Establishment of a Conditioned Culture System to Maintain the Activity

of Neutrophils In Vitro for a Long Time

Neutrophil transfusion is a clinical adjunctive anti-infective therapy, and the maintenance of in vitro activity of neutrophils used for transfusion is a key point affecting the effectiveness of this therapy, which is currently a challenging aspect of related research. Therefore, this study explored the combined use of MSC-conditioned medium and some drug molecules to delay neutrophil in vitro senescence and maintain immune function, to overcome the above difficulties. Neutrophils were cultured in vitro using various conditions (including multiple supernatants of MSC culture, pH regulation of culture medium, various drug molecules and their formulas affecting the survival of neutrophils). The in vitro lifespan and biological functions of neutrophils were measured. The experimental results showed that compared with the control group (RPMI-1640 medium), the conditioned medium cultured for three days with MSCs could better prolong the in vitro lifespan of neutrophils while maintaining neutrophil functions such as ROS release and phagocytosis, with significant differences between the two groups (P<0.01). Further experimental screening and verification revealed that compared with the use of conditioned medium cultured for three days with MSCs alone, the optimized neutrophil conditioning medium (MSC-conditioned medium, pH6.0, 1 μmol/L nicotine, 10 ng/mL G-CSF, 0.6 mmol/L NAC, 10 μmol/L DFP) significantly prolonged the in vitro lifespan of neutrophils (P<0.01). In conclusion, this study found that the combination of mesenchymal stem cell-conditioned medium and multiple drug molecules could better maintain the in vitro activity of neutrophils, providing a good experimental basis and theoretical basis for the in vitro preservation and transfusion research of neutrophils.

CSTR: 32200.14.cjcb.2024.07.0006