Avian cochlea organ culture
The goal is to establish primary cultures of the avian sensory epithelium
Artificial perilymph: 1 mL pipette tips 1 mL pipetter 5 mg/mL collagen/chondroitin-6-sulfate solution transwell 6 well plates (30 mm, 3 um pores): Corning CoStar 1x HBSS 0.237 mg/mL Leupeptin 1.2 mg/mL AEBSF 0.5 mg/mL Thermolysin in HBSS 4 2-week old white leghorn chicks 70% ethanol sterile paper towels ultra-fine, fine, transfer, gross forceps 26 gauge syringe sterile 60 mm culture dishes
(1) coat transwell membrane with 200 uL collagen/GAG solution (t=0). store at room temperature (2) thaw Leupeptin, AEBSF and thermolysin (3) add 700 uL AEBSF and 700 uL Leupeptin to 5.4 mL HBSS in 15 mL tube. place in ice bath. (4) add 160 uL AEBSF and 160 uL Leupeptin to 1.5 mL thermolysin. place in 37C incubator. (5) retreive chickens from animal facility (6) spray primary culture hood with ethanol (7) place HBSS in 60 mm culture dish. place artificial perilymph in 10 mL tube. (7) anesthetize chickens, harvest heads. (8) place heads in 70% ethanol (9) remove head from ethanol, place on paper towel (10) remove basilar papillae (11) place basilar papillae in HBSS (12) remove tegmentum vasculusum (13) repeat for all basilar papillae (14) remove thermolysin from incubator, place in 60 mm culture dish (15) transfer basilar papillae to thermolysin (16) treat in thermolysin for 20 min, 37C (17) add 1 mL artificial perilymph to transwell membranes. withdraw artificial perilymph. add 1 ml again. (t=2h) (18) place 1 ear into each well of the transwell setup (17) using 26 gauge syringe, peel cells off of basement membrane (18) repeat step 17 for all ears (19) remove ears from each well, leaving just sensory epithelia (20) drain artificial perilymph from above the transwell membrane surface, bringing sensory epithelium to the surface. the surface should be slightly hydrated, and tissue should have white appearance. (21) wait for 3 min. (22) SLOWLY add 2 mL artificial perilymph to the transwell surface. (23) repeat steps 21-22 for all ears (24) place in incubator
Artificial Perlilymph should be made fresh once per month. Store collagen/GAG solution in refrigerator. Should be sonicated once per month.
1. Warchol ME (2002) Cell density and N-cadherin interactions regulate cell proliferation in the sensory epithlelia of the inner ear J Neurosci 22(7), 2607-2616. 2. Torchinsky C, Messana E, Arsura M, Cotanche DA (1999). Regulation of p27Kip1 during gentamicin mediated hair cell death. J Nuerocytol 28: 913-924.