Use this two-step protocol to reproducibly expand thousands of CD4+CD8+ double-positive (DP) T cells in cultures initiated with CD34+ human cord blood (CB) cells.
In step one, CD34+ CB cells are cultured for 14 days in medium containing expansion supplement to promote their proliferation and differentiation into CD7+CD5+ pro-T cells. In step two, pro-T cells generated during those first 14 days are cultured for another 4 weeks in medium containing maturation supplement to promote their maturation into DP cells. This medium kit is serum- and feeder-free, eliminating variation introduced by serum and stromal cell lines.
For your convenience, all kit components are also available for purchase individually. StemSpan™ Lymphoid Progenitor Expansion Supplement (10X)  contains a combination of recombinant human cytokines and other additives formulated to selectively promote the expansion and differentiation of CD34+ cells isolated from human CB samples to CD7+CD5+ progenitor T (pro-T) cells. Additionally, StemSpan™ T Cell Progenitor Maturation Supplement (10X)  enables further maturation of pro-T cells to DP cells. It also supports the maturation of DP cells to CD8 single-positive (SP) T cells with additional stimuli. StemSpan™ Lymphoid Progenitor Expansion Supplement (10X) and StemSpan™ T Cell Progenitor Maturation Supplement (10X) are intended for use in combination with StemSpan™ SFEM II  medium, and on plates coated with StemSpan™ Lymphoid Differentiation Coating Material (100X) .
An optional protocol is available to further mature DP cells to CD8 SP T cells. This extended protocol uses a complete T cell progenitor maturation medium made with reagents included in the StemSpan™ T Cell Generation Kit and must be combined with ImmunoCult™ CD3/CD28/CD2 T Cell Activator  and IL-15 .