Qualification of a multidonor mixed lymphocyte reaction assay for the functional characterization of immunomodulatory extracellular vesicles

EV products obtained from iPSC-derived MSCs show batch-to-batch variations in their ability to modulate allogeneic immune responses in vitro

“…Here, upon pooling mononuclear cells from 12 different donors, strong allogenic immune responses are induced resulting in the activation of CD4 and CD8 T cells that can be recognized by their cell surface expression of CD25 and CD54. As reported in detail previously, the content of activated CD4 as well as of activated CD8 cells can be suppressed partially by immunomodulatory competent MSC-EV preparations ( Bremer et al, 2023 ; Nardi Bauer et al, 2023 ).…”

“…Activities of variable extends were also monitored for the iMSC-EV preparations of all E9 EV preparations. Thus, even though iMSCs obtained in our experiments are capable to release bona fide MSC-EVs, the obtained EV preparations varied non-predictable in their monitored immunomodulatory activity ( Bremer et al, 2023 ; Madel et al, 2023 ). Thus, both, the metric data as well as the data of the functional analyses reveal inconsistencies among the given iMSC-EV preparations but no statistical differences.…”

“…As already mentioned in the introduction, we have observed that EV preparations being manufactured from CM of primary BM-derived MSCs frequently differ in their in vitro studied immunomodulatory capabilities as well as their therapeutic capabilities that have been studied in different animal models ( Wang et al, 2020 ; Van Hoecke et al, 2021 ; Bremer et al, 2023 ; Madel et al, 2023 ; Nardi Bauer et al, 2023 ). In addition to donor-to-donor variations, we observe batch-to-batch variations with some MSC stocks having a higher and others a lower likeliness for the production of potent EV preparations ( Madel et al, 2023 ; Nardi Bauer et al, 2023 ).…”

“…The immunomodulatory activities of samples of the obtained iMSC-EV preparations were evaluated individually within the mdMLR assay exactly as described previously ( Bremer et al, 2023 ; Nardi Bauer et al, 2023 ). Briefly, peripheral blood mononuclear cells (PBMCs) from 12 different donors were isolated by conventional Ficoll density gradient centrifugation and pooled using identical cell numbers from each donor.…”

Exosomes: The New Kid on the Block in Orthobiologics

“…This assay measures the ability of an EV preparation to suppress T-cell activation and to affect monocyte subpopulations. 1 Since modulation of inflammation and immune cell subpopulations is highly relevant to musculoskeletal tissue pathology, such an assay holds promise for orthopaedic applications of EVs.…”