Three-dimensional culture of immortalized skin cells on the superficial layer of which a stratum corneum is formed, method for producing said three-dimensional culture, and method for evaluating test substance using said three-dimensional culture
Provided is a three-dimensional culture of immortalized skin cells, comprising a plate-like substrate and a culture layered product of immortalized skin cells adhering to the surface of the plate-like substrate, in which the superficial layer of the culture layered product is composed of a stratum corneum. The three-dimensional culture can be produced as follows: immortalized skin cells are seeded on a top surface of a culture medium-impregnated plate-like substrate, and three-dimensional culture is performed while applying stress on a superficial layer of an immortalized skin cell layer formed by layering of the immortalized skin cells. The three-dimensional culture can be used to evaluate the sensitization of a test substance.
1 . A method for producing a three-dimensional culture of HaCaT cells with stratum corneum formed on a superficial layer thereof, the method comprising:
performing three-dimensional culture of HaCaT cells including a gene of first luciferase controlled by a stable expression promoter and a gene of second luciferase controlled by a promoter that is capable of evaluating skin sensitization, with enough culture medium not to dry out a superficial layer of a HaCaT cell layer formed by layering of the HaCaT cells, signals from the first luciferase and the second luciferase being detected distinguishably from each other.
2 . The method for producing according to claim 1 , further comprising:
seeding the HaCaT cells on a top surface of a plate-like substrate, wherein
the three-dimensional culture of the HaCaT cells is performed with enough culture medium not to dry out the superficial layer of the HaCaT cell layer.
3 . The method for producing according to claim 2 , further comprising:
storing an inner container in an outer container, the inner container including the plate-like substrate on a bottom thereof, wherein the three-dimensional culture of the HaCaT cells is performed with enough culture medium not to dry out the superficial layer of the HaCaT cell layer being charged in the outer container.
4 . The method for producing according to claim 1 , wherein the HaCaT cells further include a gene of third luciferase controlled by a promoter that is capable of evaluating physiological activity, a signal from the third luciferase being detected distinguishably from either of the signals from the first luciferase and the second luciferase.