A New Method for Primary Culture of Mouse Dorsal Root Ganglion Neurons

In order to establish a simple and highly purified method for primary culture of mouse dorsal root ganglion neurons(DRGn), in this study, the DRGn of young mice were obtained by collagenase type I and trypsin digestion. Then the DRGn were obtained by immunocytochemical staining of mouse neuron specific enolase (NSE) monoclonal antibody, while using flow cytometry to further detect the positive rates of DRGn. The cultured primary DRGn grew well and had a purity of about 83.72%. The DRGn survival time was 60 days when cultured in DMEM medium containing nerve growth factor (NGF). The culture scheme is simple and stable, and a large number of high purity DRGn could be cultured, which provides a reliable model for further study of nerve cells.


Introduction
Spinal DRGn are afferent neurons of peripheral nervous system and primary neurons of sensory conduction system. With the increasing research on the nervous system, such as the pain mechanism of diseases [1][2][3], spinal cord injury repair [4,5], electrophysiological research [6][7][8][9], it is necessary to establish a simple and easy-to-obtain primary nerve cell culture scheme.
Based on the previous studies [10][11][12][13], this scheme simplifies the cultivation process, aiming to cultivate a large number of high-purity nerve cells and providing a stable and convenient model for neuropathophysiology research.

Methods and procedures Animals
The healthy male C57BL/6 mice aged 6~8 weeks were selected from the animal center of China Three Gorges University. All procedures in the animal studies were approved by the Animal Care and Use Committee at China Three Gorges University.

Isolation and acquisition of DRGn
C57BL/6 mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 3 minutes. The mice were dissected from the back in order to take out the spinal column and were put in the plate of 4℃ Hank's solution. The spinal columns were divided into two halves by ophthalmic scissors along the midline of the spine. The spinal cords were gently moved by microforceps to make the DRG exposure. Each DRG was collected in a centrifuge tube while the nerve roots were cut off. An appropriate amount of 0.125% collagenase type I was added into centrifuge tube to digest DRG and was shaken at 37℃ for 30-50 minutes, and then it was centrifuged to remove collagenase type I.
Next, the previous step was repeated before the collagenase type I was removed again. Finally, a proper amount of 0.25% trypsin was added into the centrifuge tube before it was shaken for about 20-30 minutes at 37℃.
After 24 hours of culture, the medium in the culture dishes was change entirety, and the medium was changed every 3 days. The DRGn at different time stages were observed under an Olympus microscope and their morphological changes were also recorded.

Immunocytochemistry
After 7 days of culture, the DRGn were washed for 3 times with phosphate-buffered saline(PBS), 0.01M, pH7.4, and were fixed with 4% paraformaldehyde at room temperature for 40 minutes, then were washed for 3 times with PBS for 5 minutes each time. 0.1% Triton solution was submerged in for 30 minutes at room temperature, and was washed for 3 times with PBS for 5 minutes each time. To block non-specific protein, 10% goat serum was submerged in for 30 minutes at room temperature before it was then removed.
The DRGn were incubated overnight at 4°C with NSE monoclonal antibody (Abcam, ab16808) diluted at the ratio of 1:100. The primary antibody was recovered and washed for 3 times with PBS, and was incubated with goat anti-mouse IgG (KERUI, 125435, China) at the ratio of

Morphological observation of neurons in DRGn
The primary cultured DRGn adhered slowly and most of them adhered to the wall after 24 h. The DRGn presented a clear spherical shape in the first day. When cultured to the 5th day, most of the DRGn protruded synapses and the characteristic morphology of neurons was obvious. On the 7th day, the DRGn grew vigorously in good condition. The DRGn intertwined into a network with axons and dendrites, and had larger cell bodies, which were round or polygonal. After 15 days, the bodies of DRGn gradually shrank and became irregular, who showed an aging trend, and the cross-links among cells were disordered. The medium was changed semi-quantitatively every 3 days until a plenty of DRGn were still visible on the 60th day ( Figure.1).

Immunocytochemistry of DRGn
The nuclei of all DRGn were stained blue by hematoxylin under an inverted phase contrast microscope ( Figure. The DRG of young mice was extracted and digested by collagenase type I and trypsin to form single cell suspension for culture. Fibroblasts were the common impurity cells when cultured DRGn. Differential adherence method is commonly used to purify DRGn, meanwhile some DRGn will be lost in this process. Another purification method is to add 5-fluoro-2-deoxyuridine nucleotide and uridine after 24 hours of cultivation to clear the proliferative non-neuronal cells [14,15]. Although it exerts a certain effect, it also causes partial damage to DRGn. This program, however, has undergone a large number of experiments so as to improve the methods of extracting the primary DRGn of the predecessors. The biggest improvement in this program was that the digestion time was prolonged. The digestion time was 60-90 minutes with collagenase type I twice, and the digestion time of trypsin was 20-30 minutes. The purity of the primary DRGn extracted by this program can reach more than 80%, without the need to add purified drugs. It is concluded that the enzymatic method removes some impurity cells to a certain extent.
10% horse serum and 100μg/L NGF were added to DMEM medium for culturing DRGn. Fetal bovine serum contained mitotic factors, which were often used as serum for culturing proliferating cells. But horse serum can avoid this defect. Therefore, if horse serum were used in nerve cell culture, the growth of fibroblasts could be inhibited to some extent. NGF can maintain the survival of nerve cells, and promote the growth and elongation of axons and dendrites as well, so it is also an essential component in the medium [16][17][18]. With regard to the selection of Petri dishes, it is advisable to use small Petri dishes, such as 35 mm Petri dishes, because the growth of nerve cells requires certain density, and the cross-linking between axons and dendrites is conducive to the transmission of information among nerve cells.
NSE is a glycolytic enzyme existing in nerve cells and neuroendocrine tissues. It is often used as a marker to identify primary nerve cells [19,20]. It mainly exists in cytoplasm. Immunochemical staining showed that the cell bodies of the positive neurons were brown, and the positive rate was 83.72% by flow cytometry detection.
The programme of this experiment is easy to operate and obtain materials, which are adult mice instead of embryonic mice. This programme is also convenient to culture DRGn, and can reduce other purification steps. The survival time of DRGn is long, and most of the neurons can still be seen on the 60th day of culture. In conclusion, this experimental method can provide DRGn from adult mice, and provide a new model for the further study of neurology.