Ex vivo T lymphocyte expansion for retroviral transduction: influence of serum-free media on variations in cell expansion rates and lymphocyte subset distribution.
OBJECTIVE
In the setting of allogeneic stem cell transplantation, suicide gene-manipulated donor T cells that can be selectively inactivated in vivo would potentially allow optimal control of the GVL (graft-vs-leukemia)/GVHD (graft-vs-host disease) balance. Retroviral T-cell transduction requires ex vivo cell expansion, which is often achieved by IL-2 and anti-CD3 stimulation. Traditionally, culture media for cell expansion are supplemented with fetal bovine serum (FBS) or human serum. While these sera promote cell growth and viability, they contain uncharacterized elements that may yield inconsistent results from batch to batch. Cell expansion in serum-free media would therefore be preferable.


METHODS
We compared T-cell expansion rates in three commercially available serum-free culture media (X-VIVO 15, AIM-V, and Cellgro SCGM), with or without the addition of human serum (HS, 5%). We also aimed to evaluate how the in vitro expansion affected the composition of the various T-cell subsets. Buffy-coats from four healthy donors were expanded for 21 days. The media were compared to standard RPMI 1640 medium, supplemented with HS (5%) or FBS (10%). For retroviral transductions, the LN vector carrying the neomycin- resistance gene was used in four additional donors.


RESULTS
In our hands, X-VIVO 15 gave the highest rate of serum-free expansion (a median of 79-fold expansion, range 20-117). For serum-free expansion, activation with OKT3 for 21 days gave slightly higher expansion rates than a 5-day course (however, without statistical significance). When serum was added, this discrepancy was not seen. Cytokine analysis (IFN-gamma, IL-10, and IL-4) showed a distinct type1 cytokine pattern with elevated IFN-gamma levels during the whole period of culture. Flow cytometric analyses showed substantial inter-media, but also some inter-donor, variability in T-cell subset compositions. Transduction of cells with the LN vector and G418 selection resulted in a 14-fold increase (range 3-18) for serum-free X-VIVO 15 based cultures. Cell phenotypes remained unchanged by the transduction procedure as compared to nontransduced cells.


CONCLUSIONS
Among the tested serum-free media, X-VIVO 15 has shown to best support the in vitro expansion of T cells, resulting in equal percentages of CD4(+) and CD8(+) cells. These cells can easily be transduced and selected. There seem to be no significant benefits, regarding absolute cell numbers or T-cell subset compositions, with OKT3-stimulation for more than five days. The addition of low levels of HS increases the consistencies in the cell expansion rates for all media.
Carlens
S
S
Centre for Allogeneic Stem Cell Transplantation, Huddinge University Hospital, and Departments of, Huddinge, Sweden. stefan.carlens@impi.ki.se
Gilljam
M
M
Remberger
M
M
Aschan
J
J
Christensson
B
B
Dilber
M S
MS
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes; D019002:CD56 Antigen / Q000032:analysis; D018414:CD8-Positive T-Lymphocytes; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D020544:Graft vs Leukemia Effect; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis; D016753:Interleukin-10 / Q000032:analysis; D015847:Interleukin-4 / Q000032:analysis; D017493:Leukocyte Common Antigens / Q000032:analysis; D008213:Lymphocyte Activation; D016131:Lymphocyte Subsets; D051379:Mice; D015375:Receptors, Interleukin-2 / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology*; D013997:Time Factors; D014162:Transfection
D000906:Antibodies; D017252:CD3 Complex; D019002:CD56 Antigen; D016895:Culture Media, Serum-Free; D006684:HLA-DR Antigens; D015375:Receptors, Interleukin-2; D016753:Interleukin-10; D015847:Interleukin-4; D007371:Interferon-gamma; D017493:Leukocyte Common Antigens