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Schwann cells are the major seed cells to repair nerve injury and nerve disease in peripheral nervous system^[1-7]. Not only can they form the myelin^[8] to provide access for peripheral nerve repair, but also secrete a variety of neurotrophic factors and extracellular matrix to provide appropriate microenvironment for peripheral nerve repair^[9-16]. Recent studies have shown that Schwann cells also play an important role in the repair of spinal cord injury^[17-21]. However, the source of Schwann cells is limited, although some people with fetal peripheral nerve have nurtured Schwann cells successfully, the purity of Schwann cells is affected due to the pollution of fibroblasts. Some researchers suggest a variety of purification methods^[22-27], but each has its disadvantages and cannot well meet clinical applications. The present study compared four methods of purifying rat Schwann cells in vitro.

Schwann cell transplantation has been proved to be effective in the treatment of peripheral nerve injury. A large number of high-purity Schwann cells are required for clinical application, which demands Schwann cells cultures in vitro. The present study utilized hemi-explants single enzyme digestion method^[26] for primary culture to obtain a large number of cells in a short period of time and compared the four purification methods. It is impossible to remove never membrane completely because the outer membrane of nerve fibers and perineurium are formed by fiber cells. And compared with Schwann cells, proliferation and growth factor of fibroblast is faster. Therefore, it is difficult to delete fibroblasts completely.   Differential adhesion method purifies Schwann cells according to the different adherent speed and capacity between fibroblasts and Schwann cells. The Schwann cells were treated with low concentration (0.125%) trypsin, when the cells shrinks standard medium was added to terminate digestion. The low concentration trypsin can reduce Schwann cells damage, and short period of time also has little impact on cell activity. Therefore, the activity of Schwann cells is high. However, due to the adherent speed and capacity differences between cells, some fibroblasts are absorbed or deform. Moreover, as the strength to pipette cells is not easy to control, Schwann cell purity is always affected by the mixed cells. Immunomagnetic beads selection method labels Schwann cells using special cell surface markers, and isolated Schwann cells using immunomagnetic beads coated second antibody in a magnetic field. This method is reported by Maurice and Norbert^[27] for the first time, and it is also used in domestic ^[31], in which the purity of Schwann cells can reach 99%. But this method requires repeated centrifugation, and positive cells should be quickly eluted from the separation column at the final step, so the mechanical compression to the cells will significantly affect cells activity, which results in a lot of debris after the process of purification. Moreover, the cells take some time to return to the original state after purification. Cold jet method separates Schwann cells and fibroblasts according to the different adherent capacity and temperature-sensitivity. This method is first reported by Jirsova^[29]. The Schwann cell purity is more than 99% after twice purification. The operation process is monitored under the microscope. The pipetting can be terminated according to Schwann cells shape changes after adding 4 ℃ PBS and floating condition of pipetted Schwann cells. After cold temperature stimulation, cells were placed immediately in standard medium at 37 ℃ so that cells continue to grow at appropriate temperature. The cell viability is slightly affected. This method has been proved to be effective by later studies ^[32]. G418 selection method purifies Schwann cells according to the faster proliferation of fibroblasts than Schwann cells, and the cytotoxic drugs acting on the faster proliferation cells. This method has been widely used ^[33]. However, the viability of Schwann cells is also affected as the cytotoxic drugs have no specificity to cells. But G418 selection method can obtain high purity Schwann cells when combining with low concentration of trypsin digestion. Differential adhesion method sorts cells by speed of cell adhesion and has been also frequently used to sort cells. This method just uses trypsin to digest cells within short time, and mildly affect cell viability, so cell viability is high. However, cell adhesion capacity of single cell from different species is not very clear. Consequently, the purity of cells sorted by differential adhesion method is affected, leading to insufficient purity. Immunomagnetic beads selection method can get high purity cells as the specificity of antibody binding to antigen. But it significantly damages cell viability by repeated centrifugalization. Cold jet method uses the temperature sensitivity of cells to purify Schwann cells. Because the temperature sensitivity of Schwann cells is much greater than fibroblasts, Schwann cells float when fibroblasts remain adhering. Therefore, high purity Schwann cells are harvested by drawing the supernatant. Cell viability is affected after a short cold shock treatment. The cell viability of G418 selection method is impacted as the cytotoxic drug used. However, the purity of Schwann cells is fair after recovery a few days from cytotoxic drug condition. Therefore, this method can also get high purity cells.  In conclusion, compared with other three methods, cold jet method can obtain Schwann cells with high purity and high viability. Moreover, it is cost effective, and simple to operate. It is a applicable method to purify Schwann cells in vitro.

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