Cell Culture Media
Aug 09, 2022
| 5 min read

The Importance of Finding the Right Media for Your CHO Subtype

CHO cell lines are diverse and possess many genetic variations between subtypes and even within subtypes. These differences require careful consideration of the culture conditions for each cell line. Learn more about the origin of CHO cell lines and how testing different media can help optimize your upstream production process. Look out for helpful tips from our cell culture media expert, Maverick Lau.

This article is posted on our Science Snippets Blog

Why are CHO Cells so Widespread in Biopharmaceutical Manufacturing?

Chinese hamster ovary (CHO) cells are an epithelial cell line originally derived from the ovary of the Chinese hamster in the 1960s. CHO cells are popular in research laboratories and the biopharmaceutical industry. In fact, 70% of protein biotherapeutics are produced in CHO cells (1).

CHO cells have various features that make them ideally suited for the production of recombinant proteins:

  • Easy to culture and can reach high densities
  • Produce high yields of protein products
  • Can catalyze human-like post-translational modifications  
  • Established track record
  • Adapted to adherent and suspension culture, increasing production scale and capabilities
  • Can be grown in serum-free, chemically defined media

CHO Cell Variants

There are now various cell lines derived from the original CHO cells, including CHO-K1, CHO-DXB11, CHO-DG44, CHO-S, and CHO-GS (Figure 1). Each has slightly different attributes, growth requirements, and responsiveness to environmental conditions. Therefore, the choice of media can strongly influence their performance. Their history is lengthy and often confusing.

Figure  1 - The origin of CHO cell line variants referenced in this article. 

CHO-K1 Cell Line

CHO-K1 is a lineage subcloned from a clonal population of the parental CHO cells in the late 1960s (2). CHO-K1 cells lack a gene required for proline biosynthesis. Therefore, proline is added to the media

CHO-DXB11 Cell Line

To create production cell lines, a reliable selection system is required. In 1980, CHO-K1 cells were subject to random chemical mutagenesis to generate mutations in both copies of the dihydrofolate reductase (Dhfr) gene, thus creating a new cell line: CHO-DXB11 (also known as CHO-DUKX).

The DHFR protein encoded by the Dhfr gene converts dihydrofolate to tetrahydrofolate, which is essential for the synthesis of nucleic acids and amino acids. With such an important role, an exogenous copy of the Dhfr gene is required for CHO-DXB11 cell viability. This functional gene can - conveniently - be co-transfected alongside the gene of interest. Cells that successfully take in the plasmid containing the Dhfr gene and the gene of interest will have restored DHFR activity and can now grow. This represents a simple selection mechanism for successfully transfected cells.  

CHO-DG44 Cell Line

CHO-DG44 is a distinct yet also DHFR-deficient cell line. Mutagenesis of a different CHO cell starting population (CHO-MtxRIII, a mutant of the original parental CHO cell line) was performed to create the CHO-DG44 lineage. Both copies of the gene are fully deleted (rather than one deletion and one missense mutation present in CHO-DXB11 variants). This was carried out to avoid problems of reversion with the CHO-DBX11 cell line, which made them tricky to use in selection (2).  

CHO-DG44 cells are among the most widely used CHO cell lines in the industry, and are the foundation of Sartorius’ cell line development platform.  

CHO-S Cell Line

The CHO-S lineage was derived from a sister population of CHO-K1 cells (2) and adapted for suspension growth in 1971. There is some confusion about the true origin of the CHO-S cell line (2).

CHO-GS Cell Line

CHO-GS cells (CHO-K1SV) are based on a different selection system – the glutamine synthase (GS) expression system. Derived from CHO-K1 cells, the CHO-GS line is suspension adapted and can be grown under serum-free conditions.

Differences Between CHO Variants

There is substantial genetic heterogeneity among the different CHO cell lineages, creating cell line-specific differences in cell behavior, nutrient requirements, favorable culture conditions, protein production capabilities, and product quality (2). For example, research has shown that CHO-K1 cells favored cell-specific productivity, whereas CHO-S had a preference for biomass production but lower protein expression, comparable to CHO-DG44 cells in the same experimental setup (3,4). These differences are not just the case between cell lineages but also within cell populations of the same variant (2).

To further complicate things, the cell culture medium also significantly impacts cell performance and product quality. But this also provides an opportunity to optimize the performance of your specific CHO cell line.

A media benchmarking is necessary to find a truly compatible medium for your unique cell line. After finding a compatible medium, testing different feeding strategies can further enhance cell growth and productivity.

Find the Best CHO Medium for Your Process

While all CHO cell lines share a common ancestor, their vast (and often confusing) history means variants are now distinct, genetically diverse populations with their own culture requirements. A one-size-fits-all approach might yield satisfactory results, but you might achieve significant improvements in cell performance by employing a more tailored strategy. Screening multiple media types is the best way to ensure you maximize yield, stability, reproducibility, and product quality. While screening multiple culture media can be time-consuming, the cost-benefit of media screening is not to be underestimated: the product yield can be improved by up to 30%.   

Sartorius’ fully chemically defined, serum-, and hydrolysate-free CHO media support the robust and reproducible culture of CHO cells. Four uniquely formulated CHO media are each designed to meet the needs of the different CHO subtype.  

Our CHO Media sample kits simplify your approach to testing, identifying, and selecting the optimum media and feeds for your requirements and process.

    +      Request your free sample kit

    +      Learn more about Cell Culture Media


1. Walsh G. Biopharmaceutical benchmarks 2018. Nat Biotechnol. 2018 Dec 6;36(12):1136–45.  

2. Wurm FM. CHO quasispecies-Implications for manufacturing processes [Internet]. Vol. 1, Processes. MDPI AG; 2013 [cited 2022 Jul 21]. p. 296–311. Available from: http://www.mdpi.com/2227-9717/1/3/296

3. Reinhart D, Damjanovic L, Kaisermayer C, Sommeregger W, Gili A, Gasselhuber B, et al. Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis. Biotechnol J. 2019 Mar 1;14(3).  

4. Tihanyi B, Nyitray L. Recent advances in CHO cell line development for recombinant protein production. Vol. 38, Drug Discovery Today: Technologies. Elsevier Ltd; 2020. p. 25–34. 

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A Comprehensive Guide to Finding the Right Cell Culture Media for Your Bioprocesses

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