Process Development in Isolation and Culture of HumanMonocytes from Cord Blood; Simple and Cos t-EffectiveMethod

Objective: Macrophages are multifunctional immune cells thatare widely used for immunological research. To s tudy the functionof macrophages in vitro, umbilical cord blood is a convenientand non-invasive source that provides sufficient numbersof monocytes and macrophages. Monocyte isolation proceduresvary widely in terms of purity, yield, cos t, and technical difficulty.We aimed to find an optimized protocol for monocyte isolationfrom cord blood and compare the effect of three differentvariables in monocyte differentiation and culture techniques: ۱.Cell culture media; ۲. Serum sources and ۳. Cell culture systemson cellular phenotype, adhesion, and viability.Materials and Methods: We used double density gradient centrifugationtechniques (Ficoll® and hyperosmotic Percoll®) formonocyte isolation and flow cytometry analysis of light scatteringand/or expression of pan surface markers, such as CD۳,CD۱۴, and CD۱۹ to determine cell viability and purity. Afterpurification, cells were cultured in adhesion preventing tissueculture plates in either RPMI-۱۶۴۰ or DMEM-F۱۲ or IMDMculture medium supplemented with either ۰.۵-۱۰% fetal bovineserum (FBS), human serum (HS), or human platelet lysate(hPL).Results: We introduce a novel, reproducible, and inexpensiveisolation method that yields monocytes with high purity(from۷۰ to ۹۸%). Also, the results showed that monocyte adhesionto the plate is significantly increased in the presence of alow concentration of serum, and IMDM containing ۵% hPL canbe the bes t option for macrophage cultivation and differentiation.Conclusion: This optimized method is a simple and cos t-effectivescale-up method to provide uniform and pure monocytesfor s tudies of the innate defense sys tem.

کلیدواژه ها:

Cell Viability ، Innate Immune Sys tem ، Monocyte ، Umbilical Cord Blood