Viability assessment of human peripheral blood-derived stem cells after three methods of nebulization

Abstract

Background and objectives: Drug delivery by nebulization has become a crucial strategy for treating different respiratory and lung diseases. Emerging evidence implicates stem cell therapy as a promising tool in treating such conditions, not only by alleviating the related symptoms but by improving the prognosis. However, delivery of human peripheral blood-derived stem cells (hPBSCs) to the respiratory airways remains an innovative approach yet to be realized. This study is an analytic, translational, and in vitro research to assess the viability and morphological changes of identified cell populations in hPBSCs cocktail derived from COVID-19 patients.

Methods and results: Peripheral blood (PB) samples were obtained from patients enrolled in the SENTAD-COVID Study (ClinicalTrials.gov Reference: NCT04473170). hPBSCs cocktails (n=15) were provided by the Cells Processing Laboratory of Abu Dhabi Stem Cells Center, and were nebulized by three different methods of nebulization: compressor (jet), ultrasonic, and mesh. Our results reported that nucleated CD45dim cell count was significantly lower after the three nebulization methods, but nucleated CD45- cells show a significant decrease only after mesh nebulization. Mesh-nebulized samples had a significant reduction in viability of both CD45dim and CD45- cells.

Conclusions: This study provides evidence that stem cells derived from PB of COVID-19 patients can be nebulized without substantial loss of cell viability, cell count, and morphological changes using the compressor nebulization. Therefore, we recommend compressor nebulizers as the preferable procedure for hPBSCs delivery to the respiratory airways in further clinical settings.

Keywords: COVID-19; Peripheral blood stem cells; cell survival; lung diseases; nebulizers.

Conflict of interest statement

None.

AJSC Copyright © 2021.

Figures

Figure 1

Flow cytometry sequential, logic and manual gating strategy for immunophenotypic characterization of hPBSCs. Visualized hPBSC on dot plots showed their FSC and SSC signals related to the cell’s size and granularity, respectively (upper panel gates). The cells were stained with Hoechst 33342, following 7ADD dye exclusion criteria, and immunofluorescence staining for CD45, CD34, CD133, and CD90. hPBSCs: human peripheral blood-derived stem cells; 7ADD: 7-aminoactinomycin D; APC: allophycocyanin; ECD: phycoerythrin-Texas Red conjugate; FITC: fluorescein isothiocyanate; FSC: forward scatter; PE: phycoerythrin; SSC: side scatter.

Figure 2

Changes in nucleated absolute cells counts (cells/µL) analyzed by flow cytometry. Cells were stained with Hoechst 33342 before and after three nebulization methods; each color represents one nebulization method [black: compressor (n=5); red: mesh (n=5); and blue: ultrasound nebulization (n=5)]. A. CD45- nucleated cells. B. CD45dim nucleated cells. *Statistically significant differences (P<0.05). **Statistically highly significant differences (P<0.01).

Figure 3

Changes in viable absolute cells counts (cells/µL) analyzed by flow cytometry. Cells were stained with 7ADD before and after three nebulization methods; each color represents one nebulization method [black: compressor (n=5); red: mesh (n=5); and blue: ultrasound nebulization (n=5)]. A. CD45- nucleated cells. B. CD45dim nucleated cells. *Statistically significant differences (P<0.05). 7ADD: 7-aminoactinomycin D.

Figure 4

Effect of nebulization on cell morphology and viability. Representative immunofluorescence images of hPBSCs cocktail before (A) and after ultrasound (B), mesh (C) and compressor (D) nebulization. hPBSCs were stained with FITC-conjugated monoclonal surface antibody CD45 (1:100) and Hoechst nucleic acid dye 33342 (10 µg/mL). Images were acquired using Leica SP8 confocal microscope using 63× objective. Two main populations were found: hematopoietic (*) and non-hematopoietic cells (arrow). Compressor nebulization shows no adverse effects on hPBSCs morphology. Please note three, one, and two hematopoietic cells along with one, one, and two non-hematopoietic cells were pointed out after ultrasound, mesh, and compressor nebulization, respectively. hPBSCs: Human peripheral blood-derived stem cells; FITC: fluorescein isothiocyanate.