Mana Shojapour1, Ghasem Mosayebi2, Reza Hajihossein3, Farshid Noorbakhsh4, Aram Mokarizadeh5, Mohammad Hossein Ghahremani6. 1. Department of molecular Medicine. School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. 2. Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran. 3. Department of Parasitology, Arak University of Medical Sciences, Arak, Iran. Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 4. Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 5. Department of Immunology, Faculty of Medicine, Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran. 6. Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Abstract
BACKGROUND: The purification of Schwann cells has proven to be a difficult process, with most methods requiring the use of special equipment. However, obtaining a sufficient number and high purity of Schwann cells is an integral aspect in their use for clinical application. Therefore, the aim of this study was to establish a simple and effective protocol for the isolation and purification of Schwann cells from the sciatic nerve of C57BL/6 mice. Furthermore, we aimed to provide a protocol for the isolation of exosomes from these cells. METHODS: To purify Schwann cells, we used a combination of in situ nerve pre-degeneration and fetal bovine serum. To determine the most effective method of cell purification, we treated the culture with varying concentrations of fetal bovine serum and examined which concentration provided the highest Schwann cell purity. Exosomes were then isolated from Schwann cells through a process of repeated centrifugation and filtration steps. RESULTS: We were able to increase the purified population of Schwann cells from C57BL/6 mice by reducing the concentration of FBS. The purity of Schwann cells at FBS concentrations of 10%, 5%, and 2% were 93.42%, 91.25%, and 97.83%, respectively. CONCLUSION: When using a concentration of 2% FBS, we obtained the highest purification yield of Schwann cells. Our protocol does not require special equipment or materials. We have created a protocol that is simple, fast, and safe while providing a high yield of purified Schwann cells. The exosome isolation method described in this paper is an appropriate approach with a high quality and yield.
BACKGROUND: The purification of Schwann cells has proven to be a difficult process, with most methods requiring the use of special equipment. However, obtaining a sufficient number and high purity of Schwann cells is an integral aspect in their use for clinical application. Therefore, the aim of this study was to establish a simple and effective protocol for the isolation and purification of Schwann cells from the sciatic nerve of C57BL/6 mice. Furthermore, we aimed to provide a protocol for the isolation of exosomes from these cells. METHODS: To purify Schwann cells, we used a combination of in situ nerve pre-degeneration and fetal bovine serum. To determine the most effective method of cell purification, we treated the culture with varying concentrations of fetal bovine serum and examined which concentration provided the highest Schwann cell purity. Exosomes were then isolated from Schwann cells through a process of repeated centrifugation and filtration steps. RESULTS: We were able to increase the purified population of Schwann cells from C57BL/6 mice by reducing the concentration of FBS. The purity of Schwann cells at FBS concentrations of 10%, 5%, and 2% were 93.42%, 91.25%, and 97.83%, respectively. CONCLUSION: When using a concentration of 2% FBS, we obtained the highest purification yield of Schwann cells. Our protocol does not require special equipment or materials. We have created a protocol that is simple, fast, and safe while providing a high yield of purified Schwann cells. The exosome isolation method described in this paper is an appropriate approach with a high quality and yield.
Authors: Ruenn Chai Lai; Fatih Arslan; May May Lee; Newman Siu Kwan Sze; Andre Choo; Tian Sheng Chen; Manuel Salto-Tellez; Leo Timmers; Chuen Neng Lee; Reida Menshawe El Oakley; Gerard Pasterkamp; Dominique P V de Kleijn; Sai Kiang Lim Journal: Stem Cell Res Date: 2010-01-04 Impact factor: 2.020