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eISSN: 1643-3750

Impact of cell culture media on the expansion efficiency and T-cell receptor Vbeta (TRBV) repertoire of in vitro expanded T cells using feeder cells

Andrea Block, Maria Rohde, Ulrike Erben, Markus Hammer, Michael Hummel, Katja Blunert, Heinz-Peter Schultheiss, Hans-Dieter Volk, Michel Noutsias

Med Sci Monit 2008; 14(5): BR88-95

ID: 855745

Available online: 2008-04-29

Published: 2008-04-29

Background: The effects of different cell culture media on expansion efficiency and alterations in T-cell receptor V beta (TRBV) expression of in vitro expanded lymphocytes are not well established.
Material and Method: Low numbers of CD3+ T cells from peripheral blood lymphocytes of healthy donors were subjected to polyclonal in vitro expansion in the presence of autologous CD3-depleted mononuclear cells as feeder cells (FCs) and their numbers and TRBV expressions were compared in media containing human (HS-RPMI) or fetal bovine serum (FBS-RPMI), Panserin413, or X-Vivo 15TM designed for lymphocyte culture.
Results: During three courses of restimulation within 28 days with CD3-antibody (OKT-3), IL-2, and initial CD3+, T-cell: FC ratios of 1:50 lowered to 1:5 and T cells expanded more than 1,000-fold in the media containing complete sera. Loss of cluster formation, associated with expansion failure, was only observed in cultures using synthetic media and resulted in only about 70-fold expansion. Whereas TRVB expression as determined by real-time PCR was substantially altered after 14 days of culture in X-Vivo 15, at day 28 only T cells from long-term culture in HS-RPMI presented the initial TRBV composition.
Conclusions: Culture media have substantial impact on in vitro T-cell expansion. In the presence of FCs, medium containing human serum is superior to synthetic media and FBS-RPMI for long-term culture regarding T-cell number and TRBV repertoire. In contrast, the synthetic media Panserin413 and XVivo15 show lower expansion efficiency and reproducibility and, as RPMI1640+10%FBS, can contribute to overstimulation of certain TRBVs at advanced culture time points.

Keywords: T-Lymphocytes - physiology, Reverse Transcriptase Polymerase Chain Reaction, Receptors, Antigen, T-Cell, alpha-beta - metabolism, Monocytes - cytology, Macrophages - cytology, Lymphocyte Activation, Immunohistochemistry - methods, DNA Primers - chemistry, Culture Media, Serum-Free - pharmacology, Cells, Cultured, Cattle, Cell Culture Techniques - methods, Antigens, CD3 - biosynthesis, Animals