CHO cell line. A 60-year-old story.

Working in his lab at the University of Colorado Medical School, Theodore was facing a serious issue. Since he successfully cloned HeLa cells in 1955 (1), Theodore Puck had focused his attention on cytogenetic changes, but the high number and large size of human chromosomes didn't make his job any easier. Rodents were already in use in laboratories at that time, but could not help Puck. Mice have forty chromosomes. Rats have forty-two.

In 1948, Watson was establishing research in China on malaria and epidemiology, when the Chinese civil war knocked on his door. Forced to flee the country, Watson took in his suitcases the only gift he had received during his years of work in Nanking: ten couples of Chinese hamsters offered by Dr. Hu of the Peking Union Medical College.

Based in New York, Victor Schwentker had a natural gift for animal breeding and asked Watson for access to this new species, freshly landed at San Francisco airport. Within two years, Schwentker established the first colony outside of China, when all other breeders had failed to raise the animal in captivity. Word spread and researchers started placing orders. 

George Yerganian, a graduate student at Harvard, was one of them. Working on plant genetics, George stumbled upon the papers of Pontecorvo and Matthey who had found that Chinese hamster, or Cricetulus griseus, had only 22 chromosomes (2). And when Schwentker discontinued sales of Chinese hamsters in 1954, Yerganian became the sole distributor of the species for cytogenetic studies.

In 1957, aware of the success story of Yerganian, Puck asked him to send a single female Chinese hamster.

But this time, Puck had a different idea in mind…

In 1940s, growing ex-vivo mammalian cell lines was a nightmare.

Even when antibiotics were discovered to fight multiple fungus and bacteria infections, the viability of the mammalian cells remained the central problem. Puck had managed to provide HeLa continuous cell line to many research centers, but for many inquiries, HeLa cells had proven to be useless.

Puck decided to make a biopsy from the ovary of his Chinese hamster and put it in a petri dish. He observed that the cells grew quickly, in a pretty stable way (3), in monolayer adherent mode, and didn’t exhibit the limitations on doubling times (Hayflick Limit) observed in primary cells. Furthermore, due to the hamster origin, the risk of propagation of human viruses is decreased, reducing production loss and increasing biosafety.

In 1968, after subcloning, Puck observed a particular clone that was not able to synthesize proline, so paving the way for selection methods utilizing specific components in media. Co-developed with his young colleague Kao, the new cell line was named “CHO-K1” (4).

In the 80’s, Genentech was very busy making a name for itself. Thanks to its mastery of recombinant DNA techniques in E.Coli expression system, Genentech was becoming the most promising biotech of the market.

Having already developed and successfully marketed several drugs, the company decided to tackle the generation of tissue plasminogen activator (tPA) - a protein to dissolve blood clots in the heart - as the next pipeline priority.

But that time, the Genentech cloners hit a wall.

Only a small quantity of protein was detected and even worse, it was misfolded. In fact, after translation, proteins undergo post-translational modifications, which affect their efficacy and immunogenicity. And the Genentech team rapidly figured out that bacteria were not able to perform complex human glycosylations.

On the contrary, CHO expression system revealed itself to do the job with excellent pharmaceutical activity and biocompatibility, and Genentech rapidly adopted this new tool. In 1987, the FDA approved tPA as the first CHO derived recombinant protein, for use as a biotherapeutic drug. Since then, 374 individual biopharmaceutical products have gained a license in the United States and the European Union (Walsh, 2018).

CHO became known as “The mammalian E. coli”, as Puck loved to called it amongst the scientific community. 

The increasing demand for CHO-derived processes provoked the necessity for new developments. Among them, it was critical to cultivate cells in suspension mode, to optimize cell density and increase secreted titers.

In 1980, Gail Urlaub and Lawrence Chasin at Columbia University generated CHO lacking dihydrofolate reductase (DHFR) activity in one locus and a missence mutation in the other. DHFR-deficient strains (CHO-UA21) cannot grow unless transfected with a functional copy of DHFR or in media supplemented with thymidine (5).

In 1983, Gail deleted both dhfr alleles by mutagenesis of a different CHO cell starting population, thus generating CHO‐DG44 lineage (6). In 1991, the CHO‐S cell line was generated from another CHO cell starting population. Adapted for growth in suspension liquid culture, this cell line is ideal for scale-up activity and was selected for growth and transfection efficiency. Moreover, its tolerance to variations in pH, oxygen levels, temperature or pressure make them the ideal cell for large-scale bioreactor culture.

Since then, many different CHO cell lines have been developed in order to increase protein productivity, and remains the ideal platform for recombinant antibody manufacturing.

REFERENCES 

(1) Puck T.T., Marcus P.I. A rapid method for viable cell titration and clone production with HeLa cells in tissue culture: the use of X-irradiated cells to supply conditioning factors. Proc. Natl. Acad. Sci. U.S.A. 41:432-437(1955)

(2) George Yerganian. Chromosomes of the Chinese Hamster, Cricetulus griseus. I. The normal complement and identification of sex chromosomes. From The Children's Cancer Research Foundation, The Children's Medical Center, and the Department of Pathology, Harvard Medical School, Boston, Massachusetts, U. S. A.. Received July 13, 1958

(3) J. H. TJIO AND THEODORE T. PUCK, PH.D. GENETICS OF SOMATIC MAMMALIAN CELLS II. From the Department of Biophysics, Florence R. Sabin University of Colorado Medical Center, Denver. Received for publication, March 29, 1958

(4) Kao FT, Puck TT. Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells. Proc Natl Acad Sci U S A. 1968;60(4):1275-1281. doi:10.1073/pnas.60.4.1275

(5) Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980;77(7):4216-4220. doi:10.1073/pnas.77.7.4216

Urlaub, Gail & Käs, Emmanuel & Carothers, Adelaide & Chasin, Lawerence. (1983). Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell. 33. 405-12. 10.1016/0092-8674(83)90422-