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. 2022 Jun 23;11(13):2008.
doi: 10.3390/cells11132008.

Effect of 3D Synthetic Microscaffold Nichoid on the Morphology of Cultured Hippocampal Neurons and Astrocytes

Clara Alice Musi  1   2 , Luca Colnaghi  3   4 , Arianna Giani  2 , Erica Cecilia Priori  1   2 , Giacomo Marchini  2 , Matteo Tironi  2 , Claudio Conci  5 , Giulio Cerullo  6 , Roberto Osellame  6 , Manuela Teresa Raimondi  5 , Andrea Remuzzi  7 , Tiziana Borsello  1   2
Affiliations

Effect of 3D Synthetic Microscaffold Nichoid on the Morphology of Cultured Hippocampal Neurons and Astrocytes

Clara Alice Musi et al. Cells. .

Abstract

The human brain is the most complex organ in biology. This complexity is due to the number and the intricate connections of brain cells and has so far limited the development of in vitro models for basic and applied brain research. We decided to create a new, reliable, and cost-effective in vitro system based on the Nichoid, a 3D microscaffold microfabricated by two-photon laser polymerization technology. We investigated whether these 3D microscaffold devices can create an environment allowing the manipulation, monitoring, and functional assessment of a mixed population of brain cells in vitro. With this aim, we set up a new model of hippocampal neurons and astrocytes co-cultured in the Nichoid microscaffold to generate brain micro-tissues of 30 μm thickness. After 21 days in culture, we morphologically characterized the 3D spatial organization of the hippocampal astrocytes and neurons within the microscaffold, and we compared our observations to those made using the classical 2D co-culture system. We found that the co-cultured cells colonized the entire volume of the 3D devices. Using confocal microscopy, we observed that within this period the different cell types had become well-differentiated. This was further elaborated with the use of drebrin, PSD-95, and synaptophysin antibodies that labeled the majority of neurons, both in the 2D as well as in the 3D co-cultures. Using scanning electron microscopy, we found that neurons in the 3D co-culture displayed a significantly larger amount of dendritic protrusions compared to neurons in the 2D co-culture. This latter observation indicates that neurons growing in a 3D environment may be more prone to form connections than those co-cultured in a 2D condition. Our results show that the Nichoid can be used as a 3D device to investigate the structure and morphology of neurons and astrocytes in vitro. In the future, this model can be used as a tool to study brain cell interactions in the discovery of important mechanisms governing neuronal plasticity and to determine the factors that form the basis of different human brain diseases. This system may potentially be further used for drug screening in the context of various brain diseases.

Keywords: 3D co-cultures; brain models; brain on dish; dendritic spines; primary culture.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustration of the primary hippocampal astrocyte—neuron co-culture set-up in the 3D synthetic microscaffold Nichoid.
Figure 2
Figure 2
Confocal microscopy of DIV21 hippocampal astrocyte and neuron co-cultures in 2D flat culture and in the 3D synthetic microscaffold Nichoid. (A–C) 2D co-cultures formed by (A) hippocampal astrocytes, marked with GFAP in red, and (B) hippocampal neurons, marked with MAP2 in green. Nuclei were stained with Hoechst, in blue. (D–F) Co-culture in the 3D Nichoid formed by (D) hippocampal astrocytes, marked with GFAP in red, and (E) hippocampal neurons, marked with MAP2 in green. Nuclei were stained with Hoechst, in blue. Images were taken with a 100× (oil immersion) objective. Scale bar of 10 μm.
Figure 3
Figure 3
Confocal microscopy of DIV21 hippocampal astrocyte and neuron co-cultures in 2D and in the 3D synthetic microscaffold Nichoid. Lateral views of (A) 2D and (B) 3D co-cultures in which hippocampal astrocytes were marked with GFAP in red and neurons were marked with MAP2 in green. Nuclei were stained with Hoechst in blue. Images were taken with a 100× (oil immersion) objective.
Figure 4
Figure 4
SEM microscopy of DIV21 hippocampal astrocyte and neuron co-cultures in 2D and in the 3D synthetic microscaffold Nichoid. (A) The 2D hippocampal astrocyte and neuron co-culture. Scale bar of 10 μm. (B) Higher magnification of 2D hippocampal astrocyte and neuron co-culture. Scale bar of 2 μm. (C) The 3D hippocampal astrocyte and neuron co-culture in the Nichoid. Scale bar of 10 μm. (D) Higher magnification of 3D hippocampal astrocyte and neuron co-culture in the Nichoid. Scale bar of 2 μm.
Figure 5
Figure 5
SEM microscopy of DIV21 hippocampal neurons co-cultured with astrocytes in 2D and in the 3D synthetic microscaffold Nichoid. (A) The 2D hippocampal astrocyte and neuron co-culture. Scale bar of 2 μm. (B) Higher magnification of 2D hippocampal astrocyte and neuron co-culture. Filopodia and spines were indicated by arrows. (C) The 3D hippocampal astrocyte and neuron co-culture. Scale bar of 2 μm. (D) Filopodia (arrow) and spine (triangle) numbers increased in 3D hippocampal astrocyte and neuron co-cultures. Scale bar of 2 μm.
Figure 6
Figure 6
SEM microscopy and protrusion quantification of DIV21 hippocampal neurons co-cultured with astrocytes in 2D and in the 3D synthetic microscaffold Nichoid. (A) The 2D hippocampal astrocyte and neuron co-culture. Scale bar of 2 μm. (B) Higher magnification of 2D hippocampal astrocyte and neuron co-culture. Scale bar of 1 μm. Arrows indicate the protrusions. (C) The 3D hippocampal astrocyte and neuron co-culture in the Nichoid. Scale bar of 2 μm. (D) Higher magnification of 3D hippocampal astrocyte and neuron co-culture in the Nichoid. Scale bar of 1 μm. Arrows indicate examples of filopodia that were considered in the quantification. (E) Results of filopodia counts. Conditions were compared using t-tests. Statistical significance: *** p < 0.001. Data are the means ± SEM of 20 dendrites from three independent conditions.
Figure 7
Figure 7
Confocal microscopy of DIV21 hippocampal co-cultures of astrocytes and neurons in 2D and in the 3D synthetic microscaffold Nichoid. (A–D) Grayscale images from horizontal sections of 2D hippocampal co-cultures of astrocytes and neurons marked with Hoechst (A), MAP2 (B), GFAP (C), and drebrin (D). (E) Merged fluorescence image of 2D hippocampal co-cultures with Hoechst in blue, MAP2 in green, GFAP in red, and drebrin in violet. (F,G) Lateral views of 2D hippocampal astrocyte and neuron co-cultures displayed in (E). (H–K) Grayscale horizontal sections of 3D hippocampal astrocyte and neuron co-cultures marked with Hoechst (H), MAP2 (I), GFAP (J), and drebrin (K). (L) Merged fluorescence image of 3D hippocampal astrocyte and neuron co-cultures with Hoechst in blue, MAP2 in green, GFAP in red, and drebrin in violet. (M,N) Lateral sections of 3D hippocampal astrocyte and neuron co-cultures displayed in L. Images were taken with a 100× (oil immersion) objective.
Figure 8
Figure 8
Confocal microscopy of DIV21 hippocampal co-cultures of astrocytes and neurons in 2D and in the 3D synthetic microscaffold Nichoid. (A–D) Grayscale images from horizontal sections of 2D hippocampal astrocyte and neuron co-cultures marked with Hoechst (A), MAP2 (B), GFAP (C), and PSD95 (D). (E) Merged fluorescence image of 2D co-culture with Hoechst in blue, MAP2 in green, GFAP in red, and PSD95 in violet. (F,G) Lateral views of the 2D co-cultures displayed in (E). (H–K) Grayscale images of horizontal sections of 3D hippocampal co-cultures of astrocytes and neurons marked with Hoechst (H), MAP2 (I), GFAP (J), and PSD95 (K). (L) Merged fluorescence image of the 3D hippocampal co-cultures with Hoechst in blue, MAP2 in green, GFAP in red, and PSD95 in violet. (M,N) Lateral view of 3D co-cultures displayed in L. Images were taken with a 100× (oil immersion) objective.
Figure 9
Figure 9
Confocal microscopy of DIV21 hippocampal astrocyte and neuron co-cultures in 2D and in the 3D synthetic microscaffold Nichoid. (A–D) Grayscale images of horizontal sections of 2D hippocampal co-cultures of astrocytes and neurons marked with Hoechst (A), MAP2 (B), GFAP (C), and synaptophysin (D). (E) Merged fluorescence image of 2D hippocampal co-cultures with Hoechst in blue, MAP2 in green, GFAP in red, and synaptophysin in violet. (F,G) Lateral sections of 2D hippocampal astrocyte and neuron co-cultures displayed in (E). (H–K) Grayscale horizontal sections of 3D hippocampal astrocyte and neuron co-cultures marked with Hoechst (H), MAP2 (I), GFAP (J), and synaptophysin (K). (L) Merged fluorescence image of 3D hippocampal astrocyte and neuron co-cultures with Hoechst in blue, MAP2 in green, GFAP in red, and synaptophysin in violet. (M,N) Lateral sections of 3D hippocampal astrocyte and neuron co-cultures displayed in L. Images were taken with a 100× (oil immersion) objective.
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T.B.: Ricerca Finalizzata 2016 RF-2016-02361941, MIUR, -PON “Ricerca e Innovazione” PerMedNet id project ARS01_01226-PROGETTI DI RICERCA DI RILEVANTE INTERESSE NAZIONALE Prot. 2017MYJ5TH; European Commission’s Horizon 2020 research and innovation program No. 847749; MIUR Bando PRIN 2017 cod. 2017MYJ5TH. M.T.R: European Research Council (ERC projects NICHOID, G.A. 646990, NICHOIDS, G.A. 754467, and MOAB, G.A. 825159); European Commission (FET-OPEN project IN2SIGHT, G.A. 964481); European Space Agency (ESA project NICHOID-ET, G.A. 4000133244/20/NL/GLC); Italian Ministry of University and Research (MIUR-FARE project BEYOND, G.A. R16ZNN2R9K). L.C. gratefully acknowledges support from BrightFocus Foundation (Grant A2019296F), the Fondo di Beneficenza- Gruppo Intesa Sanpaolo, and Vita-Salute San Raffaele University.
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