从新生大鼠脊髓神经中分离和纯化施万细胞

Jinkun Wen; Dandan Tan; Lixia  Li; Jiasong  Guo

doi:10.21769/BioProtoc.2588

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Peer-reviewed

Isolation and Purification of Schwann Cells from Spinal Nerves of Neonatal Rat

从新生大鼠脊髓神经中分离和纯化施万细胞

JW Jinkun Wen

DT Dandan Tan

LL Lixia Li

JG Jiasong Guo email

发布: 2017年10月20日第7卷第20期 DOI: 10.21769/BioProtoc.2588 浏览次数: 12140

评审: Sébastien GillotinAnonymous reviewer(s)

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Cover of Molecular Neurobiology, featuring study using the protocol.

Mar 2017

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Abstract

Primary cultured Schwann cells (SCs) are widely used in the investigation of the biology of SC and are important seed cells for neural tissue engineering. Here, we describe a novel protocol for harvesting primary cultured SCs from neonatal Sprague-Dawley (SD) rats. In the present protocol, dissociated SCs are isolated from the spinal nerves of neonatal rats and purified by the treatment of cytosine arabinoside (AraC).

Keywords: Schwann cell (施万细胞)

Background

SCs are the glial cells of the peripheral nervous system (PNS). Isolation and purification of primary SCs are crucial steps for studying the biology of SC. In addition, purified primary cultured SCs are important seed cells for neural tissue engineering. To date, various methods of culturing SCs have been reported based on the method of Brockes (Brockes et al., 1979). By reported methods, sciatic nerves are mostly used for SC isolation because they are large in size and can be easily obtained. However, SCs from sciatic nerves are easily contaminated with fibroblasts because the connective tissue is difficult to be cleared off. Especially the epineurium and perineurium are the main source of fibroblasts. Without special treatment, contaminating fibroblasts proliferate much faster than SCs and will soon be the predominant cells in the cultures. In the past decades, numerous purification methods have been developed for isolating SCs from the contaminating fibroblasts. The details of these purification methods included single or combination of antimitotic treatment (Wood, 1976), antibody-mediated cytolysis (Brockes et al., 1979), immunoselection (Assouline et al., 1983; Vroemen and Weidner, 2003), repeated explantation (Oda et al., 1989), cold jet technique (Haastert et al., 2007), differential adhesion (Pannunzio et al., 2005) and differential detachment (Jin et al., 2008). These purification methods involve either complicated techniques with high cost or long harvested period with low cell yield. Therefore, obtaining a large number of purified SCs is still a challenging work for basic research and further clinical use. Here, we describe a method that uses spinal nerves from neonatal SD rats as a cell source to efficiently obtain highly purified SCs in a short period.

Materials and Reagents

Pipette tips (Corning, Axygen^®, catalog numbers: T-1000-B-R-S , T-200-Y-R-S )
Cell culture dish (35 x 10 mm) (3.5-cm dish) (Corning, catalog number: 430165 )
1.5 ml centrifuge tubes (Corning, Axygen^®, catalog number: MCT-150-C )
50 ml centrifuge tubes (Corning, catalog number: 430290 )
100 µm cell strainer (Corning, catalog number: 431752 )
Neonatal Sprague-Dawley (SD) Rat (Postnatal 2-4 days, P2-4)
Distilled water
75% ethanol
Hank’s balanced salts solution (HBSS) (Thermo Fisher Scientific, Gibco^TM, catalog number: C14175500BT )
Poly-L-lysine hydrobromide (PLL) (Sigma-Aldrich, catalog number: P1274 )
0.25% trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200072 )
Fetal bovine serum (FBS) (Mediatech, catalog number: 35-076-CV )
Dulbecco’s modification of Eagle’s medium/Ham’s F-12 50/50 Mix (DMEM/F12) (Mediatech, catalog number: 10-092-CV )
Cytosine Arabinoside (AraC) (Sigma-Aldrich, catalog number: C1768 )
Forskolin (Sigma-Aldrich, catalog number: F6886 )
Recombinant Human Heregulin β-1 (Heregulin) (PeproTech, catalog number: 100-03 )
Dimethyl sulfoxide (DMSO) (MP Biomedicals, catalog number: 196055 )
Penicillin/streptomycin (Pen/Strep) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Phosphate-buffered saline (PBS) (Beyotime Biotechnology, catalog number: C0221A )
Paraformaldehyde (PFA) (Guangdong Guanghua Sci-Tech, catalog number: 1.17767.014 )
Gelatin (Sigma-Aldrich, catalog number: G7041 )
Triton X-100 (Sigma-Aldrich, catalog number: V900502 )
4,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542 )
Anti-glial fibrillary acidic protein antibody produced in rabbit (GFAP) (Sigma-Aldrich, catalog number: G9269 )
Anti-S-100 protein antibody, clone 15E2E2, produced in mouse (S100) (Merck, catalog number: MAB079-1 )
Anti-nerve growth factor receptor antibody, p75, produced in rabbit (P75) (Merck, catalog number: AB1554 )
Alexa Fluor^® 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, Invitrogen, catalog number: A-11001 )
Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, Invitrogen, catalog number: A-11008 )
100x PLL (see Recipes)
10% FBS (see Recipes)
1,000x AraC (see Recipes)
500x heregulin (see Recipes)
10,000x forskolin (see Recipes)
SC culture medium (see Recipes)
4% paraformaldehyde (PFA) (see Recipes)
0.1% Triton X-100 (see Recipes)
Blocking buffer (see Recipes)
1,000x DAPI (see Recipes)

Equipment

Pipettes (Eppendorf, model: Research Plus^® , 200 μl, 1000 μl)
CO₂ incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Model 3100 Series , catalog number: 3111)
Tissue culture hood (AIRTECH, catalog number: SW-CJ-1F )
Surgical scissors and forceps (RWD Life Science, catalog numbers: S14001-15 , F13024-13 , S12003-09 , F13029-10 , see Figure 1A)
Note: S14001-15 and F13024013 are scissors and forceps used for decapitation, S12003-09 and F13029-10 are scissors and forceps used for skin dissection.
Spring scissors (66 Vision, catalog number: 54053B , see Figure 1B)
Fine forceps (Fine Science Tools, Dumont, model: #5, catalog number: 11252-23 , with tips of 0.1 x 0.06 mm, see Figure 1B)
Superfine forceps (Fine Science Tools, Dumont, model: #5, catalog number: 11252-20 , with tips of 0.05 x 0.02 mm, see Figure 1B)
Dissecting board, needles and ice packs
Water bath (Ningbo Scientz Biotechnology, model: GH-15 )
Stereomicroscope (Olympus, model: SZ61 )
Centrifuge (Eppendorf, model: 5430 )

Figure 1. Dissection tools. A. Surgical scissors and forceps used to decapitate the rat and to dissect skin. B. Spring scissors and fine forceps used to separate muscle, open vertebral laminae and collect spinal nerves.

Procedure

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Wen, J., Tan, D., Li, L. and Guo, J. (2017). Isolation and Purification of Schwann Cells from Spinal Nerves of Neonatal Rat. Bio-protocol 7(20): e2588. DOI: 10.21769/BioProtoc.2588.