Small extracellular vesicles derived from human adipose‐derived mesenchymal stromal cells cultured in a new chemically‐defined contaminate‐free media exhibit enhanced biological and therapeutic effects on human chondrocytes in vitro and in a mouse osteoarthritis model

Abstract

Human small extracellular vesicles (sEVs) derived from adipose-derived mesenchymal stromal cells (ASC) have been reported to suppress the progression of osteoarthritis (OA) in animal studies and subsequently, translation of this potential to assess their clinical efficacy is anticipated. However, fabrication protocols for sEVs to eliminate potential contamination by culture medium-derived components need to be established prior to their clinical use. The purpose of the present studies was to elucidate the influence of medium-derived contaminants on the biological effects of sEVs, and to establish isolation methods for sEVs using a new clinical grade chemically-defined media (CDM). The quantity and purity of ASC-derived sEVs cultured in four different CDMs (CDM1, 2, 3 and 4) were evaluated. The concentrates of the four media incubated without cells were used as the background (BG) control for each set of sEVs. The biological effect of sEVs fabricated in the four different CDMs on normal human articular chondrocytes (hACs) were evaluated in vitro using a variety of methodological assessments. Finally, the sEVs with the highest purity were tested for their ability to suppress the progression of knee OA mouse model. Analysis of the BG controls revealed that CDM1-3 contained detectable particles, while there was no visible contamination of culture media-derived components detected with CDM4. Accordingly, the sEVs fabricated with CDM4 (CDM4-sEVs) exhibited the highest purity and yield. Notably, the CDM4-sEVs were the most efficient in promoting the cellular proliferation, migration, chondrogenic differentiation, and anti-apoptotic activity of hACs. Furthermore, CDM4-sEVs significantly suppressed the osteochondral degeneration in vivo model. Small EVs derived from ASCs cultured in a CDM without detectable contaminants demonstrated enhanced biological effects on hACs and the progression of OA. Thus, sEVs isolated with CDM4 most optimally meet the requirements of efficacy and safety for assessment in their future clinical applications.

Comparison of the ability of exosomes and ectosomes derived from adipose-derived stromal cells to promote cartilage regeneration in a rat osteochondral defect model

“…The proliferation of cells was monitored using the CCK8 kit (Dojindo, Japan) as recommended by the manufacturer. In light of the scarcity of research concerning the utilization of ASC-Ectos in cell treatment, three concentrations of exosomes (1, 2, and 5 × 10 9 particles/ml) were chosen for cell proliferation assessments, drawing upon previous investigations pertaining to ASCs and chondrocytes [ 23 , 24 ]. The concentration of 2 × 10 9 particles/ml (approximately equivalent to 20 μg/ml) was determined as the optimal choice for cell experiments based on the observed highest cell viability in the CCK8 assay.…”

Human Infrapatellar Fat Pad Mesenchymal Stem Cell–derived Extracellular Vesicles Purified by Anion Exchange Chromatography Suppress Osteoarthritis Progression in a Mouse Model

“…In addition to the cell source, the cell culturing process and extracellular vesicle purification method are critical for controlling quality and producing scalable MSC-extracellular vesicles for clinical use. Thus, a chemically modified medium that is free from serum contamination enables the production of clear MSC-extracellular vesicles and retains their biological effects [9]. Anion exchange chromatography is appropriate for managing large amounts of extracellular vesicle-enriched media in a clinical laboratory.…”