Mass production of extracellular vesicles

Improving cell culture yields

To improve cell culture yields at low cost, the aim is to have the largest possible surface area in the smallest possible volume. A distinction is made between scale-out and scale-up processes. Scale-out is a way to optimize the culture when the platform space is not expandable by using a system allowing the addition of material in parallel, in opposite to scale-up.

Among the techniques used in scale-out, we find, for example, the culture of cells in two-dimensional flasks or hyperflasks, in which exosome production has already been achieved [2]. There is also the use of Hollow Fiber Reactor [1]. That consists of culturing the cells on semi-permeable fibers. The complemented culture medium flows through these fibers, and the exosomes released by the cells accumulate in the external medium, which is not complemented in order to avoid unwanted exogenous EVs, and this medium is then harvested.

In scale-up, it is the use of spinner flasks and bioreactors with cell culture on microcarriers that has mainly developed in recent years [3]. The principle of this technique is as follows: the cells will attach and grow on microcarriers (which are small plastic beads) suspended in a bioreactor containing the culture medium. This greatly increases the surface area available to the cells in a small space and is also scalable and compatible with good manufacturing practices.

Improving exosome production yields

To improve the number of extracellular vesicles produced per cell, the principle is to stimulate them. This can be done by physical or chemical stimulation. The final quantity of EVs produced depends on the number of EVs produced per cell, but also on the uptake of these vesicles by the cells. Both factors can be influenced, although it is sometimes difficult to know which one the stimulation actually affects.

There are many ways of stimulating the cells, and they do not provide the same final output in production. One of the most classic means of stimulation is serum deprivation. Flask-cultured cells are placed in serum-free medium and left for one to several days. The amount of EV produced during this period, is greater than the amount of EV produced by the cells during the same period in the presence of serum [4]. The advantage of this method is that exogenous exosomes are not present.

This gold-standard-method is often used as a comparison for other production methods.

Other existing stimulation methods include hypoxia [5], which consists of placing the cells in a medium with very low oxygen content (between 0.1 and 1% oxygen). There is also placement in acidic and non-neutral pH or heat shock, which places the cells at a high temperature for a few minutes or hours. Moreover, one finds laser irradiation or the addition of certain substances such as anti-inflammatories. 

All these methods show an improvement factor (stimulation/control) between 1.2 and 10.2, except for one anti-inflammatory, Celecoxib 20Um, for which stimulation shows an improvement factor of 23.7 [1]. In comparison, physical stimulation in a 3D bioreactor by agitation inducing shear stresses generates an improvement factor of 22, which is twice as high [6]. Although these figures must be analyzed finely because they vary according to the isolation methods, and the quantity of extracellular vesicles is not the only criterion for judging the viability of a method.

Indeed, the slightest stress inflicted on a cell can impact the characteristics of the exosomes it will produce, either on its physical and chemical properties (size, number, zeta potential…), biological content (proteins, RNA, lipids, metabolites…), purity, stability or therapeutic effect [7]. It is therefore essential to characterize them with different methods. One must check that they are usable afterwards and have the desired therapeutic effect or characteristics. The method used to increase yields must not cause any undesirable. The field has only recently had common recommended standards for EV characterization [8]. This demand for standards of purity, stability, and efficacy testing is increasing in order to develop quality controls.

If the aim is to use these vesicles for therapeutic purposes, it is also necessary to optimize production, bearing in mind that they will have to be produced in accordance with good manufacturing practice [9]. If this is not possible, then the method is not viable for providing EVs for clinical trials.

References 

[1] 10.1016/j.addr.2021.113843 ; Technological advances towards extracellular vesicles mass production, Grangier et Al

[2] R.J. Madel et al., Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially affect symptoms in an advanced murine Graft-versus-Host-Disease model. bioRxiv 2020.12.21.423658 (2020) doi:10.1101/2020.12.21.423658.

[3] De Almeida Fuzeta M et al., 2020, Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System

[4]  J. Li et al., Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles, J. Extracell. Vesicles 4 (2015) 26883.

[5] King, H. W., Michael, M. Z. & Gleadle, J. M. Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer 12, 421 (2012)

[6] Piffoux M et al., Extracellular vesicles for personalized medicine: The input of physically triggered production, loading and theranostic properties, 2019

[7] Colao IL, et al., Manufacturing Exosomes: A Promising Therapeutic Platform 2018

[8] Théry C et al., Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines, 2018

[10] Exosomes in clinical trial and their production in compliance with good manufacturing practice 2020Horng-Jyh Harn, Yu-Shuan Chen, En-Yi Lin, Tzyy-Wen Chiou10.4103/tcmj.tcmj_182_19Tzu Chi Medical Journal