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. 2020 Jul 3;10(1):11007.
doi: 10.1038/s41598-020-67691-2.

Functional and transcriptional characterization of complex neuronal co-cultures

Heather A Enright  1 , Doris Lam  2 , Aimy Sebastian  2 , Ana Paula Sales  3 , Jose Cadena  3 , Nicholas R Hum  2   4 , Joanne J Osburn  2 , Sandra K G Peters  2 , Bryan Petkus  2 , David A Soscia  3 , Kristen S Kulp  2 , Gabriela G Loots  2   4 , Elizabeth K Wheeler  3 , Nicholas O Fischer  5
Affiliations

Functional and transcriptional characterization of complex neuronal co-cultures

Heather A Enright et al. Sci Rep. .

Abstract

Brain-on-a-chip systems are designed to simulate brain activity using traditional in vitro cell culture on an engineered platform. It is a noninvasive tool to screen new drugs, evaluate toxicants, and elucidate disease mechanisms. However, successful recapitulation of brain function on these systems is dependent on the complexity of the cell culture. In this study, we increased cellular complexity of traditional (simple) neuronal cultures by co-culturing with astrocytes and oligodendrocyte precursor cells (complex culture). We evaluated and compared neuronal activity (e.g., network formation and maturation), cellular composition in long-term culture, and the transcriptome of the two cultures. Compared to simple cultures, neurons from complex co-cultures exhibited earlier synapse and network development and maturation, which was supported by localized synaptophysin expression, up-regulation of genes involved in mature neuronal processes, and synchronized neural network activity. Also, mature oligodendrocytes and reactive astrocytes were only detected in complex cultures upon transcriptomic analysis of age-matched cultures. Functionally, the GABA antagonist bicuculline had a greater influence on bursting activity in complex versus simple cultures. Collectively, the cellular complexity of brain-on-a-chip systems intrinsically develops cell type-specific phenotypes relevant to the brain while accelerating the maturation of neuronal networks, important features underdeveloped in traditional cultures.

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Conflict of interest statement

All authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Immunofluorescence characterization of cortical cultures in simple and complex systems at DIV31. Neurons were identified by staining for Tuj-1 (Neuron-specific class III beta-tubulin, a, e). Glial fibrillary acidic protein (GFAP) was used to identify astrocytes (b, f) and myelin basic protein (MBP) was used to identify mature oligodendrocytes and myelin (white arrowheads) (c, g). Merged images with nuclear stain (DAPI, blue) are shown in (d) and (h). Figure has been modified to remove electrode autofluorescence. Scale bar = 50 µm.
Figure 2
Figure 2
Immunofluorescence characterization of myelination in simple and complex systems at DIV31. Mature axons were identified by staining for p-NF-H (phosphorylated neurofilament H, a, e, i). Myelin basic protein (MBP) was used to identify mature oligodendrocytes and myelin (b, f, j). Merged images with nuclear stain (DAPI, blue) are shown in (c), (g) and (k). Zoomed-in images of MBP/axon co-localization (white boxes) from panels (g) and (k) are shown in (h) and (l), respectively. White arrowheads indicate areas of co-localization. Quantification of MBP in both simple and complex systems is shown in (d). Scale bar = 50 µm. ****p < 0.0001.
Figure 3
Figure 3
Single-cell RNAseq data. (a) t-SNE plot showing eleven different cell clusters. Different cell types (and sub-types) are color-coded. (b) t-SNE plot of all cells for both simple and complex groups at each DIV. Gene markers for specific cell types are shown in (c–h); Tubb3 and Nrgn for neurons (c, d); Gfap and Aqp4 for astrocytes (e, f); Pdgfra and Mbp for oligodendrocytes (g, h).
Figure 4
Figure 4
Gene ontology enrichment analysis of cluster 1 neurons at DIV14 and DIV31. (a) t-SNE plot illustrating cluster 1 neurons. (b) Venn diagram illustrates the number of distinct and similar biological processes at DIV14 and DIV31. Bar graphs (c–e) illustrates the top 10 biological processes that are distinct at DIV14 (c), common between time points (d) and distinct to DIV31 (e).
Figure 5
Figure 5
Gene ontology enrichment analysis of cluster 1 and 6 neurons at DIV31. (a) t-SNE plot illustrating cluster 1 and 6 neurons. (b) Venn diagram illustrating distinct and similar biological processes identified by GO between cluster 1 and 6. (c) Representative distinct processes from the 89 identified for cluster 1. (d) Representative similar processes from the 37 identified between cluster 1 and cluster 6 and (e) Representative distinct processes from the 100 identified from cluster 6.
Figure 6
Figure 6
Characterization of astrocytes and oligodendrocytes. Dot plots represent key cell type marker gene expression within simple and complex cultures at DIV14 and DIV31. (a, i) t-SNE plot illustrating cluster 3 of astrocytes; (a, ii) Dot plot of cluster 3 astrocytes. (b, i) t-SNE plot illustrating cluster 2 of astrocytes. (b, ii) Dot plot of cluster 2 astrocytes (c, i) t-SNE plot illustrating clusters of oligodendrocytes. (c, ii) Dot plot of oligodendrocytes. Dot size indicates proportion of cells in cluster that express a gene; the shading indicates the average level of expression (low to high indicated as light to dark purple).
Figure 7
Figure 7
Baseline characteristics of simple and complex cultures. Two representative electrodes with neuronal activity are shown for simple and complex recordings in (a) and (b) at DIV31, respectively. Comparisons of activity and firing features over days in vitro (DIV) are shown including: (c), % active electrodes, (d), firing rate, (e), % spikes in bursts, (f), burst duration, (g), interspike interval within bursts (ISI) and (h), synchrony. Data are shown as mean ± SEM. Asterisks (*) indicate significance in two-way ANOVA. *p < 0.05, **p < 0.01. Asterisks associated with DIV indicate significance for culture type. Asterisks alone indicate significance for media type.
Figure 8
Figure 8
Responses of simple and complex cultures to bicuculline (BIC) at DIV32. Two representative electrodes for baseline and bicuculline evoked activity are shown for simple and complex recordings in (a) and (b), respectively. Comparisons of changes in specific features upon BIC exposure expressed as the fold change are shown, including: (c) # bursts, (d) bursts/min, (e) burst duration and (f) synchrony. Data are shown as mean ± SEM.
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