High-Content Imaging and Analysis of 2D and 3D Neuronal Cultures Grown in Optimized Media

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The ability to grow neurons in culture, and to monitor and quantify neurite outgrowth, aids in both the understanding of nervous system function and the development of treatments for neurological disorders such as Alzheimer’s and Parkinson’s diseases [1]. Synaptic growth and neural toxicity can be monitored by growing neurons in culture with the aid of optimized neuronal media and supplements, followed by fluorescent labeling and imaging. Recent developments in 3D culture systems and supports have enhanced the ability to grow neurons in a matrix that more closely resembles the state of nervous system tissue in intact organisms, without the need to use expensive and time-consuming animal models. High-content analysis (HCA) systems can be used to obtain high-resolution images of 2D and 3D neuronal cultures and perform quantitation and complex analysis of key parameters of neurite outgrowth for hundreds of samples in a relatively short period of time [2,3].

The Thermo Scientific CellInsight CX7 LZR HCA Platform is a 7-laser high-content imaging instrument that reduces scanning times and improves axial resolution in 2D and 3D cultures. These features provide the speed and sensitivity necessary to analyze neuronal culture systems for more physiologically relevant drug discovery assays, and allow tracking and quantification of the dynamic changes in neurites and synapses underlying synaptogenesis or synaptic damage. Here we discuss use of the new-generation Gibco B-27 Plus Neuronal Culture System to generate 2D and 3D neuronal cultures with enhanced growth. These neuronal cultures can then be stained with a variety of fluorescent markers and probes for high-throughput imaging so that neuronal growth, length, and toxicity can be quantified using HCA, allowing data to be obtained from hundreds of neuronal samples over a short period of time.
 

• Gibco Primary Rat Cortex Neurons (Cat. No. A1084002)
• Gibco Primary Rat Hippocampal Neurons (Cat. No. A36513)
• Gibco StemPro Neural Stem Cells (Cat. No. A15654)
• B-27 Plus Neuronal Culture System (Cat. No. A3653401, contains Gibco Neurobasal Plus Medium and B-27 Plus Supplement)
• Gibco Neurobasal Medium (Cat. No. 21103049)
• Gibco B-27 Supplement (50X) (Cat. No. 17504044)
• Gibco CultureOne Supplement (100X) (Cat. No. A3320201)
• Gibco N-2 Supplement (100X) (Cat. No. 17502048)
• Gibco BDNF Recombinant Human Protein (Cat. No. 10908010)
• Gibco GDNF Recombinant Human Protein (Cat. No. PHC7044)
• Gibco CNTF Recombinant Human Protein (Cat. No. PHC7015)
• Invitrogen MAP2 Polyclonal Antibody (Cat. No. PA5-17646)
• Invitrogen HuC/HuD Monoclonal Antibody (Cat. No. A21271)
• Invitrogen Alexa Fluor 488 Goat Anti–Mouse IgG (H+L) Secondary Antibody (Cat. No. A11001)
• Invitrogen Alexa Fluor 555 Goat Anti–Mouse IgG (H+L) Secondary Antibody (Cat. No. A21424)
• Invitrogen Alexa Fluor 488 Goat Anti–Rabbit IgG (H+L) Secondary Antibody (Cat. No. A11034)
• Invitrogen Alexa Fluor 647 Goat Anti–Rabbit IgG (H+L) Secondary Antibody (Cat. No. A21244)
• Thermo Scientific Nunclon Sphera 96-Well Round U-Bottom Plates (Cat. No. 174925)
• Invitrogen Tubulin Tracker Deep Red (Cat. No. T34077)
• Invitrogen Live Cell Imaging Solution (Cat. No. A14291DJ)
• CellInsight CX7 LZR HCA Platform (Cat. No. CX7A1110LZR)
   

Growth of neuronal cultures in B-27 and B-27 Plus systems

Cryopreserved rat cortical or hippocampal neurons were cultured in the B-27 system (Neurobasal Medium with B-27 Supplement) or B-27 Plus system (Neurobasal Plus Medium with B-27 Plus Supplement) for 3–4 weeks. The cells were fixed with 4% formaldehyde for 15 min, permeabilized with 0.5% Triton™ X-100 detergent for 10 min, and then immunostained with HuC/HuD and MAP2 primary antibodies followed by Alexa Fluor 555 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit secondary antibodies, respectively. The samples were imaged on a CellInsight CX7 LZR HCA Platform.

Monitoring neuronal toxicity

Cryopreserved rat cortical neurons were grown for 2 days and then treated with cadmium chloride or paclitaxel for 24 hr. The cells were then fixed and stained with HuC/HuD and MAP2 primary antibodies followed by Alexa Fluor 488 goat anti-mouse and Alexa Fluor 647 goat anti-rabbit secondary antibodies, respectively. The samples were imaged on a CellInsight CX7 LZR HCA Platform.

3D neurosphere culture and Tubulin Tracker Deep Red staining

Neural stem cells (NSCs) were grown on Nunclon Sphera U-bottom plates (1.3 x 10⁴ cells/well) to form neurospheres. The cells were differentiated in the B-27 system or B-27 Plus system, each with or without CultureOne Supplement. N-2 Supplement, growth factors BDNF, GDNF, and CNTF (all 10 μg/mL), and 10 μM ROCK inhibitor (Y-27632) were also added to the cultures. The neurons were differentiated for 2 weeks and stained with 1 μM Tubulin Tracker Deep Red for 1 hr. The spheroids were then washed with Live Cell Imaging Solution and imaged on the CellInsight CX7 LZR HCA Platform.

High-content imaging and analysis

The neuronal cells were analyzed on a CellInsight CX7 LZR HCA Platform using the neuronal profiling bioapplication at 10x magnification. A total of 200 cells were imaged per well, and neuronal cell bodies and neurite outgrowth were measured using their respective parameters. The nuclei were segmented with DAPI, and neuronal cell bodies were identified with HuC/HuD-positive staining. Neurite length was measured with MAP2 staining. For 3D neurosphere imaging, confocal mode was used, where each image represents a maximum intensity projection (MIP) of thirty Z optical slices of 10 μm each.
 

Results

2D neuronal cultures immunostained with MAP2 and HuC/HuD antibodies to label dendrites and neuronal cell bodies were imaged on the CellInsight CX7 LZR HCA Platform and analyzed with Thermo Scientific HCS Studio 2.0 Cell Analysis Software to obtain measurements of neurons per field and neurite length. Both rat cortical and hippocampal neurons had increased growth, as indicated by neurons per field, when grown in the B-27 Plus Neuronal Culture System compared to the original B-27 system (Figure 1). 

Neuronal toxicity can also be quantitated by imaging and analysis on the CellInsight CX7 LZR HCA Platform and HCS Studio 2.0 Cell Analysis Software as demonstrated by treating rat cortical neurons with 10 mM cadmium chloride or 10 μM paclitaxel. Both treatments significantly decreased the total neurite length per field and percentage of neurons positive for HuC/HuD, demonstrating the neuronal toxicity of these compounds (Figure 2).

Neurons were also grown in 3D culture to form neurospheroids that were stained with Tubulin Tracker Deep Red to label neuronal processes in live cells without the need for fixation or immunostaining. As demonstrated by staining with Tubulin Tracker Deep Red and imaging on the CellInsight CX7 LZR HCA Platform, the 3D neurospheroids showed enhanced growth when grown in the B-27 Plus system with CultureOne Supplement, compared to neurospheroids grown in the original B-27 system, or without the supplement (Figure 3).

High-content imaging and analysis with the CellInsight CX7 LZR HCA Platform and HCS Studio 2.0 Cell Analysis Software enable fast, high-resolution imaging of both 2D and 3D neuronal cell cultures along with automated quantitation and complex analysis of several parameters of neuronal growth and health, including identification of neuronal cell bodies, neurons per field, and neurite length. The B-27 Plus Neuronal Culture System allows superior growth of 2D and 3D neuronal cultures over longer periods of time than the original B-27 culture system. Tubulin Tracker Deep Red, a docetaxel-based fluorescent reagent for live-cell labeling of microtubules, provides neuronal labeling throughout live 3D neurospheroids without the need for fixation or immunostaining. These optimized neuronal culture systems and fluorescent staining reagents combined with high-resolution automated imaging and analysis allow for investigation of neuronal growth and toxicity of hundreds of samples over a relatively short period of time, helping to enable new discoveries in neuronal function and neuropharmacology. These discoveries may potentially be applied to understanding of the nervous system and treatment of neurological disorders.

1. Chang KH, Lee-Chen GJ, Huang CC et al. (2018) Modeling Alzheimer’s disease by induced pluripotent stem cells carrying APP D678H mutation. Mol Neurobiol Sep 20. doi:10.1007/s12035-018-1336-x. [Epub ahead of print]
2. van Deijk AF, Broersen LM, Verkuyl JM et al. (2017) High content analysis of hippocampal neuron-astrocyte co-cultures shows a positive effect of Fortasyn Connect on neuronal survival and postsynaptic maturation. Front Neurosci 11:440.
3. Radio N, Frank S (2018) Neuronal cell morphology in primary cerebellar granule cells using high-content analysis. Methods Mol Biol 1727:227–237.