Phototoxicity test method
A test method and phototoxicity technology, applied in the field of phototoxicity test, to achieve the effect of easy search
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Embodiment 1
[0130] Example 1 (Induction of differentiation of human ES cells into human retinal pigment epithelial cells)
[0131] As human embryonic stem cells (ie, ES cells), a KhES-1 strain established by Kyoto University was used.
[0132] The above-mentioned KhES-1 strain was cultured in ES cell maintenance culture medium (containing DMEM / F12, KSR (Knockout serum replacement), non-essential amino acids, L -Glutamine, 2-mercaptoethanol, etc.) on mouse embryonic fibroblast (MEF) feeder cells treated with mitomycin C to maintain the undifferentiated state of the KhES-1 strain . After culturing, colonies of human embryonic stem cells (ie, ES cells) were obtained by recovering from the culture dish using a dissociation solution containing trypsin / collagenase IV.
[0133] Use a micropipette to suck up the obtained colony, crush it into a size of about tens of cells per cell mass, and add 1 μM to 10 μM SB431542, 1 μM Colonies of human embryonic stem cells (i.e., ES cells) with hundreds t...
Embodiment 2
[0137] Example 2 (cultivation of human retinal pigment epithelial cells)
[0138] The colony containing human retinal pigment epithelial cells with melanin induced by differentiation in Example 1 was aspirated with a micropipette, and then treated with 0.05% trypsin to disperse the cells.
[0139] Into a 60mm Petri dish coated with Matrigel in advance, add a medium made by adding 2-20ng / ml bFGF to the DMEM / F12 culture solution containing N2 supplement, and inoculate the obtained dispersed cells on this medium . The dispersed cells seeded in this way also contained unspecified cells other than human retinal pigment epithelial cells.
[0140] Incubate the cells at 37°C, 5% CO 2 After cultured and fused in an incubator, the cells were treated with 0.25% trypsin, and the cells were recovered from the culture dish. The recovered cells were seeded into 10 cm Petri dishes pre-coated with Matrigel, and then, at 37 °C, 5% CO 2 Cultured in an incubator until the cells were confluent...
Embodiment 3
[0142] Example 3 (The first step, the second step and the third step of the phototoxicity test method of the present invention under light-shielding conditions as a control for non-light irradiation)
[0143] The human retinal pigment epithelial cells prepared in Example 2 were treated with 0.25% trypsin, and the cells were recovered from the culture dish. Seed the recovered cells at 100,000 or 120,000 cells per well in a 96-well culture dish pre-coated with Matrigel, and then incubate at 37°C, 5% CO 2 Cultured in an incubator.
[0144] After culturing for 2 to 4 hours, use a pipette to remove the medium from the 96-well culture dish, and then add Earle’s Balanced Salt Buffer Solution (EBSS) to the above-mentioned 96-well culture dish at a rate of 150 μl per well. Wash the cells.
[0145] Next, a test substance solution or a solvent control solution previously adjusted to the following concentration using EBSS was added to the above-mentioned petri dish at a rate of 100 μl p...
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