Middle ear mucosa-like cell sheet, process of producing the same and method of using the same
a cell sheet and middle ear technology, applied in the field of middle ear mucosa-like cell sheet, can solve the problems of largely losing the function of cilia, unable to preserve the mucosa in the mastoid antrum, and delay in the conversion of the mucosa to epithelium after surgery
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example 1
[0068]A tissue fragment of rabbit nasal mucosa. collected under the germfree condition was ex-planted onto a culture dish coated with type 1 collagen, and a primary culture was done with a KCM medium (Keratinocyte culture medium). Consequently, the nasal mucous epithelial cells crawled out of the surrounding area of the tissue fragment, and increased rapidly. Several days after the culture, the interfusion of the cells displaying the fibroblast cytoid form in part was observed. After culturing up to about 80% confluent, the nasal mucosa epithelial cells were recovered from the culture dish, and disseminated at a cell density of 5×104 cells / cm onto the temperature-responsive culture dish (poly (N-isopropylacrylamide), coat volume 2.0 μg / cm2) coated with collagen for tissue (CELLGEN). At that time, the temperature-responsive culture dish was obtained by applying 0.35 ml of N-isopropylacrylamide monomer dissolved in isopropyl alcohol at 35% onto the Falcon 3002 petri dish (6 cm diamete...
example 2
[0072]By the same method as Example 1, the section of inferior turbinate mucosa tissue in human nasal cavity (FIG. 12 upper left photograph) collected under the germfree condition was explanted on the culture dish coated with type I collagen, and a primary culture was done with KCM medium (Keratinocyte culture medium) (FIG. 12 lower left photograph). As illustrated in FIG. 10, the human nasal mucosa cell was disseminated on the cell insert whose porous membrane portion was coated with 1.5 μg / cm2 volume of poly (N-isopropylacrylamide) by the same method as Example 1, and the obtained nasal mucosa cell was cultivated with the culture surface touched to air. After the culture for six days, it was transferred to the CO2 incubator at 20° C., and recovered successfully as the human nasal mucosa cell sheet by performing low temperature treatment for 20 minutes (FIG. 12 lower right photograph). The process of operation is illustrated in FIG. 11. Also, the situation of each process was illus...
example 3
[0074]The portion of nasal mucosa epithelial tissue was detached from inferior turbinate mucosa tissue in human nasal cavity which was collected under the germfree condition, the nasal mucosa epithelial tissue was explanted on commercially-supplied culture dish, and the primary culture was done with KCM medium. The obtained nasal mucosa epithelial tissue is illustrated in FIG. 17, and the rest detached the nasal mucosa epithelial tissue is illustrated in FIG. 18. The human nasal mucosa cell was disseminated on the cell insert whose porous membrane portion was coated with 1.2 μg / cm2 volume of poly (N-isopropylacrylamide) by the same method as Example 1, and cultivated with the culture surface touched to air. At that time, the KCM medium containing human serum 10% was utilized as a medium. After the culture for seven days, it was transferred to the CO2 incubator at 20° C., and recovered successfully as the human nasal mucosa cell sheet by performing low temperature treatment for 20 mi...
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Abstract
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