Functional assays to assess the therapeutic potential of extracellular vesicles

Abstract

An important aspect in the development of extracellular vesicle (EV) therapeutics is identifying and quantifying the key features defining their identity, purity, sterility, potency and stability to ensure batch‐to‐batch reproducibility of their therapeutic efficacy. Apart from EV‐inherent features, therapeutic efficacy depends on a variety of additional parameters, like dosing, frequency of application, and administration route, some of which can be addressed only in clinical trials. Before initiating clinical trials, EV‐inherent features should be tested in well‐standardized quantitative assays in vitro or in appropriate animal models in vivo. Ideally, such assays would predict if a particular EV preparation has the potential to achieve its intended therapeutic effects, and could be further developed into formal potency assays as published by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines. Furthermore, such assays should facilitate the comparison of EV preparations produced in different batches, on different manufacturing platforms or deriving from different cell sources. For now, a wide spectrum of in vitro and in vivo assays has been used to interrogate the therapeutic functions of EVs. However, many cannot accurately predict therapeutic potential. Indeed, several unique challenges make it difficult to set up reliable assays to assess the therapeutic potential of EVs, and to develop such assays into formal potency tests. Here, we discuss challenges and opportunities around in vitro and in vivo testing of EV therapeutic potential, including the need for harmonization, establishment of formal potency assays and novel developments for functional testing.

Optimizing extracellular vesicles’ isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant

“…Functional assay for evaluation of EV isolates. As extensively reviewed by Nguyen et al (50), several assays with an aspect of proliferation assessment have been developed, many of which are very specific to the cells/ biological systems being investigated and dependent on the use of primary cells (83)(84)(85)(86)(87). Primary cells have the disadvantages of limited life span, the need for special supplements for their maintenance, and their limited expansion capacity (50).…”

“…As extensively reviewed by Nguyen et al (50), several assays with an aspect of proliferation assessment have been developed, many of which are very specific to the cells/ biological systems being investigated and dependent on the use of primary cells (83)(84)(85)(86)(87). Primary cells have the disadvantages of limited life span, the need for special supplements for their maintenance, and their limited expansion capacity (50). On the other hand, many biological in vitro experiments have also utilized standardized cell lines like the stromal cell line HS5 where EV doses are incubated with these cells and proliferation is assayed by addition of MTS tetrazolium compound after 72 or 96 hours (26,54).…”

Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production

“…Comparable results were obtained in our study with the improved protocol. Sophisticated microfluidic organ-on-a-chip models are currently also under development enabling technically advanced bone marrow construction that may further advance platelet manufacture at clinical scale [ 13 , 39 ]. A nature-inspired system for functional MK and platelet production using silk-based vascular tubes was already introduced a decade ago [ 37 ].…”

The Role and Application of Salivary Exosomes in Malignant Neoplasms

“…Exosome RNAs that can travel between cells are called "esRNAs", and may involve miRNAs, a class of noncoding mRNAs with sizes that vary from 19 to 25 nucleotides in length and that mediate cell-to-cell communication in the microenvironment, as well as play an important role in tumor diagnosis. [16][17][18]…”