Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

1. Glia Cell Culture

Glial cells can be cultured from different regions of central nervous system. The whole process is shown in process figure.

Day 1 Coating culture plate and coverslips

1. Dry sterilized glass microscope round coverslips in an autoclave.
2. Plate the sterilized coverslips into sterilized 24-well culture plate.
3. Coat the coverslips with poly-lysine and incubate for 2 hours under root temperature.
4. Coat 6-well culture plates in the same way as step 3.
5. Wash the coverslips and 6-well culture plate twice with distilled water and air dry in culture hood.
6. Add 200 μL DMEM medium(with 10% FBS) in each well in 24-well plate and 2 mL in each well in 6-well plate and put them into incubator under 37 degree with 5% CO₂

Day 2 Isolating cortex and glial cell culture

1. Sterilize the positive flow dissection hood.
2. Turn on UV light for 20 min.
3. Spray all surfaces with 70% ethanol and wait 15 min before use.
4. change tense: Anesthetize rat pups (P2-P6) by hypothermia, and decapitate at the base of the framen magnum using operating scissors.
5. Open the cranium along the sagittal suture using an iris scissor and peel off the skull.
6. Remove the forebrain and put them into chilled L15 medium, under stereomicroscope, carefully clean the dura and pia membrane with blood vessels, isolate the cortex and wash several times with L15 medium.
7. Cut the cortex into small pieces with microsurgery scissor.
8. Add 0.125% trypsin-EDTA prepared with L15 medium into cortex pieces and incubate in 37 degree for 15 min.
9. Transfer tissue blocks into 20mL DMEM medium with 20% FBS in 50mL tube, add DNase stock solution to final concentration 10ug/mL, aspirate the tissue solution up and down for 20 times with fire polished glass Pasteure pipette, collect the single cells suspension.
10. Wash the cell suspension one time with DMEM medium, re-suspend cells in DMEM with 10% FBS, count cells under microscope with a hemocytometer, seed cells at 5000-10000/cm². Maintain cells in a 37 degree 5% incubator, change half of the medium 2 times per week.

2. Dorsal Root Ganglia Neurons Isolation, Culture and Purification

Day 1 Prepare Culture Material

1. Coat the sterilized coverslips with poly-lysine, wash coverslips twice with distill water and air dry, put them into 24-well plates.
2. Add 100μL Neurobasal medium with 2% B27 and 2.5S NGF (50 ng/mL), put plate into 37 degree 5% CO₂.

Day 2 Isolate DRGs from embryos

1. Sterilized the flow hood in the same way as glial cell culture.
2. Euthanize a pregnant rat (E15) by overdose with CO₂, sterilized abdomen by 70% ethanol, embryos were isolated from uterus and put into chilled L15 medium.
3. Isolate Spinal cords with connected dorsal root ganglia under stereomicroscope and transfer to 35mm dishes with chilled L15 medium.
4. Collect single cell suspension and wash once with NBF medium (Neurobasal medium containing 2% B27 and 2.5S NGF (50 ng/mL)).

Day 3 Purify DRG Neurons

1. 18h-24h after neuron seeding, add a 1 mM concentrated FUDR stock solution to the neuron culture to a final concentration of 20 μM and a final volume of 200 μL.
2. 72h later, replace half the medium with Neurobasal medium containing 2% B27 and 2.5S NGF (50ng/mL) without FUDR. After that, change the medium every other day.

3. Coculture DRG neurons with glial cells

1. When glial cell culture reached confluence (around 20 days after seeding), they were ready for coculture with neurons.
2. 24 hours before adding the purified neurons, glial cells medium were changed to Neurobasal medium containing 2% B27 and 2.5S NGF(50 ng/mL).
3. Purified neurons were collected from culture wells, single cells suspension were obtained by mechanical passing through fire polished Pasteure pipette.
4. After counting the number, neurons were seeded at 500/cm² on confluent glial cells in Neurobasal medium containing 2% B27 and 2.5S NGF (50ng/mL).
5. Neurons adhesion and neurite growth on glial cells could be recorded and analyzed at different time points by image analysis software and immunocytochemistry using cell type specific antibodies.

4. Representative Results

1. Glial cell culture: After seeding, glial cells will become confluent around 20 days. Under phase contrast microscope, it was easily identified that different morphological glial cell subpopulations formed different growth pattern substructures and the GFAP positive astrocytes accounted for more than 90% as shown by immuocytochemisty technique (Figure 1, Figure 2).
2. DRG neurons culture: In our method, after 72 hours FUDR treatment, the purity of DRG neurons will reach as high as 99% within 6 days, DRG neurons showed their unique morphologies and with high density neurite growth(Figure 3, Figure 4).
3. Glial cell and neurons coculture: DRG neurons adhesion and neurite outgrowth occurred readily on glial cells within 4 hours after seeding, careful observations showed that neuron adhesion and neurite growth were influenced by glial cell subpopulations which formed special growth pattern substructures, this could be easily identified under phase contrast microscope and by immunocytochemistry(Figure 5, Figure 6).

Figure 1. Morphology of confluent glial cells. Cortical glial cells were plated on polylysine coated coverslip and cultured for 20 days, note the different growth pattern of glial cells, cells on the left side arranged in a radiated way.

Figure 2. Confluent glial cells labeled by Glial fibrillary acidic protein (GFAP) antibody. Note the radiated arrangement of GFAP (red) positive cells on the right side.

Figure 3. Dorsal root ganglia neurons grew in vitro without FUDR treatment. The contaminating cells formed DRG neurons’ background.

Figure 4. Dorsal root ganglia neurons grew in vitro after FUDR treatment. The background contaminating cells had been eliminated completely.

Figure 5. Dorsal root ganglia neurons grown on glial cells. Neurons adhesion and neurite growth were inhibited on the radiated arranged cells and limited on the right side glial cells.

Figure 6. Dorsal root ganglia neurons growing on glial cells labeled by neurofilament antibody. The neurite(green) were inhibited on the radiated arranged left side glial cells and limited on the right side, all the glial cells were labeled by GFAP antibody(red).