Identification of an appropriate substrate for HCI of human primary immune cells. Four human primary immune cell types (B cells, monocytes, neutrophils, and CD4+ T cells) were independently cultured in 384-well plates in which well bottoms were coated with one of eight substrates: MS-1, MS-2, MS-3, 3D Hydrogel, PDL, SPA, 3D Hydrogel PDL, or 3D Hydrogel SPA. Each cell type was plated at three concentrations: 100,000, 50,000, or 25,000 cells/well. (A) Representative images of each cell type and substrate at a concentration of 100,000 cells/well. Cells were fixed with 4% PFA and stained with DAPI prior to imaging. Magnification: 60×. Scale bars: 20 microns. (B) Cell attachment for four human primary immune cells in the eight substrates shown in A. Cells were plated at 100,000 cells/well. The y-axis represents the number of cells counted after fixation and automated liquid handling in conditions similar to those of the hcHCR protocol. Each dot represents one biological replicate (one unrelated healthy human donor). Colored points and error bars display the mean ± SD for three technical replicates of each biological replicate. Black error bars display the mean ± SD of the biological replicates. Significance values are from a linear mixed-effects model. (C) TNF, IL6, and IFNB expression in primary human monocytes cultured in the presence or absence of PDL substrate. Cells were plated at 100,000 cells/well. LPS stimulation was used as a positive control. Gene expression was measured by qPCR. Each data point represents one biological replicate. Error bars display the mean ± SD. Significance values are for a Wilcoxon signed-rank test. (D) Retention ratios for four primary human immune cells at three cell concentrations. Cells were plated in PDL substrate and underwent fixation and automated liquid handling in conditions similar to those of the hcHCR protocol. The retention ratio for each biological replicate was calculated as the estimated number of cells per well [(number of cells counted)/(number of fields of view imaged) × (total number of fields of view per well)] divided by the number of cells plated per well. Each dot represents one biological replicate (one unrelated healthy human donor). Colored points and error bars display the mean ± SD for three technical replicates of each biological replicate. Black error bars display the mean ± SD of the biological replicates. Significance values are from a linear mixed-effects model.
