Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile

Abstract

Human mesenchymal stromal cells (hMSCs) are mechanically sensitive undergoing phenotypic alterations when subjected to shear stress, cell aggregation, and substrate changes encountered in 3D dynamic bioreactor cultures. However, little is known about how bioreactor microenvironment affects the secretion and cargo profiles of hMSC-derived extracellular vesicles (EVs) including the subset, "exosomes", which contain therapeutic proteins, nucleic acids, and lipids from the parent cells. In this study, bone marrow-derived hMSCs were expanded on 3D Synthemax II microcarriers in the PBS mini 0.1L Vertical-Wheel bioreactor system under variable shear stress levels at 25, 40, and 64 RPM (0.1-0.3 dyn/cm²). The bioreactor system promotes EV secretion from hMSCs by 2.5-fold and upregulates the expression of EV biogenesis markers and glycolysis genes compared to the static 2D culture. The microRNA cargo was also altered in the EVs from bioreactor culture including the upregulation of miR-10, 19a, 19b, 21, 132, and 377. EV protein cargo was characterized by proteomics analysis, showing upregulation of metabolic, autophagy and ROS-related proteins comparing with 2D cultured EVs. In addition, the scalability of the Vertical-Wheel bioreactor system was demonstrated in a 0.5L bioreactor, showing similar or better hMSC-EV secretion and cargo content compared to the 0.1L bioreactor. This study advances our understanding of bio-manufacturing of stem cell-derived EVs for applications in cell-free therapy towards treating neurological disorders such as ischemic stroke, Alzheimer's disease, and multiple sclerosis.

Keywords: 3D microcarriers; Cargo profile; Extracellular vesicles; Human mesenchymal stromal cells; Shear stress; Vertical-Wheel bioreactor.

Graphical abstract

Fig. 1

hMSC expansion and metabolism on Synthemax II microcarriers in PBS Vertical-Wheel Bioreactors at 25, 40, and 64 RPM. (A) Process flow for hMSC expansion on Synthemax II microcarrers in PBS Vertical-Wheel Bioreactors; (B) hMSC growth kinetics during expansion and EV collection stages; (C) Glucose and Lactate metabolism: (i) Glucose concentration kinetics; (ii) Lactate concentration kinetics. (iii) Glucose consumed normalized to cell number; (iv) Lactate produced normalized to cell number; (v) Lactate production to glucose consumption ratios. (D) Glutamine metabolism: (i) Glutamine concentration kinetics. The extracellular vesicle (EV) collection medium is formulated with Glutamax. Therefore, the glutamine concentration at day 5 is 0. (ii) Glutamic acid concentration kinetics; (iii) Ammonia concentration kinetics.

Fig. 2

Metabolic and extracellular vesicle (EV) biogenesis pathways and reactive oxygen species (ROS) analysis for hMSCs in PBS Vertical-Wheel Bioreactors at 25, 40, and 64 RPM. Various genes were measured by RT-qPCR on the mRNAs isolated from the harvested cells at the end of EV collection (n = 6, results were from two independent bioreactor runs), relative to 2D control. (A) Glycolytic genes; (B) Autophagy genes; Flow cytometry histograms for (C) Total ROS; (D) Mitochondrial ROS; and (E) Autophagosomes. Red line: negative control; blue line: 2D culture control; orange line: 25 RPM bioreactor culture; light green: 40 RPM, and dark green: 64 RPM. (F) ESCRT-dependent EV biogenesis markers; (G) ESCRT-independent EV biogenesis markers. * indicates p < 0.05; ** indicates p < 0.01, *** indicates p < 0.001.

Fig. 3

Characterizations of extracellular vesicles (EVs) secreted by 0.1L bioreactor-expanded hMSCs. (A) Western Blot of exosomal marker expression, including HSC70, TSG101, CD81, Calnexin (negative marker); HRS, Syntenoin-1, CD81, and CD63. (B) Comparison of EV yield for the number of EVs per mL spent medium (n = 3), (C) Number of EVs normalized to the cell number (n = 3). (D) EV size and distribution determined based on TEM images (n = 50–80). (E) TEM images of EVs for various groups. Scale bars: 100 nm. (F) miRNA expression in the isolated EVs determined by RT-qPCR (n = 6). The value shows ΔΔCt. Results were combined from two independent bioreactor runs. *p < 0.05; **p < 0.01, ***p < 0.001.

Fig. 4

Bioreactor-expanded hMSCs and the secreted EV protein cargo analysis by proteomics. (A) Venn diagram of the differentially expressed proteins (DEPs) from hMSCs of bioreactor culture vs. 2D culture. (B) GO annotation of the hMSC DEPs (558 as in A) for the enriched pathways. (C) Venn diagram of the DEPs for hMSC-EVs. (D) GO annotation of the hMSC-EV DEPs (616 as in C) for the enriched pathways. (E) GO annotation of the DEPs (863 as in C) for the Bioreactor only subset of hMSC-EV protein cargo.

Fig. 5

hMSC expansion and metabolism on Synthemax II microcarriers in 0.5L PBS Vertical-Wheel Bioreactors. (A) Process flow for hMSC expansion on Synthemax II microcarrers in PBS Vertical-Wheel Bioreactors; (B) hMSC numbers during expansion and EV collection stages; (C) Glucose and Lactate metabolism: (i) Glucose concentration kinetics; (ii) Lactate concentration kinetics. (iii) Glucose consumed normalized to cell number; (iv) Lactate produced normalized to cell number; (v) Lactate production to glucose consumption ratios. (D) Glutamine metabolism: (i) Glutamine concentration kinetics. The EV collection medium is formulated with Glutamax. (ii) Glutamic acid concentration kinetics; (iii) Ammonia concentration kinetics.

Fig. 6

Metabolic and extracellular vesicle biogenesis pathways for hMSCs in 0.5L PBS Vertical-Wheel Bioreactors. Various genes were measured by RT-qPCR on the mRNAs isolated from the harvested cells at the end of EV collection (n = 6, results were from two independent bioreactor runs), relative to 0.1L bioreactor control. (A) Glycolytic genes; (B) Autophagy genes; (C) ESCRT-dependent EV biogenesis markers; (D) ESCRT-independent EV biogenesis markers. * indicates p < 0.05; ** indicates p < 0.01, *** indicates p < 0.001.

Fig. 7

Characterizations of extracellular vesicle secreted by 0.5 L bioreactor-expanded hMSCs. (A) Comparison of EV production yield as the number of EVs normalized to the cell number. (B) Protein content of isolated EVs normalized to EV number. (C) Western Blot of exosomal marker expression. (D) miRNA expression in the isolated EVs determined by RT-qPCR (n = 6, results were from two independent bioreactor runs). (E) In vitro wound healing assay for the human fibroblasts that received the 2D and bioreactor hMSC-EVs. Scale bar: 100 μm. VWB: vertical-wheel bioreactor. * indicates p < 0.05; ** indicates p < 0.01, *** indicates p < 0.001.