Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Summary

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Abstract

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Introduction

Improving our understanding of the central nervous system (CNS) is critical to improve therapeutic options for many neuroinflammatory and neurodegenerative diseases. The CNS, a complex network of interconnected cells within the brain, spinal cord, and optic nerves, comprises neurons, oligodendrocytes, astrocytes, and their innate immune cells, the microglia¹. An in vitro approach can often drastically reduce the number of mice required to perform meaningful research; however, the complex nature of the CNS makes it impossible to recapitulate the in vivo situation using cell lines. Mixed neural cell cultures provide an extremely valuable research tool to investigate neuro(immuno)logy questions in a relevant model, in line with the Replacement, Reduction and Refinement (3Rs) principles^2,3.

Thomson et al. described a cell culture method using prenatal spinal cord cells that differentiate into all the aforementioned main CNS cell types⁴. This system also has synapse formation, myelinated axons, and nodes of Ranvier. The main limitation of this culturing method is that, being spinal cord, it does not usefully model the brain, and the cell yields from embryonic day 13 (E13) spinal cords are constricting. Thus, this limits the number of experimental conditions that can be investigated. Therefore, this study aimed to develop a new cell culture system that recapitulates the characteristics of the brain with increased cell yield to reduce the requirements for animals.

Using Thomson et al. as a starting point, we developed a cell culture model derived purely from prenatal mouse brains. These cultures have the same cell populations, interconnectivity, and treatment options as the spinal cord cultures, except there is less myelination by comparison. However, having a CNS in vitro model with an approximately threefold higher cell yield is more efficient, requiring fewer mice and less time processing embryos. We optimized this unique culture system for multiple downstream applications and scales, including using glass coverslips for microscopy analysis and various sizes of plastic well plates, including 96-well plates for high throughput research.

Protocol

All animal experiments complied with local laws and guidelines for animal use, and were approved by the local Ethical Review Committee at the University of Glasgow. Animals were housed in specific pathogen-free conditions in accordance with the UK Animals Scientific Procedures Act 1986, under the auspices of a UK Home Office Project License. For this study, in-house bred adult C57BL/6J mice were used. The use of young females (8-12 weeks) is recommended due to the higher success rate of pregnancy; males can be reused for multiple rounds of breeding. Figure 1 represents a schematic overview of the described method to generate mixed neuronal and glial cultures.

1. Preparation of the tissue culture consumables

1. Prepare the plates and/or dishes containing microscopy coverslips inside a Class 2 Safety cabinet. Sterilize all reagents or autoclave to ensure sterility during the culture period.
2. Add the appropriate volume of BA-PLL (13.2 µg/mL poly-L-lysinehydrobromide [PLL] in boric acid buffer [BA] [50 mM boric acid, 12.5 mM sodium tetraborate, pH 8.5]; see Table of Materials) to each well (1,000 µL/well in a 6-well plate, 100 µL/well in a 96-well plate; volumes are summarized in Table 1). For microscopy coverslips, add 20 mL of BA-PLL to a 9 cm diameter tissue culture dish containing 200 sterile coverslips, and swirl to distribute evenly.
3. Incubate at 37 °C for 1-2 h.
4. Remove the BA-PLL solution from each well or dish containing microscopy coverslips, and wash by adding 20 mL of sterile water, swirling the coverslips, and then removing the water. Repeat this wash step three times. For a dish containing coverslips, leave sterile water in the tissue culture dish on the final wash to easily remove the coverslips.
5. Remove as much liquid as possible with a sterile pipette and allow to dry for at least 2 h to overnight.
6. Store the coated plates at 4 °C for up to 2 months.
   NOTE: The prepared boric acid with poly-l-lysine (BA-PLL) solution can be reused up to three time, adding new PLL each time. Store the BA-PLL at 4 °C. Dishes are treated with BA-PLL, as the PLL allows the cells to stick down and grow. Without this treatment, the cells will lift after approximately 1 week of culture and no longer be able to differentiate.

2. Dissection of E17 embryonal brains

1. Co-house one or multiple female mice with a male mouse. Check the females daily for a mucus plug, indicating mating has taken place.
   NOTE: Any "plugged" female mice need to be separated from the male to ensure the correct start date of gestation. Mice can be weighed to confirm pregnancy or monitored visually.
2. Cull the pregnant mouse at E17 using appropriate methods in compliance with local animal welfare guidance and laws, for example, by raising carbon dioxide (CO₂) concentration, a lethal anesthetic overdose, or dislocation of the neck.
   NOTE: The chosen method must not disrupt the embryos. For this study, exposure to a rising concentration of carbon dioxide gas followed by confirmation of death by severing the femoral artery was used to cull pregnant dams.
3. Place the culled, pregnant mouse on its back on a dissection board; while pinning it down is not required, it might make it easier for inexperienced researchers. Pinch the midline of the abdomen using forceps. Using sharp scissors, cut open the abdomen through the skin and the peritoneum over the midline from the genitalia to the ribcage, being careful not to puncture the uterus.
   1. The mouse uterus has two horns, each typically containing one to five embryos. Remove the uterus containing the embryos from the mother and immediately place it on ice.
4. Cut through the yolk sack on the side of the placentas, being careful not to damage the embryos, and remove the embryos from their yolk sack.
5. Immediately decapitate the embryos. Add the decapitated heads into a dish with Hanks' balanced salt solution (HBSS) without calcium (Ca²⁺) and magnesium (Mg²⁺) (HBSS-/-) on ice.
   NOTE: If genotyping is required, one can remove the tail at this stage for genetic analysis. When multiple genotypes are expected, the heads of each embryo should be kept separately for culturing.
6. Using angled forceps, position the head on its side facing left.
7. Pierce the eye with one edge of the forceps, firmly holding the chin with the other.
8. Starting at the nape, gently tear the skin of the scalp along the midline toward the tip of the snout.
9. Entering through the spinal cord, noticeable as a white oval, use the angled forceps to crack open the skull along the midline, exposing the brain.
10. Gently peel the skull away on the side facing upward, exposing the brain.
11. Lift the brain out of the skull, disposing of the skull once the brain has been completely removed.
12. Using the forceps, remove the meninges, which are noticeable as a thin membrane with dense blood vessels.
13. Place the brains into a bijou (see Table of Materials) containing 2 mL of HBSS-/- on ice.
14. Repeat steps 2.6 to 2.13 with the remaining brains, adding up to four brains per bijou.
15. Add 250 µL of 10x trypsin to the bijou and triturate the brains by shaking the bijou. Incubate for 15 min at 37 °C.
    NOTE: All steps from this point forward should be performed in a sterile tissue culture hood.
16. Thaw 2 mL of soybean trypsin (SD) inhibitor (Leibovitz L-15, 0.52 mg/mL trypsin inhibitor from soybean, 40 µg/mL DNase I, 3 mg/mL bovine serum albumin [BSA] fraction V; see Table of Materials) from -20 °C by placing it at 37 °C.
17. Add 2 mL of SD inhibitor to each bijou containing brains (per bijou containing up to four brains), shaking the bijou again to disperse it evenly.
    NOTE: SD inhibitor decreases the trypsin activity to prevent unnecessary digestion of the samples and preserve cell viability.
18. Without centrifugation, remove 2 mL of the supernatant from each bijou and transfer into a 15 mL centrifuge tube, being careful not to transfer cell clumps.
19. Triturate the remaining cells in the bijou with a 19 G needle attached to a 5 mL syringe by aspirating the suspension twice. This will create a thick mucus-like mixture.
    1. Repeat twice more using a 21 G needle. If there are clumps remaining, triturate once more with the 21 G needle.
20. Transfer the cells from the bijou to the same 15 mL centrifuge tube (from step 2.18) using a 23 G needle.
21. Centrifuge at 200 x g at room temperature (RT) for 5 min.
22. Remove all the supernatant using a 5 mL serological pipette and transfer it to another 15 mL centrifuge tube, being careful not to disturb the loose pellet at the bottom containing the required cells.
23. Centrifuge the supernatant again at 200 x g at RT for 5 min.
    NOTE: This step is not essential, but if one requires many cells or has few embryos, one could perform this step to recover as many cells as possible from the supernatant.
24. Using 10 mL of plating media (PM) (49% Dulbecco's modified eagle medium [DMEM], 1% penicillin/streptomycin [Pen/Strep], 25% horse serum, 25% HBSS with Ca²⁺ and Mg²⁺ [HBSS+/+]; see Table of Materials), combine and resuspend the two pellets together to create a whole cell suspension.
25. Count the cells using trypan blue and either a hemocytometer or digital cell counter, and dilute the cell suspension with PM to a concentration of 1.8 x 10⁶ cells/mL.

3. Plating the cells

1. Add the required volume of the cell suspension to the required format as detailed in Table 1: 1,000 µL per well in 6-well format, 50 µL per well in 96-well format, or 100 µL per coverslip.
2. Incubate for 2-4 h at 37 °C with 5%-7% CO₂. Check the cells have adhered using an inverted microscope.
3. Remove the media and top up with new differentiation media (DM+: DM- including 10 µg/mL insulin. DM-: DMEM, 1% Pen/Strep, 50 nM hydrocortisone, 10 ng/mL biotin, 2.5 mL 100x N1 media supplement; see Table of Materials). Volumes are detailed in Table 1. Press down any floating coverslips using a sterile pipette tip.

4. Maintaining the cultures

NOTE: These cultures require feeding thrice weekly to support optimal growth and differentiation. The cultures will reach optimum health and maturity for experiments on DIV (days in vitro) 21. Cells can be kept in culture for up to 28 days, after which the cultures quickly degenerate.

1. Three times per week until DIV12, replace part of the supernatant with fresh DM+ by removing 500 µL per well in 6-well format, 50 µL per well in 96-well format, or 500 µL per dish containing three coverslips, and adding 600 µL per well in 6-well format, 60 µL per well in 96-well format, or 600 µL per coverslip dish (Table 1).
2. Three times per week from DIV13 onwards, replace part of the supernatant with fresh DM- by removing 500 µL per well in 6-well format, 50 µL per well in 96-well format, or 500 µL per dish containing three coverslips, and adding 600 µL per well in 6-well format, 60 µL per well in 96-well format, or 600 µL per coverslip dish (Table 1).

Results

Microscopy
Cultures grown on glass coverslips are ideal to analyze by microscopy. To visualize the development of the cultures, the coverslips were fixed in 4% paraformaldehyde (PFA) at multiple timepoints from DIV0 (once cells were attached) until DIV28. The cultures were stained for immunofluorescence imaging as previously described⁵ using three different staining combinations: NG2 (immature oligodendrocytes) and nestin (neuronal stem/progenitor cells) as developmental mar...

Discussion

The CNS is a complex network that spans from the brain down to the spinal cord and consists of many cell types, predominantly neurons, oligodendrocytes, astrocytes, and microglia¹. As each cell has an important role in maintaining homeostasis and generating appropriate responses to challenges in the CNS^9,10,11, a culture system that contains all these cell types is a useful and versatile tool to investiga...

Materials

Name	Company	Catalog Number	Comments
10x Trypsin	Sigma	T4549-100ML	To digest tissue
140 mm TC Dish	Fisher	11339283	Put 8 35 mm dishes per 1 140 mm dish
15 mL Falcon	Sarstedt	62554502	To collect cells into pellet for resuspension in plating media
18 G needle	Henke Sass Wolf	4710012040	For trituration of sample
21 G needle	BD	304432	For trituration of sample
23 G needle	Henke Sass Wolf	4710006030	For trituration of sample
35 mm TC Dish	Corning	430165	Plate out 3 PLL coated coverslips per 1 35 mm dish
5 mL syringe	Fisher	15869152	For trituration of sample
6 well plate	Corning	3516	To plate out cells for RT-qPCR, and flow cytometry
7 mL Bijoux	Fisher	DIS080010R	To put brains intp
96 well plate	Corning	3596	To plate out cells for high-throughput testing
ACSA-2 Antibody, anti-mouse, PE	Miltenyi	130-123-284	For flow cytometry staining of astrocytes
Angled forceps	Dumont	0108-5/45-PO	For dissection
Biotin	Sigma	B4501	For DM+/-
Boric Acid	Sigma	B6768-500G	For boric acid buffer
Brilliant Violet 421 anti-mouse CD24 Antibody, clone M1-69	Biolegend	101825	For flow cytometry staining of neurons and astrocytes
Brilliant Violet 605 anti-mouse CD45 Antibody, clone 30-F11	Biolegend	103139	For flow cytometry staining of microglia
Brilliant Violet 785 anti-mouse/human CD11b Antibody, clone M1/70	Biolegend	101243	For flow cytometry staining of microglia
BSA Fraction V	Sigma	A3059-10G	For SD Inhibitor
CNP	Abcam	AB6319	Mature oligodendrocytes
Coverslip	VWR	631-0149	To plate out cells for microscopy
Dissection Scissors	Sigma	S3146-1EA	For dissection
DMEM High glucose, sodium pyruvate, L-Glutamine	Gibco	21969-035	For DM+/-, and for plating media
DNase I	Thermofisher	18047019	For SD Inhibitor, can use this or the other Dnase from sigma
DNase I	Sigma	D4263	For SD Inhibitor, can use this or the other Dnase from thermofisher
eBioscience Fixable Viability Dye eFluor 780	Thermofisher	65-0865-14	Live / Dead stain
Fine forceps	Dumont	0102-SS135-PO	For dissection
GFAP	Invitrogen	13-0300	Astrocytes
HBSS w Ca Mg	Sigma	H9269-500ML	For plating media
HBSS w/o Ca Mg	Sigma	H9394-500ML	For brains to be added to
Horse Serum	Gibco	26050-070	For plating media
Hydrocortisone	Sigma	H0396	For DM+/-
Iba1	Alpha-Laboratories	019-1971	Microglia
Insulin	Sigma	I1882	For DM+
Leibovitz L-15	GIbco	11415-049	For SD Inhibitor
MBP	Bio-Rad	MCA409S	Myelin
Mouse CCL5/RANTES DuoSet ELISA Kit	BioTechne	DY478-05	ELISA kit for quantifying concentration of CCL5 in supernatants of 96 well plate
N1 media supplement	Sigma	N6530-5ML	For DM+/-
Nestin	Merck	MAB353	Neuronal stem/progenitor cells
NeuN	Thermofisher	PA578499	Neuronal cell body
NG2	Sigma	AB5320	Immature oligodendrocytes
O4 Antibody, anti-human/mouse/rat, APC	Miltenyi	130-119-155	For flow cytometry staining of oligodendrocytes
Pen/Strep	Sigma	P0781-100ML	For DM+/-, and for plating media
Poly-L-Lysinehydrobromide	Sigma	P1274	For Boric acid / poly-L-lysine solution to coat coverslips
SMI31	BioLegend	801601	Axons
Sodium Tetraborate	Sigma	221732-100G	For boric acid buffer
Trizol	Thermofisher	15596026	For lysing cells for RT-qPCR
Trypsin inhibitor from soybean	Sigma	T9003-100MG	For SD Inhibitor