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

Abstract

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.

A high-throughput template for optimizingDrosophilaorgan culture with response-surface methods

“…Additional efficiency in exploring the parameter space was achieved with multifactorial screens based on design-ofexperiments methodology, an approach frequently used in the optimization of processes (Jeon et al, 2010). Multifactorial screens can provide efficient identification of key medium components.…”

Chinese hamster ovary cell performance enhanced by a rational divide-and-conquer strategy for chemically defined medium development

“…The statistical analysis for the response surface design and quadratic mixture design were performed using Design-Expert as previously reported (19,21,24). Multivariate data analysis [principal component analysis (PCA) and partial least squares (PLS)] was performed using the SIMCA software (Unmetric AB).…”

Characteristics of the heme catabolic pathway in mild unconjugated hyperbilirubinemia and their associations with inflammation and disease prevention

“…Cell count per test tube was adjusted to 250.000. Cells were kept overnight in supplemented RPMI medium 69 . After washing, cells were fixed (1% formalin, 10 min, RT), washed again, and permeabilised (70% ice-cold ethanol, 10 min, on ice).…”