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KMID : 1177720130070010029
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Journal of Alternatives to Animal Experiments
2013 Volume.7 No. 1 p.29 ~ p.34
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Development of skin sensitization alternative test through co-culture of THP-1 dendritic cell line and HaCaT keratinocyte cell line
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Yeo Kyeong-Uk
Cha Won-Seok
Yeon Dong-Eun
Choi Sung-Hee
Lee Ji-Youn
Shin Kyeong-Min
Jo Ji-Hoon
Heo Yong
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Abstract
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Co-culture system of THP-1 dendritic cell line and HaCaT keratinocyte cell line was developed for its usefulness in sorting out chemicals with skin allergic potential. THP-1 cells were plated onto wells of 24-well culture plate followed by placement of culture plate insert to each well. Thereafter HaCaT cells were added into the culture plate inserts. Levels of macrophage inflammatory protein-1β (MIP-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) production were determined using culture supernatants from HaCaT cell or THP-1 cell culture area. The optimal culture duration was 48 hour among 24, 48, and 72 hour for production of those cytokines. In addition, complete RPMI medium was better than complete DMEM medium. Co-culture of THP-1 and HaCaT cells was maintained in the presence or absence of 4 doses, 0.01X, 0.1X, 0.5X, or 1X CV75 (75% cell viability concentration) of 2 sensitizing chemicals (dinitrochlorobenzene, hexyl cinnamic aldehyde) and an irritant chemical (lactic acid) for 48 hour. Stimulation index (SI) was calculated through dividing each cytokine amount at each chemical concentration by vehicle control`s cytokine amount. When SI 3 is considered as a threshold for categorizing chemicals into skin-sensitizer, dinitrochlorobenzene and hexyl cinnamic aldehyde were positive for MIP-1β, but no chemicals were above SI 3 for both IL-6 and IL-8. Further evaluations for other various cytokines and cytokine determination using cell lysates should be performed for examining usefulness of the co-culture system as a skin sensitizing alternative method.
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KEYWORD
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skin sensitization, HaCaT keratinocyte cell, THP-1 dendritic cell, macrophage inflammatory protein-1β
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