7-AAD/annexin V 동시 염색법을 이용한 제대혈의 세포생존율 평가
Assessment of Cell Viability in Umbilical Cord Blood by Using 7-AAD/annexin V Dual Staining
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배경: 세포생존율은 제대혈의 품질을 결정하는 중요한 지표이다. 제대혈의 세포생존율을 측정하기 위해 trypan blue나 7-aminoactinomycin (7-AAD)를 이용한 방법이 일반적으로 사용되고 있으나, 이들 방법은 초기세포사멸은 검출할 수 없다. 저자들은 7-AAD와 초기세포사멸을 반영하는 annexin V를 동시에 이용하여 제대혈 채취 후 시간 경과에 따른 세포생존율을 측정하고, 기존의 trypan blue 및 7-AAD를 이용한 방법과 비교하고자 하였다. 방법: 2012년 7월부터 2013년 3월까지 분당차병원에서 출산한 산모 30명에서 채취한 제대혈을 대상으로 하였다. 제대혈은 채취 직후, 24, 48시간이 경과한 시점에서 trypan blue, 7-AAD, 7-AAD/annexin V를 이용하여 세포생존율을 반복 측정하였다. 결과: 7-AAD/annexin V를 이용하여 제대혈 채취 직후, 24, 48시간 경과 후에 측정한 총유핵세포의 생존율은 측정시간 순서대로 각각 92.8±4.5%, 78.4±7.8%, 65.5±8.1%, 단핵구는 94.4±1.7%, 90.8±4.2%, 84.2±6.7%, CD34 양성세포군은 92.4±3.0%, 90.7±4.7%, 89.3±7.0%였다. Trypan blue법으로 측정한 세포생존율은 48시간까지 90% 이상을 보였다. 결론: 7-AAD/annexin V를 이용해 측정한 제대혈의 세포생존율은 총유핵세포의 경우 24시간 경과 시점부터 평균 80% 이하였으나, 제대혈이식에서 생착에 가장 중요한 CD34 양성세포의 생존율은 48시간 경과 시점까지 85% 이상을 유지하였다. 저자들은 본 연구 결과가 향후 제대혈의 품질을 평가하는 기초자료로 활용될 수 있을 것으로 기대한다.
Abstract
Background: The quality of cord blood largely depends on cell viability. Viability assessments using trypan blue or 7-aminoactinomycin (7-AAD) staining, which are commonly used methods, may not reflect early apoptosis of cord blood cells. We aimed to investigate early apoptosis in cord blood cells following elapsed time after collection using double staining with annexin V and 7-AAD and to compare the result with that of viability evaluation using trypan blue or 7-AAD staining. Methods: Umbilical cord blood samples were obtained from 30 pregnant women at the time of delivery between July 2012 and March 2013. Viability of cord blood cells was determined at 0 (T0), 24, and 48 hr after collection by using trypan blue exclusion assay, 7-AAD staining, and 7-AAD/annexin V staining. Results: Viabilities defined by 7-AAD/annexin V staining at T0, 24, and 48 hr after collection were respectively as follows: total nucleated cells, 92.8±4.5%, 78.4±7.8%, and 65.5±8.1%; mononuclear cells, 94.4±1.7%, 90.8±4.2%, and 84.2±6.7%; and CD34-positive cells, 92.4±3.0%, 90.7±4.7%, and 89.3±7.0%. The viability using trypan blue was more than 90% until 48 hr after collection. Conclusions: The mean viability of total nucleated cells using 7-AAD/annexin V staining decreased to less than 80% at 24 hr after collection; however, the viability of CD34-positive cells was more than 85% until 48 hr. Our study’s data will provide useful information for the assessing the quality of cord blood products.
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