EXPERIMENTAL STUDY ON CULTIVATION AND PURIFICATION OF Schwann CELLS AND ITS COMPOSITION WITH SMALL INTESTINAL SUBMUCOSA IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Hongyin , FENG Zhe , BAI Lin , HOU Jianglong , LUO Jingcong , YANG Zhiming , XIE Huiqi.

Laboratory of Stem Cell and Tissue Engineering, Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China. Corresponding author: XIE Huiqi, E-mail: xiehuiqi@163.com;

Corresponding author：

Keywords：

Tissue engineering; Schwann cells; Small intestinal submucosa; Nerve growth factor; Biocompatibility Nerve repair

DOI：

10.7507/1002-1892.20130165

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To obtain highly purified and large amount of Schwann cells (SCs) by improved primary culture method, to investigate the biocompatibility of small intestinal submucosa (SIS) and SCs, and to make SIS load nerve growth factor (NGF) through co-culture with SCs. Methods Sciatic nerves were isolated from 2-3 days old Sprague Dawley rats and digested with collagenase II and trypsin. SCs were purified by differential adhesion method for 20 minutes and treated with G418 for 48 hours. Then the fibroblasts were further removed by reducing fetal bovine serum to 2.5% in H-DMEM. MTT assay was used to test the proliferation of SCs and the growth curve of SCs was drawn. The purity of SCs was calculated by immunofluorescence staining for S-100. SIS and SCs at passage 3 were co-cultured in vitro. And then the adhesion, proliferation, and differentiation of SCs were investigated by optical microscope and scanning electron microscope (SEM). The NGF content by SCs was also evaluated at 1, 2, 3, 4, 5, and 7 days by ELISA. SCs were removed from SIS by repeated freeze thawing after 3, 5, 7, 10, 13, and 15 days of co-culture. The NGF content in modified SIS was tested by ELISA. Results The purity of SCs was more than 98%. MTT assay showed that the SCs entered the logarithmic growth phase on the 3rd day, and reached the plateau phase on the 7th day. SCs well adhered to the surface of SIS by HE staining and SEM; SCs were fusiform in shape with obvious prominence and the protein granules secreted on cellular surface were also observed. Furthermore, ELISA measurement revealed that, co-culture with SIS, SCs secreted NGF prosperously without significant difference when compared with the control group (P gt; 0.05). The NGF content increased with increasing time. The concentration of NGF released from SIS which were cultured with SCs for 10 days was (414.29 ± 20.87) pg/cm2, while in simple SIS was (4.92 ± 2.06) pg/cm2, showing significant difference (P lt; 0.05). Conclusion A large number of highly purified SCs can be obtained by digestion with collagenase II and trypsin in combination with 20-minute differential adhesion and selection by G418. SIS possesses good biocompatibility with SCs, providing the basis for further study in vivo to fabricate the artificial nerve conduit.

Citation： LIU Hongyin,FENG Zhe,BAI Lin,HOU Jianglong,LUO Jingcong,YANG Zhiming,XIE Huiqi.. EXPERIMENTAL STUDY ON CULTIVATION AND PURIFICATION OF Schwann CELLS AND ITS COMPOSITION WITH SMALL INTESTINAL SUBMUCOSA IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(6): 743-747. doi: 10.7507/1002-1892.20130165 Copy

1.	Terenghi G, Calder JS, Birch R, et al. A morphological study of Schwann cells and axonal regeneration in chronically transected human peripheral nerves. J Hand Surg (Br), 1998, 23(5): 583-587.
2.	Wei RQ, Tan B, Tan MY, et al. Grafts of porcine small intestinal submucosa with cultured autologous oral mucosal epithelial cells for esophageal repair in a canine model. Exp Biol Med (Maywood), 2009, 234(4): 453-461.
3.	Caione P, Capozza N, Zavaglia D, et al. In vivo bladder regeneration using small intestinal submucosa: experimental study. Pediatr Surg Int, 2006, 22(7): 593-599.
4.	Lee EW, Shindel AW, Brandes SB. Small intestinal submucosa for patch grafting after plaque incision in the treatmentof Peyronie’s disease. Int Braz J Urol, 2008, 34(2): 191-197.
5.	Fiala R, Vidlar A, Vrtal R, et al. Porcine small intestinal submucosa graft for repair of anterior urethral strictures. Eur Urol, 2007, 51(6): 1702-1708.
6.	Voytik-Harbin SL, Brightman AO, Kraine MR, et al. Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem, 1997, 67(4): 478-491.
7.	Rich KM, Alexander TD, Pryor JC, et al. Nerve growth factor enhances regeneration through silicone chambers. Exp Neurol, 1989, 105(2): 162-170.
8.	Whitworth IH, Brown RA, Doré CJ, et al. Nerve growth factor enhances nerve regeneration through fibronectin grafts. J Hand Surg (Br), 1996, 21(4): 514-522.
9.	Santos X, Rodrigo J, Hontanilla B, et al. Evaluation of peripheral nerve regeneration by nerve growth factor locally administered with a novel system. J Neurosci Methods, 1998, 85(1): 119-127.
10.	Piotrowicz A, Shoichet MS. Nerve guidance channels as drug delivery vehicles. Biomaterials, 2006, 27(9): 2018-2027.
11.	Luo JC, Chen W, Chen XH, et al. A multi-step method for preparation of porcine small intestinal submucosa (SIS). Biomaterials, 2011, 32(3): 706-713.
12.	Millesi H. Techniques for nerve grafting. Hand Clin, 2000, 16(1): 73-91.
13.	Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997, 14(1-2): 67-116.
14.	Terenghi G. Peripheral nerve regeneration and neurotrophic factors. J Anat, 1999, 194(Pt 1): 1-14.
15.	Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv, 2008, 26(1): 1-21.
16.	Gu X, Ding F, Yang Y, et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol, 2011, 93(2): 204-230.
17.	Hart A, Terenghi G, Wiberg M. Tissue engineering for peripheral nerve regeneration//Tissue Engineering. Berlin: Springer, 2011: 245-262.
18.	Ansselin AD, Fink T, Davey DF. Peripheral nerve regeneration through nerve guides seeded with adult Schwann cells. Neuropathol Appl Neurobiol, 1997, 23(5): 387-398.
19.	Kim DH, Connolly SE, Kline DG, et al. Labeled Schwann cell transplants versus sural nerve grafts in nerve repair. J Neurosurg, 1994, 80(2): 254-260.
20.	Guénard V, Kleitman N, Morrissey TK, et al. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J Neurosci, 1992, 12(9): 3310-3320.
21.	黄小强, 罗卓荆, 李明全. 新生SD大鼠坐骨神经雪旺细胞培养与纯化的方法学研究. 中华创伤骨科杂志, 2005, 7(4): 341-343, 348.
22.	Jin YQ, Liu W, Hong TH, et al. Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment. J Neurosci Methods, 2008, 170(1): 140-148.
23.	Pannunzio ME, Jou IM, Long A, et al. A new method of selecting Schwann cells from adult mouse sciatic nerve. J Neurosci Methods, 2005, 149(1): 74-81.
24.	Vroemen M, Weidner N. Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve. J Neurosci Methods, 2003, 124(2): 135-143.
25.	Kreider BQ, Messing A, Doan H, et al. Enrichment of Schwann cells cultures from neonatal rat sciatic nerve by differential adhesion. Brain Res, 1981, 207(2): 433-444.
26.	张勇杰, 金岩, 聂鑫, 等. 多步法分离和纯化雪旺细胞的实验研究. 中华神经外科疾病研究杂志, 2002, 1(4): 347-350．.
27.	Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduct. J Neurosci Methods, 2003, 122(2): 195-200.
28.	苏琰, 张长青, 孙鲁源, 等. 小肠粘膜下层桥接周围神经缺损的实验研究. 中华创伤骨科杂志, 2004, 6(10): 1136-1139.
29.	Lu H, Hoshiba T, Kawazoe N, et al. Autologous extracellular matrix scaffolds for tissue engineering. Biomaterials, 2011, 32 (10): 2489-2499.

1. Terenghi G, Calder JS, Birch R, et al. A morphological study of Schwann cells and axonal regeneration in chronically transected human peripheral nerves. J Hand Surg (Br), 1998, 23(5): 583-587.
2. Wei RQ, Tan B, Tan MY, et al. Grafts of porcine small intestinal submucosa with cultured autologous oral mucosal epithelial cells for esophageal repair in a canine model. Exp Biol Med (Maywood), 2009, 234(4): 453-461.
3. Caione P, Capozza N, Zavaglia D, et al. In vivo bladder regeneration using small intestinal submucosa: experimental study. Pediatr Surg Int, 2006, 22(7): 593-599.
4. Lee EW, Shindel AW, Brandes SB. Small intestinal submucosa for patch grafting after plaque incision in the treatmentof Peyronie’s disease. Int Braz J Urol, 2008, 34(2): 191-197.
5. Fiala R, Vidlar A, Vrtal R, et al. Porcine small intestinal submucosa graft for repair of anterior urethral strictures. Eur Urol, 2007, 51(6): 1702-1708.
6. Voytik-Harbin SL, Brightman AO, Kraine MR, et al. Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem, 1997, 67(4): 478-491.
7. Rich KM, Alexander TD, Pryor JC, et al. Nerve growth factor enhances regeneration through silicone chambers. Exp Neurol, 1989, 105(2): 162-170.
8. Whitworth IH, Brown RA, Doré CJ, et al. Nerve growth factor enhances nerve regeneration through fibronectin grafts. J Hand Surg (Br), 1996, 21(4): 514-522.
9. Santos X, Rodrigo J, Hontanilla B, et al. Evaluation of peripheral nerve regeneration by nerve growth factor locally administered with a novel system. J Neurosci Methods, 1998, 85(1): 119-127.
10. Piotrowicz A, Shoichet MS. Nerve guidance channels as drug delivery vehicles. Biomaterials, 2006, 27(9): 2018-2027.
11. Luo JC, Chen W, Chen XH, et al. A multi-step method for preparation of porcine small intestinal submucosa (SIS). Biomaterials, 2011, 32(3): 706-713.
12. Millesi H. Techniques for nerve grafting. Hand Clin, 2000, 16(1): 73-91.
13. Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997, 14(1-2): 67-116.
14. Terenghi G. Peripheral nerve regeneration and neurotrophic factors. J Anat, 1999, 194(Pt 1): 1-14.
15. Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv, 2008, 26(1): 1-21.
16. Gu X, Ding F, Yang Y, et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol, 2011, 93(2): 204-230.
17. Hart A, Terenghi G, Wiberg M. Tissue engineering for peripheral nerve regeneration//Tissue Engineering. Berlin: Springer, 2011: 245-262.
18. Ansselin AD, Fink T, Davey DF. Peripheral nerve regeneration through nerve guides seeded with adult Schwann cells. Neuropathol Appl Neurobiol, 1997, 23(5): 387-398.
19. Kim DH, Connolly SE, Kline DG, et al. Labeled Schwann cell transplants versus sural nerve grafts in nerve repair. J Neurosurg, 1994, 80(2): 254-260.
20. Guénard V, Kleitman N, Morrissey TK, et al. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J Neurosci, 1992, 12(9): 3310-3320.
21. 黄小强, 罗卓荆, 李明全. 新生SD大鼠坐骨神经雪旺细胞培养与纯化的方法学研究. 中华创伤骨科杂志, 2005, 7(4): 341-343, 348.
22. Jin YQ, Liu W, Hong TH, et al. Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment. J Neurosci Methods, 2008, 170(1): 140-148.
23. Pannunzio ME, Jou IM, Long A, et al. A new method of selecting Schwann cells from adult mouse sciatic nerve. J Neurosci Methods, 2005, 149(1): 74-81.
24. Vroemen M, Weidner N. Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve. J Neurosci Methods, 2003, 124(2): 135-143.
25. Kreider BQ, Messing A, Doan H, et al. Enrichment of Schwann cells cultures from neonatal rat sciatic nerve by differential adhesion. Brain Res, 1981, 207(2): 433-444.
26. 张勇杰, 金岩, 聂鑫, 等. 多步法分离和纯化雪旺细胞的实验研究. 中华神经外科疾病研究杂志, 2002, 1(4): 347-350．.
27. Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduct. J Neurosci Methods, 2003, 122(2): 195-200.
28. 苏琰, 张长青, 孙鲁源, 等. 小肠粘膜下层桥接周围神经缺损的实验研究. 中华创伤骨科杂志, 2004, 6(10): 1136-1139.
29. Lu H, Hoshiba T, Kawazoe N, et al. Autologous extracellular matrix scaffolds for tissue engineering. Biomaterials, 2011, 32 (10): 2489-2499.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON CULTIVATION AND PURIFICATION OF Schwann CELLS AND ITS COMPOSITION WITH SMALL INTESTINAL SUBMUCOSA IN VITRO

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content