Kushida Taketoshi, Inaba Muneo, Ikebukuro Kazuya, Ichioka Naoya, Esumi Takashi, Oyaizu Haruki, Yoshimura Tomoo, Nagahama Takashi, Nakamura Kouichi, Ito Tomoki, Hisha Hiroko, Sugiura Kikuya, Yasumizu Ryoji, Iida Hirokazu, Ikehara Susumu
First Department of Pathology, Transplantation Center, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan.
Stem Cells. 2002;20(2):155-62. doi: 10.1634/stemcells.20-2-155.
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.
我们之前使用食蟹猴建立了一种新的骨髓细胞（BMCs）采集方法，可使BMCs受外周血T细胞污染降至最低。我们最初在食蟹猴的长骨（肱骨、股骨和胫骨）中进行这种新的“灌注法”。在此，我们将灌注法应用于从食蟹猴的髂骨获取BMCs，因为在人类中通常通过传统抽吸法从髂骨采集BMCs。灌注法包括两种方式：髂骨横向灌注和髂骨纵向灌注。将通过灌注法从长骨和髂骨采集的BMCs与通过抽吸法从髂骨采集的BMCs进行比较。通过CD4⁺和CD8⁺T细胞频率测定的外周血对BMCs的污染，在通过灌注法从髂骨或长骨获得的BMCs中（CD4⁺加CD8⁺T细胞<4%）显著低于通过髂骨抽吸法获得的BMCs（CD4⁺加CD8⁺T细胞>20%）。然而，通过髂骨灌注法获得的BMCs中未成熟髓样细胞（如原粒细胞、早幼粒细胞、中幼粒细胞和晚幼粒细胞）的数量高于通过髂骨抽吸法获得的BMCs。体外培养集落形成单位测定显示，通过灌注法获得的BMCs的祖细胞活性显著高于通过抽吸法获得的BMCs。这些发现表明，使用灌注法时BMCs受外周血污染比使用抽吸法时少得多。为了确定通过灌注法采集BMCs的最佳部位，研究了通过灌注法从长骨和髂骨采集的BMCs的年龄依赖性变化。长骨中BMCs的数量各不相同（肱骨>股骨>胫骨），且呈现年龄依赖性减少，而在3至6岁的食蟹猴髂骨中数量保持相似。然而，在食蟹猴中，发现通过灌注法从髂骨（而非长骨）采集BMCs存在脂肪栓塞风险，尤其是在高压下快速灌注BMCs时。这些发现表明，即使在人类中，灌注法也优于抽吸法，且采集BMCs的最佳部位是肱骨。
