目的 驗證基于吖啶橙(AO)/碘化丙啶(PI)熒光染色原理的自動細胞分析儀檢測細胞因子誘導的殺傷細胞(CIK)數(shù)量及活率的可行性,并對 CIK 細胞培養(yǎng)全過程及凍存復蘇后至使用前過程進行檢測,建立相應的數(shù)量及活率標準。方法 采集健康供者的外周血分離、培養(yǎng) CIK 細胞,取培養(yǎng)過程中的細胞使用 AO/PI 熒光染色法、應用自動細胞分析儀檢測細胞數(shù)量及活率,對結果的專屬性、準確度、精密度、數(shù)量線性及范圍、活率線性及范圍進行驗證。應用建立的方法對外周血分離后的外周血單個核細胞(PBMC)、培養(yǎng)過程中、凍存前及復蘇后的 CIK 細胞數(shù)量及活率進行全過程檢測。結果 專屬性、準確度、精密度、數(shù)量線性及范圍、活率線性及范圍驗證均符合預期要求。CIK 細胞培養(yǎng)全過程活率均不低于 80%,樣本平均倍增時間為(42.26±1.17)h。凍存前的 CIK 細胞在加入含 5% DMSO 的冷凍保護劑后 90 min 內活率穩(wěn)定,復蘇后未經(jīng)稀釋或洗滌處理 120 min 內活率穩(wěn)定,復蘇后經(jīng)洗滌去除 DMSO 處理后 12 h 內活率穩(wěn)定。結論 基于AO/PI熒光染色原理的自動細胞分析儀可以用于檢測 CIK 細胞的數(shù)量及活率,其結果可以用于細胞放行評價及穩(wěn)定性研究。

Objective To verify the feasibility of the automatic cell analyzer based on the fluorescent staining principle of acridine orange (AO)/propyl iodide (PI) to detect the number and viability of cytokine induced killer cells (CIK), and to detect the whole process of CIK cell culture and the process from freezing resuscitation to pre-use, and establish the corresponding number and viability standards. Methods Peripheral blood samples collected from the healthy donors were used to induce CIK cells. The quantity and viability of the cells during culturing were evaluated using AO/PI fluorescence staining method, and the specificity, accuracy, precision, quantity linearity, viability linearity and range were validated. Furthermore, the quantity and viability of PBMC separated from peripheral blood, CIK cells during the cultivation, before freezing and after thawing were evaluated to provide a basis for the monitoring and release of CIK cells. Results The specificity, accuracy, precision, quantity linearity, viability linearity and range all met with the expected criterions. The viability of the entire process of the CIK cell culturing was greater than 80%, and the average population doubling time was (42.26 ± 1.17) h. Before cryopreservation, the viability of CIK cells was stable within 90 min after adding cryoprotectant containing 5% DMSO. After thawing, the viability was stable within 120 min without dilution or washing treatment, and it was stable within 12 hours after washing and removing DMSO. Conclusion Automatic cell analyzer based on AO/PI fluorescence staining can be used to measure the number and viability of CIK cells, and the results can be used for cell release evaluation and stability study.

R965.2

科諾醫(yī)學細胞質量檢測技術公共服務平臺(S-2020-M74-500513)