Cell culture must go vegetarian

I used to work next door to a microbiology lab where bacteria was grown on brain and heart broth. The smell alone was enough to persuade me to stay with synthetic organic chemistry and keep well away from the black art of cell culture. Brain and heart broth is still used in path labs, but, when it comes to biopharmaceutical manufacturing, cells are having to learn to do without animal components whenever possible. This has undoubtedly been a challenge for the industry, but is leading to a better understanding of cell metabolism and physiology, which, in turn, allows cleaner and more efficient production of recombinant proteins and other biologics.

It was Harry Eagle of the US National Institutes of Health who pioneered cell culture from the mid-1950s. The medium he devised contains all the different nutrients cells need to grow, such as amino acids, salts, vitamins and glucose. To this he added a liberal dose of horse serum, which contains various growth factors without which the cells die. Eagle's medium was an important breakthrough because a variety of cells were able to grow on it, allowing research in molecular biology to progress and eventually, in the late 1970s, to open the door to industrial cell culture and the biopharmaceutical industry.

Foetal bovine serum (FBS) became the research and industry standard for supplementing cell culture media, but to this day, no one is quite sure what it contains. This lack of precision and certainty sits uneasily with the demands of good manufacturing practice (GMP) manufacture. So, although cells love to grow on serum, they have to be weaned off it. The BSE epidemic naturally led to concerns about the safety of medicines, such as recombinant proteins, made using bovine serum. In the UK, over 1000 people with haemophilia were infected with HIV through blood-derived Factor VIII (FVIII) treatment. Recombinant FVIII, produced by mammalian cells, is a much safer option; it would be a terrible irony if patients were put at risk again through contaminated FBS. Prions — the infectious agents causing BSE and variant CJD — are not the only potential contaminant in serum; manufacturers must also demonstrate removal of viruses, fungi, bacteria and endotoxins.

But in fact, the trend towards serum-free cell culture dates from before BSE, and was driven primarily by concern over the cost of serum and the difficulties of obtaining a reliable supply. Moreover, unlike a chemical solvent, serum is not well-defined. Each batch is different, and must be checked and validated. Serum also causes problems in downstream processing because of its high protein content. This can overwhelm the protein product being manufactured during the purification process. That is why foetal, rather than adult, bovine serum has often been preferred for monoclonal antibody production, because foetal serum contains fewer interfering antibodies than adult bovine serum. But foetal serum is harvested from bovine foetuses taken from pregnant cows during slaughter — a procedure that raises animal welfare concerns.

The three vital growth factors in serum are albumin, transferrin and insulin. The first steps towards serum-free media involved getting rid of the serum and providing these protein ingredients from others sources, mainly blood. Then, with the advent of the BSE crisis, it was thought the proteins ought to be replaced too, to get rid of the animal components altogether. "This proved harder than people thought it would be," says Dr Malcolm Rhodes, technical director of bioProcessUK. "Albumin provides a number of micronutrients, and helps control pH and transferrin binds iron, without which you get oxidation in the cell culture."

Media supply companies such as Invitrogen and SAFC Biosciences have put years of development work, which is still ongoing, into this area, and there is a range of serum-free, protein-free and chemically-defined media now available for various applications. Serum-free cell culture is often used for research and may still contain proteins. If the protein comes from a recombinant source, then the medium may also be animal-free. The protein-free media could contain yeast or plant — usually wheat or soya — hydrolysates as a replacement for albumin, transferrin and insulin. These are often, but not always, free of animal components. A chemically-defined medium is totally animal-free and is, increasingly, the choice for industrial cell culture.

Cells do not like change. If they have been growing and producing in a serum-supplemented medium, a sudden transfer to a serum-free medium will be stressful. They need to go through a gradual adaptation process. "It is not well understood what happens to the cells during this weaning process," says Dr Rhodes. "There may be genetic or epigenetic changes taking place." (An epigenetic change affects gene activity, but not gene sequence.) Cells are also more sensitive to changes in pH, osmolality, temperature, mechanical forces and enzyme action in a serum-free medium. It would appear that the protein content of a conventional medium provides the cells with some protection from these changes. Moreover, attachment-dependent cell lines require some kind of extracellular matrix, which serum seems to provide. Without it, surfaces must be coated with something the cells can attach themselves to, such as laminin or fibronectin.

In brief, moving a cell culture from conventional to serum-, protein-, or animal-free media takes time and money, which is why some older biotech products are still manufactured with serum. The regulatory authorities will accept this — so long as the animal-derived components used in the process come from a source deemed to be safe. But new products must be as free of animal components as possible. Some manufacturers have invested in switching an established product. For instance, Baxter now makes FVIII in a albumin-free medium.

For conventional biopharmaceuticals, the adoption of more defined media is now well underway. The challenge now is for cell therapies — where the cell itself is the product — to go animal-free. Cells destined as therapies are often grown on a layer of mouse 'feeder' cells and are, therefore, prone to viral contamination. However, Geron (a US company that plans to begin the world's first clinical trial of human embryonic stem cells later this year) has recently published methods of growing cells without the feeder layer. Many other stem cell therapy companies are now starting to develop animal-free culture technologies.

According to SAFC Biosciences, the future of media development is towards more and more chemical definition, and will include the use of experimental tools such as RNA profiling to improve our understanding of the metabolic profile of cells during each stage of the cell cycle. "It is a hard road, but there is more potential for optimizing production if you can control individual components in a cell culture medium," argues Dr Rhodes.