Adaptation to Serum-free Culture

The goal when adapting cultured cells to serum-free conditions is to maintain the original cell phenotype and avoid selecting variant subpopulations of cells. One component of the process, therefore, wiU be to monitor the cell characteristics of interest, such as the concentration of a secreted product. 

Serum provides many different components affecting cell survival, proliferation rate and secreted or internal product concentration. 

The process of serum-free adaptation of cells currently grown in serum begins with selecting a serum batch that supports growth of the cells in low serum concentrations and selecting a rich nutrient medium that supports cell proliferation in low serum. 

The cells can then be adapted through growth in a series of progressively lower serum concentrations. To achieve the final step to serum-free condition, one of three methods can be used addition of commercial serum substitutes, addition of defined proteins or addition of only amino acids, trace elements and other small molecules to provide a protein-free medium. The last of these alternatives is the most difficult to achieve however, it may ultimately prove to be the most useful. 

Cell and Tissue Culture Laboratory Procedures in Biotechnology, edited by A. Doyle and J.B. Griffiths. 1998 John Wiley Sons Ltd. 

---

Rodrigues, ME, Costa, AR, Henriques M, Cunnah P, Melton DW, Azeredo J, Oliveira R. (2013) Advances and drawbacks of the adaptation to serum-free culture of CHO-Kl cells for monoclonal antibody production. Appl. Biochem. Biotechnol., 169(4) 1279-1291. 

Andersen et al. (1996) and Andersen (1995) have studied the effect of temperature on the recombinant protein production using a baulovinis/insect cell expression system. In Tables 17.15, 17.16, 17.17, 17.18 and 17.19 we reproduce the growth data obtained in spinner flasks (batch cultures) using Bombyx mori (Bm5) cells adapted to serum-free media (Ex-Cell 400). The working volume was 125 ml and samples were taken twice daily. The cultures were carried out at six different incubation temperatures (22, 26,28, 30 and 32 TT). 

Belloncik S, Akoury WE, Cheroutre M (1997), Importance of cholesterol for nuclear polyhedrosis virus (NPV) replication in cell cultures adapted to serum-free medium, In Maramorosh K, Mitsubashi J (Eds), Invertebrate Cell Culture Novel Directions and Biotechnology Applications, Science Publishers, Enfield, NH, pp. 141-147. 

Serum is classically added to cell cultures to provide hormones, growth factors, binding and transport proteins and other supplemental nutrients. Not all lots of serum have the same potential to support cell growth because they may have lower amounts of these components. Conversely, some lots may be toxic or inhibitory due to adulteration or contamination with microbes or viruses. Such sera may inhibit or kill cells at low serum concentrations due to high endotoxin levels or other inhibitory factors. This effect has nothing to do with the ability of the cells to adapt to serum-free conditions. The first step in serum-free adaptation is, therefore, to select a serum lot that can support growth of the cell line at low serum concentrations. 

Healthy culture of cells to be adapted to serum-free conditions... 

We have developed human cell lines from several tissues using multiple vectors and approaches new cell lines have been generated by transfection with immortalizing cellular or viral genes followed by continuous passage, subcloning and adaptation to serum-free conditions. These procedures have been carried out in designated laboratories, separated from other cell culture activities. 

Fig. 3.8 Antibody yields from batch cultures for 20 antibody-producing cell lines adapted to serum-free media.

As different cell fines require different medium compositions, adaptation of a cell fine to grow without serum is quite time consuming, and not all cell fines have been adapted to serum-free or protein-free media. Therefore, for laboratory scale, for example, basic cell culture research or cultivation of primary cells, still mostly complex serum-containing media are common. In industrial production with estabfished, optimized cell fines, serum-free, bovine-free, and chemically defined media are state of the art. 

Some of the serum-free media were ineffective and some were very expensive. Better results may be obtained after a period of adaptation after 3 days adaptation in 2% serum, cells were transferred to serum-free medium. Those in Mito + or in Costar s SF-1 continued to grow, albeit at a reduced rate, for several weeks providing a culture supernatant from which monoclonal IgG could be harvested. 

This discussion provides an overview of the methods for selecting a serum lot and balanced nutrient medium as starting points for adapting a cultured cell line to serum-free conditions. The ultimate stringency of serum-free medium attained (containing serum substitutes, defined additives or protein-free) depends on the needs of the researcher and the characteristics of the cell line. Serum provides many different functions for the cell and there are many different types of cells, each of which requires a medium derived to meet its particular needs. The information provided here should enable the development of a serum-free medium appropriate for each particular case. 

The way to create a serum-free culture is to adapt the cells to the serum-free medium. In our laboratory, we tried to adapt a human lymphoblastoid cell line, Namalwa, from a medium containing 10% serum to serum-free. We were able to adapt Namalwa cell to a ITPSG serum-free medium which contained insulin, transferrin, sodium pyruvate, selenious acid and galactose in RPMI-1640. In the case of cell adaptation for production of autocrine growth factor, we were able to grow the cell line in serum- and protein-free media as well as in K5 62-K1 (T1) which produces an autocrine growth factor, LGF-1 (leukemia derived growth factor-1). 

Fig. 3.7 Typical adaptation profiles for two different antibody-producing PER.C5 cell lines (A and B). Cells cultured in the presence of 10% FBS are transferred directly to serum-free media in Erlenmeyer shake flasks. Cells are passaged and population doubling time is calculated.

Sf9 and Sf21 cells can grow either as adherent or as suspension cultures, are easily adapted to most of the common serum free insect cell culture media [52], and it is most often possible to obtain culture titers of viral structural proteins higher than 30 mg/1 [20]. 

In Section 2, factors that could lead to particle assembly and secretion into the supernatant were discussed. At this point a deeper analysis of the factors affecting cell infection will be made. Optimisation of the production process should take into account virus-cell interactions, and more specifically viral attachment and internalisation into the cell. The impact of chemical modifications of the medium in baculovirus attachment-internalisation has not been carefully studied. It is widely known for example, that serum increases the infec-tivity of baculovirus. These reviewers have had one case where we were only able to succeed in infecting Sf9 cells adapted to growth in serum-free media [52], with a baculovirus produced by Sf9 cells (not adapted to grow in serum-free media), after adding serum to the culture (authors unpublished observations). However, since serum is not desirable for use in industrial production, its utilisation should be avoided as much as possible. 

High-Five cells (BTI-TN-5BI-4) are derived from Trichoplusia ni cells and are frequently employed due to their capacity to express high protein levels when compared with other insect cell lines, such as Sf-9 cells (Rhiel et al., 1997). This cell line shows high growth rates in adherent culture, and can easily be adapted to grow in suspension and in serum-free media. 

The cell line has been adapted for growth in serum-free media using spinner flasks and stirred tanks and cultures can be scaled up readily to produce high yields of recombinant protein (Jain et at, 1991). In batch cultures, infection of the cells with baculovirus can be monitored easily by measuring the increase in cell volume and decrease in cell viability. Several days after infection the cells lyse, releasing the recombinant protein product. Product titre is influenced by the oxygen requirement of the insect cells. 

Cells can synthesize most of the essential amino acids, but do so in amounts too low to compensate for dilution in low-density cultures. The requirement for many amino acids is, therefore, a function of cell concentration. During adaptation of cells to lower serum or serum-free conditions it may be necessary to culture the cells at high density, 3-6 x 10 cells ml until they have adapted. This can be accomplished by centrifuging the cells and resuspending at higher densities. 

The specific standard methods of a new perfusion culture will now be described for growth and maintenance of mammalian cells in suspension cultures at high density. The biofermenter was used for high density culture of Namalwa cells with serum-free medium as the model. In 1980, the parent Namalwa cells were obtained fi-om Mr. F. Klein of Frederick Cancer Research Center, Frederick, Maryland, U. S. A. In our laboratories, we were able to adapt the cells to a serum and albumin-fi ee medium and named the cells KJM-1. ITPSG and ITPSG+F68 used a serum-free medium containing insulin, 3 g/ml Transferrin, 5 g/ml sodium pyruvate, 5 mM seienious acid,... 

A limitation in the use of CHO cell lines for producing biopharmaceutical proteins has been the long time it can take to adapt such cell lines to single cell suspension culture in serum- or protein-free media. A variant of the CHO-Kl cell line that grows spontaneously in protein-free suspension culture has been described for use with the GS system [59]. The isolation of natural variants has also been exploited to isolate an NSO clone which no longer requires cholesterol [60]. This nutrient is insoluble and its addition to protein-free media is not straightforward. 

The development of in vitro techniques for culture of eukaryotic cells has clearly demonstrated the dependence of cell proliferation on cell membrane receptor-mediated interactions with macromolecular polypeptide growth factors in serum (for reviews, see Gospodarowicz and Moran, 1976 Bradshaw and Rubin, 1980). And although a few cell lines have been adapted to grow in serum-free defined media, serum is still, for most cell types, a necessary constituent of the culture media. Therefore, it must be considered that modulation of cell proliferation by retinoids might result in certain instances from an alteration by the retinoids of the response pattern of the cell to particular hormones or growth factors present in the serum. 

Adaptation of cells to low serum or serum-free medium 128 Stationary cultures... 

---