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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   

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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), Spinal nerve(脊髓神经), Isolation(分离), Purification(纯化), Rat(大鼠)

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

  1. Pipette tips (Corning, Axygen®, catalog numbers: T-1000-B-R-S , T-200-Y-R-S )
  2. Cell culture dish (35 x 10 mm) (3.5-cm dish) (Corning, catalog number: 430165 )
  3. 1.5 ml centrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
  4. 50 ml centrifuge tubes (Corning, catalog number: 430290 )
  5. 100 µm cell strainer (Corning, catalog number: 431752 )
  6. Neonatal Sprague-Dawley (SD) Rat (Postnatal 2-4 days, P2-4)
  7. Distilled water
  8. 75% ethanol
  9. Hank’s balanced salts solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: C14175500BT )
  10. Poly-L-lysine hydrobromide (PLL) (Sigma-Aldrich, catalog number: P1274 )
  11. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
  12. Fetal bovine serum (FBS) (Mediatech, catalog number: 35-076-CV )
  13. Dulbecco’s modification of Eagle’s medium/Ham’s F-12 50/50 Mix (DMEM/F12) (Mediatech, catalog number: 10-092-CV )
  14. Cytosine Arabinoside (AraC) (Sigma-Aldrich, catalog number: C1768 )
  15. Forskolin (Sigma-Aldrich, catalog number: F6886 )
  16. Recombinant Human Heregulin β-1 (Heregulin) (PeproTech, catalog number: 100-03 )
  17. Dimethyl sulfoxide (DMSO) (MP Biomedicals, catalog number: 196055 )
  18. Penicillin/streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  19. Phosphate-buffered saline (PBS) (Beyotime Biotechnology, catalog number: C0221A )
  20. Paraformaldehyde (PFA) (Guangdong Guanghua Sci-Tech, catalog number: 1.17767.014 )
  21. Gelatin (Sigma-Aldrich, catalog number: G7041 )
  22. Triton X-100 (Sigma-Aldrich, catalog number: V900502 )
  23. 4,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542 )
  24. Anti-glial fibrillary acidic protein antibody produced in rabbit (GFAP) (Sigma-Aldrich, catalog number: G9269 )
  25. Anti-S-100 protein antibody, clone 15E2E2, produced in mouse (S100) (Merck, catalog number: MAB079-1 )
  26. Anti-nerve growth factor receptor antibody, p75, produced in rabbit (P75) (Merck, catalog number: AB1554 )
  27. Alexa Fluor® 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, Invitrogen, catalog number: A-11001 )
  28. Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, Invitrogen, catalog number: A-11008 )
  29. 100x PLL (see Recipes)
  30. 10% FBS (see Recipes)
  31. 1,000x AraC (see Recipes)
  32. 500x heregulin (see Recipes)
  33. 10,000x forskolin (see Recipes)
  34. SC culture medium (see Recipes)
  35. 4% paraformaldehyde (PFA) (see Recipes)
  36. 0.1% Triton X-100 (see Recipes)
  37. Blocking buffer (see Recipes)
  38. 1,000x DAPI (see Recipes)

Equipment

  1. Pipettes (Eppendorf, model: Research Plus® , 200 μl, 1000 μl)
  2. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: Model 3100 Series , catalog number: 3111)
  3. Tissue culture hood (AIRTECH, catalog number: SW-CJ-1F )
  4. 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.
  5. Spring scissors (66 Vision, catalog number: 54053B , see Figure 1B)
  6. Fine forceps (Fine Science Tools, Dumont, model: #5, catalog number: 11252-23 , with tips of 0.1 x 0.06 mm, see Figure 1B)
  7. Superfine forceps (Fine Science Tools, Dumont, model: #5, catalog number: 11252-20 , with tips of 0.05 x 0.02 mm, see Figure 1B)
  8. Dissecting board, needles and ice packs
  9. Water bath (Ningbo Scientz Biotechnology, model: GH-15 )
  10. Stereomicroscope (Olympus, model: SZ61 )
  11. 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

  1. Preparation
    1. Coat a 3.5-cm dish with 1 ml PLL solution (0.1 mg/ml in distilled water, see Recipes) at 37 °C in a CO2 incubator overnight.
    2. Immerse the scissors and forceps in 75% ethanol for 30 min to sterilize them and then expose them to UV light for 30 min to air dry them.
    3. Sterilize the dissection room (in where the rat to be dissected) by UV light for 30 min.
    4. Remove PLL solution through aspiration and thoroughly rinse the dish surface with distilled water twice. Air dry the PLL-coated dish in the tissue culture hood before dissection.
    5. Prepare two 3.5-cm dishes, pipette 1.5 ml cold HBSS to each dish and place them on ice packs.
    6. For each rat pup to be used, place a 1 ml aliquot of 0.25% trypsin-EDTA in a 37 °C water bath to prewarm it.

  2. Tissue collection
    1. Anaesthetize the neonatal rat (postnatal 2-4 days) by cooling it on ice until it stops moving.
      Note: Animal use was approved by the Southern Medical University Animal Care and Use Committee in accordance with the guidelines for the ethical treatment of animals. All efforts were made to minimize animal sacrifice and suffering.
    2. Sterilize the rat with 75% ethanol.
    3. Rapidly decapitate the rat with big surgical scissors and forceps, dispose the head into a collection bag.
    4. Place the rat on the dissecting board in prone posture (dorsal side up), and pin the extremities with needles (see Figure 2A).
    5. Using a set of small scissors and forceps, cut the back skin along the median line from rostral to caudal, separate the skin from the body from medial to lateral (see Figure 2B).
    6. Using the spring scissors and fine forceps, separate the shoulder blades from backbone, and pin the shoulder blades on the dissecting board.
    7. Using the spring scissors and fine forceps, carefully expose the brachial plexuses and dissociate the connective tissue from the nerves. Then cut the nerves at the distal end (see Figures 2C and 2D).
    8. Using the spring scissors and fine forceps, carefully separate the hamstring muscles from the knee to the hipbone to expose the sciatic nerves. Then cut the sciatic nerves at the distal end (see Figure 2E).
      Note: Do not cut the proximal end of the nerves in steps B7 and B8. The nerves are not directly dissected out in these steps. Instead, the nerves should be dragged out from intervertebral foramina as described in the following step B11.
    9. Using the spring scissors and fine forceps, cut the bilateral vertebral arch and remove the vertebral laminae from rostral to caudal to expose the spinal cord (see Figure 2F).
    10. Using the spring scissors and fine forceps, remove the spinal cord carefully to expose the underlying dorsal root ganglia (see Figures 2G and 2H).
    11. Using the fine forceps, grasp the ganglion carefully and then drag out the connecting spinal nerve and put it in a 3.5-cm dish with ice-cold HBSS. Repeat this procedure to harvest all of the spinal nerves from caudal to rostral (mainly including brachial plexus nerves, intercostal nerves and sciatic nerves) (see Figures 2I and 2J).
      Notes:
      1. By grasping the ganglia in the inner of canalis vertebralis (spinal canal), all of the spinal nerves can be easily found and collected.
      2. Slowly and gently drag the spinal nerves out through the intervertebral foramina will help to obtain the nerves longer.
      3. Harvesting of the nerves is most easily done under a stereomicroscope. It is highly suggested.
    12. Use the superfine forceps to remove the connective tissue that adheres to the nerve, and cut off the ganglion with fine scissors. Transfer the clean nerve into another 3.5-cm dish with ice-cold HBSS. Repeat this procedure to clear all of the nerves (see Figures 2K and 2L).
      Notes:
      1. About 30 nerves including sciatic nerves, branches of brachial plexuses and intercostal nerves can be obtained from each rat pup.
      2. Each collected nerve is with a ganglion in the proximal end. The ganglion looks like a head of the nerve and more transparent than the nerve (see Figure 2K, arrows). Meanwhile, rare connective tissue which appears as membranaceous tissue with blood vessels may adhere to the nerves (see Figure 2K, arrowheads).
      3. Nerves harvested by dragging out through intervertebral foramina are always free from epineuria because the epineuria adhere tightly to the intervertebral foramina. This is a major advantage of this protocol. As traditional methods inevitably bring in the epineuria which tightly wrap the nerves (Weinstein et al., 2001; Tao, 2013). And the epineuria are the main source of contaminating cells (such as fibroblasts) in the SC cultures.
      4. Another advantage of the present protocol is that a large number of nerves can be obtained from each rat pup, rather than just two sciatic nerves in traditional methods. Thus, the sacrificed animals can be significantly reduced.


        Figure 2. Step-by-step procedures of harvesting spinal nerves. A. Decapitated rat pup is pinned on the dissecting board. B. The back skin is cut and separated from the body. C. The shoulder blades are separated from the backbone and pinned on the dissecting board. D. The brachial plexuses are exposed and its distal ends are cut. E. The sciatic nerve is exposed and its distal ends are cut. F. Open vertebral canal by laminectomy using a pair of spring scissors. G. The spinal cord is dragged and removed from rostral to caudal. H. The dorsal root ganglia are exposed (arrows) after spinal cord is removed off. I. Schematic diagram shows the spinal nerves (in blue) mainly including brachial plexus nerves (BPN), intercostal nerves (ICN) and sciatic nerves (SN) available for harvesting. Spinal cord (SC) is indicated in yellow. J. A representative image shows the nerve is dragged out through an intervertebral foramen (arrow) while some dorsal root ganglia remain in the foramina (arrowheads). K. The collected nerves are with ganglia (arrows) and sometimes with connective tissue (arrowheads). L. The nerves after the ganglia and connective tissue are removed off.

  3. Dissociation and cell seeding
    1. Transfer the clean nerves to a 1.5 ml tube and cut the nerves into small pieces of 0.5-1 mm long.
    2. Add 1 ml of prewarmed 0.25% trypsin-EDTA into the tube. Cap the tube and incubate it in a 37 °C water bath for 30 min with intermittent vibration every 10 min.
    3. Terminate the trypsinization by supplementing with 100 μl FBS into the medium. And then gently triturate the sample 30-35 times using a 1 ml pipette tip to make single cell suspension.
      Note: Triturate gently to avoid bubble formation and splashing. After 30-35 times of pipetting up and down, some residual clumps remain in the cell suspension.
    4. Filter the cell suspension through a 100 μm cell strainer to remove the residual clumps.
      Note: The clumps are mainly axonal debris and un-separated tissues.
    5. Centrifuge the cell suspension at 106 x g (Rcf) for 10 min at room temperature (RT), and discard the supernatant.
    6. Resuspend the cell pellet in 300 μl 10% FBS in DMEM/F12 (see Recipes). Plant the cell suspension in the middle of the dried PLL-coated 3.5-cm dish. Do not shake the dish lest the cell suspension spread out to adhere to the wall of the dish. Culture the cells in a CO2 incubator for 2 h to facilitate cell attachment.
      Notes:
      1. A total number of ~1 x 106 cells can be obtained from each rat for one dish in our routine practice.
      2. Minimize the volume of plating medium (300 μl 10% FBS) can increase the cell density and facilitate cell attachment to the dish.
    7. Add 1 ml 10% FBS to the dish after the cells attach to the dish.

  4. Purification and expansion
    1. In the next day, replace the culture medium with DMEM/F12 containing 10% FBS and 10 μM AraC (see Recipes) to eliminate contaminating fibroblasts.
      Note: In the absence of mitogenic factors, such as heregulin and forskolin, SCs proliferate quite slowly while fibroblasts do proliferate quickly. AraC is an antimetabolic agent that impairs DNA synthesis and kills dividing cells. Therefore, fibroblasts are eliminated efficiently while SCs survive well in an appropriate treating time span (48 h).
    2. After 48 h, replace the medium with SC culture medium (see Recipes) to expand SCs.
    3. When the culture reaches 90% confluence, cells are routinely passaged at a ratio of 1:3 to expand the cells. And cells from the 3-5th passages are used for experiments in our lab.
      Note: Primary cultured SCs of the 3-5th passages show bipolar and tripolar shapes (see Figure 3A). The purity of these cells can reach 98% identified by immunostaining of SC specific markers (see Figures 3B-3D). We found that SCs proliferate much slower after they were passaged for more than 5 times. In addition, most published papers about SC biology stated that primary SCs from neonatal rat were used in the 3-5th passages. So, in our lab, all SCs used for studies are from the 3-5th passages.

Data analysis

To identify the purity of SCs in our cultures, GFAP, P75 and S100 were used as the specific markers of SCs to perform immunostaining as our previous report (Wen et al., 2017). Briefly, cultured cells were fixed with 4% PFA (see Recipes) for 20 min. Then cells were penetrated with 0.1% Triton X-100 (see Recipes) for 30 min and incubated with blocking buffer (see Recipes) at RT for 1 h. And then the cells were incubated with primary antibodies (1:400 diluted in blocking buffer) at 4 °C overnight, followed by an incubation with Alexa-488 fluorescent conjugated secondary antibodies (1:400 diluted in blocking buffer) at RT for 2 h. After immunostaining, cells are incubated with 1 μg/ml DAPI (see Recipes) for 5 min at RT to stain nuclei. As shown in Figure 3, purified SCs show bipolar or tripolar shapes, and are positive for specific markers including S100, GFAP, and P75.


Figure 3. Identification of cultured Schwann cells. A and A’. Primary cultured SCs under phase contrast microscope show bipolar or tripolar shapes. B-D’. Primary SCs are identified by immunostaining with specific markers including S100 (B, B’), GFAP (C, C’) and P75 (D, D’).

Recipes

  1. 100x poly-L-lysine hydrobromide (PLL)
    Dissolve 500 mg PLL powder in 50 ml distilled water to make 100x stock solution of 10 mg/ml
  2. 10% fetal bovine serum (10% FBS) DMEM/F12 media
    Dilute 5 ml FBS and 500 μl Pen/Strep into 44.5 ml Dulbecco’s modification of Eagle’s medium/Ham’s F-12 50/50 Mix (DMEM/F12)
  3. 1,000x cytosine arabinoside (AraC)
    Dissolve 4.86 mg AraC in 2 ml distilled water to make 1,000x stock solution of 10 mM and sterilize the solution by filtration
    Store at -20 °C and protect from light
  4. 5,000x heregulin
    Dissolve 50 μg heregulin in 1 ml sterile 0.1% bovine serum albumin (BSA) to make 5,000x stock solution of 50 ng/μl
  5. 10,000x forskolin
    Dissolve 10 mg forskolin in 812 μl DMSO to make 10,000x stock solution of 30 mM
  6. SC culture medium
    DMEM/F12 containing 3% FBS supplement with 10 ng/ml heregulin, 3 μM forskolin, and 1% Pen/Strep
    Dilute 1.5 ml FBS, 10 μl 5,000x Heregulin, 5 μl 10,000x Forskolin and 500 μl Pen/Strep into 48 ml DMEM/F12
  7. 4% paraformaldehyde (PFA)
    Dilute 4 g PFA in 100 ml PBS
    Stir at 65 °C until complete dissolution, and store at 4 °C
  8. 0.1% Triton X-100
    Dilute 100 μl Triton X-100 into 100 ml PBS
  9. Blocking buffer
    Dissolve 0.5 g gelatin (5%, w/v) in 10 ml PBS, and add 300 μl Triton X-100 (0.3%) to the buffer
  10. 1,000x 4, 6-diamidino-2-phenylindole (DAPI)
    Dissolve 5 mg DAPI in 5 ml distilled water to make 1,000x stock solution of 1 mg/ml, dilute it to 1x with PBS before used

Acknowledgments

This protocol is adapted from the previously published paper (Wen et al., 2017). This work was supported by the National Key Basic Research Program of China (2014CB542202 and 2014CB542205), National Natural Science Foundation of China (30973095, 81371354 & 81571182); Science and Technology Project of Guangzhou (12C32121609) and Natural Science Foundation of Guangdong Province (S2013010014697) to J Guo.

References

  1. Assouline, J. G., Bosch, E. P. and Lim, R. (1983). Purification of rat Schwann cells from cultures of peripheral nerve: an immunoselective method using surfaces coated with anti-immunoglobulin antibodies. Brain Res 277(2): 389-392.
  2. Brockes, J. P., Fields, K. L. and Raff, M. C. (1979). Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve. Brain Res 165(1): 105-118.
  3. Haastert, K., Mauritz, C., Chaturvedi, S. and Grothe, C. (2007). Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol. Nat Protoc 2(1): 99-104.
  4. Jin, Y. Q., Liu, W., Hong, T. H. and Cao, Y. (2008). Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment. J Neurosci Methods 170(1): 140-148.
  5. Oda, Y., Okada, Y., Katsuda, S., Ikeda, K. and Nakanishi, I. (1989). A simple method for the Schwann cell preparation from newborn rat sciatic nerves. J Neurosci Methods 28(3): 163-169.
  6. Pannunzio, M. E., Jou, I. M., Long, A., Wind, T. C., Beck, G. and Balian, G. (2005). A new method of selecting Schwann cells from adult mouse sciatic nerve. J Neurosci Methods 149(1): 74-81.
  7. Tao, Y. (2013). Isolation and culture of Schwann cells. Methods Mol Biol 1018: 93-104.
  8. Vroemen, M. and Weidner, N. (2003). Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve. J Neurosci Methods 124(2): 135-143.
  9. Weinstein, D. E. and Wu, R. (2001). Isolation and purification of primary Schwann cells. Curr Protoc Neurosci Chapter 3: Unit 3.17.
  10. Wen, J., Qian, C., Pan, M., Wang, X., Li, Y., Lu, Y., Zhou, Z., Yan, Q., Li, L., Liu, Z., Wu, W. and Guo, J. (2017). Lentivirus-mediated RNA interference targeting RhoA slacks the migration, proliferation, and myelin formation of Schwann cells. Mol Neurobiol 54(2): 1229-1239.
  11. Wood, P. M. (1976). Separation of functional Schwann cells and neurons from normal peripheral nerve tissue. Brain Res 115(3): 361-375.

简介

原代培养的雪旺氏细胞(SCs)广泛用于SC的生物学研究,是神经组织工程的重要种子细胞。 在这里,我们描述了从新生Sprague-Dawley(SD)大鼠中收获原代培养的SC的新方案。 在本方案中,解离的SCs从新生大鼠的脊神经中分离并通过治疗阿糖胞苷(AraC)进行纯化。
【背景】SC是周围神经系统(PNS)的神经胶质细胞。主要SCs的分离和纯化是研究SC生物学的关键步骤。此外,纯化的原代培养的SC是用于神经组织工程的重要种子细胞。迄今为止,已经基于Brockes(Brockes等人,1979)的方法报道了培养SC的各种方法。通过报道的方法,坐骨神经主要用于SC隔离,因为它们尺寸大并且可以容易地获得。然而,坐骨神经的SC容易被成纤维细胞污染,因为结缔组织难以清除。特别是神经管和神经束是成纤维细胞的主要来源。没有特殊处理,污染的成纤维细胞比SCs快得多,并且将很快成为培养物中主要的细胞。在过去几十年中,已经开发出许多用于从污染成纤维细胞中分离SC的纯化方法。这些纯化方法的细节包括单一或组合的抗有丝分裂处理(Wood,1976),抗体介导的细胞溶解(Brockes等人,1979),免疫选择(Assouline等人,,1983; Vroemen和Weidner,2003),反复去除(Oda等人,1989),冷喷射技术(Haastert等人,2007),差异粘附(Pannunzio et al。,2005)和差异分离(Jin et al。,2008)。这些纯化方法涉及成本高或收获期长的复杂技术,细胞产量低。因此,获得大量纯化的SCs仍然是基础研究和进一步临床应用的挑战性工作。在这里,我们描述了一种使用新生SD大鼠脊髓神经作为细胞源的方法,以在短时间内高效地获得高纯度的SC。

关键字:施万细胞, 脊髓神经, 分离, 纯化, 大鼠

材料和试剂

  1. 移液器提示(Corning,Axygen ®目录号:T-1000-B-R-S,T-200-Y-R-S)
  2. 细胞培养皿(35×10mm)(3.5cm培养皿)(Corning,目录号:430165)
  3. 1.5ml离心管(Corning,Axygen ,目录号:MCT-150-C)
  4. 50ml离心管(Corning,目录号:430290)
  5. 100μm细胞过滤器(Corning,目录号:431752)
  6. 新生儿Sprague-Dawley(SD)大鼠(出生2-4天,P2-4)
  7. 蒸馏水
  8. 75%乙醇
  9. Hank的平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:C14175500BT)
  10. 聚-L-赖氨酸氢溴酸盐(PLL)(Sigma-Aldrich,目录号:P1274)
  11. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200072)
  12. 胎牛血清(FBS)(Mediatech,目录号:35-076-CV)
  13. Dulbecco修改Eagle's Medium / Ham's F-12 50/50 Mix(DMEM / F12)(Mediatech,目录号:10-092-CV)
  14. 胞嘧啶阿拉伯糖苷(AraC)(Sigma-Aldrich,目录号:C1768)
  15. 福斯柯林(Sigma-Aldrich,目录号:F6886)
  16. 重组人类胰腺β-1(Heregulin)(PeproTech,目录号:100-03)
  17. 二甲基亚砜(DMSO)(MP Biomedicals,目录号:196055)
  18. 青霉素/链霉素(Pen / Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  19. 磷酸盐缓冲盐水(PBS)(Beyotime Biotechnology,目录号:C0221A)
  20. 多聚甲醛(PFA)(广东光华科技,目录号:1.17767.014)
  21. 明胶(Sigma-Aldrich,目录号:G7041)
  22. Triton X-100(Sigma-Aldrich,目录号:V900502)
  23. 4,6-二脒基-2-苯基吲哚(DAPI)(Sigma-Aldrich,目录号:D9542)
  24. 兔抗体(GFAP)产生的抗胶质纤维酸性蛋白抗体(Sigma-Aldrich,目录号:G9269)
  25. 抗-S-100蛋白抗体,小鼠中产生的克隆15E2E2(S100)(Merck,目录号:MAB079-1)
  26. 抗兔神经生长因子受体抗体p75(P75)(Merck,目录号:AB1554)
  27. Alexa Fluor 488山羊抗小鼠IgG(H + L)(Thermo Fisher Scientific,Invitrogen,目录号:A-11001)
  28. Alexa Fluor 488山羊抗兔IgG(H + L)(Thermo Fisher Scientific,Invitrogen,目录号:A-11008)
  29. 100x PLL(见配方)
  30. 10%FBS(见配方)
  31. 1,000x AraC(见配方)
  32. 500x蛋黄(见食谱)
  33. 10,000x毛喉素(见食谱)
  34. SC培养基(见食谱)
  35. 4%多聚甲醛(PFA)(见配方)
  36. 0.1%Triton X-100(参见食谱)
  37. 阻塞缓冲区(见配方)
  38. 1,000x DAPI(见配方)

设备

  1. 移液器(Eppendorf,型号:Research Plus ,200μl,1000μl)
  2. CO 2/2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Model 3100 Series,目录号:3111)
  3. 组织培养罩(AIRTECH,目录号:SW-CJ-1F)
  4. 手术剪刀和镊子(RWD Life Science,目录号:S14001-15,F13024-13,S12003-09,F13029-10,见图1A)
    注意:S14001-15和F13024013 是剪刀和用于斩首的镊子,S12003-09和F13029-10是用于皮肤解剖的剪刀和镊子。
  5. 春季剪刀(66 Vision,目录号:54053B,参见图1B)
  6. 精镊子(Fine Science Tools,Dumont,型号:#5,目录号:11252-23,尖端为0.1×0.06mm,见图1B)
  7. 超细镊子(Fine Science Tools,Dumont,型号:#5,目录号:11252-20,尖端为0.05×0.02mm,见图1B)
  8. 解剖板,针和冰袋
  9. 水浴(宁波科技生物科技,型号:GH-15)
  10. 立体显微镜(Olympus,型号:SZ61)
  11. 离心机(Eppendorf,型号:5430)


    图1.解剖工具。 A.手术剪刀和镊子用于斩断大鼠和解剖皮肤。 B.弹簧剪刀和细镊子用于分离肌肉,开放脊椎椎板并收集脊髓神经

程序

  1. 制备
    1. 在37℃,CO 2培养箱中,将一个3.5厘米的培养皿与1毫升PLL溶液(0.1毫克/毫升的蒸馏水,见食谱)一起覆盖。
    2. 将剪刀和镊子浸入75%乙醇中30分钟灭菌,然后将其暴露于紫外线下30分钟,风干。
    3. 用紫外线灭菌30分钟的解剖室(待解剖的大鼠)
    4. 通过抽吸去除PLL溶液,并用蒸馏水彻底冲洗盘表面两次。在剥离前将组织培养罩中的PLL涂层盘空气干燥。
    5. 准备两个3.5厘米的菜肴,将1.5毫升冷HBSS吸取到每个菜肴上,并将它们放在冰袋上
    6. 对于要使用的每只大鼠小鼠,将1ml 0.25%胰蛋白酶-EDTA的等分试样置于37℃的水浴中预热。

  2. 组织收集
    1. 通过在冰上冷却使其停止移动来麻醉新生大鼠(出生2-4天)。
      注意:动物使用由南方医科大学动物护理使用委员会根据动物道德治疗指南批准。所有努力都是为了尽量减少动物的牺牲和痛苦。
    2. 用75%乙醇灭菌大鼠。
    3. 用大手术剪刀和镊子快速斩首大鼠,将头部放入收集袋中。
    4. 将大鼠放置在解剖板上,并以倾斜姿势(背侧向上),并用针将四肢插入(参见图2A)。
    5. 使用一套小型剪刀和镊子,沿着中线从头部到尾部切割背部皮肤,将皮肤从身体分离为内侧至外侧(见图2B)。
    6. 使用弹簧剪刀和细镊子,将肩胛骨与骨架分开,并将肩胛骨固定在解剖板上。
    7. 使用弹簧剪刀和细镊子,仔细暴露臂丛,并将结缔组织与神经分离。然后切断远端的神经(参见图2C和2D)。
    8. 使用弹簧剪刀和精细镊子,将腿筋肌肉从膝盖小心地分离到髋骨以暴露坐骨神经。然后切断远端的坐骨神经(见图2E)。
      注意:不要在步骤B7和B8切割神经的近端。在这些步骤中神经没有被直接解剖出来。相反,如以下步骤B11所述,神经应该从椎间孔被拉出。
    9. 使用弹簧剪刀和细镊子切割双侧椎弓并将脊椎椎板从头端移至尾部以露出脊髓(见图2F)。
    10. 使用弹簧剪刀和精细镊子,小心地取出脊髓,露出背根神经节(见图2G和2H)。
    11. 使用细镊子,小心抓住神经节,然后将连接的脊髓神经拉出,并放入带有冰冷HBSS的3.5厘米盘中。重复这一步骤,从尾部收获所有的脊神经,到脊柱(主要包括臂丛神经,肋间神经和坐骨神经)(见图2I和2J)。
      注意:
      1. 通过抓住椎管(椎管)内侧的神经节,可以方便地找到并收集所有脊神经。














      2. 神经的收获最容易在立体显微镜下完成。非常建议。
    12. 使用超细镊子去除粘附于神经的结缔组织,用精细剪刀切断神经节。将干净的神经转移到冰冷的HBSS的另一个3.5厘米的盘中。重复此过程以清除所有神经(见图2K和2L)。
      注意:


















      1. 每个收集的神经在近端具有神经节。神经节看起来像神经的头部,比神经更透明(见图2K,箭头)。同时,出现作为血管膜质组织的罕见结缔组织可能会粘附于神经(见图2K,箭头)。
      2. 通过椎间孔牵引收获的神经总是没有神经尿,因为神经尿与椎间孔紧密相连。这是该协议的主要优点。由于传统的方法不可避免地带来紧紧包裹神经的神经尿(Weinstein et al。,2001; Tao,2013)。神经尿是SC培养物中污染细胞(如成纤维细胞)的主要来源。
      3. 本方案的另一个优点是可以从传统方法中获得大量的神经,而不仅仅是两个坐骨神经。因此,牺牲的动物可以显着减少。


        图2.收获脊髓神经的逐步程序。 A.斩断的大鼠幼鼠被固定在解剖板上。 B.背部皮肤被切割并与身体分离。 C.肩胛骨与骨干分离并固定在解剖板上。 D.臂丛被暴露,其远端被切割。 E.坐骨神经被暴露,其远端被切割。 F.通过椎板切除术使用一对弹簧剪刀打开椎管。 G.将脊髓从颈部拖到尾部。 H.脊髓被去除后,背根神经节被暴露(箭头)。示意图显示脊髓神经(蓝色),主要包括可用于收获的臂神经神经(BPN),肋间神经(ICN)和坐骨神经(SN)。脊髓(SC)以黄色表示。一个代表性的图像显示神经通过椎间孔(箭头)被拖出,而一些背根神经节留在孔中(箭头)。收集的神经有神经节(箭头),有时带有结缔组织(箭头)。离开神经节和结缔组织后的神经。

  3. 解离和细胞播种
    1. 将清洁的神经转移到1.5ml管中,将神经切成0.5-1mm长的小块。
    2. 将1ml预热的0.25%胰蛋白酶-EDTA加入管中。盖上管子,并在37°C水浴中孵育30分钟,间歇振动每10分钟一次
    3. 通过将100μlFBS补充到培养基中终止胰蛋白酶消化。然后用1 ml移液管吸头轻轻地将样品30-35次,使单细胞悬浮注意:轻轻地研磨以避免气泡形成和飞溅。在上下移动30-35次之后,细胞悬浮液中残留有一些残留物。
    4. 通过100μm的细胞过滤器过滤细胞悬浮液以除去残留的块 注意:团块主要是轴突碎片和未分离的组织。
    5. 在室温(RT)下将细胞悬浮液以106 x(Rcf)离心10分钟,弃去上清液。
    6. 将细胞沉淀重悬于DMEM / F12中的300μl10%FBS中(参见食谱)。在干燥的PLL涂覆的3.5厘米盘的中间装入细胞悬浮液。不要摇动盘子,以免细胞悬浮液散开,粘在盘子的墙壁上。将细胞培养在CO 2培养箱中2小时以促进细胞附着 注意:
      1. 在我们的常规实践中,可以从每只大鼠获得总数为〜1×10 6 的细胞。
      2. 最小化电镀培养基(300μl10%FBS)的体积可以增加细胞密度并促进细胞附着于培养皿。
    7. 细胞附着于盘后,将1 ml 10%FBS加入盘中
  4. 净化和膨胀
    1. 在第二天,用含有10%FBS和10μMAraC的DMEM / F12替代培养基(参见食谱)以消除污染的成纤维细胞。
      注意:在没有促有丝分裂因子的情况下,如肌肉和毛喉素,SCs增殖相当缓慢,而成纤维细胞快速增殖。 AraC是一种抗代谢剂,会损害DNA合成并杀死分裂细胞。因此,成纤维细胞被有效地消除,而SC在适当的治疗时间(48小时)内良好地存活。
    2. 48小时后,用SC培养基更换培养基(参见食谱)以扩大SC。
    3. 当培养物达到90%汇合时,细胞以1:3的比例常规传代以扩增细胞。来自3-5 th 段的细胞用于实验中的实验。
      注意:3-5代的原代培养的SCs代表双极和三极体形状(参见图3A)。这些细胞的纯度可以达到98%,通过SC特异性标志物的免疫染色鉴定(见图3B-3D)。我们发现,SCs传播5次以上后,慢慢增长。此外,关于SC生物学的大多数发表的论文表明,来自新生大鼠的原代SC用于3-5 段落中。所以,在我们的实验室里,所有用于研究的SC都来自3-5 th 段落。

数据分析

为了确定我们文化中SCs的纯度,使用GFAP,P75和S100作为SC的特异性标记物,作为我们以前的报告(Wen等人,2017)进行免疫染色。简言之,培养的细胞用4%PFA固定(参见食谱)20分钟。然后用0.1%Triton X-100(参见食谱)将细胞穿透30分钟,并在室温下与封闭缓冲液(参见Recipes)一起孵育1小时。然后将细胞在4℃下与一抗(1:400稀释在封闭缓冲液中)孵育过夜,随后在室温下与Alexa-488荧光偶联的二抗(1:400稀释在封闭缓冲液中)孵育2小时。免疫染色后,将细胞与1μg/ ml DAPI(参见食谱)在室温下孵育5分钟以染色核。如图3所示,纯化的SCs显示双极或双极形状,对于S100,GFAP和P75等特异性标志物是阳性的。


图3.鉴定培养的雪旺氏细胞。 A和A'。在相差显微镜下的原代培养的SCs显示双极或三极的形状。 B-D”。通过用包括S100(B,B'),GFAP(C,C')和P75(D,D')的特异性标记的免疫染色鉴定原代SC。

食谱

  1. 100倍聚-L-赖氨酸氢溴酸盐(PLL)
    将500毫克PLL粉末溶于50ml蒸馏水中,制成100克/毫升的100倍储备溶液
  2. 10%胎牛血清(10%FBS)DMEM / F12培养基 将5ml FBS和500μlPen / Strep稀释到44.5ml Eagle's培养基/ Ham's F-12 50/50 Mix(DMEM / F12)的Dulbecco's修饰物中。
  3. 1,000x胞嘧啶阿拉伯糖苷(AraC)
    将4.86 mg AraC溶解于2 ml蒸馏水中,制成10 000份1000x储备溶液,并通过过滤消毒溶液。 储存于-20°C,避光保存
  4. 5,000x asgulin
    将50μg蛋黄溶解在1ml无菌的0.1%牛血清白蛋白(BSA)中以制备50ng /μl的5,000x储备溶液
  5. 10,000x福斯柯林
    将10毫克的毛喉素溶解在812微升的DMSO中以制备10,000x的30mM的储备溶液
  6. SC培养基
    含有10ng / ml蛋黄,3μM毛喉素和1%Pen / Strep的3%FBS补充的DMEM / F12
    稀释1.5ml FBS,10μl5,000x Heregulin,5μl10,000x Forskolin和500μlPen / Strep到48ml DMEM / F12中
  7. 4%多聚甲醛(PFA)
    在100ml PBS中稀释4 g PFA 在65℃下搅拌直到完全溶解,并在4℃下储存
  8. 0.1%Triton X-100
    将100μlTriton X-100稀释到100ml PBS中
  9. 阻塞缓冲区
    将0.5 g明胶(5%,w / v)溶于10ml PBS中,加入300μlTriton X-100(0.3%)至缓冲液
  10. 1,000x4,6-二脒基-2-苯基吲哚(DAPI)
    将5mg DAPI溶解在5ml蒸馏水中,制成1mg / ml的1,000x储备溶液,然后用PBS稀释至1x,然后使用

致谢

该协议改编自以前发表的文章(Wen等人,2017)。这项工作得到了国家重点基础研究计划(2014CB542202和2014CB542205),国家自然科学基金(30973095,81371354& 81571182)的支持。广州科技项目(12C32121609)和广东省自然科学基金(S2013010014697)至郭国。

参考

  1. Assouline,J.G.,Bosch,E.P。和Lim,R。(1983)。 从周围神经培养物中纯化大鼠雪旺氏细胞:使用抗免疫球蛋白包被的表面的免疫选择性方法抗体。 Brain Res 277(2):389-392。
  2. Brockes,J.P。,Fields,K.L。和Raff,M.C。(1979)。 研究培养的大鼠雪旺氏细胞。 I.建立来自外周神经的培养物的纯化群体。脑组织165(1):105-118。
  3. Haastert,K.,Mauritz,C.,Chaturvedi,S.and Grothe,C。(2007)。 人和大鼠成人雪旺氏细胞培养物:快速有效的富集和非常有效的非病毒转染方案。 Nat Protoc 2(1):99-104。
  4. Jin,Y. Q.,Liu,W.,Hong,T.H。和Cao,Y。(2008)。 通过使用多重胶原酶处理的差异细胞脱离进行有效的施旺细胞纯化。 Neurosci Methods 170(1):140-148。
  5. Oda,Y.,Okada,Y.,Katsuda,S.,Ikeda,K.and Nakanishi,I。(1989)。 从新生大鼠坐骨神经制备雪旺细胞的简单方法。 J Neurosci Methods 28(3):163-169。
  6. Pannunzio,M.E.,Jou,I.M.,Long,A.,Wind,T.C.,Beck,G.andBalian,G。(2005)。 从成年小鼠坐骨神经中选择雪旺氏细胞的新方法。 Neurosci Methods 149(1):74-81。
  7. 陶,Y.(2013)。 施万细胞的分离和培养。方法Mol Biol 1018:93-104。
  8. Vroemen,M。和Weidner,N。(2003)。 通过从成年周围神经选择p75低亲和力神经生长因子受体表达细胞来纯化雪旺氏细胞。 Neurosci Methods 124(2):135-143。
  9. Weinstein,D.E.and Wu,R。(2001)。 主要施万细胞的分离和纯化。 Curr Protoc Neurosci 第3章:单位3.17。
  10. Wen,J.,Qian,C.,Pan,M.,Wang,X.,Li,Y.,Lu,Y.,Zhou,Z.,Yan,Q.,Li,L.,Liu,Z., Wu,W.and Guo,J.(2017)。 靶向RhoA的慢病毒介导的RNA干扰减轻了施旺细胞的迁移,增殖和髓鞘形成。 / a> Mol Neurobiol 54(2):1229-1239。
  11. Wood,P.M。(1976)。 从正常周围神经组织分离功能性雪旺氏细胞和神经元。脑 Res 115(3):361-375。
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引用: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.
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