血细胞分离机大量采集实验猕猴外周血单核细胞方法探讨

Collection of Large Quantity of PBMCs of Laboratory Rhesus Macaque by Blood Separator

目的使用COBE Spectra血液成分分离机大量采集实验猕猴外周血单核细胞（PBMCs）,继而可从中分离得到外周血造血干细胞（PBSC）,为进一步利用猕猴开展免疫学研究、基因治疗提供足够的目的细胞,探讨采集的关键技术,建立安全、有效的采集方法。方法体重为4～5 kg的实验猕猴5只,采集前一个月内分3次进行自体或异体血液于4℃储备共120 mL,用于采集时填充管路。5只猴采集前接受rhGM-CSF 20μg/kg皮下注射动员4～5 d,麻醉动物后行股动脉穿刺,选择自动外周血干细胞收集程序（Auto-PBSC）进行采集。采集结束后管路中血液以10 mL/min回输给动物3～5 min。结果生长因子连续注射第4天外周血白细胞数增至最高,收获细胞数量随循环血量和采集次数增加而增多。经动员的所有猴能够采集到需要的PBMC,最多达9.9×108,采集次数1～3次,循环血量达750～1420 mL,实验结束后1只猕猴因心脏衰竭死亡。结论人用血细胞分离机可用于4～5 kg实验猕猴PBMC的大量采集。由于动物不同于人体,为保证采集成功需要选用适合于猕猴的程序,采集前做好储血和生长因子动员准备,稳定的麻醉保定,提高抗凝剂比例,积极处理并发症是关键。

Objective A large quantity of peripheral blood mononuclear cells （PBMCs） of laboratory rhesus macaque was isolated by a COBE Spectra blood separator, subsequently more peripheral blood stem cells （PBSC） could be obtained from PBMCs. In this study, we examined the key procedures and to establish a safe and efficient method to get enough cells for immunological research and gene therapy of many diseases. Methods Leukapheresis procedure was performed in 5 male rhesus monkeys with a body mass of 4 - 5 kg. A total of 120 mL autologous or heterogenous blood donation were stored in a bag containing anticoagulant at 4℃ prior to use for priming the apheresis, rhGM-CSF in a dose of 20μg/kg was subcutaneously administered to the animals daily for 4 - 5 days prior to leukapheresis. The femoral artery was cannulated with an intraeath cannula after the monkey was anesthetized. All cases were collected by Anto-PBSC program. After completion of the collection procedure, the whole blood left in the catheter was infused back into the monkeys with a speed of 10 mL/min for 3 - 5 min. Results Peripheral WBC count was increased to a highest value at 4 days after rhGM-CSF administration. PBMCs were collected from 5 eytokine-mobilized monkeys in which the amount of PBMCs was increased along with the increase of peripheral blood volume and harvest times. The collection procedure was successful in all the 5 monkeys. Up to 9.9 × 10^8 PBMCs were obtained after 1 - 3 times of harvesting. The processed blood volume was 750 - 1420 mL. One monkey died of cardiac failure. Conclusion COBE Spectra blood separator for human use can be used to collect a large quantity of PBMCs of monkey with body weight of 4 - 5 kg. To ensure success in the collection, adaptive collection program, donation blood preservation, eytokine-mobilization, raisingthe ratio of anticoagulant, stable aneasthesia, and active treatment of complications during the collection are required.