Effect of Pre-Processing Storage Condition of Cell Culture-Conditioned Medium on Extracellular Vesicles Derived from Human Umbilical Cord-Derived Mesenchymal Stromal Cells

Abstract

EVs can be isolated from a conditioned medium derived from mesenchymal stromal cells (MSCs), yet the effect of the pre-processing storage condition of the cell culture-conditioned medium prior to EV isolation is not well-understood. Since MSCs are already in clinical trials, the GMP-grade of the medium which is derived from their manufacturing might have the utility for preclinical testing, and perhaps, for clinical translation, so the impact of pre-processing storage condition on EV isolation is a barrier for utilization of this MSC manufacturing by-product. To address this problem, the effects of the pre-processing storage conditions on EV isolation, characterization, and function were assessed using a conditioned medium (CM) derived from human umbilical cord-derived MSCs (HUC-MSCs). Hypothesis: The comparison of three different pre-processing storage conditions of CM immediately processed for EV isolation would reveal differences in EVs, and thus, suggest an optimal pre-processing storage condition. The results showed that EVs derived from a CM stored at room temperature, 4 °C, −20 °C, and −80 °C for at least one week were not grossly different from EVs isolated from the CM immediately after collection. EVs derived from an in pre-processing −80 °C storage condition had a significantly reduced polydispersity index, and significantly enhanced dot blot staining, but their zeta potential, hydrodynamic size, morphology and size in transmission electron microscopy were not significantly different from EVs derived from the CM immediately processed for isolation. There was no impact of pre-processing storage condition on the proliferation of sarcoma cell lines exposed to EVs. These data suggest that the CM produced during GMP-manufacturing of MSCs for clinical applications might be stored at −80 °C prior to EV isolation, and this may enable production scale-up, and thus, and enable preclinical and clinical testing, and EV lot qualification.

Procoagulant Activity of Umbilical Cord-Derived Mesenchymal Stromal Cells’ Extracellular Vesicles (MSC-EVs)

“…Similar trends were observed when they were plotted as nanoparticles released per MSC cell (Supplementary Figure S2G-K). The median of about 20,000 nanoparticles per canine MSC in early passages (Supplementary Figure S2H) was less than what we reported for human MSCs in the early passages following 24 h of medium conditioning [57].…”

“…PHG0264) was replaced with DMEM (0.33 mL/cm 2 ) without fetal bovine serum (FBS) or other supplements when flasks reached 60-70% cell confluence. After 24 h, CM was collected and clarified to remove cells and cellular debris as described [57]. Before EV isolation, CM was stored at −80 • C since previous work found this to be the optimal storage condition for MSC-derived EVs [57].…”

“…After 24 h, CM was collected and clarified to remove cells and cellular debris as described [57]. Before EV isolation, CM was stored at −80 • C since previous work found this to be the optimal storage condition for MSC-derived EVs [57]. EV consistency between cell lines was assured using cell lines with viability ≥ 95% during passaging.…”

Extracellular Vesicles as Therapeutic Resources in the Clinical Environment

“…Regarding EVs produce from in vitro cell cultures, culture media can be stored without altering EV concentration, which remained stable after one week of storage at 4 • C, −20 • C, and −80 • C. However, EV miRNA levels decreased during this period, especially when the media were stored at 4 • C and −20 • C. After 30 days of storage, the EV miRNA content dropped to below 50% compared to the initial amount [125,126].…”

Modern aspects of external application and prospects of using the secretome of mesenchymal stem cells (review)

“…Некоторые авторы [27] описывают условия хранения при -20 °C и при +4 °C, что, по их данным, не влияло активность СМСК. Ингибиторы протеазы были добавлены к секретомам лишь в нескольких исследованиях [24,28].…”