Cadelano Francesca, Giannasi Chiara, Gualerzi Alice, Gerli Martina, Niada Stefania, Della Morte Elena, Brini Anna Teresa
Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20100 Milan, Italy.
Laboratory of Biotechnological Applications, IRCCS Istituto Ortopedico Galeazzi, 20157 Milan, Italy.
Biology (Basel). 2025 Feb 10;14(2):181. doi: 10.3390/biology14020181.
Batch-to-batch reproducibility and robust quality assessment are crucial for producing cell-free biologics, such as conditioned medium (CM) derived from mesenchymal stem/stromal cells (MSCs). This study investigated the effects of freezing CM at -80 °C prior to concentration, a step that could occur in large scale pipelines, compared to freshly processed CM. Quality assessment included total protein quantification; extracellular vesicle evaluation using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and cytofluorimetry; and biochemical analysis using Raman spectroscopy. The freezing process resulted in a 34% reduction in total protein content, as confirmed for selected bioactive mediators, and significant depletion of specific particle types, particularly larger ones. Interestingly, the total particle concentration and polydispersity remained stable. Alterations in Raman spectra highlighted changes in protein, lipid, and nucleic acid content. These findings demonstrate that even routine steps like freezing can alter CM composition, likely due to temperature-induced structural changes in biological molecules. Careful consideration of pre- and intra-processing handling temperatures is critical for preserving the integrity of CM and ensuring consistent quality. This study emphasizes the importance of refining manufacturing protocols in the production of cell-free biologics.
批次间的可重复性和稳健的质量评估对于生产无细胞生物制品至关重要，例如源自间充质干/基质细胞（MSC）的条件培养基（CM）。本研究调查了与新鲜处理的CM相比，在浓缩之前于-80°C冷冻CM（这一步骤可能发生在大规模生产流程中）的影响。质量评估包括总蛋白定量；使用纳米颗粒跟踪分析（NTA）、透射电子显微镜（TEM）和细胞荧光分析进行细胞外囊泡评估；以及使用拉曼光谱进行生化分析。冷冻过程导致总蛋白含量降低34%，这在选定的生物活性介质中得到证实，并且特定颗粒类型显著减少，尤其是较大的颗粒。有趣的是，总颗粒浓度和多分散性保持稳定。拉曼光谱的变化突出了蛋白质、脂质和核酸含量的变化。这些发现表明，即使是像冷冻这样的常规步骤也会改变CM的组成，这可能是由于温度诱导生物分子的结构变化所致。仔细考虑处理前和处理过程中的温度对于保持CM的完整性和确保质量一致性至关重要。本研究强调了完善无细胞生物制品生产中的制造方案的重要性。
