CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities.

BACKGROUND AIMS: Extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) show immunomodulatory activity in different assays both in vitro and in vivo. In previous work, the authors compared the immunomodulatory potential of independent MSC-EV preparations in a multi-donor mixed lymphocyte reaction (mdMLR) assay and an optimized steroid-refractory acute graft-versus-host disease (aGVHD) mouse model. The authors observed that only a proportion of the MSC-EV preparations showed immunomodulatory capabilities and demonstrated that only MSC-EV preparations with mdMLR immunomodulating activities were able to suppress aGVHD symptoms in vivo and vice versa. Since the mdMLR assay is complex and depends on primary human cells of different donors, the authors sought to establish an assay that is much easier to standardize and fulfills the requirements for becoming qualified as a potency assay. METHODS: The bona fide MSC antigen CD73 possesses ecto-5'-nucleotidase activity that cleaves pro-inflammatory extracellular adenosine monophosphate into anti-inflammatory adenosine and free phosphate. To test whether the ecto-5'-nucleotidase activity of the MSC-EV preparations reflected their immunomodulatory potential, the authors adopted an enzymatic assay that monitors the ecto-5'-nucleotidase activity of CD73 in a quantitative manner and compared the activity of well-characterized MSC-EV preparations containing or lacking mdMLR immunomodulatory activity. RESULTS: The authors showed that the ecto-5'-nucleotidase activity of the MSC-EV preparations did not correlate with their ability to modulate T-cell responses in the mdMLR assay and thus with their potency in improving disease symptomatology in the optimized mouse aGVHD model. Furthermore, the ecto-5'-nucleotidase activity was resistant to EV-destroying detergent treatment. CONCLUSIONS: Ecto-5'-nucleotidase activity neither reflects the potency of the authors' MSC-EV preparations nor provides any information about the integrity of the respective EVs. Thus, ecto-5'-nucleotidase enzyme activity is not indicative for the immunomodulatory potency of the authors' MSC-EV products. The development of appropriate potency assays for MSC-EV products remains challenging.

Scaled preparation of extracellular vesicles from conditioned media.

Extracellular vesicles (EVs) especially of mesenchymal stem/stomal cells (MSCs) are increasingly considered as biotherapeutic agents for a variety of different diseases. For translating them effectively into the clinics, scalable production processes fulfilling good manufacturing practice (GMP) are needed. Like for other biotherapeutic agents, the manufacturing of EV products can be subdivided in the upstream and downstream processing and the subsequent quality control, each of them containing several unit operations. During upstream processing (USP), cells are isolated, stored (cell banking) and expanded; furthermore, EV-containing conditioned media are produced. During downstream processing (DSP), conditioned media (CM) are processed to obtain concentrated and purified EV products. CM are either stored until DSP or are directly processed. As first unit operation in DSP, clarification removes remaining cells, debris and other larger impurities. The key operations of each EV DSP is volume-reduction combined with purification of the concentrated EVs. Most of the EV preparation methods used in conventional research labs including differential centrifugation procedures are limited in their scalability. Consequently, it is a major challenge in the therapeutic EV field to identify appropriate EV concentration and purification methods allowing scale up. As EVs share several features with enveloped viruses, that are used for more than two decades in the clinics now, several principles can be adopted to EV manufacturing. Here, we introduce and discuss volume reducing and purification methods frequently used for viruses and analyze their value for the manufacturing of EV-based therapeutics.