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

Summary

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Abstract

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Introduction

The brain is an intricate circuitry of neuronal and non-neuronal cells. For years, scientists have been trying to gain insight into this complex machinery. To do so, neuroscientists initially resorted to various transformed nerve-based cell lines for investigations. However, the inability of these clonal cell lines to form strong synaptic connections and proper axons or dendrites have shifted scientific interest to primary neuron cultures^1,2. The most exciting aspect of primary neuron culture is that it creates an opportunity to observe and manipulate living neurons³. Moreover, it is less complex compared to neural tissue, which makes it an ideal candidate for studying the function and transport of various neuronal proteins. Recently, several developments in the fields of microscopy, genomics, and proteomics have generated new opportunities for neuroscientists to exploit neuron cultures⁴.

Primary cultures have allowed neuroscientists to explore the molecular mechanisms behind neural development, analyze various neural signaling pathways, and develop a more coherent understanding of synapsis. Though a number of methods have reported cultures from primary neurons (mostly from the hippocampal origin^5,6,7), a unified protocol with a chemically defined medium that enables long-term culture of neurons is still needed. However, neurons plated at a low density are most often observed, which do not survive long-term, likely due to the lack of trophic support⁸ that is provided by the adjacent neurons and glial cells. Some methods have even suggested co-culturing of the primary neurons with glial cells, wherein the glial cells are used as a feeder layer⁹. However, glial cells pose a lot of problems due to their overgrowth, which sometimes override the neuronal growth¹⁰. Hence, considering the problems above, a simpler and more cost-effective primary neural culture protocol is required, which can be used by both neurobiologists and neurochemists for investigations.

A primary neuron culture is essentially a form of 2D culture and does not represent the plasticity, spatial integrity, or heterogeneity of the brain. This has given rise to the need for a more believable 3D model called neurospheres^11,12. Neurospheres present a novel platform to neuroscientists, with a closer resemblance to the real, in vivo brain¹³. Neurospheres are non-adherent 3D clusters of cells that are rich in neural stem cells (NSCs), neural progenitor cells (NPCs), neurons, and astrocytes. They are an excellent source for the isolation of neural stem cells and neural progenitor cells, which can be used to study differentiation into various neuronal and non-neuronal lineages. Again, variability within neurosphere cultures produced using the previously reported protocols presents a barrier to the formulation of a unified neurosphere culture protocol¹⁴.

This manuscript presents a protocol in which it is possible to generate both 2D and 3D platforms by alternating cell plating densities from a mixed cortical and hippocampal culture. It is observed that within 7 days free-floating neurospheres are obtained from high-density plated neurons isolated from E14-E16 Sprague Dawley rat embryo, which upon further culture, form bridges and interconnections through radial glial-like extensions. Similarly, in the low density plated neurons, a primary neuron culture that can be maintained for up to 30 days is obtained by changing the maintenance medium twice per week.

Protocol

All experimental procedures involving animal were approved by the Institutional Animal Ethics Committee of CSIR-Indian Institute of Chemical Biology (IICB/AEC/Meeting/Apr/2018/1).

1. Reagent and media preparation

1. Poly-D-lysine (PDL) solution: prepare PDL solutions at concentrations of 0.1 mg/mL in deionized water and store in 4 °C until use.
2. Dissociation medium: To 1 L of sterile, filtered deionized water, combine the following components in the respective concentrations: sodium chloride (8 mg/mL), potassium chloride (0.4 mg/mL), potassium phosphate monobasic (0.06 mg/mL), D-glucose (1 mg/mL), sodium phosphate dibasic (0.479 mg/mL), and 1 M HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 10 mM]. Vortex all the components to aid proper mixing and store in 4 °C until use.
   NOTE: Use the dissociation medium in ice-cold form during dissociation but at room temperature (RT) for washing and other purposes.
3. Plating medium: plating medium consists of the following: minimum essential medium (MEM) Eagle's with Earle's Balanced Salt Solution (BSS; 88.4%), D-glucose (0.6%), horse serum (10%), and penicillin/streptomycin (1%). Combine the components in the respective ratios and perform the procedure inside a hood under sterile conditions.
   NOTE: Always use freshly prepared plating medium to avoid degradation of any component.
4. Maintenance medium: prepare the maintenance medium by combining the following in the respective ratios: neurobasal medium (97%), 0.5 mM commercially obtained glutamine sample, B27 serum-free supplement (2%), and penicillin/streptomycin (1%). Combine the components in the respective ratios and perform the procedure inside a hood under sterile conditions. Ensure that all components are freshly prepared.

2. Preparation of coverslips

1. Take a 12 mm diameter round glass coverslip and soak it in 1 M hydrochloric acid (HCl) for 4 h.
2. Transfer the coverslips in distilled water using a pair of forceps and swirl it gently to get rid of the acid completely.
3. Transfer the washed coverslips for an additional round of cleaning in a beaker containing 100% ethanol.
4. Before using the coverslips, dry them well in the laminar hood by keeping them on tissue paper.

3. Preparation of poly-D-lysine coated plates for neuron culture

1. Take two 24 well plates: one for high density plating and another for low density plating. Open the sterile packets only inside the laminar hood.
2. Transfer the 12 mm of sterile glass coverslips in the 24 well plates.
3. Pour 300 µL of PDL solution (0.1 mg/mL in deionized water) in each well so that it fully covers the surface of the coverslips.
4. Wrap the plates with aluminum foil to prevent drying and keep it in the CO₂ incubator overnight.
5. The next day (before plating), aspirate the PDL solution and wash properly with 300 µL of sterile deionized water two to three times.
6. Add 200 µL of freshly prepared plating medium and return the plates to the incubator until plating.

4. Removal and decapitation of the fetus

NOTE: Sterilize all surgical instruments packed in aluminum foil in an autoclave at 121 °C (15 psi) for 30 min. This includes a pair of blunt-end scissors, forceps, fine forceps, two fine scissors, and one artery forceps for the entire procedure.

1. For generating neurons and neurospheres, use a timed-pregnant Sprague Dawley rat and mark the day with vaginal plug detection as E0.
   ​NOTE: The culture must be performed between E14-E16.
2. On the day of culture, place a sterile glass Petri plate on ice and fill it with cold Hank's Balanced Salt Solution (HBSS).
3. Anesthetize an E14-E16 pregnant rat with an intraperitoneal (i.p.) injection of 90 mg ketamine/kg of body weight and 10 mg xylazine/kg, then sacrifice by performing cervical dislocation.
   NOTE: Rats can also be euthanized by an overdose of pentobarbital or overdose of ketamine with xylazine or diazepam.
4. Sterilize the dam's abdomen by spraying 70% ethanol and make a V-shaped cut in the abdominal area using sterile forceps and a pair of blunt-end scissors.
5. Take the embryonic sacs carefully on the Petri plate with cold HBSS solution.
   NOTE: Do not use the same forceps and scissors that were just used for the skin, as this will contaminate the internal organs. Use a different set of scissors/forceps for the internal organs.
6. Take the embryos out of the embryonic sacs in fresh, cold HBSS.
7. Decapitate the head with sterile scissors.

5. Removal of brain and dissection of the cortex with hippocampus

1. Before starting, fill 90 mm sterile Petri dishes with cold, sterile HBSS.
2. Transfer the heads in the sterile dishes using sterile, blunt-ended dressing forceps.
3. Under the stereomicroscope, hold the head from the snout region with sterile, serrated forceps and remove the brain by cutting the skin and skull open.
4. Collect all the embryo brains in the same manner in the HBSS solution.
5. Remove all meninges from the hemispheres and midbrain by holding the brainstem.
6. Carefully remove the intact hemispheres resembling mushroom caps that contain the hippocampus and cortex.
7. Collect the hemispheres containing cortex and intact hippocampus in a 15 mL conical tube containing 10 mL of dissociation medium.

6. Dissociation of cortical and hippocampal tissue into single neurons

1. Allow the collected tissues to settle down and aspirate the dissociation medium, leaving 5%-10% of medium in it.
2. Add 10 mL of fresh dissociation medium to the tissue, and repeat step 6.1 twice.
3. Add 4.5 mL of dissociation medium and 0.5 mL of 0.25% (1x) trypsin EDTA (ethylene diamine tetraacetate) solution.
4. Keep the tissue in the incubator at 37 °C for 20 min for the digestion to proceed.
5. Aspirate the medium and add 10 mL of dissociation and plating medium consecutively to the digested tissues.
6. Allow the digested tissues to settle down and aspirate the dissociation medium. Add 2.5 mL of plating medium and pour into the base of a 90 mm sterile dish.
7. Triturate the digested tissues in the corner base of the dish using a 1,000 µL pipette tip to occupy the minimal volume.
8. Pass the obtained cell suspension through the 70 µm cell strainer, excluding any chunks of tissue.
9. Determine the density of viable cells using the trypan blue dye exclusion method and count the number of cells in an automated cell counter.
   1. For trypan blue dye exclusion method, take 10 µL of the cell suspension and 10 µL of 0.4% trypan blue stain, mix thoroughly, and add 10 µL of the mixture in one of the two enclosed chambers of the disposable chamber slides.
   2. Insert the slide containing the mixture into the cell counter and obtain the reading.
      NOTE: The trypan blue dye exclusion method is based on the principle that live cells (due to their intact membranes) will exclude trypan blue dye and will hence show a clear cytoplasm, compared to a non-viable cell that will easily take up trypan blue and appear blue in color¹⁵.
10. Dilute the number of cells obtained to plate 1.5 x 10⁵ cells/mL for high density and 20,000 cells/mL for low density in two separate tubes containing 30 mL each of the plating medium.
11. Aspirate the previously added plating medium from each well and plate 500 µL of cells dispersed in plating medium in each well.
12. After that return the plates to the incubator at 37 °C and 5% CO₂ for 4 h.
13. Examine the cells for adherence under the microscope 4 h after plating.
14. If there is proper adherence of the cells in both plates, replace the medium in each well with 500 µL of fresh maintenance medium and incubate at 37 °C.
15. Culture these neurons grown at low density for 30 days by changing the maintenance medium 2x per week.
16. Culture the neurospheres obtained from the high-density plated neurons in the same maintenance medium by transferring them to the ultra-low attachment plates.
17. Characterize the neurons and the neurospheres by immunostaining them with important markers. For immunocytochemistry, first fix the cells/neurospheres using 4% formaldehyde for 30 min on the plate itself, then permeabilize the cells with 0.1% non-ionic detergent for 10 min.
18. Add primary antibodies for both neurons (anti-Tuj1, GFAP, O4, tau) and neurospheres (anti-Nestin, GFAP, Tuj1) in phosphate-buffered saline (PBS) at 1:300 concentrations and incubate overnight at 4 °C¹⁶.
    NOTE: The Tuj1 (class III β-tubulin) and tau are positive markers for the primary neurons, while GFAP (glial fibrillary acidic protein) and O4 (oligodendrocyte marker) are negative markers for primary neurons^17,18. In the case of neurospheres, Tuj1, GFAP, and Nestin all serve as positive markers^19,20.
19. The next day, wash the cells with PBS once or twice and add appropriate secondary antibodies in PBS at 1:600 concentrations at RT for 2 h.
    NOTE: The anti-Mouse or anti-Rabbit secondary antibodies are selected depending on the host species of the primary antibody added. It should be kept in mind that the secondary antibodies must be conjugated to fluorescence derivatives suitable for fluorescence microscopy purposes.
20. Wash the cells again with PBS once or twice.
    1. Perform nuclear staining of the cells with Hoechst 33258 (1 mg/mL stock solution in deionized water). Prepare 0.1% Hoechst solution in PBS from the stock solution and add it to the cells.
    2. Incubate the cells with 0.1% Hoechst solution for 30 min, then wash again with PBS.
21. Add 20 µL PBS (or mounting medium) on the slide and slowly mount the coverslip containing the stained cells over the area of the slide containing PBS. Seal the margins of the coverslip with dibutylphthalate polystyrene xylene (DPX).
22. Perform imaging of the fixed cells under a microscope at 10x and 40x magnification.

Results

In this protocol, a simple strategy has been elucidated in which variable cell plating densities from two different neural screening platforms are obtained. Figure 1A,B illustrates the adherence of cells after 4 h of plating the neurons in high and low density plated cells, respectively. On observing the proper adherence of the neurons as shown in Figure 1, the plating medium was replaced by maintenance medium in...

Discussion

This protocol describes that how by altering the cell plating densities of primary neurons, two variable neuronal platforms are obtained. Though this is a simple method, each step must be meticulously performed to achieve the desired results. Other previous methods have either reported long-term primary neuron cultures or neurosphere cultures. Most primary neuron culture protocols have involved the culturing of hippocampal neurons for 3-5 weeks, but most have failed, as the neurons die and wither away due to lo...

Materials

Name	Company	Catalog Number	Comments
Anti-GFAP	Abcam	AB7260
Anti- Nestin	Abcam	AB92391
Anti-O4	Millipore	MAB345
Anti-Tau	Abcam	AB76128
Anti-Tuj1	Millipore	MAB1637
B27 Serum Free Supplement	Gibco	17504-044
Cell Counter	Life technologies	Countess II FL
CO2 Incubator	Eppendorf	Galaxy 170 R
D-glucose	SDFCL	38450-K05
Ethanol	Merck Millipore	100983
Fluorescence Microscope	Olympus	IX83 Model
Formaldehyde	Sigma Aldrich	47608
GlutaMax-I Supplement	Gibco	35050-061
GtXMs IgG Fluor	Millipore	AP1814
GtXMs IgG (H+L)	Millipore	AP124C
HEPES	SRL	16826
Hoechst 33258	Calbiochem	382061
Horse Serum	HiMedia	RM10674
Hydrochloric Acid	Rankem	H0100
Laminar Hood	BioBase	BBS-V1800
MEM Eagle’s with Earle’s BSS	Sigma Aldrich	M-2279
Microscope	Dewinter	Victory Model
Neurobasal Medium	Gibco	21103-049
Plasticware (24 well plate, cell strainers, and low adherence plates)	BD Falcon	353047, 352350 and 3471
90 mm Petridishes	Himedia	PW001
Penicillin/Streptomycin	Gibco	15140-122
Poly-D-Lysine	Millipore	A.003.E
Potassium Chloride	Fisher Scientific	BP366-500
Potassium Phosphate Monobasic	Merck	MI6M562401
Sodium Chloride	Qualigem	15918
Sodium Phosphate Dibasic	Merck	MI6M562328
Stereomicrosope	Dewinter	Zoomstar Model
Triton-X 100	SRL	2020130
Trypan Blue Solution	Gibco	15250-061
0.25 % Trypsin-EDTA	Gibco	25200-072