Engineered Exosomes–Stimulation

CD Bioparticles is a leading manufacturer and supplier of various drug delivery products and services. Utilizing CD Bioparticles' cutting-edge manufacturing platform for extracellular vesicles, we specialize in EV-based therapies, particularly those utilizing exosomes as carriers. Our comprehensive services extend to enhancing exosome production, characterizing, purifying, and conducting potency assays.

Introduction to How to Increase Exosomes Production

Figure 1. Identity and the heterogeneity of extracellular vesicles and exosomes¹

Exosomes have emerged as promising strategies for treating various diseases, including cancer and COVID-19, and promoting tissue regeneration, such as in the heart and spinal cord. Despite the tremendous potential of exosome-based therapies, a significant challenge to overcome in their translation to treatment and clinical practice is the low production yield and the inability to scale up production. CD Bioparticles is making significant progress in the direction of mass-producing EVs.

How to Increase Exosomes Production:

Physical, chemical, or biological stimulation can be employed to enhance the yield of exosomes per cell. Research has shown significant potential in improving exosome secretion rates and bringing exosome-based therapies closer to clinical translation^2,3,4,5.

• Physical stimulation: Methods like heat shock and shear stimulation using turbulent flow can be used.

Heat shock: Heat shock can cause changes in the folding state of intracellular proteins, thereby activating intracellular stress response pathways to promote the production of exosomes. Alternatively, it may affect exosome production by modulating intracellular signaling pathways, such as the expression of heat shock proteins and the release of other cytokines. This method results in a twofold increase in exosome production compared to conventional conditions.

Shear stimulation: The shear stimulation may increase the secretion of exosomes by activating the cell signaling pathways. It could also lead to the release of more exosomes by influencing the characteristics of the cell membrane. For example, turbulent flow method has been shown to increase exosome secretion by 20 times compared to conventional methods.

• Chemical stimulation: Hypoxia, glucose deprivation, and ethanol are examples of chemical stimuli that can boost exosome production.

Hypoxia In a low oxygen environment, certain regulatory factors within the cells, such as HIF-1α (hypoxia-inducible factor-1α), may stimulate the production of exosomes, potentially increasing it by up to 1.5 times.

Glucose deprivation: When cells are deprived of glucose, they undergo metabolic stress, triggering adaptive responses that can impact exosome production. One potential mechanism is the activation of cellular stress response pathways, such as the unfolded protein response (UPR) and the autophagy pathway. These pathways can be activated in response to glucose deprivation, leading to increased exosome production as part of the cellular stress response. Under conditions of glucose deprivation, cells can increase exosome secretion by up to 3-fold.

Ethanol Proper stimulation with ethanol can indeed lead to cellular stress responses, triggering intracellular signaling pathways that affect exosome production. Additionally, ethanol may influence cell membrane characteristics, impacting exosome release, and can also alter cell metabolism, thereby affecting exosome production and release. Reports suggest that ethanol stimulation can result in up to a 5-fold increase in exosome content.

• Biological stimulation: Cytokines and anti-inflammatory drugs are examples of biological stimuli that can enhance exosome secretion.

Cytokines: Cytokines may influence exosome production by regulating intracellular signaling pathways, affecting immune responses, and modulating intercellular signaling. Compared to normal conditions, cytokine stimulation can potentially increase exosome content by approximately 3-fold.

Anti-inflammatory drugs: Similar to cytokines, anti-inflammatory drugs also influence exosome production by regulating intracellular signaling pathways, affecting immune responses, and modulating intercellular signaling. They can potentially increase exosome content by up to 10-fold.

• Genetic engineering: Genetic engineering technology allows for the introduction of specific genes into cells to increase the production of exosomes. For example, overexpressing genes that encode for exosome production can promote their secretion.

Our Stimulation EV Featured Services:

CD Bioparticles is specialized in the development of drug delivery systems and customizing exosomes for drug delivery utilizing our core technologies. In addition to our regular services of exosome manufacturing, purification/isolation, and characterization, we also provide exosome stimulation options such as turbulent flow to enhance the yield of extracellular vesicles for our partners.

Figure 2. Illustration of the EV production process.

We aim to increase the secretion rate of extracellular vesicles (EVs) using a variety of methods, delivering high-purity, appropriately sized exosome products.

• Physical: We utilize techniques such as mechanical loading, geometry, acoustics, and electrical stimulation. These methods significantly enhance secretion rates compared to traditional 2D culture techniques. We carefully calibrate the loading intensity to meet the stimulation threshold and avoid excessive cellular stress. Our turbulent flow stimulation enables a massive release of EVs. The benefits include:

1. High yield: A tenfold increase in speed compared to cell starvation.

2. Flexibility: Turbulent flow is highly tunable and easy to implement.

3. Compatibility: The process works well with various cell lines (15 cell lines have been tested, both in adherence and suspension).

• Chemical: We offer customization based on product requirements and cargo content, maintaining active chemical factors to increase targeted EV production. This method avoids indiscriminate secretion and provides robust support for your experiments.
• Biological stimulation: We boost exosome secretion with administration of appropriate cytokines or anti-inflammatory drugs to stimulate cells.

By using these state-of-the-art techniques, we try to enhanced production of high-quality exosomes to support your research and development needs.

Quotations and Ordering

References

1. R. Kalluri, V. S. LeBleu, The biology, function, and biomedical applications of exosomes. Science. 2020, 367, eaau6977.
2. Hamish W King, et al.; Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer. 2012, 12:421.
3. Garcia NA, et al.; Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells. PLoS One. 2015, 10(9): e0138849.
4. Raghu Kalluri, Valerie S. LeBleu. Exosomes derived from alcoholtreated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS. Science. 2020, 367, eaau6977.
5. Headland SE, et al.; Cutting-Edge Analysis of Extracellular Microparticles using ImageStreamX Imaging Flow Cytometry. Scientific Reports. 2014, 4(1).