The cold-shock response in mammalian cells: investigating the HeLa cell cold-shock proteome

Self Cite

Chinese hamster ovary cells (CHO) are routinely used in industry to produce recombinant therapeutic proteins and a number of studies have reported increased recombinant mRNA levels at temperatures <37 degrees C. Surprisingly, the effect of reduced temperature on mRNA translation in CHO cells has not been investigated despite this process being highly responsive to environmental stresses. The relationship between low temperature culturing of CHO cells and mRNA translation was therefore investigated using labeling studies and dual luciferase reporter gene technology. Global protein synthetic capacity was not greatly affected at 32 degrees C but was diminished at lower temperatures. The expression of both cap-dependent and cap-independent (IRES driven) mRNA translated luciferase reporter gene activity was highest at 32 degrees C on a per cell basis and this was partially accounted for by increased mRNA levels. Importantly, post-translational events appear to proceed with higher fidelity and accuracy at 32 than 37 degrees C resulting in increased yield of active protein as opposed to an increase in total polypeptide synthesis. Therefore at 32 degrees C recombinant cap-dependent mRNA translation appears sufficient to maintain recombinant protein yields on a per cell basis and this is associated with improved post-translational processing.

Section: Gel Electrophoresis and Western Blot Analysismentioning

confidence: 99%

Post‐translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells

Masterton¹,

Roobol²,

Al-Fageeh³

et al. 2009

Self Cite

“…In addition to cell metabolic adjustments induced by mild hypothermia, which affected the availability of key NSDs for glycoform formation, the expression of the glycosyltransferases that perform the glycan processing might also be affected. A number of studies have reported changes in the host cell proteome upon sub‐physiological temperature culturing [Baik et al, ; Dietmair et al, ; Underhill and Smales, ], including changes to ER chaperone levels, metabolism, and transport. It is therefore worthwhile to examine the effect of mild hypothermia on the expression of proteins involved in N‐linked glycosylation.…”

Section: Resultsmentioning

confidence: 99%

How does mild hypothermia affect monoclonal antibody glycosylation?

Sou

Sellick²,

Lee³

et al. 2015

The application of mild hypothermic conditions to cell culture is a routine industrial practice used to improve recombinant protein production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimization. In this study, we investigated the impact of mild hypothermia on protein glycosylation. Chinese hamster ovary (CHO) cells expressing a monoclonal antibody (mAb) were cultured at 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32°C and was accompanied by a reduction in intracellular nucleotide sugar donor (NSD) concentrations and a decreased proportion of the more processed glycan structures on the mAb constant region. To better understand CHO cell metabolism at 32°C, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggested reduced fluxes of carbon species towards nucleotide and NSD synthesis and more energy was used for product formation. Expression of the glycosyltransferases that are responsible for N‐linked glycan branching and elongation were significantly lower at 32°C. As a result of mild hypothermia, mAb glycosylation was shown to be affected by both NSD availability and glycosyltransferase expression. The combined experimental/FBA approach generated insight as to how product glycosylation can be impacted by changes in culture temperature. Better feeding strategies can be developed based on the understanding of the metabolic flux distribution. Biotechnol. Bioeng. 2015;112: 1165–1176. © 2014 Wiley Periodicals, Inc.

“…Conversely, the activity of cells detached by the proposed method might be lowered due to the temperature being set at 10 C. Underhill and Smales (2007) reported that proliferation of HeLa cells cultured at 10 C for 24 h decreased by half compared to cells cultured at 37 C. Kaufmann et al (1999) demonstrated that the shift of culture temperature from 37 C to 30 C caused growth arrest, especially in the G1 phase of the cell cycle. Thus, temperature modulation may decrease cell vitality.…”

Section: Discussionmentioning

confidence: 99%

Enzyme‐free cell detachment mediated by resonance vibration with temperature modulation

Kurashina

Hirano

Imashiro

et al. 2017

Cell detachment is an essential process in adherent cell culture. However, trypsinization, which is the most popular detachment technique used in culture, damages cellular membranes. Reducing cellular membrane damage during detachment should improve the quality of cell culture. In this article, we propose an enzyme-free cell detachment method based on resonance vibration with temperature modulation. We developed a culture device that can excite a resonance vibration and control temperature. We then evaluated the cell detachment ratio and the growth response, observed the morphology, and analyzed the cellular protein of the collected cells-mouse myoblast cell line (C2C12). With the temperature of 10°C and the maximum vibration amplitude of 2 μm, 77.9% of cells in number were successfully detached compared with traditional trypsinization. The 72-h proliferation ratio of the reseeded cells was similar to that with trypsinization, whereas the proliferation ratio of proposed method was 12.6% greater than that of trypsinization after freezing and thawing. Moreover, the cells can be collected relatively intact and both intracellular and cell surface proteins in the proposed method were less damaged than in trypsinization. These results show that this method has definite advantages over trypsinization, which indicates that it could be applied to subcultures of cells that are more susceptible to trypsin damage for mass culture of sustainable clinical use. Biotechnol. Bioeng. 2017;114: 2279-2288. © 2017 Wiley Periodicals, Inc.