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Schwann cells have been shown to be closely related to the development and regeneration of peripheral nerves and play an extremely important role in maintaining peripheral nervous system function and repairing peripheral nerve injury^[1-3]. For this reason, Schwann cells have been widely used in peripheral nerve tissue engineering^[4]. A basis for research is to harvest a large number of purified Schwann cells. Many methods have been presently developed to culture Schwann cells, such as explant culture^[5], enzyme digestion^[6], anti-mitosis^[7], specific adhesion^[8], immunoselection^[9-10], differential attachment^[11], and inhibition of fibroblasts^[12]. Different methods have different advantages and drawbacks^[13-15]. The explant culture was first reported by Askanas et al ^[16] in 1980. Its advantage is to be able to harvest considerable high-purity Schwann cells, but it wastes long time, and cell proliferative capacity is influenced. The enzyme digestion was first reported by Brockes et al ^[17] in 1979. Its advantage is to harvest considerable cells within short time, but it can not ensure cell purity. The immunoselection method can acquire high-purity cells but costs high. Based on the characteristics of nerve structure, the present study adopted double enzyme digestion and two incubations, followed by differential attachment to discard fibroblasts to enhance the purity of Schwann cells. A set of feasible methods were finally determined to harvest considerable Schwann cells with high purity and activity.

The in vitro proliferation and purity of Schwann cells are relatively difficult owing to poor proliferative capacity and fibroblast contamination. With further research, pre-injury has been used to solve the problem of poor proliferative capacity of Schwann cells^[20]. However, there has been no optimal treatment of fibroblast contamination. Based on the histological structure characteristics of the nerve and different attaching speeds of cells, the present study developed the extraction and purification methods of Schwann cells.   Removal of epineurium and perineurium The nerve contains epineurium, perineurium, and endoneurium. Each layer has considerable fibroblasts. Neonatal rat sciatic nerve is very tiny, so it is very difficult to excise the epineurium, in particular the perineurium, under the microscope. Adult rat sciatic nerve is very thick, so it is easy to excise the epineurium, but it remains difficult, however feasible, to excise the perineurium. The present study excised rat epineurium and perineurium to reduce the source of fibroblasts as far as possible, which is different from previous findings^[21]. Because perineurium and endoneurium reside in the lateral myelin sheath, so fibroblast abscission is first primarily performed. In the present study, in the first inoculation after digestion, nerve tissue should be preserved as far as possible, and the suspended fibroblasts were removed by attachment (Figure 5a); in the second inoculation, nerve tissue blocks were rapidly re-suspended to separate the fibroblasts from tissue for later differential attachment, which reduces the source of fibroblasts and avoids decreased Schwann cell activity caused by long-term retention in in vitro tissue (Figure 5b). These are different from previous findings^[22-24].   Schwann cell morphology and attaching capacity After attachment, neonatal rat Schwann cells exhibited a well-stacked, splendent cell body. Other cell bodies or cell processes were seen passing through the passage between many Schwann cell bodies and bottom wall, which indicates a relatively weak attaching capacity. The large processes had intumescent, irregular, thin and flat ends, which attached to bottom wall. In addition, large processes stretched out small spinous processes, which also attached to bottom wall. Adjacent processes were interwoven by small processes, demonstrating that Schwann cell attachment primarily depends on the ends of large processes and small processes. Adult rat Schwann cells exhibited similar morphology to neonatal rat Schwann cells, but cell body was larger, and fraction was weaker, demonstrating that neonatal and adult rat Schwann cells exhibited attaching capacity. Attachment speed A key to in vitro culture of Schwann cells is to discard the mixed fibroblasts^[25]. Differential attachment has been a common method at home and abroad. Results from this study revealed that compared with the fibroblasts, neonatal Schwann cells exhibited a faster attaching speed, which was different from previous findings^{[6, 23, 26]}, and the underlying mechanisms need to be investigated, while adult Schwann cells showed a slower attaching speed, which was consistent with previous results^{[13, 21, 23]}.  Generally, the proliferative capacity of cells would be decreased with aging. The present study detected the proliferative capacity of Schwann cells by MTT microcolorimetry, demonstrating that neonatal rat Schwann cells exhibited stronger proliferative capacity than adult rat Schwann cells and that the use of MTT microcolorimetry can acquire high-activity Schwann cells. In addition, during the process of culture, purification, the drugs inhibiting fibroblast growth was not used, which avoids the drug toxicity-caused injury to Schwann cells.

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