As the developing nervous system is very susceptible to toxicant exposure, developmental neurotoxicity (DNT) of environmental chemicals is hazardous to human health. The consequent neurological deficits may have long lasting emotional and financial impact on families and society.
Current DNT testing guidelines make use of animal models, especially rodents. The huge quantities needed can be highly cost- and time-consuming, especially due to excess of chemicals that require testing1,2,3.
In addition to this demand, the current and future proposed regulations on the utilization of animal test models make the need to find new models that minimize animal experimentation while offering an appropriate means of testing new chemicals, highly imperative.
3D cell models that incorporate human neural stem cells (hNSCs) combined into neurospheres have been presented as a suitable substitute for use in DNT testing. Various processes of brain development such as differentiation, migration, proliferation, and apoptosis2 can be recapitulated by the in vitro system.
The system also fulfills the recommendations to circumvent the disadvantages of species differences between actual exposure effects and in vivo testing.
This article demonstrates a 3D neurosphere model comprising of hNSCs, to perform toxicity testing of many potential neurotoxicants. In order to create and retain cells in the 3D model, a specialized spheroid microplate was employed.
3D neurosphere multipotency, proliferation, and continued differentiation into astrocytes, oligodendrocytes, and neurons were validated first.
Neurospheres maintained in the 3D spheroid plate were then used to conduct neurotoxicity testing. Induced levels of oxidative stress, necrotic, and apoptotic activity in the treated neurospheres were calculated and then, compared to negative control spheres.
Materials and methods
StemPro® Neural Stem Cells were acquired from ThermoFisher (Waltham, MA).
Corning Life Sciences (Corning, NY) donated 384-Well High Content Imaging Film Bottom Microplates, 384-Well Black, Clear, Round Bottom Ultra-Low Attachment Spheroid Microplates, and 6-Well Clear, TC-Treated Microplates.
Neurotoxicants and plate coatings
Mercury (II) chloride, hydrogen peroxide solution, methylmercury (II) chloride, laminin, and poly-L-ornithine hydrobromide were bought from Sigma-Aldrich (Saint Louis, MO).
Media and antibodies
1% fetal bovine serum and glutaGRO™ DMEM were acquired from Corning Life Sciences. Neurobasal media, 2% B-27 supplement, 1x glutaMAX™, and 1% N-2 supplement were acquired from Life Technologies. dcAMP and T3 supplement were purchased from Sigma-Aldrich.
Enzo Life Sciences (Farmingdale, NY) donated Nestin (human) monoclonal antibody (2C1 3B9), βIII-Tubulin (human) monoclonal antibody (TU-20), Goat anti-mouse IgG1 (ATTO 590 conjugate), Oct4 monoclonal antibody (9B7), GFAP monoclonal antibody, and Goat anti-mouse IgG (ATTO 647N conjugate). Antioligodendrocyte specific protein antibody was purchased from abcam (Cambridge, MA).
Enzo Life Sciences (Farmingdale, NY) donated GFP CERTIFIED® Apoptosis/Necrosis detection kit, NUCLEAR-ID® Blue/ Red cell viability reagent (GFP CERTIFIED), and ROS-ID™ Total ROS detection kit. Accutase® Cell Detachment Solution was acquired from Corning Life Sciences.
Cytation™ Cell Imaging Multi-Mode Reader
Cytation 5 is a modular multi-mode microplate reader combined with automated digital microscopy. Monochromator-and filter-based microplate reading are available, and the microscopy module offers up to 60x magnification in brightfield, color brightfield, fluorescence, and phase contrast.
Fluorescence imaging in up to four channels in a single step can be performed using this instrument. Cytation 5 places special emphasis on live cell assays, featuring CO2/ O2 gas control, temperature control to 65°C and dual injectors for kinetic assays.
The instrument is controlled by integrated Gen5™ Data Analysis Software. Using fluorescence and brightfield microscopy, along with individual differentiated cells plated in 2D format, the instrument was able to image spheroids.
Specialized media optimization
glutaGRO™ DMEM was supplemented with 1% fetal bovine serum and 1% N-2 supplement to create astrocyte media. In order to create oligodendrocyte media, 1x glutaMAX, 2% B-27 supplement and 30ng/mL T3 supplement to neurobasal media were added.
2% B-27 supplement, 1x glutaMAX, and neurobasal media were combined to create neuron media. In addition, 1mM dcAMP was added after two days.
6-well plates that were coated with laminin and poly-L-ornithine, beforehand, were used to propagate neural stem cells. Then, cells were extracted and added to wells of a 384-well spheroid microplate at concentrations of 32,000-100 cells/well, with 12 replicates per concentration.
Single neurospheres formed after a period of 48-hour. Cytation 5, set to 37°C/5% CO2 with a 4x brightfield and objective imaging was used to image the wells containing spheroids.
Neurosphere proliferation and multipotency validation
In order to separate the wells containing cultured neurospheres, primary antibodies specific for Oct4 and Nestin were added. Then, fluorescently labeled secondary antibodies were added to wells, as well as to negative control wells that lack a primary antibody.
The Cytation 5, previously set to 37°C/5% CO2 with a 20x objective and Texas Red and RFP fluorescent imaging channels beforehand, was used to image the wells.
Neural stem cell differentiation
In order to break apart neurospheres, Accutase (50μL) was added to particular wells. In order to differentiate the cells into astrocytes, oligodendrocytes, and neurons, individual neural stem cells were added to separate wells of a poly-L-ornithine/laminin coated 384-well high content imaging plate when the optimized media mentioned earlier is present.
Simultaneously, the entire neurospheres were also moved to wells of the 384-well plate containing differentiation media. While the astrocytes were incubated for 4-5 days, and the oligodendrocytes were incubated for 5-6 days, the neurons were incubated for 7 days.
After completing the prescribed differentiation protocols, immunofluorescence was performed again with primary antibodies for markers exhibited in each lineage (GFAP: astrocyte; βIII-Tubulin: Neuron; Oligodendrocyte specific protein: oligodendrocyte) and secondary antibodies described earlier.
Following this, wells were imaged using the Cytation 5, previously set to 37°C/5% CO2 using a 20x or 40x objective and DAPI, RFP, Texas Red fluorescent imaging channels, along with a phase contrast channel overlay.
Neural stem cells with 2000 cells/well concentration were added to a 384-well spheroid plate and the allowed to aggregate. The consequent neurospheres were then exposed to different concentrations of mercury chloride, hydrogen peroxide, and methylmercury chloride, for 1, 2, 4, or 7 days.
Spheres were subjected to daily dosage of compound and fresh media. After incubation, media containing compound was extracted, the wells were washed using fresh media, and media comprising of apoptosis, total ROS, and dead/live cell probes was added for 4 hours.
In order to remove unbound probe, the wells were washed twice using fresh media. This was then imaged with Cytation 5, previously set to 37°C/5% CO2, with a 4x objective and GFP, DAPI, RFP, and Texas Red fluorescent imaging channels.
Results and discussion
Image-based tracking of neurosphere growth
To determine the ability of neural stem cells to propagate in a 3D configuration, replicate cell concentrations cultured into spheroids were imaged at regular intervals (Figure 1), starting at day 0, after the completion of spheroid formation, and repeating again on Days 1, 2, 4, 5, and 7.
While performing brightfield imaging in Cytation 5, the integrated Gen5 Data Analysis Software was employed to automatically place object masks around each spheroid.
Figure 1. Imaging and area analysis of 3D neurosphere growth. Thumbnail 4x aggregated neurosphere brightfield images. Twelve replicates per row of neurospheres formed from neural stem cells dispensed at 32,000, 16,000, 8000, 4000, 2000, 1000, 500, and 100 cells/well (top to bottom).
Figure 2. Plot of average percent growth in spheroid area compared to initial area measurement per dispensed cell concentration during incubation period.
The average growth percentage in area within the object masks for the 12 imitation neurospheres, which were calculated by the software, was plotted over time (Figure 2). Active growth in the spheroid plate was exhibited by all neurospheres, which can be deduced from the raise in the average percent spheroid growth from Day 0 to Day 7.
Compared to original values, percent growth in area ranged between about 100% for neurospheres initially consisting of 32,000 cells and 275% for neurospheres, initially consisting of 100 cells. This shows that the highest degree of proliferation is exhibited by spheroids starting from a lower number of aggregated cells, over time.
Neurosphere proliferation and multipotency validation
Oct4 and Nestin fluorescent probes were used to test the ability of neurospheres derived from neural stem cells to retain high multipotency capability and proliferative through immunofluorescence. While Oct4 is a transcription factor associated with the multipotency in stem cells, Nestin is an intermediate filament protein that is essential for self-renewal of neural stem cells.
The fluorescent signal emanating from primary and secondary antibody binding in Figures 3A and C confirm the expression of Nestin and Oct4 proliferation and multipotency proteins within 3D cultured neural stem cells.
No fluorescence is observed from negative control wells containing no primary antibody, indicating that secondary antibody binding takes place only in the presence of bound primary antibody, and further validating these results.
Figure 3. Detection of proliferation and multipotency markers. Overlaid 20x brightfield and fluorescence images of positive and negative control wells. (A) Proliferation positive control: Nestin (human) monoclonal antibody (2C1 3B9) plus Goat anti-mouse IgG1 (ATTO 590 conjugate) antibody. (B) Proliferation negative control: Goat anti-mouse IgG1 (ATTO 590 conjugate) antibody alone. (C) Multipotency positive control: Oct4 monoclonal antibody (9B7) plus Goat anti-mouse IgG (ATTO 647N conjugate) antibody. (D) Multipotency negative control: Goat anti-mouse IgG (ATTO 647N conjugate) antibody alone. RFP Channel: ATTO 590 goat anti-mouse IgG1; Texas Red Channel: ATTO 647 goat anti-mouse IgG.
Neural stem cell differentiation
Two separate methods were used to evaluate the ability of 3D cultured neural stem cells to differentiate between glial cells and neurons. First, the neurospheres were broken into individual neural stem cells using Accutase, and were added to separate wells of the imaging plate that contains specific media.
This enables the cell to differentiate into astrocytes, oligodendrocytes, and neurons. Whole neurospheres were separately moved to the imaging plate wells containing individual differentiation medias.
After the prescribed differentiation protocols were completed, immunofluorescence was again conducted, including primary antibodies for markers exhibited in each lineage (GFAP: astrocyte; βIII-Tubulin: Neuron; Oligodendrocyte specific protein: oligodendrocyte) and previously described secondary antibodies.
Figure 4. 2D and 3D neural stem cell differentiation. (A) 2D differentiated neurons and (B) 3D neurogenesis; 40x objective; RFP Channel: βIII-Tubulin 1° Ab/ATTO 590 2° Ab; DAPI Channel: Hoechst 33342. (C) 2D differentiated astrocytes, 40x objective and (D) 3D astrogenesis, 20x objective; Texas Red Channel: GFAP 1° Ab/ATTO 647 2° Ab; DAPI Channel: Hoechst 33342. (E) 2D differentiated oligodendrocytes and (F) 3D oligodendrogenesis; 20x objective; Texas Red Channel: oligodendrocyte specific protein 1° Ab/ ATTO 647 2° Ab; DAPI Channel: Hoechst 33342; Phase contrast channel overlay also shown in (F).
Differentiation into neurons, astrocytes, and oligodendrocytes (Figures 4A, 4C and 4E, respectively) was observed from 3D cultured neural stem cells differentiated in 2D format. When exposed to similar incubation conditions, neuro-, astro-, and oligodendrogenesis (Figures 4B, 4D and 4F, respectively) were also seen from differentiated neurospheres.
The overall neurosphere validation experimental outcomes confirms that neural stem cells express no ill effects from 3D culture, maintain multipotency, proliferative, and differential capabilities, and therefore represent a viable model for neurotoxicity testing.
Finally, the toxic effect of compounds on 3D neurospheres was determined using the established neurotoxins: mercury chloride (HgCl), hydrogen peroxide (H2O2), and methylmercury chloride (MeHgCl).
Cytation 5’s integrated Gen5 Data Analysis Software was used to perform the cellular analysis to accurately identify the signal from each probe emanating from the neurospheres (Figure 5).
In spite of potential changes in all other probes, fluorescence from the live cell probe remains consistent. Therefore, this threshold signal which was measured using the DAPI channel was employed to automatically draw object masks around each neurosphere (Figure 5A).
In order to ensure that the spheres registered as single objects instead of as individual cells, cellular analysis criteria were set, and minimum and maximum object size values were increased accordingly.
Using the GFP, RFP, and Texas Red imaging channels (Figures 5B-G), total fluorescent signal inside the object mask was captured, and calculated to determine the effect of each compound treatment on oxidative stress, apoptosis, and necrosis levels, respectively, within 3D cultured neural stem cells. The following formula was used to calculate the fold change.
RFUTreated (Time X) /RFUUntreated (Time X)
Here, RFUTreated (Time X) is the fluorescence value emanating from neurotoxicant treated spheroids and RFUUntreated (Time X) is the fluorescence from negative control wells containing untreated spheroids after 1, 2, 4, or 7 days of incubation.
Images in Figure 5 show how incubation with the known neurotoxicants causes a dramatic increase in signal generated by fluorescent probes when compared to signal from untreated wells.
When cellular analysis is incorporated, generated object masks focus solely on fluorescence emanating from each neurosphere and eliminate background signal, creating a highly sensitive measurement. Fold change can then be calculated as described earlier.
Figure 5. Image-based cellular analysis using 4x images following neurotoxin treatment and fluorescent probe incubation. (A) Object mask automatically placed around Nuclear-ID live cell probe signal emanating from treated neurosphere captured using DAPI channel. Fluorescent signal from positive or negative control wells following four-day treatment with 250 or 0μM H2O2, respectively, shown for (B,C) total ROS probe (GFP channel); (D,E) apoptosis detection reagent (RFP channel); and (F,G) dead cell probe (Texas Red channel).
To calculate the fold change, the test well signal from each specific compound at various compound incubation times was divided by the average from the untreated wells (Figure 6).
Figure 6. Test compound neurotoxic effect calculations. Results shown for (A) total ROS; (B) apoptosis; and (C) necrosis.
As per results in Figure 6A, methylmercury chloride exhibits the highest and earliest effect, peaking on day 2, while an eventual increase in oxidative stress on the neurospheres is elicited by each compound by day 7 of incubation.
In comparison, treatment with mercury chloride results only in a peak fold change in signal from the Total ROS detection kit that is 2x lower than hydrogen peroxide and 4x lower than methylmercury chloride.
The data generated by the apoptosis reagent (Figure 6B) shows that there is an ultimate increase in the apoptotic activity within the cells of the same spheres as a result of oxidative stress caused by each test molecule.
The highest apoptosis induction peaks after four days of incubation and similar results can be seen after incubations with each neurotoxicant. Then, signal from the assay reagent decreases by day 7 because the cells have then become mostly necrotic.
The results from the necrosis reagent (Figure 6C) confirm this. Fold changes from the signal created by this fluorescent probe raise as the amount of time each neurotoxicant is incubated with the neurospheres, peaking on day 7.
This article shows that neural stem cells cultured into 3D neurospheres, with a Corning 384-well spheroid microplate, present a robust, viable cell model that is both easily developed and reproducible.
The efficient microplate configuration enables compound dosing, assay performance and simple media replacement, while at the same time allowing for cellular imaging without transfer to a separate plate.
Users are presented with a sensitive technique for assessing the presence of essential proteins in cultured neurospheres, when primary antibodies from Enzo Life Sciences, specific for unique targets, are combined with fluorescently labeled secondary antibodies.
In addition, Enzo’s fluorescent probes provide a quick and easily discernible technique for detection of changes in significant toxic biomarkers. The Cytation™ 5 Cell Imaging Multi-Mode Reader is a flexible and sensitive system when performing phase contrast, fluorescent imaging of 2D neural stem cells and 3D neurospheres, and brightfield by employing a wide magnification range.
Integrated Gen5™ Data Analysis Software can accurately detect and analyze changes in multiple whole spheroids in real time. With the combination of microplate and 3D cell model, instrumentation and assessment methods, a powerful solution to perform high throughput, accurate assessments of potential neurotoxic effects on test molecules, is created.
Produced from materials originally authored by: Brad Larson1, Peter Banks1, Hilary Sherman2, Hannah Gitschier2, Alexandra Wolff3 and Wini Luty3 from:
1BioTek Instruments, Inc., Winooski, VT
2Corning Incorporated, Life Sciences, Kennebunk, ME
3Enzo Life Sciences, Farmingdale, NY
- U.S. Environmental Protection Agency. Guidelines for Neurotoxicity Risk Assessment. Washington, D.C. The National Academy Press, 1998.
- Organization for Economic Co-operation and Development. OECD Guidelines for Testing of Chemicals, Section 4: Health Effects. Paris, France. OECD iLibrary. 2007.
- Lein, P.; Silbergeld, E.; Locke, P.; Goldberg, A.M. In vitro and other alternative approaches to developmental neurotoxicity testing (DNT). Environ Toxicol Pharmacol. 2005, 19(3), 735-744.
- Salama, M.; Lotfy, A.; Fathy, K.; Makar, M.; El-emam, M.; El-gamal, A.; El-gamal, M.; Badawy, A.; Mohamed, W.M.Y.; Sobh, M. Developmental neurotoxic effects of Malathion on 3D neurosphere system. Applied & Translational Genomics. 2015, 7, 13-18.
About BioTek Instruments, Inc.
BioTek Instruments, Inc., headquartered in Winooski, VT, USA, is a worldwide leader in the design, manufacture, and sale of microplate instrumentation and software.
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