Abstract: Objective To establish a method for the cultivation of keratinocytes in human dermal fibroblast (HDF) feeder layer. Methods The seeding efficacy, growth and confluence of normal human epidermal keratinocytes (NHEK) in mitomyci n C-treated HDF feeder layers from various passages of same HDF line were obse rved. Results ① The concentration of mitomycin C suitable for HDF feeder layer w as 10μg/mL.② The first to 20th passages of the same HDF line were preferable to establish different allogenic keratinocyte cultures. There was no correlation between the number of passages and periods needed for NHEK to reach 80% confluence (r=-0.27,P>0.05).③ HDF feeder layer could improve the efficient growth of primary keratinocyte culture plated at very low seeding densities (0.1×10⁴ cells/cm²). Conclusions Different allogenic keratinocytes can grow in HDF feeder layers from various passages of the same HDF line. HDF feeder layer is able to improve the seeding efficacy of human keratinocytes, which may be the reasonte culture plated at very low seeding densities.

Key words: Fibroblast, Cell culture, Keratinocytesy