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. Author manuscript; available in PMC: 2018 May 2.
Published in final edited form as: Cell Rep. 2018 Apr 17;23(3):741–755. doi: 10.1016/j.celrep.2018.03.084

Figure 1. Generation of a Chemically Defined, Customizable Media that Can Expand Human T Cell Subsets in the Absence of Serum.

Figure 1

(A) The first phase of generating a serum-free medium that can expand all human T cell subsets consisted of creating 10 prototype media by mixing different ratios of 3 base media that contain different concentrations of amino acids, vitamins, trace elements, antioxidants, metal ions, polyamines, and lipids. These prototype media were tested for their ability to expand activated primary human T cells using anti-CD3/CD28-coated beads and reiterated with a design of experiments (DOE) statistical quadratic model through Design-Expert 9.0.1 software with a desired response to maximally expand human T cells without serum supplementation. Phase 2 consisted of eliminating xenogeneic components, examining metabolites consumed in serum-supplemented X-VIVO-15 medium and prototype media from phase 1, and modifying media so that concentrations of metabolites are maintained upon feeding of activated T cells. The final phase focused on optimizing carbon sources, lipid concentrations, lentiviral transduction efficiency, and cytokine production post-activation on activated human T cells.

(B) Total CD4 T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and activated by anti-CD3/CD28-coated beads in 1B2H medium containing optimal glucose, no glucose, or optimal glucose without glutamine. T cell proliferation was measured by CFSE dilution by flow cytometry. Data are representative of 3 independent experiments.

(C–E) Primary human CD4 T cells were sort-purified into TN (CD25−CD45RA+CCR7+CD27+) (C), TCM (CD25−CD45RO+CCR7+CD27+) (D), and TEM cells (CD25−CD45RO+CCR7−CD27−) (E) and stimulated with anti-CD3/CD28-coated beads in 1B2H medium with or without 5% human serum (see Figure S2 for gating strategy). Cell expansion was monitored by Coulter counter on the indicated days. Data are representative of 2–3 donors and independent experiments. *p < 0.05, **p < 0.01, paired two-tailed Student’s t test on day 9 population doublings; ns, not significant.