Comparison of Bone Marrow Cells Harvested from Various Bones of Cynomolgus Monkeys at Various Ages by Perfusion or Aspiration Methods: A Preclinical Study for Human BMT

Abstract

Using cynomolgus monkeys, we have previously established a new method for harvesting bone marrow cells (BMCs) with minimal contamination of the BMCs with T cells from the peripheral blood. We originally conducted this new "perfusion method" in the long bones (the humerus, femur, and tibia) of cynomolgus monkeys. Here, we apply the perfusion method to obtain BMCs from the ilium of cynomolgus monkeys, since BMCs are usually collected from the ilium by the conventional aspiration method in humans. The perfusion method consists of two approaches: transverse iliac perfusion and longitudinal iliac perfusion. BMCs harvested by the perfusion method from the long bones and ilium were compared with those collected from the ilium by the aspiration method. The contamination of BMCs with peripheral blood, determined by the frequencies of CD4+ and CD8+ T cells, was significantly lower in BMCs obtained from the ilium or long bones by the perfusion method (CD4+ plus CD8+ T cells <4%) than in those obtained by the iliac aspiration method (CD4+ plus CD8+ T cells >20%). However, the numbers of immature myeloid cells, such as myeloblasts, promyelocytes, myelocytes, and metamyelocytes, were higher in BMCs obtained by the iliac perfusion method than in those obtained by the iliac aspiration method. The assays for in vitro colony-forming unit in culture revealed that progenitor activity was significantly higher in BMCs obtained by the perfusion method than in those obtained by the aspiration method. These findings suggest that the contamination of BMCs with peripheral blood is much less when using the perfusion method than when using the aspiration method. To determine the best site for harvesting BMCs by the perfusion method, age-dependent changes in BMCs harvested by the perfusion method from the long bones and ilium were examined. The numbers of BMCs varied in the long bones (humerus > femur > tibia) and showed age-dependent decreases, whereas they remained similar in the ilium of cynomolgus monkeys from 3 years to 6 years of age. However, in cynomolgus monkeys, BMC harvesting by the perfusion method from the ilium (but not from the long bones) is found to involve the risk of fat emboli, particularly when the BMCs are quickly perfused under high pressure. These findings suggest, even in humans, that the perfusion method is better than the aspiration method, and that the best site for collection of BMCs is the humerus.