This protocol can be used to obtain large number of sensor and the motor fibroblast and Schwann cells rapidly. The cells obtained through this message can be used in the study of nerve regeneration and nervous system diseases. We show demonstration can represent the experimental to better control the time of differential digestive step and make the experimental steps easier to understand.
To burst use scissors to make a three centimeter incision, in the skin of the back. Remove the spinal column. Carefully open the vertebral canal to expose the spinal cord.
Lace the spinal cord in a 60 millimeter Petri dish, with three to four milliliters of ice cold D Hanks balanced salt solution under a dissecting microscope, excise the ventral root and the dorsal root. Place them in ice cold D Hanks balanced salt solution. After removing the HBSS, slice the nerves into three to five millimeter pieces, with scissors.
Add one milliliter of 0.25%trypsin and transfer the nerve pieces to five milliliter centrifuge tubes. Incubate at 37 degrees Celsius for 18 to 20 minutes. Next, to stop the digestion, add at three to four milliliters of DMEM containing 10%fetal bovine serum.
Pipette the mixture up and down gently about 10 times. Centrifuge at 800 times G for five minutes. After centrifugation discard the supernatant.
And re-suspend the precipitate in two to three milliliters of DMEM supplemented with 10%FBS. Filter the cell suspension through a 400 mesh filter, and then use the suspension to inoculate a 60 millimeter Petri dish. Incubate the Petri dishes for four to five days at 37 degrees Celsius, in the presence of 5%carbon dioxide.
When the culture has reached 90%confluence, wash the cells once using 1XPBS. Then, to digest the Schwann cells, add one milliliter of 0.25%trypsin at 37 degrees Celsius per 60 millimeter dish. After eight to 10 seconds at room temperature, stop the digestion, by adding three milliliters of DMEM supplemented with 10%FBS.
To detach the Schwann cells, gently blow on the cell with a pipette. Verify by microscope that the cells are detached. Then collect the medium, which contains the Swan cells, and centrifuge at 800 times G for five minutes.
Discard the supernatant, and re-suspend the precipitate in three milliliters of medium. Use this suspension to inoculate uncoated 60 millimeter Petri dishes, and incubate the dishes at 37 degrees Celsius for 30 to 45 minutes. Brands for the medium, which will contain the Schwann cells to a Poly L lysine coated medium dish, and incubate the dish at 37 degrees Celsius for two days.
After removing the medium containing the Schwann cells wash the fibroblasts, adhering to the Petri dishes with 1XPBS. To digest the fibroblasts, add one milliliter of 0.25%trypsin at 37 degrees Celsius. After following digestion to proceed for two minutes at 37 degrees Celsius, end the digestion by adding DMEM supplemented with 10%FBS.
To detach the fibroblasts, use a pipette to blow on the bottom of the dish. Collect the medium, including the fibroblasts, and transfer it to centrifuge tubes. Centrifuge the tubes at 800 times G for five minutes.
Discard the supernatant, and resuspend the precipitate with two milliliters of DMEM containing 10%FBS. Inoculate the cells in un-coded, 60 millimeter, Petri dishes, and incubate at 37 degrees Celsius for 30 to 45 minutes. Isolate the fibroblasts which will adhere to the Petri dishes, by removing and discarding the growth medium.
Add three milliliters of DMEM supplemented with 10%FBS. Incubate at 37 degrees Celsius for two days until the passage one cells reach 90%confluence. Repeat the differential digestion and adherence.
After culturing the passage to fibroblasts and Schwann cells for two days, digest the cells, collect them, and count them. Inoculate PLL coated slides at a concentration of one times 10 of these cells per well for ICC staining. Bay's contrast microscopy shows the morphology of cultured Schwann cells and fibroblasts throughout the isolation process.
After prolonged culture time, the Schwann cells were clustered together between the fibroblasts or located on the surface of fibroblasts. After digestion for 10 seconds, the Schwann cells, indicated by the red arrows, were round while the fibroblasts remained flat and were attached to the bottom of the dish. After isolation by differential digestion and differential adherence typical cell morphologies were observed for all four cell types.
Motor fibroblasts, sensory fibroblasts, motor Schwann cells and sensory Schwann cells. The sensory and motor fibroblasts were visualized using a cone focal laser scanning microscope. The fibroblasts were stained with antibodies against CD 90 X33342 dye was used to label the cell nuclei.
The merged images of fibroblasts immunostaining and nuclear staining, indicated that 92.51%and 92.64%of CD 90 and hexed co-labeled cells, were present in the motor and sensory fibroblasts respectively. The sensory and motor Schwann cells were visualized using a confocal laser scanning microscope. The Schwann cells were stained with antibodies against S100 X33342 dye was used to label the cell nuclei.
The merge images of Swan cell immunostaining and nuclear staining indicated the presence of 91.61%and 93.56%of S100 and hexed co-labeled cells in motor and sensory Schwann cells, respectively. Cell purity was also analyzed using flow cytometry. In the fibroblast cultures, approximately 90%of cells were fibroblasts, indicated by M2 in the FCA graphs while the remaining cells were Schwann cells.
In the Schwann cell cultures, more than 92%of cells were Schwann cells, again indicated by M2 in the FCA graphs, while the remaining cells were fibroblasts. This protocol is rarely useful for starting the mechanism of sensor and motor nerve regeneration of fibroblasts, and the Schwann cells transplantation, to promote nerve regeneration.
