An immune cell spray (ICS) formulation allows for the delivery of functional monocyte/macrophages

Abstract

Macrophages are key cells of the innate immune system and act as tissue resident macrophages (TRMs) in the homeostasis of various tissues. Given their unique functions and therapeutic use as well as the feasibility to derive macrophages in vitro from hematopoietic stem cell (HSC) sources, we propose an "easy-to-use" immune cell spray (ICS) formulation to effectively deliver HSC-derived macrophages. To achieve this aim, we used classical pump spray devices to spray either the human myeloid cell line U937 or primary murine HSC-derived macrophages. For both cell types used, one puff could deliver cells with maintained morphology and functionality. Of note, cells tolerated the spraying process very well with a recovery of more than 90%. In addition, we used osmotic preconditioning to reduce the overall cell size of macrophages. While a 800 mosm hyperosmolar sucrose solution was able to reduce the cell size by 27%, we identified 600 mosm to be effective to reduce the cell size by 15% while maintaining macrophage morphology and functionality. Using an isolated perfused rat lung preparation, the combinatorial use of the ICS with preconditioned and genetically labeled U937 cells allowed the intra-pulmonary delivery of cells, thus paving the way for a new cell delivery platform.

Figure 1

Spraying of U937 cells. (a) Schematic overview of the cell spraying procedure and (b) image of the pump spray devices used for our studies. (c) Numbers of viable and dead U937 cells after the spraying process, analyzed via trypan blue staining. Initial concentrations of 6 × 10⁶ cells/mL (left) and 12 × 10⁶ cells/mL (right) were used (significance of **P < 0.01 or ****P < 0.0001 by two-tailed paired student t-test, n = 3–6 mean ± SEM). (d) Cell numbers/puff at a cell concentration of 6 × 10⁶ cells/mL (n = 6, mean ± SEM). (e) Evaluation of cell viability after the spraying process by propidium iodide (PI) staining (Left: Representative flow cytometry analysis. Right: Quantification of cell viability n = 3 mean ± SEM). (n.s: denotes not significant. Statistical analysis: analyzed by two-way repeated measurements (RM) ANOVA with Bonferroni post-hoc-test).

Figure 2

Functionality of bone marrow (BM)-derived macrophages after spraying. (a) Flow cytometric analysis of cell viability by propidium iodide (PI) staining of unsprayed and sprayed BM-derived macrophages (Left: representative images, right: quantification of n = 3, mean ± SEM). (b) Papenheim staining of cytospin preparations of sprayed and unsprayed BM-derived macrophages (scale bar: 20 µm) (c) Flow cytometric analysis of CD45.1, CD11b and F4/80 expression of unsprayed and sprayed BM-derived macrophages. Overlays of representative histograms; grey filled line: isotype, red/blue line: surface marker of unsprayed or sprayed macrophages. Lower panels: Delta mean fluorescent intensity (MFI) of surface marker expression of unsprayed and sprayed BM-derived macrophages (delta MFI was calculated by subtracting isotype (negative) MFI from surface marker (positive) MFI; n = 3, mean ± SEM). (d) Phagocytic activity of unsprayed and sprayed BM-derived macrophages. Upper panels: representative fluorescent microscopy images of BM-derived macrophages 2 hours after incubation with pHRodo E. coli BioParticles (scale bar: 200 µm). Lower panel: Representative flow cytometric analysis (grey filled line: cells without E. coli BioParticles, red line: unsprayed cells with E. coli BioParticles, blue line: sprayed cells with E. coli BioParticles) and quantification of n = 3 (mean ± SEM). (e) Analysis of MHC-II surface marker expression by flow cytometry of unsprayed and sprayed BM-derived macrophages before and after stimulation with IFNγ. Left: Representative histograms (grey filled line: isotype, green line: non-stimulated control, blue line: IFNγ stimulated) and right: Quantification of the fold change in MFI, n = 3, mean ± SEM). (n.s: denotes not significant. Statistical analysis: a: two-way repeated measurements (RM) ANOVA with Bonferroni post-hoc-test; (c–e) two-tailed paired student t-test).

Figure 3

Shrinking of BM-derived macrophages using hyperosmolar solutions. (a) Flow cytometric analysis using FSC-H/SSC-H plot discrimination (left panel) and mean of FSC-H (right panel) for control (PBS) and hypertonic solutions (n = 4–5, biological repeats, mean ± SEM, significance of ***P < 0.001 by one-way ANOVA with Tukey’s post-hoc-test). (b) Representative brightfield images of BM-derived macrophages after incubation in control medium and 1000 mosm solution (scale bar: 200 µm) and ImageJ analysis of cell area (right panel; n = 105–482, technical repeats, mean with 95% CI, significance of ***P < 0.001 by one-way ANOVA with Tukey’s post-hoc-test). (c) Flow cytometric analysis of cell viability by propidium iodide (PI) staining of macrophages after incubation (60 min) in control (PBS) and hypertonic solutions. (d) Quantification of cell viability after incubation in control (PBS) and hypertonic solutions (n = 4–5 mean ± SEM). (n.s: denotes not significant analyzed by two-way ANOVA with Bonferroni post-hoc-test).

Figure 4

Functional analysis of BM-derived macrophages after incubation in hyperosmolar solutions. (a) Flow cytometric analysis CD45.1, CD11b and F4/80 surface marker expression on BM-derived macrophages after incubation in control (PBS) and hypertonic solutions (grey filled line: isotype, red line: surface marker). (b) Delta mean fluorescent intensity (MFI) of surface marker expression (delta MFI was calculated by subtracting isotype (negative) MFI from surface marker (positive) MFI; n = 3 biological repeats, mean ± SEM, n.s: not significant by one-way ANOVA with Tukey’s post-hoc-test). (c) Cytospins of BM-derived macrophages after incubation in control (PBS) and hypertonic solutions (scale bar: 20 µm). (d) Phagocytic activity of unsprayed and sprayed BM-derived macrophages. Left: Representative flow cytometric analysis (grey filled line: cells without S. aureus BioParticles, red line: cells with S. aureus BioParticles) and quantification of n = 3 (mean ± SEM) (n.s denotes not significant, significance of ***P < 0.001 by one-way ANOVA with Tukey’s post-hoc-test).

Figure 5

Local administration of GFP-labeled U937 cells into isolated perfused rat lung (IPL) (a) Representative fluorescent microscopy images of U937 cells transduced with lentiviral vectors expressing GFP from a CMV early enhancer/chicken beta-actin promoter (Lv.CAG.GFP) (scale bar: 100 µm; BF: brightfield) (b) Flow cytometric analysis of GFP expression in Lv.CAG.GFP transduced U937 cells (blue line: untransduced cells, red line: cells transduced with Lv.CAG.GFP) (c) Analysis of U937 cell localization after application to the IPL. Upper panels: Immunofluorescence staining of cryostat sections generated from the lobus accessorius of the IPL (left: scale bar 50 µm, right: scale bar 10 µm), U937 cells were detected by anti-GFP antibody staining. Lower panel: Representative images of horizontal 2-Photon microscopy of lobus accessorius (right: scale bar: 60 µm).