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

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.
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.
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 10⁶cells 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)
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.

Notes

Most steps should be performed in a sterile ventilation hood.
All reagents should be pre-warmed at 37 °C before use. Exception is for washing olfactory mucosa tissue with cold PBS.
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.

Recipes

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

Acknowledgments

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).

References

Escada, P. A., Lima, C. and da Silva, J. M. (2009). The human olfactory mucosa. Eur Arch Otorhinolaryngol 266(11): 1675-1680.
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.
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.
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.
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.
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.