EXPERIMENTAL STUDY ON CULTURING Schwann CELLS OF RATS BY SINGLE-ENZYME DIGESTION AND EXPLANT-CULTURE METHOD_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

QIN Yiwu ¹ , ZHAO Jinmin ¹ , SU Wei ¹ , LI Xiaofeng ¹ , LUO Shixing ² , LIU Junting ¹

1Department of Trauma Orthopedics and Hand Surgery, the First Affiliated Hospital of Guangxi Medical University, Nanning Guangxi, 530021, P.R.China;;
2Department of Orthopedics, Beihai People’s Hospital. Corresponding author: ZHAO Jinmin, E-mail: zhaojinmin@126. com;

Corresponding author：

Keywords：

Schwann cells; Sciatic nerve; Cell culture; Peripheral nerve repair; Rat

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ObjectiveTo establish an efficient method of isolating and culturing high activity and high purity of Schwann cells, and to identify the cells at the levels of transcription and translation. MethodsThe sciatic nerves harvested from a 4-week-old Sprague Dawley rat were digested in the collagenase I for 15 minutes after dissecting, and then the explants were planted in culture flask directly. The cells were cultured and passaged in vitro, the growth state and morphological changes of the cells were observed under inverted phase contrast microscope. MTT assay was used to test the proliferation of cells and the cells growth curve was drawn. RT-PCR and immunohistochemistry staining were used to detect S100 and glial fibrillary acidic protein (GFAP) at the levels of transcription and translation, respectively. The purity of cells was caculated under microscope. ResultsAfter the digestion of collagenase I, fibroblast-like cells appeared around explants within 24 hours, with slender cell body and weak refraction. After tissues were transferred to another culture flask, a large number of dipolar or tripolar cells were seen after 48 hours, with slender ecphyma, plump cell body, and b refraction, and the cells formed colonies within 72 hours. The cells were covered with the bottom of culture flask within 48-72 hours after passaging at a ratio of 1∶2, and spiral colonies appeared. Cells showed vigorous growth and full cytoplasm after many passages. MTT assay results showed that the cells at passage 3 entered the logarithmic growth phase on the 3rd day, reached the plateau phase on the 7th day with cell proliferation, and the growth curve was “S” shape. RT-PCR results showed that the cells expressed S100 gene and GFAP gene, and immunohistochemistry staining showed that most of the cells were positively stained, indicating that the majority of cells expressing S100 protein and GFAP protein. The purity of Schwann cells was 98.37% ± 0.30%. ConclusionHigh activity and high purity of Schwann cells can be acquired rapidly by single-enzyme digestion and explant-culture method.

Citation： QIN Yiwu,ZHAO Jinmin,SU Wei,LI Xiaofeng,LUO Shixing,LIU Junting. EXPERIMENTAL STUDY ON CULTURING Schwann CELLS OF RATS BY SINGLE-ENZYME DIGESTION AND EXPLANT-CULTURE METHOD. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(9): 1107-1111. doi: Copy

1.	Feneley MR, Fawcett JW, Keynes RJ. The role of Schwann cells in the regeneration of peripheral nerve axons through muscle basal lamina grafts. Exp Neurol, 1991, 114(3): 275-285.
2.	Torigoe K, Tanaka HF, Takahashi A, et al. Basic behavior of migratory Schwann cells in peripheral nerve regeneration. Exp Neurol, 1996, 137(2): 301-308.
3.	Ara J, Bannerman P, Shaheen F, et al. Schwann cell-autonomous role of neuropilin-2. J Neurosci Res, 2005, 79(4): 468-475.
4.	Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997, 14(1-2): 67-116.
5.	Wang JM, Zeng YS, Wu JL, et al. Cograft of neural stem cells and schwann cells overexpressing TrkC and neurotrophin-3 respectively after rat spinal cord transection. Biomaterials, 2011, 32(30): 7454-7468.
6.	Buss A, Pech K, Kakulas BA, et al. Growth-modulating molecules are associated with invading Schwann cells and not astrocytes in human traumatic spinal cord injury. Brain, 2007, 130(Pt 4): 940-953.
7.	Webber CA, Christie KJ, Cheng C, et al. Schwann cells direct peripheral nerve regeneration through the Netrin-1 receptors, DCC and Unc5H2. Glia, 2011, 59(10): 1503-1517.
8.	Tabesh H, Amoabediny G, Nik NS, et al. The role of biodegradable engineered scaffolds seeded with Schwann cells for spinal cord regeneration. Neurochem Int, 2009, 54(2): 73-83.
9.	Jungnickel J, Haastert-Talini K, Claus P, et al. Polysialyltransferase overexpression in Schwann cells mediates different effects during peripheral nerve regeneration. Glycobiology, 2012, 22(1): 107-115.
10.	Murray-Dunning C, McArthur SL, Sun T, et al. Three-dimensional alignment of schwann cells using hydrolysable microfiber scaffolds: strategies for peripheral nerve repair. Methods Mol Biol, 2011, 695: 155-166.
11.	赵喆, 赵斌, 王玉, 等. 化学去细胞异体神经添加不同组织来源雪旺细胞对周围神经损伤修复的功能评价. 中国修复重建外科杂志, 2010, 24(11): 1281-1287.
12.	Schmitte R, Tipold A, Stein VM, et al. Genetically modified canine Schwann cells—In vitro and in vivo evaluation of their suitability for peripheral nerve tissue engineering. J Neurosci Methods, 2010, 186(2): 202-208.
13.	McGrath AM, Novikova LN, Novikov LN, et al. BDTM PuraMatrixTM peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration. Brain Res Bull, 2010, 83(5): 207-213.
14.	Kalbermatten DF, Erba P, Mahay D, et al. Schwann cell strip for peripheral nerve repair. J Hand Surg Eur Vol, 2008, 33(5): 587-594.
15.	Bockelmann J, Klinkhammer K, von Holst A, et al. Functionalization of electrospun poly (ε-caprolactone) fibers with the extracellular matrix-derived peptide GRGDS improves guidance of schwann cell migration and axonal growth. Tissue Eng Part A, 2011, 17(3-4): 475-486.
16.	Niapour A, Karamali F, Nemati S, et al. Co-transplantation of human embryonic stem cell-derived neural progenitors and Schwann cells in a rat spinal cord contusion injury model elicits a distinct neurogenesis and functional recovery. Cell Transplant, 2012, 21(5): 827-843.
17.	Askanas V, Engel WK, Dalakas MC, et al. Human schwann cells in tissue culture: histochemical and ultrastructural studies. Arch Neurol, 1980, 37(6): 329-337.
18.	Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J Neurosci Methods, 2003, 122(2): 195-200.
19.	翁慧兰, 孙淑明, 吴丽娥. 雪旺细胞体外培养和纯化的新方法. 中国基层医药, 2005, 12(10): 1299-1300.
20.	王世清, 范志海, 沈忆新. 不同部位取材的雪旺细胞培养与纯化研究. 中国修复重建外科杂志, 2011, 25(2): 166-170.
21.	刘炎, 张莉, 宫旭, 等. 双向差速法制备高纯度雪旺细胞的实验研究. 中国矫形外科杂志, 2011, 19(16): 1373-1376.
22.	Graham A. Melanocyte production: dark side of the Schwann cell. Curr Biol, 2009, 19(24): R1116-1117.

1. Feneley MR, Fawcett JW, Keynes RJ. The role of Schwann cells in the regeneration of peripheral nerve axons through muscle basal lamina grafts. Exp Neurol, 1991, 114(3): 275-285.
2. Torigoe K, Tanaka HF, Takahashi A, et al. Basic behavior of migratory Schwann cells in peripheral nerve regeneration. Exp Neurol, 1996, 137(2): 301-308.
3. Ara J, Bannerman P, Shaheen F, et al. Schwann cell-autonomous role of neuropilin-2. J Neurosci Res, 2005, 79(4): 468-475.
4. Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997, 14(1-2): 67-116.
5. Wang JM, Zeng YS, Wu JL, et al. Cograft of neural stem cells and schwann cells overexpressing TrkC and neurotrophin-3 respectively after rat spinal cord transection. Biomaterials, 2011, 32(30): 7454-7468.
6. Buss A, Pech K, Kakulas BA, et al. Growth-modulating molecules are associated with invading Schwann cells and not astrocytes in human traumatic spinal cord injury. Brain, 2007, 130(Pt 4): 940-953.
7. Webber CA, Christie KJ, Cheng C, et al. Schwann cells direct peripheral nerve regeneration through the Netrin-1 receptors, DCC and Unc5H2. Glia, 2011, 59(10): 1503-1517.
8. Tabesh H, Amoabediny G, Nik NS, et al. The role of biodegradable engineered scaffolds seeded with Schwann cells for spinal cord regeneration. Neurochem Int, 2009, 54(2): 73-83.
9. Jungnickel J, Haastert-Talini K, Claus P, et al. Polysialyltransferase overexpression in Schwann cells mediates different effects during peripheral nerve regeneration. Glycobiology, 2012, 22(1): 107-115.
10. Murray-Dunning C, McArthur SL, Sun T, et al. Three-dimensional alignment of schwann cells using hydrolysable microfiber scaffolds: strategies for peripheral nerve repair. Methods Mol Biol, 2011, 695: 155-166.
11. 赵喆, 赵斌, 王玉, 等. 化学去细胞异体神经添加不同组织来源雪旺细胞对周围神经损伤修复的功能评价. 中国修复重建外科杂志, 2010, 24(11): 1281-1287.
12. Schmitte R, Tipold A, Stein VM, et al. Genetically modified canine Schwann cells—In vitro and in vivo evaluation of their suitability for peripheral nerve tissue engineering. J Neurosci Methods, 2010, 186(2): 202-208.
13. McGrath AM, Novikova LN, Novikov LN, et al. BDTM PuraMatrixTM peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration. Brain Res Bull, 2010, 83(5): 207-213.
14. Kalbermatten DF, Erba P, Mahay D, et al. Schwann cell strip for peripheral nerve repair. J Hand Surg Eur Vol, 2008, 33(5): 587-594.
15. Bockelmann J, Klinkhammer K, von Holst A, et al. Functionalization of electrospun poly (ε-caprolactone) fibers with the extracellular matrix-derived peptide GRGDS improves guidance of schwann cell migration and axonal growth. Tissue Eng Part A, 2011, 17(3-4): 475-486.
16. Niapour A, Karamali F, Nemati S, et al. Co-transplantation of human embryonic stem cell-derived neural progenitors and Schwann cells in a rat spinal cord contusion injury model elicits a distinct neurogenesis and functional recovery. Cell Transplant, 2012, 21(5): 827-843.
17. Askanas V, Engel WK, Dalakas MC, et al. Human schwann cells in tissue culture: histochemical and ultrastructural studies. Arch Neurol, 1980, 37(6): 329-337.
18. Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J Neurosci Methods, 2003, 122(2): 195-200.
19. 翁慧兰, 孙淑明, 吴丽娥. 雪旺细胞体外培养和纯化的新方法. 中国基层医药, 2005, 12(10): 1299-1300.
20. 王世清, 范志海, 沈忆新. 不同部位取材的雪旺细胞培养与纯化研究. 中国修复重建外科杂志, 2011, 25(2): 166-170.
21. 刘炎, 张莉, 宫旭, 等. 双向差速法制备高纯度雪旺细胞的实验研究. 中国矫形外科杂志, 2011, 19(16): 1373-1376.
22. Graham A. Melanocyte production: dark side of the Schwann cell. Curr Biol, 2009, 19(24): R1116-1117.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON CULTURING Schwann CELLS OF RATS BY SINGLE-ENZYME DIGESTION AND EXPLANT-CULTURE METHOD

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