A Method For Enrichment Of CD45~+ Cells And Exosomes Based On Magnetic Compsite Particles

Compared to traditional biopsy,liquid biopsy is considered to be a more practical,real-time and non-invasive method for dignosis,monitoring and treatment of cancer.It is essentially a monitoring assay of the disease,which can be allowed to re-sample according to the patient’s response to the treatment and adjust the strategy of therapy.It plays an important role in personalized and precise medcine.Liquid biopsy markers include circulating tumor cells（CTCs）,circulating tumor DNA（ctDNA）,and Exosomes.In this study,on the one hand,we established a method for enrichment of human peripheral blood CD45⁺cells based on previous work,by optimization of sevral critical conditions including flower-like Fe₃O₄/Au magnatic nanoparticals（FMNPs）functionalization,reaction buffer and blocking buffer,etc.This work will be worthful for the development of negative enrichment method for CTCs enrichment in the furture.During this process,not only the traditional evaluating indicators such as purity,recovery rate,cell activity,and cell apoptosis were investigated.The fragmentation rate,a new indicatior for reflecting the interferrence of red cells and platelet in blood was also performed.On the other hand,we established a noval method for exsome separation from culture medium of cell lines and human plasma by using magnetic composite micro-particles.And then the products was systematically identified.For immune cell sorting system,the results showed that the purity of the target cells after seperation was increased from（83.03±1.22）%to（98.38±0.61）%,the recovery rate increased from（92.07±1.29）%to（98.50±1.35）%,and the fragmentation rate was decreased from（23.73±2.41）%to（6.18%±1.29）%.Cell activity and cell apoptosis state were also successfully comparable to the classical MACS technique（Miltenyi,Germen）.We obtained the same results in CD45⁺cell separation system,seperation purity reached to（99.68±0.27）%,recovery rate was（96.16±1.08）%,and the fragmentation rate was（4.52±0.93）%.For exosome enrichment system,the results showed that the total protein of exosome separated from culture medium of cell line was（1.3±0.09）μg/mL,and（5±0.12）μg/mL from human plasma.Exosome-specific proteins such as CD9,CD81,CD63,TSG101 were estimated to be present in products after enrichment from both of sample resources,while the cell-specific protein Calnexin was not detected.The results were successfully compared to ultracentrifugation,which demonstrating the specificity of the immunomagnetic separation of exosomes based on micron-sized magnetic composite particles.