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.
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.


METHODS
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.
Block
Andrea
A
Department of Cardiology and Pneumonology, Charite-Universitatsmedizin Berlin, Benjamin Franklin Campus, Berlin, Germany.
Rohde
Maria
M
Erben
Ulrike
U
Hammer
Markus
M
Hummel
Michael
M
Blunert
Katja
K
Schultheiss
Heinz-Peter
HP
Volk
Hans-Dieter
HD
Noutsias
Michel
M
D000818:Animals; D017252:CD3 Complex / Q000096:biosynthesis; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008213:Lymphocyte Activation; D008264:Macrophages / Q000166:cytology; D009000:Monocytes / Q000166:cytology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000502:physiology*
D017252:CD3 Complex; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D016693:Receptors, Antigen, T-Cell, alpha-beta