ISSN 2456-0235

INDEXED IN 

International Journal of Modern Science and Technology

​​​​​​​August 2019, Vol. 4, No. 8, pp 212-216. 

​​Optimization of Protocol for Maintaining and Freezing Caco-2 Cells

Rasika Pawar¹, Siddhivinayak Barve², Minal Joshi², Pranjali Dhawal², Bela Nabar¹*
¹Department of Microbiology, Smt. Chandibai Himathmal Mansukhani College, Ulhasnagar, India.
²Scientific Research Center, Vinayak Ganesh Vaze College, Mulund, Mumbai, India.

​​*Corresponding author’s e-mail: belanabar23@gmail.com

Abstract

Human Caco-2 cells are used as model of intestine for absorption studies. However standardization of growth conditions is challenging for these cells as it requires a long growth period of around twenty one days for most absorption associated studies. In this study we have standardized various growth associated parameters and procedures related to Caco-2 cell cultures. The Caco-2 protocols are recently developed in our laboratory include, the time required for trypsinization of cell  in correlation with the age of subculture, freezing mixture type, quantity of DMSO and seeding density of cells while freezing. The cells are also seeded at low density as compared to present in literature which gave better results for a 21 day old differentiated flask. Since the Caco-2 cell line is integral part of absorption and in vitro toxicology tests, standardized protocol inclusive of all the detailed procedures for growing and differentiating Caco-2 cells would help researchers in maintaining and preserving the cells.

Keywords: Caco-2; Trypsinization; Seed density; Freezing; Thawing.

References

  1. Awortwe C, Fasinu PS, Rosenkranz B. Application of Caco-2 cell line in herb-drug interaction studies: current approaches and challenges. Journal of Pharmacy and Pharmaceutical Sciences 2014;17(1):1-19.
  2. Yamashita S, Furubayashi T, Kataoka M, Sakane T, Sezaki H, Tokuda H. Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells. European Journal of Pharmaceutical Sciences 2000;10(3):195-204.
  3. Gan LS, Thakker DR. Applications of the Caco-2 model in the design and development of orally active drugs: elucidation of biochemical and physical barriers posed by the intestinal epithelium. Advanced Drug Delivery Reviews 1997;23(1-3):77-98.
  4. Artursson P, Palm K, Luthman K. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Advanced Drug Delivery Reviews 2001;46(1-3):27-43..
  5. Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biology and Toxicology 2005;21(1):1-26..
  6. Artursson P, Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochemical and Biophysical Research Communications 1991;175(3):880-5..
  7. Zucco F, Batto AF, Bises G, Chambaz J, Chiusolo A, Consalvo R, Cross H, Dal Negro G, de Angelis I, Fabre G, Guillou F. An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines. Alternatives to Laboratory Animals 2005;33(6):603-18.
  8. Briske-Anderson MJ, Finley JW, Newman SM. The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells. Proceedings of the Society for Experimental Biology and Medicine. 1997;214(3):248-57.
  9. Natoli M, Leoni BD, D’Agnano I, Zucco F, Felsani A. Good Caco-2 cell culture practices. Toxicology in vitro. 2012;26(8):1243-6.
  10. Chantret I, Barbat A, Dussaulx E, Brattain MG, Zweibaum A. Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Research 1988;48(7):1936-42.
  11. Yamashita S, Konishi K, Yamazaki Y, Taki Y, Sakane T, Sezaki H, Furuyama Y. New and better protocols for a short-term Caco-2 cell culture system. Journal of Pharmaceutical Sciences 20021;91(3):669-79.
  12. Lea T. Caco-2 cell line. The impact of food bioactives on health. Springer, Cham, 2015.103-111
  13. Fogh J, Fogh JM, Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. Journal of the National Cancer Institute 1977;59(1):221-6.
  14. Güll I, Marques Alves P, Gabor F, Wirth M. Viability of the Human Adenocarcinoma Cell Line Caco-2: Influence of Cryoprotectant, Freezing Rate, and Storage Temperature. Scientia Pharmaceutica 2009;77(1):133-42.