Production of Exosomes: Human Cell Lines and Cultivation Modes

With the advancement of biotechnology, research on exosomes has deepened, and an increasing number of research institutions and biotech companies are venturing into the production and application of exosomes. As a result, exosome production technologies have seen significant improvements. This article summarizes the currently popular human cell lines and production methods used for therapeutic exosome production.

Common Human Cell Lines

HEK293 cells:

• HEK293 cells are immortalized cells obtained from human embryonic kidney cells and are widely used in biotech applications for gene expression, protein production, and viral packaging. Now almost all drug-loaded exosomes are based on HEK293 cells. Their features, such as ease of culture, transfection efficacy and growth, has kept them at the forefront of research and biotechnology for the past 30 years. More notably, the highly exosome-producing, neutral phenotype subtypes of HEK293T and HEK293F are good candidates for modeling exosome drug carriers.
• Safety: Although HEK293 is not a cancer cell line, its immortalization can be oncogenic or toxic through exosomes. Yet, researchers have observed that exosomes derived from HEK293T contain only a few disease- or cancer-related proteins, and exert little physiological or pathological effect. Additionally, HEK293 exosomes were very nontoxic, non-immunogenic and not pro-inflammatory when applied at various concentrations to other cells (including macrophages and HepG2), indicating low toxicity and good safety both in vitro and in vivo.

Mesenchymal stem cells (MSCs):

• MSCs are multipotent stem cells that can be isolated from various human tissues, such as bone marrow, adipose, amniotic fluid, and umbilical cord blood. They can differentiate into adipocytes, chondrocytes, and osteocytes and are widely studied in clinical trials for their exceptional repair and regenerative abilities. MSC-derived exosomes (MSC-EXOs) have been clinically effective and could be therapeutic candidates. In terms of exosome production, MSCs offer an exceptional exosome yield per cell and are able to be therapeutically applied without intricate engineering. Today, 3D culture technologies have drastically enhanced exosome production.
• Safety: MSC-EXOs have a significant safety advantage, since they are less susceptible to immune reactions than other allogenic molecules. But keep in mind that while their proliferative effect aids in tissue repair, it also can help cancer develop under certain conditions. For example, the introduction of oncogenes to immortalise MSC might create tumours.

Neural stem cells (NSCs):

• NSCs are cells with self-renewal ability and multi-lineage differentiation potential, existing in the embryonic and adult nervous systems, primarily responsible for the development, maintenance, and repair of the central nervous system. They can differentiate into various neural cells such as neurons and glial cells. Their exosomes are rich in neurotrophic factors and specific miRNAs. These exosomes not only promote neural repair but also regulate neuroinflammation. Research indicates that NSCs can release exosomes to promote neurogenesis and reduce host endogenous neuronal cell death, showing tremendous potential in nerve injury repair and regenerative medicine.
• Safety: NSCs and their exosomes are generally considered safe in clinical applications. However, NSC transplantation poses safety concerns that might affect exosome safety evaluations, such as teratoma formation, abnormal cell proliferation, immune rejection, and undesirable cell phenotype differentiation, need to be considered in research. Furthermore, the cultivation of neural stem cells is relatively complex, which poses difficulties in the production of neural stem cell-derived exosomes.

Dendritic cells (DCs):

• As antigen-presenting cells, DCs play a crucial role between innate and adaptive immunity, mediating immune responses by inducing antigen-specific T cell immune reactions. DC-derived exosomes (Dex) possess unique advantages in stimulating anti-tumor immune responses due to their ability to present antigens and immune-related proteins, and have been employed in various clinical tests for cancer vaccinations and therapies. Dex can be engineered through transfection or viral transduction to display specific immunoregulatory proteins or targeting molecules on the exosome surface, increasing efficiency in targeting specific cells like neurons and microglia. However, the limited quantity of exosomes produced by immature DCs poses a challenge for their application.
• Safety: Dex has been demonstrated to have good safety in multiple clinical trials. Exosomes produced by autologous immature DCs exhibit low immunogenicity and toxicity due to the lack of immunostimulatory surface markers. However, it is noteworthy that mature DC-derived exosomes might increase risks of inflammation and arteriosclerosis.

Human amniotic epithelial cells (hAECs):

• hAECs originate from the embryonic ectoderm and possess embryonic stem cell-like differentiation capabilities and adult stem cell immunomodulatory properties. Exosomes secreted by hAECs show promising applications in regenerative medicine, enhancing lung repair after injury, expediting wound healing, and displaying significant efficacy in treating conditions such as ovarian failure.

CAR-T cells:

• CAR-T cells are genetically engineered T cells expressing specific chimeric antigen receptors that can recognize and kill cells bearing specific antigens. Exosomes derived from CAR-T cells inherit CAR-T cell targeting capabilities, allowing for precise delivery of therapeutic molecules.

Main Cellular Cultivation Modes for Exosome Production

Small-scale static culture

This refers to the method of cultivating cells in flat containers like flasks, petri dishes, and T-flasks in a static manner for exosome production, suitable for initial experiments and small-scale exosome preparation. Appropriate for adherent cells, which grow as a monolayer on the bottom of the culture vessel, it boasts simplicity, low equipment requirements, and relative cost-effectiveness. However, its drawbacks include limited yield and difficulty in controlling culture conditions, with low efficiency in nutrient and waste exchange.

Scale-up culture

• Hollow-fiber bioreactor: This equipment, composed of semi-permeable capillary fibers and a cylinder, allows cells to reside within the fibers. The media reaches cells by diffusion, with cell metabolites being discharged. This system replicates an in vivo microenvironment, efficiently providing nutrient and waste exchange, and permits continuous exosome collection. It is suitable for high-density cell culture, greatly enhancing exosome output, and is applicable for high yield and high purity exosome applications. Nonetheless, its complexity, high initial investment cost, and technical demands on operators are considerations.
• Stirred bioreactor: This device offers uniform mixing and adequate oxygenation through stirring and aeration, appropriate for exosome production from suspension-adapted cells. Stirred bioreactors allow for adjustments in stirring speed and aeration strategies to optimize cell growth and exosome release, offering high flexibility and large production capacity. However, the shear forces generated by stirring may adversely affect certain cell types, requiring careful adjustment of operational parameters to avoid cell damage.

Reference

1. Kim J, Song Y, et al. Platform technologies and human cell lines for the production of therapeutic exosomes. Extracellular Vesicles and Circulating Nucleic Acids. 2021. 2(1), 3-17.

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