Fig. 1

3D culture in high collagen density impairs T cell proliferation. a Schematic model of the 3D culture system. b T cells were cultured for 5 days in the indicated conditions, and subsequently viability was analyzed by flow cytometry. Each dot in the graph represents an individual donor. Error bars indicate standard error of the mean (SEM). c-e T cells cultured in a collagen matrix of low density (1 mg/ml, c-d) or high density (4 mg/ml, E) including fluorescently labeled collagen were imaged by confocal microscopy. c 3D projection of collagen matrix with embedded T cells. d-e Representative images of individual T cells within a low- density collagen matrix (d) or high-density collagen matrix (e). Size bars: (c-e) 10 μm. f-g T cell proliferation after 5 days in culture was measured by flow cytometry-based analysis of CellTrace Violet (CTV) dilution. f Representative histogram showing CTV dilution in T cells cultured in 2D or in 3D in a low-density collagen matrix or high-density collagen matrix. g Quantification of T cell proliferation based on CTV dilution. Three individual donors were analyzed. Connecting lines indicate measurements of the same donor. h T cells were cultured in collagen gels of high and low density and their proliferation was measured using a BrdU-based flow cytometry assay. The percentage of CD3-positive BrdU-positive cells is shown. i The breast cancer cell lines EO771.LMB, MDA-MB-231, and 4 T1 were cultured in collagen matrices of low or high density for 5 days and analyzed using a BrdU-based flow cytometry assay. The percentage of BrdU-positive cells is depicted. j-k The ratio of CD4+ to CD8+ cells was analyzed by flow cytometry after culture for 2 days (i) or 5 days (j). g-k Error bars indicate standard deviations of technical replicates