Control the Upstream Process of Exosome Production to Reduce Sample Variability
Sep 10,2026
Exosomes are an important component of small extracellular vesicles (sEVs). They are nanoscale, membrane-enclosed vesicles secreted by cells, typically ranging from 30–150 nm in diameter. Owing to their important roles in intercellular communication, molecular delivery, and disease diagnosis, exosomes have become a major research focus in regenerative medicine, liquid biopsy, drug delivery, and other fields.
As research has advanced, it has become increasingly clear that exosome sample quality depends not only on downstream purification and characterization techniques, but also on the upstream cell culture process, which can affect exosome yield, composition, and biological function. According to the MISEV2018[1] and MISEV2023[2] guidelines issued by the International Society for Extracellular Vesicles (ISEV), standardized production procedures should be established for exosome research, with cell source, culture system, culture mode, and supernatant collection conditions all being important factors affecting sample quality. Therefore, establishing a standardized upstream production process is a key foundation for obtaining stable, high-quality exosome samples.
This edition of Cell Culture Academy focuses on the entire upstream process of exosome production, breaking down the often-overlooked yet critical steps from seed cell selection, culture medium systems, and culture modes to supernatant collection and pretreatment, helping you avoid common pitfalls.
I. Seed Cell Selection: Minimize Experimental Risks at the Source
Exosomes can be derived from a wide range of cell sources, including mesenchymal stem cells (MSCs), HEK293 cells, immune cells, and tumor cells. Among these, human Wharton’s jelly-derived MSCs (hWJ-MSCs) have become one of the commonly used cell types in exosome research because of their strong proliferative capacity, high secretion levels, and low immunogenicity.
It should be noted that exosome composition is not fixed and can be influenced by cell state and the culture environment. Exosomes produced by the same cell type under different culture conditions may differ in their protein, miRNA, and lipid profiles, ultimately affecting the consistency of their biological functions.
Therefore, during exosome preparation, attention should be paid to cell source, passage number, and culture state. For example, as the number of passages increases, MSCs may exhibit reduced proliferative capacity, an increased proportion of senescent cells, and changes in exosome secretion levels. Quality control measures such as cell species identification and mycoplasma testing should also be performed to ensure the stability of exosome samples across batches.
II. Medium Selection: Reducing Interference from Exogenous Contaminants
The choice of culture medium directly determines the proportion of exogenous impurities in the system and is also one of the steps most prone to introducing experimental bias[3]. Conventional fetal bovine serum (FBS) contains large amounts of bovine-derived exosomes and lipoproteins. If used directly for conditioned culture during exosome enrichment, these components can seriously interfere with subsequent experimental results. Currently, three major solutions are commonly used in the field:
1. Exosome-depleted serum culture system: Most endogenous exosomes are removed from serum through processes such as ultracentrifugation and membrane filtration. Using commercially available exosome-depleted serum directly to prepare complete medium for cell culture is convenient and can meet routine basic research needs; however, due to limitations of current depletion processes, trace amounts of exosomes may still remain in the system.
2. Use a serum-free defined medium: A serum-free medium with chemically defined components is used without adding any animal-derived components, fundamentally avoiding contamination by animal-derived exosomes and contaminating proteins. This is a standard approach compatible with GMP manufacturing. However, special attention should be paid to the fact that some cells are prone to apoptosis during long-term serum-free culture, and the resulting apoptotic bodies can substantially reduce the purity of the final exosome preparation.
3. PBS washing followed by serum-free culture: After cell expansion in conventional serum-containing complete medium, cells are thoroughly washed with PBS 2–3 times, followed by incubation in serum-free basal medium for 24–72 h before conditioned medium collection. This approach balances cell viability and sample purity. Thorough washing can significantly reduce residual soluble proteins and free exogenous extracellular vesicles in the supernatant, providing an important foundation for improving subsequent exosome purification efficiency.
III. Upgrading the Culture Model: Overcoming Exosome Production Bottlenecks
Routine laboratory exosome preparation generally uses two-dimensional culture in culture flasks or dishes. This approach is simple to operate and suitable for small-scale research.
As exosome research progresses toward in vivo animal studies and clinical translation, traditional two-dimensional culture increasingly faces limitations such as limited culture surface area, insufficient yield, and substantial operational differences between batches. Consequently, bioreactor systems are increasingly being applied to scale-up production, including microcarrier, hollow-fiber, and fixed-bed bioreactors. Hollow-fiber bioreactors can support high-density cell culture while reducing the effects of mechanical shear, helping maintain cell state and improve exosome production efficiency.
Regardless of the culture mode used, precise control of cell state is always critical. Generally, initiating conditioned culture when cell confluence reaches 70%–80% is conducive to obtaining stable samples. Excessively high confluence can lead to contact inhibition and accumulation of metabolic waste, whereas excessively low confluence can result in insufficient exosome concentrations in the supernatant and affect purification recovery.
IV. Supernatant Processing: Preserve Sample Activity
Cell culture supernatant is the direct source material for exosomes, and its collection and pre-processing represent a critical link between upstream production and subsequent purification. During collection, the duration of conditioned culture should be strictly controlled to avoid prolonged culture, which can compromise cell condition and increase the proportion of apoptotic cells. Supernatant collection is recommended while cells remain in a stable state to minimize the generation of non-target vesicles and cellular debris.
After collection, the culture supernatant typically undergoes sequential centrifugation for pre-processing:
l 300×g for 10 min to remove residual intact viable cells;
l 2,000×g for 20 min to remove dead cells and cellular debris;
l 10,000×g for 30 min to remove larger vesicles and protein aggregates.
Pre-processed samples should be stored according to the requirements of subsequent experiments. It is important to note that repeated freeze–thaw cycles may disrupt exosome membrane structure, promote particle aggregation, and reduce functional activity. Therefore, the number of freeze–thaw cycles should be minimized whenever possible. Some studies additionally incorporate filtration through a 0.22 μm membrane; before implementation, the membrane material, protein adsorption, and potential vesicle loss during filtration should be evaluated in advance.
Exosome quality control begins with the upstream production process. Cell source, culture system, culture conditions, and supernatant collection methods can all affect the composition, stability, and reliability of exosome samples. In the face of challenges such as batch-to-batch sample variation, culture system compatibility, and process standardization, establishing standardized culture procedures, strengthening cell quality control, and optimizing sample processing conditions are important foundations for improving the reproducibility of exosome research.
References
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