Isolation of Granulocyte Colony Stimulating Factor (G-CSF) Mobilized Peripheral Blood Mononuclear Cells by Apheresis of Blood from the Rhesus Macaque
N. Lalayeva1, R. Love1, R. Manning1, R. Rose1*, N. Makori1*, S. Glaza1, T. Beck1, K. Fukuzaki1, and R. Nagata2
Apheresis facilitates collection of stem cells or other blood components (such as platelets) in the peripheral blood that are subsequently used in the research of blood production and repopulation of blood producing cells for therapies such as for acute radiation syndrome and certain types of bone marrow cancers. Mobilization and apheresis of rhesus macaque blood, however, has proven to be technically challenging due to the inefficient mobilization response after in vivo G-CSF treatment and the high extracorporeal blood volumes required for harvest of these cells. In this study, one animal was administered Neupogen® at a dose of 50 µg/kg once daily for 5 days. Apheresis was performed on the 6th day using the Amicus™ Separator with MNC Collection (Fenwal, Inc). Primary assessments of the collected blood included PBMC isolation, hematology and flow cytometry.
White blood cells in the fresh apheresis product were higher than in the pre-apheresis product. The increase in lymphocytes, monocytes and basophils in the apheresis product was greater than the neutrophil increase. Flow cytometry data showed an upward trend in the absolute counts of CD45+ CD34+, CD45+ CD34high and CD45+ CD34high CD117+ hematopoietic stem cell populations following Neupogen® administration. Using the ACK lysis buffer procedure to remove red blood cells, 1.325 x 109 PBMCs were isolated from 8 mL of apheresis product vs 2 to 2.5×107 PBMCs recovered via a standard isolation procedure. These results confirmed appropriate apheresis and product collection methods were selected, thus establishing a reliable apheresis method in the rhesus macaque.