目的 驗(yàn)證基于吖啶橙(AO)/碘化丙啶(PI)熒光染色原理的自動(dòng)細(xì)胞分析儀檢測(cè)細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞(CIK)數(shù)量及活率的可行性,并對(duì) CIK 細(xì)胞培養(yǎng)全過(guò)程及凍存復(fù)蘇后至使用前過(guò)程進(jìn)行檢測(cè),建立相應(yīng)的數(shù)量及活率標(biāo)準(zhǔn)。方法 采集健康供者的外周血分離、培養(yǎng) CIK 細(xì)胞,取培養(yǎng)過(guò)程中的細(xì)胞使用 AO/PI 熒光染色法、應(yīng)用自動(dòng)細(xì)胞分析儀檢測(cè)細(xì)胞數(shù)量及活率,對(duì)結(jié)果的專(zhuān)屬性、準(zhǔn)確度、精密度、數(shù)量線(xiàn)性及范圍、活率線(xiàn)性及范圍進(jìn)行驗(yàn)證。應(yīng)用建立的方法對(duì)外周血分離后的外周血單個(gè)核細(xì)胞(PBMC)、培養(yǎng)過(guò)程中、凍存前及復(fù)蘇后的 CIK 細(xì)胞數(shù)量及活率進(jìn)行全過(guò)程檢測(cè)。結(jié)果 專(zhuān)屬性、準(zhǔn)確度、精密度、數(shù)量線(xiàn)性及范圍、活率線(xiàn)性及范圍驗(yàn)證均符合預(yù)期要求。CIK 細(xì)胞培養(yǎng)全過(guò)程活率均不低于 80%,樣本平均倍增時(shí)間為(42.26±1.17)h。凍存前的 CIK 細(xì)胞在加入含 5% DMSO 的冷凍保護(hù)劑后 90 min 內(nèi)活率穩(wěn)定,復(fù)蘇后未經(jīng)稀釋或洗滌處理 120 min 內(nèi)活率穩(wěn)定,復(fù)蘇后經(jīng)洗滌去除 DMSO 處理后 12 h 內(nèi)活率穩(wěn)定。結(jié)論 基于AO/PI熒光染色原理的自動(dòng)細(xì)胞分析儀可以用于檢測(cè) CIK 細(xì)胞的數(shù)量及活率,其結(jié)果可以用于細(xì)胞放行評(píng)價(jià)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(xué)細(xì)胞質(zhì)量檢測(cè)技術(shù)公共服務(wù)平臺(tái)(S-2020-M74-500513)