title: 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.
abstract: 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.
authors.affiliation: Department of Cardiology and Pneumonology, Charite-Universitatsmedizin Berlin, Benjamin Franklin Campus, Berlin, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Block
authors.forename: Maria
authors.initials: M
authors.lastname: Rohde
authors.forename: Ulrike
authors.initials: U
authors.lastname: Erben
authors.forename: Markus
authors.initials: M
authors.lastname: Hammer
authors.forename: Michael
authors.initials: M
authors.lastname: Hummel
authors.forename: Katja
authors.initials: K
authors.lastname: Blunert
authors.forename: Heinz-Peter
authors.initials: HP
authors.lastname: Schultheiss
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Volk
authors.forename: Michel
authors.initials: M
authors.lastname: Noutsias
mesh_terms: 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*
chemical_list: D017252:CD3 Complex; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D016693:Receptors, Antigen, T-Cell, alpha-beta