Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures.
Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures.
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DOI：
10.1186/s12896-020-00667-2
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发表时间：
2021-01-08
影响因子：
3.5
通讯作者：
Saski CA
中科院分区：
文献类型：
--
作者：
Chitwood DG;Wang Q;Elliott K;Bullock A;Jordana D;Li Z;Wu C;Harcum SW;Saski CA
在过去的30年里，随着生物过程的强化，哺乳动物细胞过程的产量从每升10毫克增加到每升10克以上。这些生产率的提高大部分可归因于生物反应器内细胞密度的增加。因此，已制定策略，以尽量减少代谢废物的积累，如乳酸和氨。不幸的是，如果没有一些废物代谢物的积累，细胞生长和生物制药生产都不可能发生。不可避免地，代谢废物的积累导致了培养的衰退和终止。虽然我们知道这些不需要的化合物的积累会导致次优的培养环境，但我们对这些化合物可能导致全球基因组不稳定的遗传毒性知之甚少。在这项研究中，我们研究了高和中等水平的细胞外氨对中国仓鼠卵巢（CHO）细胞生理和基因组完整性的影响。通过全基因组测序，我们在功能基因中发现了2394个变异位点，包括单核苷酸多态性和插入/删除突变，这些突变是氨胁迫对功能基因产生高或中等影响的结果。此外，在维持基因组稳定性的基因中发现了一些新生突变，如Tp53、Tnfsf11、Brca1和Nfkb1。此外，我们利用CriGri-PICR中国仓鼠基因组组装对培养物的微卫星含量进行了表征，并发现了大量在氨胁迫培养物中不能忠实复制的微卫星位点。这些基因座的不忠实复制是微卫星不稳定的标志。经过严格的筛选，我们发现124个候选微卫星位点可能适合进一步研究，以确定这些位点是否可以作为预测CHO培养中基因组不稳定性的可靠生物标志物。本研究通过鉴定与基因组稳定性相关的氨敏感基因，提高了我们对氨积累对CHO细胞培养性能影响的认识，并为开发评估基因组稳定性的新诊断工具奠定了基础。在线版本包含补充材料，可在10.1186/s12896-020-00667-2获得。
As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10’s of milligrams to over 10’s of grams per liter. Most of these gains in productivity can be attributed to increasing cell densities within bioreactors. As such, strategies have been developed to minimize accumulation of metabolic wastes, such as lactate and ammonia. Unfortunately, neither cell growth nor biopharmaceutical production can occur without some waste metabolite accumulation. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a suboptimal culture environment, little is known about the genotoxic properties of these compounds that may lead to global genome instability. In this study, we examined the effects of high and moderate extracellular ammonia on the physiology and genomic integrity of Chinese hamster ovary (CHO) cells. Through whole genome sequencing, we discovered 2394 variant sites within functional genes comprised of both single nucleotide polymorphisms and insertion/deletion mutations as a result of ammonia stress with high or moderate impact on functional genes. Furthermore, several of these de novo mutations were found in genes whose functions are to maintain genome stability, such as Tp53, Tnfsf11, Brca1, as well as Nfkb1. Furthermore, we characterized microsatellite content of the cultures using the CriGri-PICR Chinese hamster genome assembly and discovered an abundance of microsatellite loci that are not replicated faithfully in the ammonia-stressed cultures. Unfaithful replication of these loci is a signature of microsatellite instability. With rigorous filtering, we found 124 candidate microsatellite loci that may be suitable for further investigation to determine whether these loci may be reliable biomarkers to predict genome instability in CHO cultures. This study advances our knowledge with regards to the effects of ammonia accumulation on CHO cell culture performance by identifying ammonia-sensitive genes linked to genome stability and lays the foundation for the development of a new diagnostic tool for assessing genome stability. The online version contains supplementary material available at 10.1186/s12896-020-00667-2.
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影响因子：
64.8
作者：
Bartkova, Jirina;Rezaei, Nousin;Gorgoulis, Vassilis G.
通讯作者：
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影响因子：
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作者：
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通讯作者：
Borth, Nicole
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作者：
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