Oct 19, 2024

Public workspaceMouse Olfactory Horizontal Basal Cell Culture

DOI
dx.doi.org/10.17504/protocols.io.j8nlkok26v5r/v1
  • 1Ottawa Hospital Research Institute
Alp Ozgun
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkok26v5r/v1
Protocol Citation: Alp Ozgun, Priya Suman, Josée Coulombe, Julianna Tomlinson, Earl G. Brown, John M. Woulfe, Michael G. Schlossmacher 2024. Mouse Olfactory Horizontal Basal Cell Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkok26v5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 27, 2023
Last Modified: October 21, 2024
Protocol Integer ID: 85597
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020625
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
This protocol describes isolation and culture of basal cells from the olfactory epithelium of mice. The process involves serial enzymatic treatment of dissected olfactory epithelia and subsequent plating in commercial airway medium with dual SMAD inhibitors. The resulting cultures express the horizontal basal cell markers CK5 and CK14.
Materials
Dissection medium:
  • Pneumacult-Ex medium

  • 1x B27 supplement without vitamin A
  • 1x N2 supplement
  • 1% penicillin/streptomycin

HBC medium:
  • Pneumacult-Ex medium
  • 1x Glutamax
  • 1x B27 supplement without vitamin A
  • 1x N2 supplement
  • 1% pen/strep
  • 10ng/mL TGF alpha
  • 1 uM A-83-01
  • 1uM DMH-1
Dissect olfactory mucosa from nasal septum TemperatureOn ice .
Induce anesthesia via intraperitoneal injection of 0.15 mL euthanyl, followed by decapitation. Peel off skin and excise the eyes and the jaw. Disinfect the skull with 70% ethanol and carefully bisect along the sagittal plane, maintaining a 1 mm distance from the interfrontal suture. Transfer the larger segment of the skull under a dissection microscope, dissect a 1 mm distal section from the nasal bone. Carefully remove the nasal bones and collect the olfactory epithelium surrounding the septal cartilage into Hank's Balanced Salt Solution.


Mince the tissue in Amount2.7 mL dissection medium, add Amount300 µL 10x Collagenase/Hyaluronidase and transfer to a 15 mL falcon.

Briefly vortex falcon and rotate at Temperature37 °C for 1h.

Spin down at Centrifigation80 x g for 30s and discard supernatant.

Triturate pellet in Amount5 mL TrypLE for 2 min and add Amount10 mL cold HBSS.

Spin down at Centrifigation350 x g for 5 min and resuspend cells in 1 mL warmed dispase and 50 uL DNase 1. Triturate for 2 min.

Add Amount10 mL cold HBSS and filter through 40 um cell strainer.

Spin down at Centrifigation350 x g for 5 min, discard supernatant, resuspend in HBC medium with Concentration10 micromolar (µM) ROCK inhibitor Y27632.

Plate on PDL and laminin coated wells, one mouse septum on 1.9 cm2 (1 well in a 24-well plate)
10. Refresh medium the next day without ROCK inhibitor.
11. Feed every 2 days. Passage with accutase and do not use any other cell dissociation products such as TryplE of trypsin. Cultures are homogenously CK5+ after 3 passages.