Primary Olfactory Ensheathing Cell Culture from Human Olfactory Mucosa Specimen

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Molecular Neurobiology
Oct 2016



The human olfactory mucosa is located in the middle and superior turbinates, and the septum of nasal cavity. Olfactory mucosa plays an important role in detection of odours and it is also the only nervous tissue that is exposed to the external environment. This property leads to easy access to the olfactory mucosa for achieving various researches. The lamina propria of olfactory mucosa consists of olfactory ensheathing cells (OECs) that cover the nerve fibers of olfactory. Here we describe a protocol for isolation of OECs from biopsy of human olfactory mucosa.

Keywords: Human olfactory mucosa (人嗅粘膜), Olfactory ensheathing cells (OECs) (嗅鞘细胞(OECs)), S100-beta antigen (S100-β抗原), Primary cell culture (原代细胞培养)


Olfactory ensheathing cells (OECs) are glial cells that express various antigens similar to astrocytes and Schwann cells such as glial fibrillary-associated protein (GFAP), S100-beta, p75 low-affinity nerve growth factor receptor, vimentin, nestin, and neuropeptide Y (Singh et al., 2013). Olfactory ensheathing cells release different neurotrophic factors and adhesion molecules that function in cellular growth and adhesions of central nervous system (Pastrana et al., 2007). In addition, these cells play an important role in the regeneration of the damaged central nervous system such as treatment of spinal cord injury and neurodegenerative diseases (Novikova et al., 2011). We select OECs as research material in our study as they have several advantage properties such as high migratory capacity, accessible source, differentiation from stem cells of nasal olfactory mucosa, and non-tumorigenicity behavior (Huang et al., 2008; Escada et al., 2009). This protocol describes a step-by-step procedure for the isolation of OECs from Human Olfactory Mucosa Specimen.

Materials and Reagents

  1. 15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352096 )
  2. 50 ml centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
  3. T25 flask culture (Nest Biotechnology, catalog number: 707003 )
  4. 24-well plates (Nest Biotechnology, catalog number: 702001 )
  5. No. 10 scalpel blade surgical tool (Aspen Surgical, catalog number: 371610 )
  6. Cell strainer sieve (Corning, Falcon®, catalog number: 352340 )
  7. Petri dish culture (Nest Biotechnology, catalog number: 704001 )
  8. Olfactory mucosa fresh specimen (Human)
  9. Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117 )
  10. Antibiotic-antimycotic (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  11. Dispase II (Sigma-Aldrich, catalog number: D4693 )
  12. Collagenase IA (Sigma-Aldrich, catalog number: C9891 )
  13. Dulbecco’s modified Eagle medium/F12 (DMEM/F12) (Thermo Fisher Scientific, GibcoTM, catalog number: 31331028 )
  14. Nerve growth factor (NGF) (Sigma-Aldrich, catalog number: N0513 )
  15. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  16. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  17. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  18. Bovine serum albumin (BSA, powder) (Sigma-Aldrich, catalog number: A2058 )
  19. Primary antibody of rabbit anti-S100-beta (Sigma-Aldrich, catalog number: S2644 )
  20. FITC-conjugated goat anti-rabbit (Abcam, catalog number: ab6717 )
  21. (Optional) Cytosine arabinoside or Cytarabine (Sigma-Aldrich, catalog number: C3350000 )
  22. Phosphate buffer saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 18912014 )
  23. Paraformaldehyde (powder) (Sigma-Aldrich, catalog number: P6148 )
  24. Culture medium (see Recipes)
  25. Phosphate buffer saline (PBS) (see Recipes)
  26. 4% paraformaldehyde (see Recipes)
  27. 0.2% Triton X-100 (see Recipes)
  28. 10% bovine serum albumin (BSA) (see Recipes)


  1. Small forceps surgical tools (Fine Science Tools, catalog number: 11050-10 )
  2. Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
  3. Inverted fluorescence microscope (Optika, model: XDS-2FL )
  4. Centrifuge machine (Hettich Lab Technology, model: Universal 320 R )
  5. Ventilation hood (Vision Scientific, model: VS-7120LV )
  6. CO2 cell culture incubator (Memmert, model: INC108 T2T3 )
  7. 37 °C water bath (Memmert, model: WNB 14 )


  1. Collection of olfactory mucosa tissue
    1. A fresh sample (diameter of 4 mm) was obtained endoscopically from the superior turbinate tissue of a patient under general anesthesia.
    2. This study was approved by the Ethical Commission of the Tehran University of Medical Sciences.
    3. Before operation, a signed written consent was given by the patient.
    4. There is no age limitation for providing fresh sample in this study.
    5. After coordination with an Ear Nose and Throat (ENT) surgeon, the olfactory mucosa tissue was placed in a 15 ml tube containing 5 ml cold HBSS with 10% antibiotics (Antibiotic-antimycotic).
    6. Olfactory mucosa tissue was delivered to the lab on dry ice.

  2. Isolation of OECs from olfactory mucosa
    1. Wash the olfactory mucosa tissue 3 times with 5 ml cold PBS each time.
    2. Incubate the olfactory mucosa tissue in 1 ml dispase II solution (2.4 IU/ml), for 1 h at 37 °C.
    3. Dissect lamina propria from the olfactory epithelium using a micro spatula under an inverted light microscope. Lamina propria is the thick orange layer and epithelium is the thin translucent gray layer.
    4. Cut the lamina propria into small pieces.
    5. Transfer the pieces to a new 15 ml Falcon tube containing 1 ml of collagenase IA and incubate for 20 min at 37 °C.
    6. Inactivate collagenase IA by adding 9 ml of PBS into the tube.
    7. Centrifuge the suspension at 250 x g for 5 min.
    8. Resuspend the resulting pellet in 1 ml of Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with neuronal growth factor (NGF, 50 ng/ml), 1% (100 U/ml) antibiotic-antimycotic.
    9. Transfer the suspension containing tiny pieces of tissue into two T25 flasks containing 5 ml of Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with neuronal growth factor (NGF, 50 ng/ml), 1% (100 U/ml) antibiotic-antimycotic each.
    10. Incubate in an incubator at 37 °C, 5% CO2 and 95% humidity for 3-4 weeks.
    11. Change culture medium every 3 days.
      Note: After 14 days culture of human olfactory lamina propria, OECs are released from periphery of spherical clusters. The morphology of most cells is spindle-shaped and bipolar with long processes and small cytoplasm or multipolar form with short processes and plentiful cytoplasm such as stellate morphology. Note that there may be fibroblast contamination in lamina propria culture. Fibroblast morphology is also spindle-shaped and bipolar with long processes. (Figures 1 and 2)
    12. After 3-4 weeks, OECs are ready for subculture.

      Figure 1. Different stages of OECs isolation from human olfactory mucosa. A. Outside view of nose; B. Location of superior turbinate in the olfactory mucosa; C. Human olfactory mucosa biopsy from superior turbinate; D. Lamina propria of olfactory mucosa after dispase II digestion; E. Lamina propria cultured after collagenase IA digestion; F. Olfactory ensheathing cells released from periphery of lamina propria, scale bar = 100 µm. 

      Figure 2. OECs after first passage. Scale bar = 100 µm.

  3. Sub-culture of primary OECs of olfactory mucosa lamina propria
    1. Check the confluency of OECs under an inverted microscope. Subculture the cells when the cell density reaches 80% confluence 3-4 weeks after seeding.
    2. Remove the medium from each T25 flask under a biological hood with a 5 ml sterile pipette.
    3. Wash the cells with 3 ml of warm (37 °C) PBS and then remove PBS.
    4. Trypsinize the cells with 1 ml of warm (37 °C) 0.05% trypsin-EDTA for 1 min in an incubator at 37 °C.
    5. Stop trypsin-EDTA reaction by adding 4 ml of DMEM/F12 containing 10% FBS and 1% antibiotic-antimycotic into each T25 flask and then transfer the cell suspension from each flask to a 15 ml Falcon tube.
    6. Centrifuge the cell suspension at 250 x g for 5 min at room temperature.
    7. Remove the medium from each tube with a 5 ml sterile pipette.
    8. Resuspend the pellet in each tube with 1 ml of fresh medium containing DMEM/F12, 10% FBS and 1% antibiotic-antimycotic.
    9. After re-suspend and counting, seed the cells into a 24-well dish (at a density of 5 x 103 cells per well) for immunocytochemistry (go to step D2) and into T25 flasks (5 x 106 cells in flask) for subculture.
    10. Subculture the T25 flasks containing OECs in (DMEM)/F12 medium containing 10% FBS and 1% (100 U/ml) antibiotic-antimycotic in the incubator at 37 °C, 5% CO2 and 95% humidity for 4-5 days. (Figure 2)

  4. Immunocytochemistry
    1. Seed 5 x 103 cells into each well of a 24-well culture dish.
    2. Wash each well three times with phosphate-buffered saline (PBS) after 24 h at room temperature.
    3. Fix cells with 4% paraformaldehyde in PBS for 20 min.
    4. Wash cells three times in PBS at room temperature for 5 min each.
    5. Permeate cell membranes with 0.2% Triton X-100 for 15 min.
    6. Wash cells three times in PBS at room temperature for 5 min each.
    7. Block cells with 10% bovine serum albumin for 1 h.
    8. Dilute the primary antibody of rabbit anti-S100-beta 1:100 in 1% BSA in PBS.
    9. Wash cells three times in PBS at room temperature for 5 min each.
    10. Dilute the secondary antibody of FITC-conjugated goat anti-rabbit 1:500 in 1% BSA in PBS.
    11. Incubate cells with secondary antibody of FITC-conjugated goat anti-rabbit for 2 h at room temperature in the dark.
    12. Wash cells three times in PBS at room temperature for 5 min each.
    13. Image cells by an inverted fluorescence microscopy (Figure 3).

      Figure 3. Purified primary olfactory ensheathing cell culture. OECs have expressed antigen of S100-beta. Scale bar = 50 µm.

Data analysis

Confirmation of cultured primary OECs was done by detecting S100-beta antigen via fluorescence immunocytochemical analysis. This analysis demonstrated that cells expressed S100-beta antigen. Test results are obtained from three independent experiments.


  1. Most steps should be performed in a sterile ventilation hood.
  2. All reagents should be pre-warmed at 37 °C before use. Exception is for washing olfactory mucosa tissue with cold PBS.
  3. If fibroblast contamination observed in the primary culture, cytosine arabinoside should be added to a final concentration of 5 µg/ml in the cell culture medium in the next 10 days. Cytosine arabinoside is a growth inhibitor of fibroblast.


  1. Culture medium (200 ml,10% FBS, 1% antibiotic-antimycotic)
    178 ml DMEM/F12
    20 ml FBS
    2 ml antibiotic-antimycotic
    Store at 4 °C
  2. Phosphate buffer saline (PBS) (500 ml)
    1 PBS tablet dissolved in 500 ml MiliQ water
    Filter and store at 4 °C
  3. 4% paraformaldehyde
    4 g paraformaldehyde dissolved in 100 ml PBS
  4. 0.2% Triton X-100
    10 µl Triton X-100 dissolved in 5 ml PBS
  5. 10% bovine serum albumin
    Dissolve 0.5 g bovine serum albumin in 5 ml PBS


This study was supported by grant number 20035 obtained from School of Advanced Technologies in Medicine and grant 19925 from Brain and Spinal Cord injury Research Center, Tehran University of Medical Sciences. The protocol was adapted from Hashemi et al. (2016).


  1. Escada, P. A., Lima, C. and da Silva, J. M. (2009). The human olfactory mucosa. Eur Arch Otorhinolaryngol 266(11): 1675-1680.
  2. Hashemi, M., Fallah, A., Aghayan, H. R., Arjmand, B., Yazdani, N., Verdi, J., Ghodsi, S. M., Miri, S. M. and Hadjighassem, M.R. (2016). A new approach in gene therapy of glioblastoma multiforme: human olfactory ensheathing cells as a novel carrier for suicide gene delivery. Mol Neurobiol 53(8): 5118-5128.
  3. Huang, Z. H., Wang, Y., Cao, L., Su, Z. D., Zhu, Y. L., Chen, Y. Z., Yuan, X. B. and He, C. (2008). Migratory properties of cultured olfactory ensheathing cells by single-cell migration assay. Cell Res 18(4): 479-490.
  4. Novikova, L. N., Lobov, S., Wiberg, M. and Novikov, L. N. (2011). Efficacy of olfactory ensheathing cells to support regeneration after spinal cord injury is influenced by method of culture preparation. Exp Neurol 229(1): 132-142.
  5. Pastrana, E., Moreno-Flores, M. T., Avila, J., Wandosell, F., Minichiello, L. and Diaz-Nido, J. (2007). BDNF production by olfactory ensheathing cells contributes to axonal regeneration of cultured adult CNS neurons. Neurochem Int 50(3): 491-498.
  6. Singh, N., Gopal, S. C., Srivastava, R. N., Chandra, T., Agarwal, S. P., Singh, S. K., Gupta, D. K. and Balapure, A. K. (2013). In vitro maintenance of olfactory mucosa: with enriched olfactory ensheathing cells. J Stem Cell Res Ther 3:1-8.



背景 嗅鞘细胞(OECs)是神经胶质细胞,其表达与星形胶质细胞和施旺细胞类似的各种抗原,例如胶质纤维相关蛋白(GFAP),S100-β,p75低亲和力神经生长因子受体,波形蛋白,巢蛋白和神经肽Y (Singh等人,2013)。嗅鞘细胞释放在中枢神经系统的细胞生长和粘连中起作用的不同神经营养因子和粘附分子(Pastrana等人,2007)。此外,这些细胞在受损的中枢神经系统的再生中起重要作用,例如治疗脊髓损伤和神经变性疾病(Novikova等人,2011)。我们选择OEC作为我们研究中的研究材料,因为它们具有诸如高迁移能力,可及源,鼻嗅粘膜干细胞分化和非致瘤性行为等优势性质(Huang et al。 ,2008; Escada 等人,2009)。该方案描述了从人类嗅觉粘膜样品中分离OEC的一步一步的过程。

关键字:人嗅粘膜, 嗅鞘细胞(OECs), S100-β抗原, 原代细胞培养


  1. 15ml离心管(Corning,Falcon ®,目录号:352096)
  2. 50ml离心管(Corning,Falcon ®,目录号:352070)
  3. T25烧瓶培养(Nest Biotechnology,目录号:707003)
  4. 24孔板(Nest Biotechnology,目录号:702001)
  5. 十号刀片手术刀(Aspen Surgical,目录号:371610)
  6. 细胞过滤筛(Corning,Falcon ®,目录号:352340)
  7. 培养皿培养(Nest Biotechnology,目录号:704001)
  8. 嗅粘膜新鲜标本(人)
  9. Hanks的平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:24020117)
  10. 抗生素 - 抗真菌药(Thermo Fisher Scientific,Gibco TM,目录号:15240062)
  11. Dispase II(Sigma-Aldrich,目录号:D4693)
  12. 胶原酶IA(Sigma-Aldrich,目录号:C9891)
  13. Dulbecco改良的Eagle培养基/F12(DMEM/F12)(Thermo Fisher Scientific,Gibco TM,目录号:31331028)
  14. 神经生长因子(NGF)(Sigma-Aldrich,目录号:N0513)
  15. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  16. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  17. Triton X-100(Sigma-Aldrich,目录号:T8787)
  18. 牛血清白蛋白(BSA,粉末)(Sigma-Aldrich,目录号:A2058)
  19. 兔抗S100-β的一级抗体(Sigma-Aldrich,目录号:S2644)
  20. FITC缀合的山羊抗兔(Abcam,目录号:ab6717)
  21. (可选)阿糖胞苷或阿糖胞苷(Sigma-Aldrich,目录号:C3350000)
  22. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:18912014)
  23. 多聚甲醛(粉末)(Sigma-Aldrich,目录号:P6148)
  24. 培养基(见食谱)
  25. 磷酸盐缓冲盐水(PBS)(见食谱)
  26. 4%多聚甲醛(见食谱)
  27. 0.2%Triton X-100(见配方)
  28. 10%牛血清白蛋白(BSA)(见食谱)


  1. 小镊子手术工具(精细科学工具,目录号:11050-10)
  2. 血细胞计数器(Sigma-Aldrich,目录号:Z359629)
  3. 倒置荧光显微镜(Optika,型号:XDS-2FL)
  4. 离心机(Hettich Lab Technology,型号:Universal 320 R)
  5. 通风罩(Vision Scientific,型号:VS-7120LV)
  6. CO 2细胞培养箱(Memmert,型号:INC108 T2T3)
  7. 37°C水浴(Memmert,型号:WNB 14)


  1. 收集嗅粘膜组织
    1. 在全身麻醉下从患者的上鼻甲组织内窥镜获得新鲜样品(直径4mm)。
    2. 本研究由德黑兰医科大学伦理委员会批准。
    3. 手术前,患者签署书面同意书。
    4. 本研究中没有提供新鲜样本的年龄限制。
    5. 在与耳鼻喉科(ENT)外科医生协调后,将嗅粘膜组织置于含有10%抗生素(抗生素 - 抗真菌剂)的5ml冷HBSS的15ml管中。
    6. 嗅粘膜组织在干冰上输送到实验室。

  2. 从嗅粘膜分离OEC
    1. 每次用5ml冷PBS洗涤嗅粘膜组织3次。
    2. 将嗅黏膜组织浸泡在1ml Dispase II溶液(2.4IU/ml)中,在37℃孵育1小时。
    3. 在倒置光学显微镜下使用微型刮刀从嗅觉上皮解剖固有层。固有层是厚橙色层,上皮是薄的半透明灰色层。
    4. 将固有层切成小块。
    5. 将片转移到含有1ml胶原酶IA的新的15ml Falcon管中,并在37℃下孵育20分钟。
    6. 通过向管中加入9ml PBS来灭活胶原酶IA
    7. 将悬浮液以250 x g离心5分钟。
    8. 将所得沉淀重悬于1ml补充神经元生长因子(NGF,50ng/ml),1%(100U/ml)抗生素 - 抗真菌剂的Dulbecco改良Eagle培养基(DMEM)/F12培养基中。
    9. 将含有微小组织的悬浮液转移到两个含有补充有神经元生长因子(NGF,50ng/ml),1%(100U/ml)抗生素的Dulbecco改良的Eagle培养基(DMEM)/F12培养基的T25烧瓶中,各种抗真菌药物
    10. 在37℃,5%CO 2和95%湿度的培养箱中孵育3-4周。
    11. 每3天更换培养基。
      注意:经过14天的人类嗅鞘固有培养后,OECs从球形簇的周围释放出来。大多数细胞的形态是纺锤形和双极的,具有长的过程和小的细胞质或具有短过程和丰富的细胞质如星状形态的多极形式。请注意,固有层固有培养物中可能存在成纤维细胞污染。 Fibrolast形态也是纺锤形和双极的长过程。 (图1和图2)
    12. 3-4周后,OEC准备进行亚文化。

      图1. OEC与人类嗅粘膜分离的不同阶段。 A.鼻子外观;嗅黏膜上鼻甲的位置; C.来自上鼻甲的人类嗅粘膜活检;唾液Ⅱ消化后嗅黏膜的固有层E.胶原酶IA消化后培养的固有层。 F.从固有固有层周围释放的嗅鞘细胞,比例尺=100μm。 

      图2.第一次通过后的OEC。 比例尺= 100μm。

  3. 嗅黏膜固有层原发性OEC的亚文化
    1. 检查OECs在倒置显微镜下的汇合。细胞密度达到80%时,培养细胞,播种3-4周
    2. 使用5ml无菌移液管在生物罩下从每个T25烧瓶中取出培养基
    3. 用3ml温热(37℃)PBS洗涤细胞,然后除去PBS。
    4. 在37℃的培养箱中用1ml温(37℃)0.05%胰蛋白酶-EDTA将细胞胰蛋白酶处理1分钟。
    5. 通过向每个T25烧瓶中加入含有10%FBS和1%抗生素抗真菌剂的4ml DMEM/F12,然后将细胞悬浮液从每个烧瓶转移到15ml Falcon管中停止胰蛋白酶-EDTA反应。
    6. 将细胞悬浮液在室温下以250×g离心5分钟
    7. 用5ml无菌移液管从每个管中取出培养基。
    8. 在每个管中用1ml含有DMEM/F12,10%FBS和1%抗生素 - 抗霉菌剂的新鲜培养基重悬。
    9. 重新悬浮和计数后,将细胞种植在24孔培养皿(每孔5×10 3个细胞密度)中,进行免疫细胞化学(转到步骤D2)和T25烧瓶(5 x 10 细胞在培养瓶中)
    10. 在含有10%FBS和1%(100U/ml)抗生素抗真菌剂的DMEM/F12培养基中,在37℃,5%CO 2和95℃的培养箱中培养含有OEC的T25培养瓶湿度为4-5天。 (图2)

  4. 免疫组化
    1. 将种子5×10 3个细胞注入24孔培养皿的每个孔中
    2. 在室温下24小时后用磷酸盐缓冲盐水(PBS)将每孔洗涤三次
    3. 用PBS中的4%多聚甲醛固定细胞20分钟
    4. 在PBS中,室温洗涤细胞三次,每次5分钟
    5. 用0.2%Triton X-100渗透细胞膜15分钟
    6. 在PBS中,室温洗涤细胞三次,每次5分钟
    7. 用10%牛血清白蛋白封闭细胞1 h
    8. 在PBS中的1%BSA中稀释兔抗S100-β1:100的一抗。
    9. 在PBS中,室温洗涤细胞三次,每次5分钟
    10. 稀释FITC-缀合的山羊抗兔1:500的二抗在PBS中的1%BSA中
    11. 在室温下在黑暗中孵育细胞与FITC-缀合的山羊抗兔二抗2小时。
    12. 在PBS中,室温洗涤细胞三次,每次5分钟
    13. 通过倒置荧光显微镜观察图像细胞(图3)

      图3.纯化的原代嗅鞘细胞培养物。 OECs表达S100-β的抗原。比例尺=50μm。




  1. 大部分步骤应在无菌通风罩内进行。
  2. 所有试剂应在37℃预热,然后再使用。异常是用冷PBS洗嗅粘膜组织。
  3. 如果在原代培养中观察到成纤维细胞污染,则应在接下来的10天内在细胞培养基中加入胞嘧啶阿拉伯糖苷至终浓度为5μg/ml。胞嘧啶阿糖胞苷是成纤维细胞的生长抑制剂


  1. 培养基(200ml,10%FBS,1%抗生素 - 抗真菌剂) 178 ml DMEM/F12
    2 ml抗生素 - 抗真菌药物 储存于4°C
  2. 磷酸盐缓冲盐水(PBS)(500 ml)
    1 PBS片剂溶于500 ml MiliQ水中 过滤并储存在4°C
  3. 4%多聚甲醛
    4g多聚甲醛溶于100ml PBS中
  4. 0.2%Triton X-100
    10μlTriton X-100溶于5ml PBS中
  5. 10%牛血清白蛋白 将0.5 g牛血清白蛋白溶解在5ml PBS中




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引用:Hashemi, M. and Hadjighassem, M. (2017). Primary Olfactory Ensheathing Cell Culture from Human Olfactory Mucosa Specimen. Bio-protocol 7(10): e2275. DOI: 10.21769/BioProtoc.2275.