Exosomes are produced and secreted out by nearly all living cells and have been found to occur in nearly every bodily fluid. They are found abundantly in the growth medium of many cultured cells, such as B lymphocytes, dendritic cells, cytotoxic T cells, intestinal epithelial cells, neurons, oligodendrocytes, platelets, mast cells and Schwann cells.

Given their important role in communicating and mediating bodily activity, exosomes are active in the immune response, reproduction and development, and neural communication, as well as cell proliferation, homeostasis, and maturation. Researchers are interested in exosomes because of their potential across a diverse range of research, diagnostic, analytic and therapeutic applications.

Exosomes have found usage in the diagnosis of cancer and other diseases using the body fluids that include plasma, serum, peritoneal fluid, and saliva. They are also being tested in clinical trials as therapeutic modalities, as well as being utilized for drug delivery.

The Production of Exosomes from Cell Cultures

Different types of cells that are cultured in standard T-flasks have been found to secrete exosomes into the spent cell culture media. As most of the cells cultured for exosome research are anchorage-dependent (that is, they must attach to attach to a surface or matrix), serum-free media is usually not recommended, and some degree and schedule of serum supplementation is also required.

Yet, there are several reports of protocols for the efficient production of exosomes by particular cell types in serum-free, even chemically defined medium. For instance, human-induced pluripotent stem cell-derived cardiovascular progenitors produced exosomes when grown on fibronectin-coated plates in a serum-free medium supplemented with only an added energy source and bFGF I.

Exosomes released from specific human T-cell clones are used to modulate immune cells’ activity, including subsets of other T cell. Studies have indicated that stem cells secrete several important growth factors, and stem cell conditioned medium is therefore required for efficient stem cell culture in vitro. When fetal bovine serum (FBS) is used in the culture, contamination with bovine exosomes is a concern. To avoid this contamination, exosome-depleted commercial versions of bovine serum are now available.

Small-scale manufacture of exosomes usually begins with a culture of adherent cells in a flask or plate in a standard culture medium. Then, it is transferred to either a medium devoid of serum, or medium containing exosome-depleted serum. These cultures are then maintained for a specific period of time and then decanted and processed for any required vesicle isolation, concentration, characterization and purification.

Exosome Production Formats

Currently, most exosome production platforms involve adherent cell culture, and as such, scaling-up activities are focusing on technologies that maximize culture surface area, such as microcarriers in stirred-tank reactors or culture in fixed-bed or hollow-fiber bioreactors.

Researchers adopt approaches such as the use of feeder cells, conditioned media, exogenous extracellular matrices and engineered producer-cell transduction. These methods are useful in some formats for large-scale exosome production.

Large Scale Exosome Production

Larger-scale manufacturing of exosomes is accomplished by using dozens of large T-225 flasks, multiple stacked array multilayer culture flasks, large fixed-bed bioreactors, stirred-tank bioreactors using microcarriers or continuous production in perfusion reactors. Some studies have reported certain challenges associated with the use of stirred-tank bioreactors for exosome production. Perfusion-based production can attenuate some of these challenges and avoid the added process and limitations associated with microcarriers.

Some membrane-based flasks or bioreactors as perfusion-capable technologies are also being used to avoid, or at least alleviate, such issues as those associated with hydrodynamic (shear) forces. These systems enable the culture over an extended period and can concentrate exosomes within a membrane segregated compartment, enabling feeding and harvest. In certain reactors, the cell-containing side of the perfusion apparatus can support the segregation of growth factors, permitting a severe reduction in added serum, added factors or conditioned medium. Fixed-bed bioreactors with published application data are now available for large-scale manufacture of exosomes.

Hollow-fiber perfusion bioreactors are capable of supporting large numbers of cells at high densities in a continuous culture mode without splitting and subculturing of the cells.

The Future of Exosome Manufacturing

In recent years, not only are native or innate exosomes being explored across biotechnology and medicine, but they also are being engineered as therapeutics and therapeutic vectors.

The type of exosome engineering used varies according to the design of production methods. End goals can include the collection of native exosomes, the production of exosomes harboring a particular pharmaceutical cargo, or producing exosomes that can present synthetic surface targeting moieties. Manufacturing of exosomes with therapeutic cargo can be accomplished either in vivo or after secretion via several chemical or physical means.

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