FIG. 1.

Basal cell culture medium formulations profoundly influence T-cell maturation in vitro. Top Panels: Cellular expansion of lymphocyte progenitor cells cultured in T-cell (OP9-DL1) or B-cell (OP9) conditions in the indicated basal culture medium. All cultures were supplemented with 1 ng/ml interleukin (IL)-7 and 5 ng/ml fms-like tyrosine kinase-3 ligand (Flt3L). Lower panel: Effects of basal medium formulations on T-lymphocyte progenitor expansion and differentiation in OP9-DL1 cocultures as shown in the top left panel. The alpha modification of minimal essential medium (αMEM)+N medium is a formulation containing nucleosides, while the αMEM−N formulation lacks nucleosides. All cultures were initiated by seeding 2×10³ fluorescence-activated cell-sorting (FACS)-sorted LSK-Thy-1.1-neg cells onto a feeder layer of stromal cells and contained 1 ng/ml IL-7 and 5 ng/ml Flt3L with otherwise identical additives as outlined in the Materials and Methods section. Cell numbers were determined by hemocytometer counting at each passage. Cultures were passaged every 3–4 days, and appropriate dilutions were made to maintain a cell density of <5×10⁵ cells/ml. The cell counts were corrected by a factor corresponding to the product of these dilutions. The percentage of CD4+CD8+ DP cells was determined by FACS analysis after 14 days of culture and was converted to absolute numbers based on the cell counts.