Culturing Microglia from the Neonatal and Adult Central Nervous System

Summary

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Abstract

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

Introduction

Microglia are the resident immune cells of the CNS, most closely resembling peripheral macrophages in structure and function ¹. It has recently been demonstrated that postnatal microglial cells derive from primitive myeloid progenitors and are generated before the eighth day of embryogenesis, upending the previous notion that postnatal hematopoietic progenitors are the source of microglia in the adult brain ². They play key roles in several neurological diseases and can quickly respond to infection or injury by releasing pro-inflammatory or anti-inflammatory cytokines ³. Thus microglia encompass a standalone unit in the CNS that can be manipulated to affect disease progression. Developing robust and reproducible methods to isolate and culture neonatal or adult microglial cells is important to future studies.

Microglia are known to be critical players in a number of brain pathologies. More recently, roles are emerging for the cells in normal brain development and function as microglia phagocytose excess neural progenitor cells from the dentate gyrus of the hippocampus ^{1, 4}. Microglia can also modulate several neurological conditions that affect the spinal cord, such as MS, neuropathic pain, and spinal cord injury ^5-7. Spinal cord microglia react differently compared to brain microglia in response to activation signals ^{8, 9}, probably due to differences in the local environment. Thus it is important to establish an appropriate in vitro system to culture and study spinal cord microglia. Neonatal microglia produce significantly more of the pro-inflammatory cytokine nitric oxide compared to adult cells after in vitro stimulation with IFN-γ or TNF-a^10,11 further highlighting the need to use adult cells to study microglia in the context of certain diseases.

The protocol we employ in the lab to culture neonatal microglia is a variation of recent methods which utilize shaking of mixed glial cultures in an effort to remove the microglia from the surface of the cell culture flask ¹². We also describe a method to culture microglia from the adult mouse spinal cord based on a protocol first described by Yip, et al¹³. This method provides a quicker way to culture adult cells compared to other available protocols ¹⁴. The resulting preparation is 70% microglia; the remaining percentage is composed of astrocytes. Although the purity of our culture is lower compared to other published methods ¹³, this culture system is useful for exploring the microglial in culture response to various activating stimuli as well as for the study of diseases that mainly affect the spinal cord and in which a strong inflammatory response is a main feature.

All the protocols described have been approved by the Stony Brook University IACUC.

Protocol

1. Dissection (Day 0)

1. Induce anesthesia to p0-2 mouse pups with hypothermia. Wipe the heads of the pups with a Kimwipe soaked in 70% ethanol to disinfect the tissue.
2. Remove the heads with a pair of scissors, using 4 pups per 10 cm culture plate. Place the heads in a petri dish containing ice-cold Hank's buffer.
3. Anchor the heads using curved forceps through the eye sockets and carefully remove the skin covering the skull with straight microforceps.
4. Remove the cranial bones with straight microforceps, using caution to not puncture or damage the cortices. The most effective way is to begin the removal starting near the cerebellum, which is located at the base of the head, as this region will be discarded.
5. Remove the brain with a small spatula, and place the (4) brains in a fresh petri dish containing ice-cold Hank's buffer.
6. All steps from this point on utilize microforceps and are performed under a dissection microscope. Starting on the ventral side of the brain, anchor the tissue by holding the cerebellum and make two small incisions on either side of the midbrain. Be careful not to cut all the way through the tissue, as the cortices cover the midbrain in this region.
7. Gently tease the midbrain and cerebellum in one piece from the two cortices. The two cortices should form a concave shape.
8. Separate the two cortices and orient one single cortex with the medial side up for further dissection. The hippocampus can be difficult to observe, but it is located opposite of the olfactory bulb which appears as a small nodule on the pointed end of the cortex.
9. Remove the hippocampus, which has a crescent-shape. Flip the cortex over to view the dorsal side.
10. Using the olfactory bulb as a starting point, remove all meninges and the olfactory bulb itself from the cortex.
11. The dissected cortices should be placed into 15 ml conical tubes with 14 ml of cold Hank's buffered saline on ice.

2. Cell Culture (Day 0)

1. Pre-coat 10 cm tissue culture dishes with 5 μg/ml of Poly-D-Lysine (PDL) diluted in autoclaved water for 3 hr at 37 °C.
2. Aspirate PDL, and wash plates once with autoclaved water. Dry plate under the UV in the tissue culture hood for 20 min. This step should be performed just prior to the dissection process.
3. Aspirate Hank's buffer from tubes containing the cortices from 4 brains each, apply 4 ml of 1x trypsin/EDTA solution, triturate tissue with p1000 tip, and place tubes in 37 °C incubator for 15 min.
4. Add 4 ml of complete microglial media per tube to stop the enzymatic digestion, mix, and spin down contents at 1.5K rpm for 5 min.
5. Aspirate supernatant and repeat wash with 4 ml of complete media. Resuspend cells in 10 ml of complete microglial media (DMEM with 10% FBS, 1% sodium pyruvate, 0.08% gentamycin), and filter through a 40 micron mesh cell strainer.
6. Plate at a density of 8 cortices per 10 ml in a 10 cm tissue culture plate and place in a tissue culture incubator at 37 °C and 5% CO₂ (See Adult Microglia protocol).

(Day 3)

1. Change media in all cell culture dishes with complete microglial media.

(Day 10)

1. Add 400 microliters of 60 mM Lidocaine in HBSS (to detach microglia) into medium of 10-cm tissue culture plates and incubate at room temperature (RT) for 10-15 min.
2. Collect the media/cell suspension from the plate, and wash the plate once with Hank's buffer. Collect the wash buffer to recover remaining microglia.
3. Add 5 mM EDTA (pH 8.0) to the cell suspension to a final concentration of 50 μM or at a 1/100 dilution.
4. Spin down cell suspension at 1,000 x g (1,500 rpm) for 5 min and re-suspend in 1 ml DMEM with 1% FBS.
5. Count viable cell number (as per Adult Microglia 3.1/3.2) and split cells into tissue culture plates at the desired experimental density. Approximately 1x10⁶ microglia are harvested from a 10 cm plate containing the cortices from 4 brains. For immunofluorescence, a density of 2.5x10⁴ cells per 18 mm coverslip is recommended.
6. Allow at least 24 hr for microglial cells to fully return to their ramified, resting state prior to use.

Protocol Text: (Adult Microglia)

1. Tissue Collection

1. Coat tissue culture plates with Poly-D-Lysine (PDL) for 3 hr at 37 °C or overnight at 4 °C. Wash the plates once with autoclaved water immediately prior to use.
2. Euthanize mouse by CO₂ asphyxiation and ascertain that euthanasia is complete. Clean spinal cord area with 70% ethanol.
3. Using a pair of scissors cut the skin on top of the spinal cord; then proceed to cut the spinal cord out from region T1 to T12. Cut the remaining muscle off of the sides of the spinal cord.
4. Slowly cut the vertebrae using microscissors as you gently hold the spinal cord with your fingertips. Be careful not to puncture the spinal cord as the tissue is very soft. Slowly extract the spinal cord using microforceps.
5. Submerge the spinal cord in a petri dish containing ice-cold HBSS. Using a light microscope remove all visible meninges using microforceps
6. Cut the spinal cord into transversal segments small enough to generate several fragments. The thickness of the fragments should not be more than 2 mm in order to facilitate efficient digestion. Transfer the spinal cord pieces to a 15 ml conical tube containing 1 ml ice-cold HBSS. Keep the tube on ice until the digestion step.

2. Digestion of Tissue

1. Aspirate HBSS from 15 ml conical tube containing spinal cord tissue. Be careful not to aspirate any piece of tissue.
2. Add 1 ml of trypsin (2.5 g/L irradiated porcine trypsin and 0.2 g/L EDTA in HBSS) and incubate at 37 °C for 30 min.
3. To stop the digestion remove the trypsin and add 3 ml of primary microglia medium which contains serum to halt the enzymatic digestion.
4. Pipet up and down to dislodge the tissue. Filter the cell suspension using a 40 μm cell strainer.

3. Cell Counting and Plating

1. Mix equal volume of cell suspension and DAPI solution.
2. Count cells using a hemocytometer.
3. Plate at a density between 5-7x10⁵ cells/ml in PDL-coated 35 x 10 mm dishes . Plating at a lower density prevented cell growth even when cells were cultured in 12 well plates which have a smaller area compared to 35 x 10 mm dishes.

Results

An example of resting and activated microglial cells is shown in Figure 1. The microglia were visualized 24 hr after plating (Figure 1a) and exhibit ramified (resting) morphology. Exposure to the priming reagent, bacterial lipopolysaccharide (LPS) results in changes in microglial morphology as the cells become activated (Figure 1b).

An example of counting the cells for plating is shown in Figure 2. Due to the presence of cell ...

Discussion

Microglia modulate CNS normal functioning as well as inflammatory responses to various pathologies. Functional synaptic remodeling by microglia has been implicated in the maintenance of normal brain homeostasis ¹⁵. During the neurogenic cascade they participate in the clearance of neural progenitor cells from the dentate gyrus of the hippocampus ^{4, 16}. Therefore, it is necessary to develop a culture system in which to study neonatal and adult microglia, which will cover the vast swath of deve...

Materials

Name	Company	Catalog Number	Comments
EDTA, 5mM	Invitrogen	15567-028
Lidocaine, 60mM	Sigma	L-5647
Trypan Blue	Sigma	T8154
Trypsin/EDTA	Cellgro	25-052-CI
DMEM, 1X	Cellgro	10-017
Sodium Pyruvate	Cellgro	25-000-Cl
Gentamycin Sulfate	Biowittaker	17-518Z
35 x 10mm tissue culture dish	Falcon	353001
Poly-D-Lysine, 100 μg/ml			Dilute 1:20
HBSS, 1X	Cellgro	20-023-CV