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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.
Subtype
Specialized Media, Supplements
Cell Type
Hematopoietic Stem and Progenitor Cells, T Cells
Species
Human
Application
Cell Culture, Differentiation, Expansion
Brand
StemSpan
Area of Interest
Cancer, Immunology, Stem Cell Biology, Cell Therapy Development
Figure 1. Frequency and Yield of CD7+CD5+ Pro-T Cells After 14 Days of Culture
CB-derived CD34+ cells (freshly isolated or frozen) were cultured for 14 days in StemSpan™ SFEM II containing Lymphoid Progenitor Expansion Supplement (Catalog #09915) on plates coated with Lymphoid Differentiation Coating Material (Catalog #09925). Cells were harvested and analyzed for CD7 and CD5 expression by (A) flow cytometry. The (B) average frequency of viable CD7+CD5+ pro-T cells on day 14 was 70%, with ~200 CD7+CD5+ cells produced per input CD34+ cell. Shown are means with 95% confidence intervals (n = 33).
Figure 2. Frequency and Yield of CD4 ISP and CD4+CD8+ DP Cells After 42 Days of Culture
CB-derived CD34+ cells (freshly isolated or frozen) were cultured with the StemSpan™ T Cell Generation Kit (Catalog #09940) for 42 days and (A) analysed by flow cytometry for the expression of CD4, CD8, CD3 and TCRαβ. The (B) frequency and (C) yield of CD4 ISP, double-positive (CD4+CD8+) and CD3+TCRαβ+-expressing double-positive cells (CD4+CD8+CD3+TCRαβ+) are shown. On average, 38% of the total viable population were DP (CD4+CD8+), of which 35% co-expressed CD3 and TCRαβ. The yields of total DP cells and CD3+TCRαβ+ DP cells per input CD34+ cell were ~23,000 and ~9,000, respectively. Shown are means with 95% confidence intervals (n = 31).
Figure 3. Frequency and Yield of CD8 SP T Cells After 49 Days of Culture
DP cells were further matured into CD8 SP T cells by culturing for an additional 7 days in StemSpan™ SFEM II with T Cell Progenitor Maturation Supplement (Catalog #09930), IL-15 (Catalog #78031) and ImmunoCult™ CD3/CD28/CD2 T Cell Activator (Catalog #10970) on coated plates. On day 49, cells were (A) analyzed by flow cytometry for the expression of CD3, TCRαβ, CD4 and CD8. The (B) frequency and yield of CD3+TCRαβ+-expressing cells and their subsets are shown. On average, 54% of the CD3+TCRαβ+ cells were DP (CD4+CD8+) and 38% were CD8 SP (CD4-CD8+). The average yield of CD8 SP T cells per input CD34+ cell was ~6,000. CD3+TCRαβ+ CD4 SP (CD4+CD8-) T cells were detected at very low frequencies (data not shown). Shown are means with 95% confidence intervals (n = 12).
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PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE.
