Mouse cochlea spiral device adherent culture method
An adherent culture and spiralizer technology, which is applied in the field of cell culture, can solve the problem that TSP-1 is not disclosed or implied, and achieve the effect of good cell adherent culture effect, easy operation and good adherence effect.
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Embodiment 1
[0040] Example 1 Isolation of mouse cochlear spiral organ
[0041] C57BL / 6J mice born 3-5 days after birth were selected as experimental materials. After the neck was cut, the skin of the auricle was cut, the auditory bulb was removed along the external auditory canal, and the mouse cochlea with vestibular base was removed.
[0042] Under a dissecting microscope, the posterior side of the cochlea roof and the thin layer of apical bone wall were removed, and the cochlea after partial bone wall removal was obtained, and the spiral ligament of the apical vascular striae was clearly exposed. The surface bone wall of the cochlea is peeled off from the top to the bottom of the cochlea to remove the peripheral bony cochlear labyrinth, remove the spiral ligament, vascular striae, covering membrane and other surrounding tissues, remove the spiral ganglion of the cochlear modiole, and along the cochlear top Spiral down, segmentally separate the basement membrane containing the spiral, a...
Embodiment 2
[0043] Example 2 Screening of substrates suitable for basement membrane attachment
[0044] The mouse cochlear basement membrane was cultured to obtain cell spheres, and the cells of the cell spheres were attached to different substrates to observe the effect of the substrate on the adhesion and growth of the cell spheres.
[0045] Reagents: DMEM / F12, N2, and B27 additives were purchased from Gibco, USA. Rat tail glue was purchased from BD Company. Polylysine, polyornithine and bovine fibronectin were purchased from Sigma, USA.
[0046] Substrate coating and grouping: In order to observe the effect of different substrates on the adhesion of cell spheres, the coverslips are first coated with different substrates. The method of coating the coverslips is: autoclave the cleaned coverslips After disinfection, put it in a 35mm petri dish in an ultra-clean bench, drop different substrates on it, and then evenly spread it on the surface of the cover glass, let it dry naturally at ro...
Embodiment 3
[0054] Example 3 Validation of substrate for adherent culture of mouse cochlear spiral organ.
[0055] Coated Petri dish:
[0056] Experimental group 1: Rat tail glue was diluted 50 times with 0.02mol / L acetic acid, and an appropriate amount was added to a 35mm petri dish to make it submerge the entire petri dish, and then placed in the incubator for about 1 hour, and the petri dish was blotted dry. Rat tail glue was rinsed with PBS, dried naturally, and set aside.
[0057] Experimental group 3: Rat tail gum was diluted 50 times with 0.02mol / L acetic acid to obtain a dilution, mixed with 100μg / ml polyornithine in an equal volume, mixed evenly, and an appropriate amount was added to a 35mm petri dish to submerge the entire After the petri dish was placed in the incubator for about 1 hour, the mixed solution of rat tail gum and polyornithine in the petri dish was blotted dry, rinsed with PBS, dried naturally, and set aside.
[0058] Serum-free medium configuration: DMEM / F12 me...
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