Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue™
Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue™
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DOI：
10.1016/s0022-1759(01)00347-7
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发表时间：
2001-06-01
影响因子：
2.2
通讯作者：
Lemke, HD
中科院分区：
文献类型：
--
作者：
Gloeckner, H;Jonuleit, T;Lemke, HD
我们描述了一种在小规模中空纤维生物反应器（培养体积：1 ml）中通过使用Aluminium Blue（TM）染料监测细胞增殖的方法。Alcohol Blue（TM）是一种非荧光化合物，其在例如通过活细胞还原后产生荧光产物。与MTT测定相反，Alcohol Blue（TM）测定不会导致细胞死亡。然而，当不从细胞中除去时，Alcohol Blue（TM）染料显示可逆的、时间和浓度依赖性生长抑制，如在白血病细胞系CCRF-CEM、HL-60和REH中观察到的。当应用于中空纤维生物反应器系统的培养基隔室中时，染料穿过中空纤维膜递送至细胞，被细胞还原并从细胞释放回到培养基隔室中。因此，可以在反映细胞隔室中细胞生长的培养基样品中测量荧光强度。这一程序有几个优点。首先，与平板中的常规培养物相比，细胞暴露于染料的量减少。第二，由于不需要取出细胞样品用于读出，因此处理步骤被最小化。此外，为了更换培养基，可以避免离心步骤，并且可以进一步培养细胞。第三，该方法允许区分在生物反应器的细胞隔室中培养的10（5）、10（6）和10（7）个增殖HL-60细胞的细胞密度。特别是对于低于10（6）个细胞/ml的细胞计数，与葡萄糖或乳酸盐测量相比，培养基隔室中的荧光测量不敏感。我们的结论是，与中空纤维生物反应器相结合的Alcohol Blue（TM）测定法为封闭系统中细胞活力和增殖的非侵入性监测提供了明显的优势。(C)出版社：Elsevier Science B. V.
We describe a method for monitoring cell proliferation in a small-scale hollow-fiber bioreactor (culture volume: 1 mi) by use of the Alamar Blue (TM) dye. Alamar Blue (TM) is a non-fluorescent compound, which yields a fluorescent product after reduction, e.g. by living cells. In contrast to the MTT-assay, the Alamar Blue (TM) assay does not lead to cell death. However, when not removed from the cells, the Alamar Blue (TM) dye shows a reversible, time- and concentration-dependent growth inhibition as observed for the leukemic cell lines CCRF-CEM, HL-60 and REH. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye call be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows discriminating between cell densities of 10(5), 10(6) and 10(7) of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is mon sensitive compared to glucose or lactate measurement for cell counts below 10(6) cells/ml, in particular. We conclude that the Alamar Blue (TM) -assay combined with a hollow-fiber bioreactor offers distinct advantages for the nan-invasive monitoring of cell viability and proliferation in a closed system. (C) 2001 Published by Elsevier Science B.V.