人类嗅粘膜样本的原代嗅鞘细胞培养

Mansoureh  Hashemi; Mahmoudreza   Hadjighassem

doi:10.21769/BioProtoc.2275

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Peer-reviewed

Primary Olfactory Ensheathing Cell Culture from Human Olfactory Mucosa Specimen

人类嗅粘膜样本的原代嗅鞘细胞培养

MH Mansoureh Hashemi email

MH Mahmoudreza Hadjighassem

发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2275 浏览次数: 8021

评审: Oneil G. BhalalaEhsan KheradpezhouhXiaoyu Liu

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The authors used this protocol in:

Cover of Molecular Neurobiology, featuring study using the protocol.

Oct 2016

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Abstract

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)

Background

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

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

Equipment

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

Procedure

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如何引用

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.