Culture Condition of Bone Marrow Stromal Cells Affects Quantity and Quality of the Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) released by bone marrow stromal cells (BMSCs) have been shown to act as a transporter of bioactive molecules such as RNAs and proteins in the therapeutic actions of BMSCs in various diseases. Although EV therapy holds great promise to be a safer cell-free therapy overcoming issues related to cell therapy, manufacturing processes that offer scalable and reproducible EV production have not been established. Robust and scalable BMSC manufacturing methods have been shown to enhance EV production; however, the effects on EV quality remain less studied. Here, using human BMSCs isolated from nine healthy donors, we examined the effects of high-performance culture media that can rapidly expand BMSCs on EV production and quality in comparison with the conventional culture medium. We found significantly increased EV production from BMSCs cultured in the high-performance media without altering their multipotency and immunophenotypes. RNA sequencing revealed that RNA contents in EVs from high-performance media were significantly reduced with altered profiles of microRNA enriched in those related to cellular growth and proliferation in the pathway analysis. Given that pre-clinical studies at the laboratory scale often use the conventional medium, these findings could account for the discrepancy in outcomes between pre-clinical and clinical studies. Therefore, this study highlights the importance of selecting proper culture conditions for scalable and reproducible EV manufacturing.

Keywords: bone marrow stromal cells (BMSCs); culture condition; extracellular vesicles (EVs); miRNAs.

Figure 1

Schematic workflow of human BMSC culture for EV isolation and analyses. Cryopreserved human BMSCs were expanded in DMEM supplemented with 10%FBS (DMEM + 10%FBS) and evenly split into 3 dishes. After 24 h incubation in DMEM + 10%FBS, the medium was switched to either fresh DMEM + 10%FBS, HPM, or HPM-XF. At 80–90% confluency, cells were re-plated onto 6 dishes at the density of 3000–4000 cells/cm². At 80–90% confluency, the number of cells was counted in 3 dishes out of 6. In the rest dishes, the media were aspirated and replaced with basal media after a thorough wash with PBS (3 times). The conditioned media were harvested after 24 h of incubation for EV analysis.

Figure 2

Doubling time. Doubling times were calculated from the number of cells at the beginning of culture and the harvest as well as the culture duration (days). Doubling times of each BMSC in three different media (DMEM, HPM, and HPM-XF) were presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Black bar: BMSCs cultured in DMEM + 10%FBS. Blue bar: BMSCs cultured in HPM. Red bar: BMSCs cultured in HPM-XF.

Figure 3

Characterization of BMSCs cultured in three different media (DMEM, HPM, and HPM-XF). (A) Flow cytometric analysis for hematopoietic markers and mesenchymal markers. Histograms of experimental samples (black solid line) were overlaid with the isotype control (filled with gray). (B) BMSCs were differentiated into adipocytes, osteoblasts, and chondrocytes and stained with Oil Red O, Alizarin Red, and Alcian Blue, respectively. Data from one representative sample are presented.

Figure 4

Characterization of EVs harvested from BMSC cultures. (A) The average size of EVs was evaluated by nanoparticle tracking analysis (NanoSight). (B) The number of EVs released from a single BMSC was calculated from the total number of EVs in the conditioned medium and the number of BMSCs in the culture. (C) The number of EVs released from a single BMSC over the doubling time of the BMSC was plotted. The trend line for EVs from DMEM was shown. Results are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Black: EVs from BMSCs cultured in DMEM + 10%FBS. Blue: EVs from BMSCs cultured in HPM. Red: EVs from BMSCs cultured in HPM-XF.

Figure 5

Fraction of RNA species contained in EVs from BMSCs cultured in either DMEM + 10%FBS (black bar) or HPM (gray bar). Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 6

Analysis of miRNA profiles. (A) Principal component analysis of miRNA profiles of each EV sample isolated from BMSCs (#3–#8) cultured in either DMEM + 10%FBS (●) or HPM (▲). (B) Heat map and hierarchical clustering analysis of each EV sample isolated from BMSCs (#3-#8) cultured in either DMEM + 10% or HPM. The sequencing data are deposited in the GEO (the accession number: GSE185942).

Figure 7

Differential gene expression analysis and ingenuity pathway analysis. (A) Heat map and hierarchical clustering analysis shows miRNAs upregulated (88 miRNAs) and downregulated (46 miRNAs) in EVs from BMSCs cultured in HPM compared to those from BMSCs cultured in DMEM + 10%FBS. (B) Ingenuity pathway analysis for the diseases & functions of 88 upregulated and 46 downregulated miRNAs identified in (A).