CHO cells: The most used cell lines in biotechnological production

Since their discovery, Chinese hamster ovary (CHO) cells have played a central role in biotechnology and other fields. Many important therapeutic agents have already been produced with the help of these special cells.

In this article you will learn more about:

  • The history of the hamster cell lineage
  • The uses of CHO cells
  • How are CHO cells analyzed in the laboratory?
  • What are the challenges in analysis and how can the fluidlab assist?  



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The history of the development of the hamster cell line


CHO cells are cells of an immortalized cell line derived from a Chinese miniature hamster (Fig. 1). The cells were isolated from the ovaries of a hamster as part of a molecular biology research series by Theodore T. Puck in 1957. The researcher observed that the cells proliferated over a period of more than 10 months without any decrease in the rate of proliferation or any visual change being detectable.

Nowadays, the cells are mainly used in the production of biopharmaceuticals and can all be traced back to the cells taken from a hamster by Theodore T. Puck.1

Common uses of CHO cells in research

CHO cells are a cell line that is often used in cell biology and biotechnology for the production of recombinant proteins and antibodies. The reason for their frequent use is the many advantages that the cells bring with them. These make them suitable host cell systems for the production of active substances to a high degree.


Use in biotechnological processes to produce pharmaceuticals

CHO cells are among the most widely used host cell systems in the industrial production of recombinant protein therapeutics.2

 An important reason for this is that the sugar chains attached to newly synthesized proteins differ little from those of a human. Indeed, the glycosylation pattern determines how well a drug works in an organism. Since that of CHO cells is so similar to that of humans, this means that the proteins produced in CHO cells have a particularly high efficacy in humans.

They also bring other advantages over other cell types:

  • Stable growth in chemically defined and serum-free suspension cultures
  • High transfection susceptibility
  • Safety regarding replication of human pathogenic viruses3
  • Easy generation of genetically modified cell clones for expression of target genes in sufficient quantity and quality for human use4


With the help of CHO cells, antibodies against cancer or rheumatism, but also growth factors can be produced. Approximately 70 % of the biomolecules used for medical purposes originate in CHO cells.5

The production process of biopharmaceuticals with the help of CHO cells is not only labor- and cost-intensive, but also takes about five months. The quality of the manufactured product depends on the selected CHO clone and its productivity. The product quality is also influenced by the composition of the medium, the pH, the passage of the culture and other factors that vary from manufacturer to manufacturer.

Nowadays, the concentration of protein-producing cells is about 10 million cells/mL, and the cultivation time is three times longer than it was twenty years ago. However, the overall cell production time can be shortened using modern technologies, such as quantitative polymerase chain reaction (PCR) or automated microplate analysis, such as enzyme-linked immunosorbent assay (ELISA). These methods lead on the one hand to a rapid selection of cells and on the other hand to the identification and cloning of the most suitable single cells.3  

 Some known drugs which are produced with the help of CHO cells are:

  • Etanercept (active ingredient for the treatment of rheumatic diseases)6
  • Infliximab (antibody for use in autoimmune diseases)7
  • Bevacizumab (antibody for the treatment of tumors)8


The molecular genetic use of CHO cells is not yet exhausted

CHO cells have been used to produce glycoprotein biologics for several decades. However, the molecular basis and cellular processes within CHO cells have not been fully elucidated, which means that their versatile properties cannot yet be fully exploited.

The therapeutic efficacy of protein biologics is mostly determined by post-translational modifications, especially glycosylation. This is a non-templated process. During the production of the glycoproteins, heterogeneous mixtures are formed, which have different proportions of different glycoforms, for example N-glycans or O-glycans. Among other things, the stability, efficacy and immunogenicity of the glycoprotein biologics depend on the glycoforms. Glycoprotein biologics produced with the help of CHO cells possess small amounts of Neu5Gc, whereas it does not occur at all in the human organism. In addition, they may or may not possess the alpha-Gal epitope. This distinguishes them from human glycoproteins. However, the amounts are so small that the glycoprotein biologics produced in CHO cells do not elicit an immune response.

Recently, researchers have started to use CHO cells for the production of therapeutic glycosaminoglycans (e.g. heparin). The challenges are similar to the production of glycoprotein biologics. Methods are lacking to control the heterogeneity of the CHO cell product and to guide the bioprocess. Therefore, research groups are still working to find a systems biology approach that agrees on different technologies, such as genetic modification, modeling and analysis of glycans and glycoproteins. The goal is to gain a complete understanding of the molecular processes and use it for the development of further cell lines.10

How CHO cells are analyzed in the laboratory

Depending on the use of the cells, different aspects of the CHO cells are analyzed. In the case of protein production, the very first step involves the cells taking up the vector with the target gene (transfection) and expressing the desired product. In such cases, it must be demonstrated that the transfection was successful.

Usually, such vectors have a selection marker, usually antibiotic resistance. After plating the cells on an antibiotic-containing plate, only those cells can grow which carry the marker and thus the target gene, whereby they are selected.11

However, what precedes all experiments with the CHO cells is the analysis in terms of cell number and viability to determine their viability. These parameters are important as they have a major impact on the subsequent success of all subsequent experiments/assays with the cells. Cell number can be determined manually or automatically. Manual cell count determination is performed using a hemocytometer (e.g. Neubauer counting chamber) (Fig.2), where the cells are counted under a microscope after applying the sample fluid and the coverslip. Based on the counted cells, it is possible to calculate how many cells per volume are contained in the sample.

 During this process, trypan blue staining is often performed as a standard procedure to determine the percentage of live and dead cells in the sample. The dye stains dead cells with permeable cell membranes blue, while living cells remain unstained. The calculated data is used to determine viability. Learn more details about cell counting here.

As mentioned above, cell counting can also be performed with automatic cell counters. In this case, appropriate devices are used that perform an automated count of CHO cells. However, to determine viability, trypan blue staining is often performed in advance in this case as well. The devices then distinguish the living from the dead cells and can also indicate the viability of the CHO cells in addition to the cell count.


What difficulties can arise when examining the cells?

On the one hand, difficulties may arise when counting the CHO cells manually. Here, human error can easily lead to deviating results. In addition, errors can occur due to lack of concentration and confusion during counting. On the other hand, the use of dyes such as trypan blue, can also lead to falsified results in the determination of viability. Since the dye is a cytotoxic substance, the counting must be performed as quickly as possible, otherwise the number of dead cells increases and incorrect conclusions can be drawn regarding the initial solution.12

Stain-free viability measurement and automatic cell counter: using the fluidlab to study hamster cells

Difficulties that arise when counting cells or determining viability can be counteracted by the fluidlab R-300 with the neural network and the holographic microscope. An additional advantage is the integrated spectrometer.

This makes the instrument applicable at various points in the analysis of CHO cells. The spectrometer can be used to monitor bacterial growth for vector preparation. For this purpose, an OD600 measurement is performed with the fluidlab. This first step in protein production is important because it provides information about bacterial quality. The resulting vectors are then used to transfect them into the CHO cells to produce the desired proteins. Furthermore, the CHO cells can be counted using the neural network of the device.

The fluidlab's digital holographic microscope creates holograms of the cells, which are analyzed using the neural network. Not only are the cells counted automatically, but additional differentiation is made between cells and other particles, and between live and dead cells. These features eliminate the need for staining with a dye and minimize human error. In addition, the field-of-view of 5.3 mm² increases statistical confidence as more objects are detected through the large field of view.

The small size of the device and its light weight of 240 g, also make it not only portable, but also usable for various work locations- regardless of the workplace.

To learn how the fluidlab R-300 can be used for different protein production steps, read our article on monoclonal antibodies.

Scientific sources

1TJIO, J. H., & PUCK, T. T. (1958). Genetics of somatic mammalian cells. II. Chromosomal constitution of cells in tissue culture. The Journal of experimental medicine, 108(2), 259–268.

2Fischer, S., Handrick, R., & Otte, K. (2015). The art of CHO cell engineering: A comprehensive retrospect and future perspectives. Biotechnology advances, 33(8), 1878–1896.

3Kim, J.Y., Kim, Y.G., Lee, G.M. (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Applied microbiology and biotechnology 93, 917-930.

4Durocher, Y., Butler, M. (2009) Expression systems for therapeutic glycoprotein production. Current opinion in biotechnology 20, 700-707.

5Ku (2013). Hamsterzellen: Fabriken der Biotechnologie. Die Presse. [26.10.2021].

6Brian Hassett, Morton Scheinberg, Gilberto Castañeda-Hernández, Mengtao Li, Uppuluri R K Rao, Ena Singh, Ehab Mahgoub, Javier Coindreau, Julie O'Brien, Steven M Vicik & Brian Fitzpatrick (2018) Variability of intended copies for etanercept (Enbrel®): Data on multiple batches of seven products, mAbs, 10:1, 166-176.

7Miao, Z., Li, Q., Zhao, J., Wang, P., Wang, L., He, H. P., Wang, N., Zhou, H., Zhang, T. C., & Luo, X. G. (2018). Stable expression of infliximab in CRISPR/Cas9-mediated BAK1-deficient CHO cells. Biotechnology letters40(8), 1209–1218.

8Barredo, G. R., Giudicessi, S. L., Martínez Ceron, M. C., Saavedra, S. L., Rodriguez, S., Filgueira Risso, L., Erra-Balsells, R., Mahler, G., Albericio, F., Cascone, O., & Camperi, S. A. (2020). A short peptide fragment of the vascular endothelial growth factor as a novel ligand for bevacizumab purification. Protein expression and purification165, 105500.

9Wippermann, A., & Noll, T. (2017). DNA methylation in CHO cells. Journal of biotechnology, 258, 206–210.

10Tejwani, V., Andersen, M. R., Nam, J. H., & Sharfstein, S. T. (2018). Glycoengineering in CHO Cells: Advances in Systems Biology. Biotechnology journal, 13(3), e1700234.

11Sautter K. (2003). Gentechnische Verfahren zur Erzeugung und Selektion von hochproduzierenden CHO-Zellen. Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg. [02.11.21]

12Crowley, L. C., Marfell, B. J., Christensen, M. E., & Waterhouse, N. J. (2016). Measuring Cell Death by Trypan Blue Uptake and Light Microscopy. Cold Spring Harbor protocols2016(7).