Method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells
A technology of bone marrow mesenchyme and inner ear hair cells, applied in artificial cell constructs, animal cells, vertebrate cells, etc.
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Embodiment 1
[0024] Example 1: Isolation of human bone marrow mesenchymal stem cells
[0025] The bone marrow of adult healthy volunteers was washed twice with a pH value of 7.4 phosphate buffered saline (PBS, purchased from Sangon Biotech Co., Ltd.), and the bone marrow was diluted to 10 with PBS. 7 cells / ml of cell suspension, gently added to the upper layer of human lymphocyte separation medium (Ficoll-Hypaque, D = 1.077 ± 0.002g / ml, purchased from Sangon Biotechnology), the volume ratio of the two was 3:2; room temperature (25 ℃), 2200rpm / min after centrifugation for 25 minutes, absorb the middle white cell layer (mononuclear cell layer); add the IMDM basal culture medium (purchased from Corning company), wash the cells, then centrifuge at 1000rpm / min for 6-8 minutes, pour off the culture medium, suspend the cells with the same culture medium, inoculate them in a culture bottle, and place at 37°C, 5% CO 2 Culture in an incubator, change the medium for the first time after 48 hours, di...
Embodiment 2
[0026] Example 2: Isolation of Chicken Embryo Utriculus Mesenchymal Cells
[0027] Utricle was isolated from fertilized 18-day chicken embryo (purchased from Hangzhou Tianyuan Breeding Co., Ltd.), digested with 0.25% trypsin at 37° C. for 15-20 minutes, and added with DMEM culture medium with a final volume concentration of 10% FBS (purchased from Corning Company) to stop, pass through a 200-mesh sieve, collect the cell suspension, centrifuge at 1000rpm / min for 6-8 minutes, suspend the cells with DMEM culture solution with a final volume concentration of 10% FBS, inoculate in a culture bottle, place at 37°C, 5%CO 2 Cultivate in an incubator, change the medium for the first time after 48 hours, discard non-adherent cells, and continue to culture adherent cells to 90% confluence, digest with 0.25% trypsin / EDTA and inoculate into cell culture flasks for culture, which are recorded as P1 generation cells, Through subculture in this way, chicken embryo utriculus mesenchymal cells ...
Embodiment 3
[0028] Example 3: Induction of human bone marrow mesenchymal stem cells in vitro differentiation of inner ear hair cells
[0029] Take the chick embryo utriculus mesenchymal cells grown to 90% confluence prepared in Example 2, soak in DMEM culture medium containing a final concentration of 2 μg / ml mitomycin C (purchased from Sigma Company), 37 ° C, 5% CO 2 After culturing in an incubator for 3 hours, the suspension was discarded and washed with PBS to obtain pretreated chicken embryo utriculus mesenchymal cells.
[0030]Take the P3-P5 generation human bone marrow MSCs obtained by the method in Example 1, and inoculate them into induction solution I, 37°C, 5% CO 2 Induced culture in the incubator for 7 days, in which the medium was changed every 2-3 days, and then changed to induction medium II, 37°C, 5% CO 2 The incubator continued to cultivate for 7-10 days, and the medium was changed every 2-3 days. After digestion with 0.25% trypsin EDTA, the 5 piece / cm 2 The density wa...
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