Journal:

Article Title: A mesenchyme-free culture system to elucidate the mechanism of otic vesicle morphogenesis

doi: 10.1111/j.0021-8782.2004.00335.x

Figure Lengend Snippet: Culture method. Hindbrain regions of E10.5 or 11.5 mouse embryos are isolated using microscissors, and incubated in 1 U mL−1 dispase. Otic vesicles are isolated using fine tungsten needles and embedded in a drop of Matrigel. The drop is allowed to gel at 37 °C in a humidified atmosphere for 30 min, then culture medium is added to the dish and culture maintained for up to 72 h.

Article Snippet: Then the otic vesicles were separated from surrounding mesenchyme with sharp tungsten needles and placed in a 50-µL drop of Matrigel (BD Biosciences) or Type I collagen (Cellmatrix I-A, Nitta Gelatin Inc.) in a tissue culture dish (Falcon) using a micropipette; their position and orientation were modified using tungsten needles before the gel solidified.

Techniques: Isolation, Incubation

Journal:

Article Title: A mesenchyme-free culture system to elucidate the mechanism of otic vesicle morphogenesis

doi: 10.1111/j.0021-8782.2004.00335.x

Figure Lengend Snippet: Otic vesicle has the ability to undergo morphogenesis without mesenchyme. (a–d) E10.5 otic vesicle cultured in Matrigel for 72 h. A limited amount of morphogenesis has occurred, and cells have emigrated from the anterior part of the otic vesicle (b, black arrowhead). (e–h) E11.5 otic vesicle cultured in Matrigel for 72 h. Neuroblasts emigrated from the anterior part of the otic vesicle within 24 h (f, black arrowhead); the semicircular canal region formed a complex epithelial structure (h, black arrow) and the cochlear duct underwent looping (f, white arrowhead). An ectopic epithelial diverticulum sometimes emerged from the cochlear region (h, white arrow). (i, j) E11.5 isolated otic vesicle cultured in type I collagen gel for 48 h. The tissue appears healthy but has not undergone morphogenesis, and neuroblast emigration is not localized. (k, l) E11.5 isolated otic vesicle cultured in serum-free medium for 48 h. There was no morphogenesis, and some loss of epithelial structure. (m–p) E11.5 otic vesicle cultured with a small amount of attached mesenchyme. The mesenchymal cells soon underwent massive proliferation and surrounded the whole otic vesicle. Their appearance is quite distinct from the emigrating neuroblasts observed in (b) and (f). Scale bars = 100 µm.

Article Snippet: Then the otic vesicles were separated from surrounding mesenchyme with sharp tungsten needles and placed in a 50-µL drop of Matrigel (BD Biosciences) or Type I collagen (Cellmatrix I-A, Nitta Gelatin Inc.) in a tissue culture dish (Falcon) using a micropipette; their position and orientation were modified using tungsten needles before the gel solidified.

Techniques: Cell Culture, Isolation