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Received November 4, 2004
Accepted January 13, 2005
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토끼 구강점막 상피세포 성장에 미치는 환경인자의 영향
Effect of Environmental Factors on the Growth of Rabbit Oral Keratinocytes
동국대학교 공과대학 생명·화학공학과, 100-715 서울시 중구 필동 3가 26 1연세대학교 공과대학 화공·생명공학부, 120-749 서울시 서대문구 신촌동 134
Department of Chemical and Biochemical Engineering, Dongguk University, 26 3-ga, Pil-dong, Chung-gu, Seoul 100-715, Korea 1School of Chemical Engineering and Biotechnology, Yonsei University, 134, Sinchon-dong, Seodaemun-gu, Seoul 120-749, Korea
jkpark@dongguk.edu
Korean Chemical Engineering Research, February 2005, 43(1), 103-109(7), NONE Epub 4 March 2005
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Abstract
토끼 구강점막 상피세포의 분리 및 일차배양 방법, 세포성장에 미치는 환경인자의 영향에 대한 연구를 T75-플라스크를 사용하여 수행하였다. 토끼의 구강점막조직을 채취(biopsy)한 후 트립신(trypsin)효소처리방법을 이용하여 0.25 cm2 점막 조직으로부터 1.92±0.59×10 6개의점막 상피세포를 회수할 수 있었다. 회수한 점막 상피세포를 50 mg/L BPE(bovine pituitary extract), 5.0 μg/L EGF(human recombinant epidermal growth factor), 0.15 mM Ca2+을 함유한 K-SFM(keratinocyte serumfree medium)을 10 mL씩 사용하여 일차 배양한 결과 8일 만에 배양용기표면에 세포가 포화(confluent)하게 성장하였고 배가시간은 2.45일이었다. 일차 배양한 세포를 회수한 후 배지종류, 배지부피, 첨가물 종류가 상피세포성장에 미치는 영향을 조사하였다. 혈청첨가배지는 세포성장에 부정적인 효과를 나타냈고, 혈청농도가 증가함에 따라 세포성장은 큰 변화가 없었다. 배지부피가 증가함에 따라 세포성장은 감소하였고, 칼슘농도가 증가할수록 세포성장은 증가하였으며 2.0 mM에서 최적치를 나타내었다. 이상으로 토끼 구강점막 상피세포를 T75-플라스크를 사용하여 배양하는 경우 50 mg/L BPE, 5.0 μg/L EGF, 2.0 mM Ca2+을 함유한 K-SFM을 10 mL씩 사용하는 조건이 가장 적합하였고 배가시간은 1.32일이었다. 이러한 연구결과는 향후 점막뿐만 아니라 피부, 각막 등 인체에 존재하는 상피세포배양을 위한 공정개발이나 생물반응기 설계에 유용한 정보를 제공할 것으로 사료된다.
Isolation and primary culture technique of rabbit oral keratinocytes, and the study for effect of environmental factors on the cell growth were carried out in T75-flask. 1.92±0.59×106 viable cells were isolated by trypsin enzymatic digestion method from 0.25 cm2 biopsy of rabbit oral mucosa. Primary culture with 10 mL of K-SFM containing 50 mg/L BPE, 5.0 μg/L EGF and 0.15 mM Ca2+ showed confluence after 8 days and doubling time was 2.54 days. Effect of medium types, medium volume and supplement types on the cell growth was investigated after the cultured keratinocytes had been harvested from primary confluence. Serum addition showed adverse effect and the increase of serum concentration didn’t have an effect on the cell growth. The increase of medium volume decreased the cell growth. The increase of calcium concentration increased the cell growth and 2.0 mM was optimum value. In conclusion, when rabbit oral keratinocytes was cultured in T75-flask, the most effective conditions was to use 10 mL of K-SFM containing 50 mg/L BPE, 5.0 μg/L EGF and 2.0 mM Ca2+, and doubling time was 1.32 days. This study can provide the useful informations to develop a process and design a bioreactor for the culture of keratinocytes in human body like skin and cornea, as well as mucosa.
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Ueda M, Ebata K, Kaneda T, Ann. Plast. Surg., 27(6), 540 (1991)
Chase DC, Gattozzi JG, Smith K, Oral. Surg., 45, 516 (1978)
Fry TL, Woods CI, Arch. Otolaryngol. Head Neck Surg., 113, 770 (1987)
Hata K, Kagami H, Ueda M, Torii S, Matsuyama M, Ann. Plast. Surg., 34, 530 (1995)
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Liu SC, Karasek M, J. Invest. Dermatol., 71, 157 (1978)
Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH, Cell, 19, 245 (1980)
Tsao MC, Walthall BJ, Ham RG, J. Cell. Physiol., 110, 219 (1982)
Hawley-Nelson P, Sullivan JE, Kung M, Hennings H, Yuspa SH, J. Invest. Dermatol., 75, 176 (1980)
Daniels JT, Kearney JN, Ingham E, Burns, 22, 35 (1996)
Wang HJ, Chen TM, Cheng LF, Cheng TY, Tung YM, Burns, 21(7), 503 (1995)
Kamata N, Yokoyama K, Fujimoto R, Ueda U, Hayashi E, Nakanishi H, Nagayama M, In Vitro Cell. Dev. Biol. -Anim., 35, 635 (1999)
Cook PW, Pittelkow MR, Shipley GD, J. Cell. Physiol., 146, 277 (1991)
Piepkorn M, Lo C, Plowman G, J. Cell. Physiol., 159, 114 (1994)
Pittelkow MR, Cook PW, Shipley GD, Derynck R, Coffey RJ, Cell Growth Differ, 4, 513 (1993)
Dulgosz AA, Cheng C, Dening MF, Dempsey PJ, Coffey RJ, Yuspa SH, Cell Growth Differ, 5, 1283 (1994)
Ristow HJ, Growth Regul, 6, 96 (1996)
Vardy DA, Kari C, Lazarus GS, Jensen PJ, Zilberstein A, Plowman GD, Rodeck U, J. Cell. Physiol., 163, 257 (1995)
Marsh BCR, Massaro-Giordano M, Marshall CM, Lavker RM, Jensen PJ, Exp. Eye Res., 74, 61 (2002)
Wheelock MJ, Jensen PJ, J. Cell Biol., 117, 415 (1992)
O'Keefe EJ, Briggaman RA, Herman B, J. Cell Biol., 105, 807 (1987)
Boyce ST, Ham RG, J. Invest. Dermatol., 81, 33s (1983)
