Pre-Concentration Freezing Alters the Composition of Mesenchymal Stem/Stromal Cell-Conditioned Medium

Conditioned medium (CM) is a complex mixture of proteins, lipids, nucleic acids, and extracellular vesicles naturally produced by cells. It holds promising potential for medical applications, but maintaining consistent quality across batches is essential to ensure reliable safety and efficacy. This study examined the impact of freezing freshly collected CM at −80 °C, a common step in larger production pipelines, compared to processing it immediately after collection. Results showed that freezing caused a 34% reduction in total protein content, a decrease in the proportion of larger vesicles, and changes in spectral and compositional fingerprints. These findings underscore the significant effects that routine manufacturing steps, such as freezing, can have on CM composition. The sensitivity of biological molecules to temperature shifts, which can disrupt their structural organization, likely accounts for these changes. Addressing such variability is crucial for developing consistent and effective CM-based applications.