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Causes of Cell Clustering
Suspended cells may adhere to each other and form clusters for various reasons. The most common reason for cell clustering is the presence of free DNA and cell fragments in the culture medium after cell lysis. The stickiness of DNA causes cells and other fragments to aggregate into large clumps. Analyzing the reasons for cell clustering in the bioprocess can be attributed to two factors:
Animal cells themselves may inherently exhibit cell clustering phenomena, such as CHOS cells.
Animal cells are prone to aggregate in unfavorable nutritional conditions and environments, and the concentration of ions like Ca2+ in the culture medium can also affect cell adhesion.
Effects of Clustering
The cell microenvironment deteriorates, leading to:
Insufficient oxygen supply
Accumulation of metabolic waste
Nutrient deficiencies
Cell death
Accumulation of metabolic waste
Nutrient deficiencies
Cell death
How to Counteract Cell Clustering?
Substances commonly used to counteract cell clustering are polysulfates, such as sodium heparin, acetylated heparin sulfate, suramin, and sodium heparan sulfate. Among various polysulfate compounds, sodium heparan sulfate is the most widely used anti-clustering agent in CHO cell suspension culture.
Principle of Sodium Heparan Sulfate Anti-Clustering
Sodium dextran sulfate (Dextran sulfate sodium salt, DSS) is an anionic derivative of dextran formed by esterification of dextran and chlorosulfonic acid, provided in the form of sodium salt, and stabilized by the addition of a small amount of phosphate.
Effects of Different Molecular Weights and Concentrations of Sodium Heparan Sulfate [1]
In 2016, scientists studied the impact of three different molecular weights of sodium heparan sulfate (4000 Da, 15000 Da, and 40000 Da, corresponding to S ratios of 3-6%, 17-19%, and 15-19%, respectively), as well as different concentrations (0.1-1.0 g/L) on the growth and monoclonal antibody production of two different recombinant CHO cells (CHO-K1 cells and DG44 cells). They also assessed the influence of adding sodium heparan sulfate on critical quality attributes of antibodies (such as aggregates, charge variants, and N-glycosylation, etc.).
Preliminary research revealed that using 200,000 Da sodium heparan sulfate, regardless of the concentration (0.1-1.0 g/L), led to a 20-30% decrease in maximum viable cell density and antibody concentration. This indicates that using 200,000 Da sodium heparan sulfate is harmful to cells. Adding different molecular weights and concentrations of sodium heparan sulfate for CHOK1 cells can significantly maintain late-stage cell density and vitality, with no corresponding increase in final antibody expression.
For DG44 cells, the addition of sodium heparan sulfate, especially with a molecular weight of 40,000 Da, significantly reduces cell clustering and significantly increases antibody product concentration. Research on the impact of sodium heparan sulfate on antibody quality attributes showed that for DG44 cells, the addition of 4000 Da and 15000 Da sodium heparan sulfate had no effect on monoclonal antibody charge variants (P<0.05), with a significant reduction in basic charge variants only observed with the addition of 40,000 Da sodium heparan sulfate [1].
Adding sodium heparan sulfate has no negative impact on monoclonal antibody quality attributes (aggregates, charge variants, and N-glycosylation), and can even have a positive impact on some quality attributes, such as influencing monoclonal antibody charge variants by reducing basic peaks.
In conclusion, the impact on cell growth, antibody production, and quality depends on the molecular weight and concentration of sodium heparan sulfate and the choice of CHO cell lines. Therefore, for different CHO cell lines, it is recommended to optimize the best molecular weight and concentration of sodium heparan sulfate to maximize its beneficial effects in monoclonal antibody production [1].
Currently, there are also many applications of sodium heparan sulfate, Mw 5,000, with an average molecular weight of about 5 kDa, to promote individual cell suspension and prevent cell clustering by altering the cell surface charge, supporting high-density cell culture, and improving cell vitality.
Seebio® offers high-quality Sodium Heparin Sulfate
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Parameter
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Specification
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Total Sulfur Content
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15~20%
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Sulfate Ion Content
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≤0.2%
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Heat Loss (105°C, 2h)
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≤10.0%
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pH
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5.0~7.5
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Optical Rotation
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+75°~ +105°
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Iron Ion
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≤2.0%
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Chloride Content
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≤1000 ppm
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Appearance
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White~Light Yellow Powder
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Microbial Limits
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Aerobic Bacteria < 10^5 cfu/g
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Mold and Yeast Count < 100 cfu/g
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Endotoxin
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<5 EU/g
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Excellent Performance: High purity with sulfur content ranging from 15% to 20%.
High Stability: Anionic complex that is soluble in water, forming a colorless solution similar to natural polysaccharides.
Low Endotoxin: Less than 5 EU/g, causing minimal harm to cells.
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Catalog Number
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Product Name
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Grade
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DCJ0018D
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Heparin Sulfate 5000
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for cell culture
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DCJ1606D
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Heparin Sulfate 40000
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for cell culture
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Seebio Sodium Heparin Sulfate Series:
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Sodium Heparin Sulfate
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Intestinal Model
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Molecular Biology
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Antiviral
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Anticoagulant
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Antilipidemic
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Cosmetics Research
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6000-8000
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√
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√
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√
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8000 (Average)
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√
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√
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√
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36000-50000
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√
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√
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√
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|
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400000-600000
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√
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√
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√
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1400000(Average)
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√
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√
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Different Types of Sodium Heparin Sulfate (DSS) for Various Applications:
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Catalog Number
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Product Name
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Average Molecular Weight
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Packaging
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DCJ0083A
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Sodium Heparin Sulfate (DSS)
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Mw 3,000
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100g, 500g, 5Kg, 25Kg
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DCJ0018A
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Mw 5,000
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100g, 500g, 5Kg, 25Kg
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|
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1840080
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Mw 8,000
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100g, 500g, 5Kg, 25Kg
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ACJ1516E
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Mw 15,000
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100g, 500g, 5Kg, 25Kg
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DCJ1606A
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Mw 40,000
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100g, 500g, 5Kg, 25Kg
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DCJ0105A
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Mw 36,000~50,000
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50g, 100g
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DCJ0016A
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Mw 500,000
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100g, 500g, 5Kg, 25Kg
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Seebio can provide sulfated heparin sodium in various specifications, with average molecular weights ranging from 3,000 to 500,000. Please feel free to call us at: service@seebio.cn or Phone: +86 21 58183719
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