Stem cell and extracellular vesicle products could pass conventional release tests while carrying damaged proteins that undermine their therapeutic performance, according to aRegenesis Repair Rehabilitationreview.
The authors argue that “proteostatic fitness” – the ability of donor cells to maintain protein homeostasis under stress – should be considered a critical quality attribute for stem cell and extracellular vesicle (EV) products. Their proposed framework includes screening for cellular senescence, profiling EV cargo, and establishing exclusion criteria for potentially harmful proteins.
Cell therapy release testing generally assesses characteristics such as identity, viability, sterility, purity, potency, and genetic stability. EV characterization can also include particle size and concentration, surface markers, morphology, contaminants, and endotoxin. However, these assessments do not routinely examine whether cells have accumulated misfolded or oxidized proteins during manufacturing.
The review focuses on senescence arising during ex vivo cell expansion. As stem cells undergo repeated divisions, their heat-shock response, proteasomal activity, and autophagic processes can deteriorate, reducing their ability to identify and clear damaged proteins. Senescent cells can also release inflammatory molecules and EVs whose cargo reflects the condition of the parent cell.
Evidence discussed in the review comes largely from neural stem cell models. In these cells, oxidized superoxide dismutase 1 (SOD1) accumulated during repeated passage alongside markers of senescence. EVs containing oxidized SOD1 were associated with mitochondrial dysfunction and secondary senescence in recipient cells.
To identify affected batches, the authors propose a multiparameter panel measuring markers such as p16 and p21 expression, senescence-associated β-galactosidase activity, and the inflammatory cytokines IL-6 and IL-8. However, no universally accepted thresholds currently exist, and the suggested limits would require harmonization between laboratories before they could support standardized release decisions.
The review also recommends combining discovery-grade mass spectrometry with targeted routine testing. Mass spectrometry could initially characterize beneficial and undesirable EV proteins, including oxidized or misfolded species. Manufacturers could then develop higher-throughput release assays using ELISA, multiplex immunoassays, EV flow cytometry, or antibody and aptamer arrays.
A further proposal involves removing harmful proteins before transplantation. The authors cite preclinical work in which a peptide targeting misfolded SOD1 reduced its burden in the spinal cords of a mouse model of amyotrophic lateral sclerosis. Applying such “cargo sanitization” during manufacturing would require further assessment of scalability, cost, immunogenicity, and off-target effects.
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