Dual Role of Dextran Sulfate 5000 Da as Anti-Apoptotic and Pro-Autophagy Agent

Abstract

Dextran sulfate 5,000 Da (DS), a sulfated polysaccharide, has been used in recombinant mammalian cell cultures to prevent cell aggregation, thereby increasing cell viability. Previous studies using Chinese hamster ovary (CHO) suspension cultures had shown that low concentrations of DS are related to an inhibition of apoptosis. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5,000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold comparing with controls, in standard batch conditions. At a molecular level, we show that DS inhibits apoptosis by DNA fragmentation delay and decrease of annexin V-labeled cells, causes a G0/G1 cell cycle arrest, decreases p53 expression and increases the pro-survival factor Hsc70 expression. DS treatment also resulted in an enhanced LC3-I to LC3-II conversion and increased autophagosomes formation employing tagged-LC3. Our data show, for the first time, that low doses of DS may promote autophagy in different cell lines. These findings suggest that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of protein production.

Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality

“…Dextran sulfate is a sulfated polysaccharide and belongs to a class of molecules with high sulfate group density which includes heparin and sumarin. These molecules have been shown to prevent cell aggregation, promote viability, and increase antibody secretion in a variety of cell types, including CHO and melanoma cells . Generally, dextran sulfate has been used to increase the speed of adaptation from fully adherent to suspension culture or as an anti‐apoptotic agent, rather than to reduce the number of very small clusters of cells to allow efficient single cell cloning .…”

“…These molecules have been shown to prevent cell aggregation, promote viability, and increase antibody secretion in a variety of cell types, including CHO and melanoma cells . Generally, dextran sulfate has been used to increase the speed of adaptation from fully adherent to suspension culture or as an anti‐apoptotic agent, rather than to reduce the number of very small clusters of cells to allow efficient single cell cloning . It can be used in routine cell culture, but addition for one passage before cloning is sufficient to increase the proportion of single cells for CHO cells.…”

6.2 High Cell Density Cultivation Process

“…In standard batch conditions, the use of dextran sulfate 5000 -a sulfated polysaccharide with demonstrated antiapoptotic activity -has shown to prolong the life of CHO cells and increase productivity of EPO by 1.8-fold comparing with controls [42]. Other supplements such as suramin [43] or insulin growth factor [44] may provide apoptotic protection.…”

Hypoxia and transforming growth factor‐beta1 pathway activation promote Chinese Hamster Ovary cell aggregation

“…In our experience, GS‐CHO recombinant cells are generally more tolerant to high aggregation conditions than DG44‐based cells. For sensitive cell lines, cell growth, viability, and protein productivity can be compromised to such an extent that anti‐aggregation agents (such as suramin, heparin, and dextran sulfate) are needed to mitigate the issue (Jing et al, ; Li et al, ; Menvielle, Safini, Tisminetzky, & Skoko, ; Zanghi, Renner, Bailey, & Fussenegger, ). While these anti‐aggregation agents improve cell culture performances in some cases, their use is not straightforward, as the same agent can have variable molecular weights between both lots and vendors, introducing the lot‐to‐lot variability into a bioprocess.…”