Kromenaker S J, Srienc F
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis 55455.
Biotechnol Prog. 1994 May-Jun;10(3):299-307. doi: 10.1021/bp00027a010.
Flow cytometry was used in combination with immunofluorescent staining for intracellular and surface-associated antibody contents to identify a significant non-producer cell fraction in a murine hybridoma cell line that had shown a decline in monoclonal antibody productivity with passaging. Viable producer cells stained for surface-associated antibody content were isolated by cell sorting on the basis of surface fluorescence intensity. The fraction of nonproducers was initially reduced from 85% to 20%. Sorting a second time, after these cells were cultivated for 2 weeks, further reduced the fraction of nonproducers to less than 4%. The stability of this purified producer hybridoma cell line during passaging and after a freeze-thaw cycle was investigated. This cell line was found to be highly unstable. A simple batch-growth model simulation was used as follows: (i) to demonstrate that nonproducers appear in hybridoma cell lines after a rare, random mutation event that results in the loss of the heavy-chain gene and/or light-chain gene expression; (ii) to show that a high rate of conversion of producer hybridomas to nonproducer hybridomas cannot entirely explain the population dynamics; (iii) to estimate the rate of conversion of producers to nonproducers to be 8.7 x 10(-5) h-1; (iv) to show that, for this conversion rate, the nonproducer cells' specific growth rates need only be 9% higher than those of the producer cells to dominate the hybridoma culture after only 25 passages; and (v) to predict that producer cells are preferentially lost during cell freezing and thawing procedures. The data suggest that particularly unstable cell lines should be analyzed and purified frequently to prevent overgrowth by nonproducers.
流式细胞术与免疫荧光染色相结合，用于检测细胞内和表面相关抗体含量，以鉴定小鼠杂交瘤细胞系中显著的非产生细胞部分，该细胞系在传代过程中显示单克隆抗体生产力下降。通过基于表面荧光强度的细胞分选，分离出表面相关抗体含量染色的活的产生细胞。非产生细胞的比例最初从85%降至20%。在这些细胞培养2周后再次分选，进一步将非产生细胞的比例降至4%以下。研究了这种纯化的产生杂交瘤细胞系在传代和冻融循环后的稳定性。发现该细胞系高度不稳定。使用了一个简单的分批生长模型模拟，如下所示：（i）证明非产生细胞在杂交瘤细胞系中出现是由于罕见的随机突变事件导致重链基因和/或轻链基因表达缺失；（ii）表明产生杂交瘤向非产生杂交瘤的高转化率不能完全解释群体动态；（iii）估计产生细胞向非产生细胞的转化率为8.7×10^(-5) h^(-1)；（iv）表明对于这种转化率，非产生细胞的比生长速率仅需比产生细胞高9%，就能在仅25次传代后主导杂交瘤培养；（v）预测在细胞冻融过程中产生细胞优先丢失。数据表明，特别不稳定的细胞系应经常进行分析和纯化，以防止非产生细胞过度生长。
