Primary culture experiments of dorsal root ganglia can be used for (1) the induced growth and development of periapical ganglia and the formation of extensive neural networks, and (2) studies of axonal growth and development using cultured DRG cells.
Operation method
Primary culture experiments on dorsal root ganglion cells
Principle
Primary culture refers to the first culture of tissues or cells removed from the body, also called primary culture. Primary culture has a short period of time away from the body, and the genetic characteristics are similar to those of the cells in the body, which is suitable for the study of cell morphology, function and differentiation. Strictly speaking, it refers to the culture before the success of the generation, at this time the cells maintain the basic properties of the original cells, if it is a normal cell, it still retains the diploid number. In practice, however, it is common to refer to cultured cells within the first to tenth generation as primary cell culture. The most commonly used primary cultures are tissue block culture and dispersed cell culture.
Materials and Instruments
Chicken embryos incubated for 8-12d. 15d gestation and neonatal 1-3d rat and mouse littermates Move The detailed steps after obtaining sterilized animals were as follows: 1. Embryos or littermates were placed supine in ice D-PBS solution under aseptic conditions, and the thoracic and abdominal cavities were opened after severing the head to remove the internal organs. Insert microscopic scissors through the opening of the cervical spinal canal, cut open the spinal canal along both sides of the dorsal midline of the spine, remove and expose the spinal cord, i.e., the dorsal root ganglia arranged along both sides of the spinal column can be seen next to the intervertebral foramen, extracted carefully by fine microphotography, and stripped of its periosteum. 2. If tissue block culture is performed, each dorsal root nodule can be dissected into 2-3 pieces by fine microscopic scissors, inoculated directly onto a substrate coated with rat tail glue, and cultured with a small amount of DMEM culture solution containing 10% fetal bovine serum. 3. If the dorsal root ganglion dispersed cell culture is needed, the isolated DRG tissue blocks can be collected into 0.25% trypsin solution and digested at 37℃ for 30 min. 10 drops of fetal bovine serum was added to terminate the digestion, and then centrifuged at 900r/min for 5 min, and the supernatant was removed. After that, according to the general method in 10% fetal bovine serum in DMEM culture medium to make a single cell suspension, adjust the cell concentration for (0.5-1) X105/ml inoculation of cells in polylysine-coated media, 37 ℃, 5% CO2 incubator culture. 4. After 24h of the above two culture methods, the planting medium in the petri dish was dumped, and the cells were cultured in serum-free medium (Neurobasal+827+glutamine+20ng/ml NGF). On the 3rd day of inoculation, the cell division inhibitor ARA-C was added to inhibit the proliferation of non-neuronal cells, and the fresh culture medium was replaced by half volume after 48h of action, and then by 1/2 volume twice a week. 5. Purified cultured DRGs can survive for as long as 2 months, and the purity can reach more than 96%. Decentralized cultured DRG neurons were seen to have halo sensation in the cytosol at 48h, round or oval, with different sizes, multipolar, bipolar and pseudo-unipolar neurons. 2d later after giving inhibitor treatment, the DRG protrusions grew rapidly and formed a sparse network-like structure. With the prolongation of culture time, the trunks and branches of neuronal protrusions were obviously lengthened and thickened, and the network of neuronal protrusions became denser, the impurity cells had been desiccated and floated, and the background of the culture became much cleaner and clearer, and the immunocytochemistry further identified the cultured cells as Neurofilaments (NF)-positive (Figs. 1-3). Caveat 1. In the culture of tissue block, it is more difficult to remove the peridium of one thousand dorsal root nodes, and it is more conducive for the cells to climb out of the block by dissecting each dorsal root node into 2-3 blocks, which will obviously improve the survival rate of the blocks. 2. The digestion and incubation time of the acquired tissues should be adjusted according to the age of the animals, i.e., it can be controlled at 20-30min in embryonic stage, and 30-50min in littermates. 3. With the popularization of serum-free culture medium, the latest literature reports that it is also used in the culture of thousand DRG. However, the difference is that serum-free medium can be directly used to inoculate the hippocampus, cortex and other central neurons, while DRG needs to be inoculated with serum-containing medium for 24h, and then changed to serum-free medium to maintain the culture, which is crucial, otherwise DRG cannot be viable. 4. When the cells are purified and cultured, pay attention to grasp the time point of ARA-C addition. If the cell density is high, it can be added after 1d of serum-free culture, while it needs to be added after 2d when the cell density is low. In addition, the gradual 1/2 exchange of serum-free culture medium containing ARA-C is a key step to ensure the high purity of DRG neurons. Common Problems 1. In terms of dissection method, ventral dissection to expose the spinal cord is crucial during DRG isolation. The author's attempt to take material from the dorsal dissection path was not easy to obtain complete DRG tissue, thus affecting the cell acquisition rate. 2. The author chose rat tail collagen as the culture substrate, and found that the neuronal cytosol and axon had stronger and more stable wall adherence, which was suitable for long-term observation; whereas neurons grown on polylysine matrix layer could only be adhered and stabilized for a short period of time, and with the prolongation of the cultivation time, neuronal protrusions floated up and lost the support of the substrate. In particular, the purified DRG will be delaminated together with the whole neural network in a short period of time. 3. The morphology, structure and growth differentiation of mouse dorsal root ganglion neurons are basically similar to those of rats, but the cytosol of mouse dorsal root ganglion neurons is slightly smaller than that of rats. In chick embryos, pseudo-single poles were more common in dorsal root ganglion neurons, and fewer neurites were branched. 4. When culturing the planted blocks, a small amount of culture medium should be added to cover each block, otherwise the block will be floated by the liquid, lose the support of the substrate and fail to grow. For more product details, please visit Aladdin Scientific website.
DMEM medium containing 10% fetal bovine serum Neuronal Maintenance Medium (Neurobasal+B27+Glutamine+20ng mlNGF) 10µM cytarabine (ARA-C)
Culture Bottle
Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press.