Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

The overall goal of this procedure is to demonstrate the development of neurospheres from mixed primary hippocampal and cortical neurons isolated from E14-E16 Sprague Dawley rat embryo, in order to offer a robust and low-cost platform for studying the effect of various neurotherapeutic leads. As you know, that brain is a complex network of neurons associated with large number of supporting cells. To understand the brain function, often most of the research is re-initiated from in vitro experiments, which involves commercially available neural lineage derived cell lines.

However, this cell lines are transformed cell lines that suffer from genotypic and phenotypic variations. To address this issues, primary neuron culture is the suitable approach. Here, we illustrated a simple and cost-effective protocol to culture primary neurons and built a robust screening platform for various neural therapeutics.

The main advantage of this protocol is that just by modulating the cell plating densities, we can showcase two contrasting neuron culture platforms where low-density leads to prolonged primary neuron culture, and high-density generates spontaneous neurospheres. Take two 24-well plates. One for high-density, and another for low-density plating.

Transfer 12 mm of sterile glass coverslips in the 24-well plates. Pour 300 microliters of PDL solution in each of the wells in a way that it fully covers the surface of the entire coverslip. Prepare PDL solution at a concentration of 0.1 mg/mL in deionized water.

Wrap the plates with aluminum foils to prevent drying, and keep it in the CO2 incubator overnight. Next day, before plating, aspirate the PDL solution. Wash properly with sterile water for two to three times.

Then add plating media, and return the plates to the incubator until plating. Sacrifice an E14-E16 timed pregnant Sprague-Dawley rat, Make a V-shaped cut in the abdominal area using sterile forceps and a pair of blunt-end scissors. Remove all the fetuses and carefully place them on the petri plate with cold HBSS solution.

Take the embryos out of the embryonic sacs and wash them carefully in a separate petri dish in fresh, cold HBSS. Decapitate the head with the help of sterile scissors. Transfer the heads in the sterile 90 mm petri plates with cold HBSS.

Under the stereo microscope, hold the head from the snout region with the sterile serrated forceps, and take out the brain by cutting the skin and the skull open. Remove all the meninges from the hemispheres and the midbrain by holding the brainstem. Carefully remove the intact hemispheres, resembling mushroom caps that contains the hippocampus and the cortex.

Collect the hemispheres containing cortex and intact hippocampus in a 15 mL conical tube containing 10 mL of dissociation media. Allow the collected tissues to settle down, and aspirate the dissociation medium, leaving five to ten percent of medium in it. Again, add 10 mL of fresh dissociation medium to it, and repeat this step twice.

Add 4.5 mL of dissociation medium and 0.5 mL of trypsin-EDTA solution. Keep it in the incubator at 37 degrees Celsius for 20 minutes for the digestion to proceed. Aspirate the medium and wash with 10 mL of dissociation medium and plating medium, consecutively.

Resuspend them in 2.5 mL of plating medium. Keep a 90 mm sterile dish over the inverted cover of the dish and pour it in the base of 90 mm sterile dish. Titrate the digested tissues in the corner base of the dish, using 1000 microliter pipette tip, so as to occupy least volume.

Pass the obtained cell suspension through the 70 micrometer cell strainer, excluding any chunks of tissue. Determine the density of viable cells using trypan blue dye exclusion, and count the number of cells in an automated cell counter. Dilute the number of cells obtained in a manner so as to plate 1.5 into 10 to the power of 5 cells per mL for high-density plating, and 20, 000 cells per mL for low-density plating in two separate tubes containing 30 mL of plating medium.

Aspirate the previously added plating medium and plate 500 microliters of cells dispersed in plating medium in each well. After that, return the plates to the incubator for four hours. Four hours after plating, examine the cells for adherence under the microscope.

After four hours of plating, in both high and low density plates, the neurons show good adherence to the plates. Replace the medium in each well with 500 microliters of fresh maintenance medium, and incubate it in an incubator at 37 degrees Celsius. We have to culture these neurons plated for at high-and low-density.

While the low-density plated neurons can be used for prolonged cultures, up to 30 days, by changing the maintenance medium twice a week, the high-density plated neurons starts to spontaneously generate neurospheres, which came day after we maintained in ultra-low attachment plate. After 24 hours of plating, both high-and low-density plated neurons are observed to display healthy morphology. A phase contrast image of the low-density plated neurons, cultured for about seven days, is displayed here.

The neurons display healthy morphology, and can be maintained up to 30 days with more vigorous routing and interconnections. The bar diagram here shows around 90 percent viability of the low-density plated neurons even after 30 days of culture, as determined by the MTT assay. The primary neurons here have been characterized by immunostaining with Tuj 1, a marker of differentiated neurons, and tau, a marker of neuronal axons.

The purity of the neurons is confirmed by the absence of staining in non-neuronal markers GFAP for astrocytes and O4 for oligodendrocytes. In all the characterization studies, nuclei have been stained with Hoechst 33258. Here, the low-density seeded cells have been stained with astrocytic marker GFAP and neuronal marker Tuj 1 to check the purity of the culture up to seven days.

It is observed that this protocol supports the preferential growth of neurons over astrocytes as indicated through the quantitative analysis. Nuclei have been stained with Hoechst 33258. Similarly, in the high-density seeded cells, the astrocytes have been stained with GFAP and neurons by Tuj 1.

Here, in the quantitative analysis, around 17 percent astrocytes are observed as compared to 83 percent neurons. The nuclei have been stained with Hoechst 33258. After seven days, spontaneously formed multiple neurospheres in the high-density neurons are observed.

Around eighth to tenth day of culture, in the high-density plated neurons, the neurospheres starts to form large projections and bridges consisting of radial glial-like extensions. The black arrows indicate the bridge between two newly formed neurospheres. Alive and dead cell assay have been performed on the neurospheres for 15 days.

In dense Calcein AM staining, with absolutely no propidium iodide staining, indicates almost 100 percent viability of the neurospheres in culture. The line graph here indicates an expansion in the size of the neurospheres with the passage of number of days. This overly stained image of neurosphere indicates a rich population of neural progenitor cells through increased expression of NPC markers Nestin and Tuj 1.

The neurospheres here show high amounts of astrocytes marked by the strong expression of GFAP, accompanied by an even higher expression of neuronal marker Tuj 1. The nuclei have been stained with Hoechst 33258. So I think our protocol is quite exciting and interesting, considering the fact that from a single strategy we are able to get two platforms:one 2-D and one 3-D.

And this will be a great platform for screening different neurotherapeutic tracks. So I guess it's really really useful to all neuroscientists. Also another important aspect is that the neurospheres we opt in are extremely rich in neural progenitor cells, the NPCs.

And they can be used to differentiate them into neurons and non-neuronal lineages. So, I feel if, really, people can master this technique by taking help from this video, it will be really really useful for everyone. Thank you.