Cargando…

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

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)...

Descripción completa

Detalles Bibliográficos
Autores principales: Jeon, Min Kyoung, Lim, Jong-Baeck, Lee, Gyun Min
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2954940/
https://www.ncbi.nlm.nih.gov/pubmed/20854694
http://dx.doi.org/10.1186/1472-6750-10-70
_version_ 1782187981887504384
author Jeon, Min Kyoung
Lim, Jong-Baeck
Lee, Gyun Min
author_facet Jeon, Min Kyoung
Lim, Jong-Baeck
Lee, Gyun Min
author_sort Jeon, Min Kyoung
collection PubMed
description 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.
format Text
id pubmed-2954940
institution National Center for Biotechnology Information
language English
publishDate 2010
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-29549402010-10-15 Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design Jeon, Min Kyoung Lim, Jong-Baeck Lee, Gyun Min BMC Biotechnol Research Article 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. BioMed Central 2010-09-21 /pmc/articles/PMC2954940/ /pubmed/20854694 http://dx.doi.org/10.1186/1472-6750-10-70 Text en Copyright ©2010 Jeon et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 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.
spellingShingle Research Article
Jeon, Min Kyoung
Lim, Jong-Baeck
Lee, Gyun Min
Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design
title Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design
title_full Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design
title_fullStr Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design
title_full_unstemmed Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design
title_short Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design
title_sort development of a serum-free medium for in vitro expansion of human cytotoxic t lymphocytes using a statistical design
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2954940/
https://www.ncbi.nlm.nih.gov/pubmed/20854694
http://dx.doi.org/10.1186/1472-6750-10-70
work_keys_str_mv AT jeonminkyoung developmentofaserumfreemediumforinvitroexpansionofhumancytotoxictlymphocytesusingastatisticaldesign
AT limjongbaeck developmentofaserumfreemediumforinvitroexpansionofhumancytotoxictlymphocytesusingastatisticaldesign
AT leegyunmin developmentofaserumfreemediumforinvitroexpansionofhumancytotoxictlymphocytesusingastatisticaldesign