Gabrijel Mateja, Kreft Marko, Zorec Robert
Celica Biomedical Center, Proletarska cesta 4, 1000 Ljubljana, Slovenia.
Biochim Biophys Acta. 2008 Feb;1778(2):483-90. doi: 10.1016/j.bbamem.2007.10.013. Epub 2007 Oct 23.
Dendritic and tumor cells are fused to produce hybridoma cells, which are considered to be used as cellular vaccines to treat cancer. Previous strategies for hybridoma cell production were based on the quantification of the electrofusion yield by labeling the cytoplasm of both parental cell types. However, a better physiological strategy would be to label subcellular structures related directly to the antigen presentation process. Therefore, we here electrofused the same amount of CHO cells stained with red and green fluorescent dextrans and have monitored the yield of hybridoma cell formation by measuring the fusion of red and green late endocytic organelles that are involved in antigen presentation. By using confocal microscopy, the level of fused, fluorescently labelled late endocytic compartments in a single hybridoma cell was determined. The results demonstrate that organellar fusion occurs in hybridomas, which is time- and temperature-dependent. This approach therefore provides a new method for the hybridoma cell vaccine evaluation, which is based on the intracellular physiological mechanism of antigen presentation.
树突状细胞与肿瘤细胞融合产生杂交瘤细胞，这些杂交瘤细胞被认为可作为细胞疫苗用于治疗癌症。以往生产杂交瘤细胞的策略是通过标记两种亲本细胞类型的细胞质来量化电融合产量。然而，一种更好的生理学策略是标记与抗原呈递过程直接相关的亚细胞结构。因此，我们在此将等量用红色和绿色荧光葡聚糖染色的CHO细胞进行电融合，并通过测量参与抗原呈递的红色和绿色晚期内吞细胞器的融合来监测杂交瘤细胞形成的产量。通过共聚焦显微镜，确定单个杂交瘤细胞中融合的、荧光标记的晚期内吞区室的水平。结果表明，杂交瘤中发生了细胞器融合，这是时间和温度依赖性的。因此，这种方法基于抗原呈递的细胞内生理机制，为杂交瘤细胞疫苗评估提供了一种新方法。
