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Fixed spheroids modulate classically polarized primary human macrophages toward less inflammatory and pro-regenerative phenotypes, and suppress the proliferation of T lymphocytes, while promoting regulatory populations. a) Scheme showing the differentiation and polarization of primary human macrophages (M1 macrophages) before incubation with fixed spheroids, for two days. b) Quantification of the MFI of multiple markers related to proinflammatory or c) reparative phenotype in human macrophages. CXCR4 is a marker of angiogenesis. Mean ± SEM, n = 3 human donors, r = 4 biological replicates. Statistical significance was determined using two-way ANOVA following Tukey’s multiple comparisons test. d) Schematic showing the incubation of CD4 + T or CD8 + T lymphocytes with fixed spheroids. e) Quantification of live cells (using Trypan blue exclusion assay) in a population of primary human CD4 + T lymphocytes activated with CD3/CD28 magnetic beads, while incubated for 4 days with an average of 8 fixed spheroids treated or not with IFN γ . Median ± min/max, n = 4 biological replicates. Statistical significance was determined using an unpaired t -test. f) Flow cytometry plots of activated CD4 + T lymphocytes stained for FOXP3 and g) MFI of FOXP3 + cells. Mean ± SD, n = 4 biological replicates. Statistical significance was determined using one-way ANOVA following Dunnett’s multiple comparisons test. h) Quantification of the total number of cells in a population of primary human CD8 + T lymphocytes activated with CD3/CD28 magnetic beads, while incubated for 4 days with an average of 8 fixed spheroids treated or not with IFN γ . Median ± min/max, n = 8 biological replicates. Statistical significance was determined using an unpaired t -test. i) Flow cytometry plots of activated CD8 + T lymphocytes stained for CD8 and j) quantification of CD8 + cells MFI. Mean ± SD, n = 4 biological replicates. Statistical significance was determined using an unpaired t -test.

Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Shape-Versatile Fixed Cellular Materials for Multiple Target Immunomodulation

doi: 10.1002/adma.202405367

Figure Lengend Snippet: Fixed spheroids modulate classically polarized primary human macrophages toward less inflammatory and pro-regenerative phenotypes, and suppress the proliferation of T lymphocytes, while promoting regulatory populations. a) Scheme showing the differentiation and polarization of primary human macrophages (M1 macrophages) before incubation with fixed spheroids, for two days. b) Quantification of the MFI of multiple markers related to proinflammatory or c) reparative phenotype in human macrophages. CXCR4 is a marker of angiogenesis. Mean ± SEM, n = 3 human donors, r = 4 biological replicates. Statistical significance was determined using two-way ANOVA following Tukey’s multiple comparisons test. d) Schematic showing the incubation of CD4 + T or CD8 + T lymphocytes with fixed spheroids. e) Quantification of live cells (using Trypan blue exclusion assay) in a population of primary human CD4 + T lymphocytes activated with CD3/CD28 magnetic beads, while incubated for 4 days with an average of 8 fixed spheroids treated or not with IFN γ . Median ± min/max, n = 4 biological replicates. Statistical significance was determined using an unpaired t -test. f) Flow cytometry plots of activated CD4 + T lymphocytes stained for FOXP3 and g) MFI of FOXP3 + cells. Mean ± SD, n = 4 biological replicates. Statistical significance was determined using one-way ANOVA following Dunnett’s multiple comparisons test. h) Quantification of the total number of cells in a population of primary human CD8 + T lymphocytes activated with CD3/CD28 magnetic beads, while incubated for 4 days with an average of 8 fixed spheroids treated or not with IFN γ . Median ± min/max, n = 8 biological replicates. Statistical significance was determined using an unpaired t -test. i) Flow cytometry plots of activated CD8 + T lymphocytes stained for CD8 and j) quantification of CD8 + cells MFI. Mean ± SD, n = 4 biological replicates. Statistical significance was determined using an unpaired t -test.

Article Snippet: Human THP-1 cell line, primary human CD4 + T cells, and CD8 + T cells were purchased from ATCC (cat.: TIB-202, PCS-800-016, and PCS-800-017, respectively).

Techniques: Incubation, Marker, Trypan Blue Exclusion Assay, Magnetic Beads, Flow Cytometry, Staining