discussion

 

Astrocytes and Neurons

Fig.1.

 

Figure 1.0 Co-culture of Astrocytes and Neurons on Jellagen® and Corning® Matrigel. BIONi010-C Astrocytes were plated onto Jellagen diluted in Cell Culture grade water (A, C) and onto Matrigel coated plates (B, D) at a density of 100k/cm2 (A, B) and 50k/cm2 (C, D) and matured for 7 days with mitomix treatment, before the addition of BIONi010-C Neurons. After further 7 days of culture the plates were fixed and stained with a neuronal marker (TUJ1 – green), an astrocyte marker (GFAP – red) and a nuclear marker (Hoechst 33342 – blue).

Two densities of BIONi010-C astrocytes were matured for 7 days on Jellagen Coated Plates or the market leading matrix before plating BIONi010-C Neurons onto the astrocytes. After a further 7 days of culture the plates were fixed and stained with a Neuronal marker (TUJ1) and an Astrocyte marker (GFAP) to analyse the adherence of the neurons.

Astrocytes were thawed and cultured on both substrates at seeding densities of 100k/cm2 and 50k/cm2. Adherence and survival were assessed and were similar between both substrates with good adherence and survival observed on Jellagen. Neurons were then plated onto the astrocyte cultures at a density of 200k/cm2. The resulting co-cultures were fixed and stained, showing the expression of the mature astrocyte marker GFAP and the neuronal marker TUJ-1.

All co-culture experiments produced acceptable levels of markers, however, in the co-cultures grown on Jellagen Coated Plates the level of neuron marker TUJ-1 and the nuclear marker Hoechst 33342 appear brighter than the market leading matrix at both seeding densities (as seen in Fig.1).


Conclusions

Jellagen is a non-mammalian, chemically simple, easy to use cell culture matrix leading to consistency and reduced variability of results when performing co-culture of astrocytes and neurons.

Jellagen successfully supports the maturation of astrocytes, neuronal adherence and neuronal outgrowth in co-culture, performing as well as, or better than Matrigel.


Reference & Note

  1. Mathias J. Aebersold, Greta Thompson-Steckel, Adriane Joutang, Moritz Schneider, Conrad Burchert, Csaba Forró, Serge Weydert, Hana Han and János Vörös. Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks. Frontiers in Neuroscience. 2018; February 2018 (12). Article 94.
  2. Jacobine Kuijlaars, Tutu Oyelami, Annick Diels, Jutta Rohrbacher, Sofie Versweyveld, Giulia Meneghello, Marianne Tuefferd, Peter Verstraelen, Jan R. Detrez, Marlies Verschuuren, Winnok H. De Vos, Theo Meert, Pieter J. Peeters, Miroslav Cik, Rony Nuydens, Bert Brône & An Verheyen. Sustained synchronized neuronal network activity in a human astrocyte co-culture system. Scientific Reports. 2016; 6:36529. DOI: 10.1038/srep36529.
  3. De Simone U, Caloni F, Gribaldo L, Coccini T. Human Co-culture Model of Neurons and Astrocytes to Test Acute Cytotoxicity of Neurotoxic Compounds. Int J Toxicol. 2017;36 (6):463-477.
  4. Jones EV, Cook D, Murai KK. A neuron-astrocyte co-culture system to investigate astrocyte-secreted factors in mouse neuronal development. Methods Mol Biol. 2012; 814:341-52.
  5. Yasmin Bar El,Sivan Kanner,Ari Barzilai and Yael Hanein. Activity changes in neuron-astrocyte networks in culture under the effect of norepinephrine. PLOS ONE. 2018;
  6. Nicola J. Allen, and Cagla Eroglu. Cell Biology of astrocyte-synapse interactions. Neuron. 2017; 96(3). 697-708.

About the Author (s)

Censo Bio is a specialist discovery services company using human stem cells (iPSC) to support neurodegenerative, neuro-inflammation and rare disease focused discovery. They provide access to a diverse range of existing human iPSC lines, and ethically sources relevant human tissue, both healthy and with disease-specific mutations, with which they reprogramme to new iPSC lines. Censo Bio can gene edit human iPSC lines to knock in, knock out or correct target genes of interest using CRISPR, and have developed reproducible differentiation protocols for a number of lineages, including microglia, astrocytes, cortical and sensory neurons.

Using these cells for neurodegenerative and neuroinflammatory disease discovery, they offer customisable phenotypic assays to deliver disease relevant compound characterisation, providing new insights and functional disease related understanding of compounds.