Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design

BMC Biotechnology

Research Article

Gyun Min Lee¹  Min Kyoung Jeon¹  Jong-Baeck Lim² 

[1] Department of Biological Sciences and Graduate School of Nanoscience & Technology (WCU), KAIST, 335 Gwahangno, Yuseong-gu, 305-701, Daejon, Korea;Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea;

关键词: Response Surface Method;    Fractional Factorial Design;    ELISpot Assay;    Antioxidant Supplement;    Normal Probability Plot;   

DOI  :  10.1186/1472-6750-10-70

 received in 2009-12-08, accepted in 2010-09-21,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundSerum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis.ResultsFrom a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM.ConclusionsThe use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

【 授权许可】

Unknown   
© Jeon et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.