title: Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design.
abstract: BACKGROUND
  Serum-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.
  
  
  RESULTS
  From 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Biological Sciences and Graduate School of Nanoscience & Technology WCU, KAIST, 335 Gwahangno, Yuseong-gu, Daejon 305-701, Korea.
authors.forename: Min Kyoung
authors.initials: MK
authors.lastname: Jeon
authors.forename: Jong-Baeck
authors.initials: JB
authors.lastname: Lim
authors.forename: Gyun Min
authors.initials: GM
authors.lastname: Lee
mesh_terms: D018929:Cell Culture Techniques; D016895:Culture Media, Serum-Free / Q000737:chemistry*; D005163:Factor Analysis, Statistical; D006801:Humans; D012984:Software; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology*
chemical_list: D016895:Culture Media, Serum-Free