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Updated: Sep 3, 2025
A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells
Published on: June 23, 2022
Effect of Pre-Processing Storage Condition of Cell Culture-Conditioned Medium on Extracellular Vesicles Derived from
Adrienne Wright1, Orman L Snyder1, Lane K Christenson2
1Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA.
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.
Insights
Storing mesenchymal stromal cell conditioned medium at -80°C before extracellular vesicle isolation preserves EV characteristics and function. This finding supports using stored medium for scalable EV production in clinical applications.
Area of Science:
- Biotechnology
- Cell Biology
- Nanomedicine
Background:
- Mesenchymal stromal cells (MSCs) are utilized in clinical trials, with their conditioned medium (CM) being a source of extracellular vesicles (EVs).
- The impact of pre-processing storage conditions for MSC-derived CM on EV isolation and characteristics remains unclear, hindering clinical translation.
- Standardizing CM storage is crucial for utilizing MSC manufacturing by-products for preclinical and clinical EV applications.
Purpose of the Study:
- To investigate the effects of different pre-processing storage conditions of human umbilical cord-derived MSC (HUC-MSC) conditioned medium on EV isolation, characterization, and function.
- To identify an optimal storage condition for CM to ensure consistent EV yield and quality for potential clinical use.
- To assess the feasibility of storing GMP-grade MSC-derived CM for scalable EV production.
Main Methods:
- Conditioned medium from HUC-MSCs was stored under various conditions (room temperature, 4°C, -20°C, -80°C) for at least one week.
- Extracellular vesicles were isolated from the stored CM, and their characteristics were analyzed (size, zeta potential, morphology, polydispersity index, dot blot staining).
- The functional impact of isolated EVs on sarcoma cell line proliferation was evaluated.
Main Results:
- EVs isolated from CM stored at room temperature, 4°C, -20°C, and -80°C showed no gross differences compared to immediate processing.
- EVs from CM stored at -80°C exhibited a significantly reduced polydispersity index and enhanced dot blot staining.
- No significant differences were observed in zeta potential, hydrodynamic size, morphology, or TEM size across storage conditions; EV function (sarcoma cell proliferation) was unaffected.
Conclusions:
- Storing GMP-grade MSC-derived CM at -80°C prior to EV isolation is a viable option that does not compromise EV characteristics or function.
- This storage strategy enables potential production scale-up for EVs derived from MSC manufacturing by-products.
- The findings support the use of -80°C stored CM for preclinical and clinical testing, facilitating EV lot qualification and translation.
