ABSTRACT

These results suggest that simultaneous transplantation of PIs and BMCs via the PV is effective in inducing persistent tolerance.

To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new "perfusion method" for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate-buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4 + and CD8 + T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this "perfusion method" are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the "perfusion method" is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft-versus-hostdisease in allogeneic BM transplantation.

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, agedependent 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 STEM CELLS 2002;20:155-162 www.StemCells.com Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara@takii.kmu.ac.jp Received October 17, 2001; accepted

No abstract