culture techniques represent a way of performing scientific, medical and toxicity
research without the use of live animals. At least, this is the view held by most
people. But is it true? Twofold NO!
Killing animals for their tissues.
In first instance, several animals have to be killed for the purpose of obtaining cells. For example, in France the number of animals killed for the obtainment of cells, tissues and organs for culturing techniques was 111.792 in 1993; in The Netherlands in 1995, this number was 114.012. Luckily, cell lines can grow for many many more generations in a plastic flask than would the cells in the original living animal, so netto less live animals are being used.
Extracting blood from unborn bovines.
To culture cells, tissues or organs in a plastic flask, one has to arrange proper growing circumstances for these cells. This is done by adding so called culture medium to the cells, often combined with fetal bovine serum (FBS). Fetal bovine serum contains many substances which are needed by cultured cells to grow and live properly. Serum is blood without cells and clotting factors. FBS is the serum of an unborn bovine i.e. a bovine fetus. It is being used extensively both in research, and (the life science-) industry, making fetal bovine serum a commercially interesting product. Let's take a closer look on its whereabouts before it is delivered in sterile plastic bottles into the cell culture laboratory.
Fetal bovine serum is obtained in those areas of the world where cows are kept in an extensive manner (e.g. Australia, Loire Valley in France, continent of America). That is, herds in which cows and bulls roam freely amongst eachother. Bulls are added to herds of cows to help maintaining the herd together, just like dogs keep a flock of sheep together. As a result however, several cows will be pregnant by the time the herd is sent to slaughter (the herd is sent in its entirety to slaughter when the average age of the cows is around two years). This is common practice, whether or not a cow is pregnant is simply not an issue! So, once at the slaughterhouse, all cows are slaughtered, including the pregnant ones. During removal of the internal organs of any cow on the slaughterline, a fetus may show up. Then, if the slaughterhouse has the right facilities, the entire reproductive tract including the fetus is cut quickly out of the carcass of the mother cow and brought to a more septic area (called: calf processing area) where the following procedure is followed to obtain its blood. First, the fetus is cut out of the uterus. The umbilical cord is tied off, the fetus is cleaned from amniotic fluid, and is desinfected. Then, a large diameter needle is inserted through the skin and between the ribs, directly into the beating heart of the unanaesthesized fetus. The blood is commonly extracted under vacuum into a sterile blood collection bag via a tube connecting the two. In the absence of a vacuum pomp, fetal blood may be obtained by means of gravity. Once the blood has been obtained, it is allowed to clot at low temperature, after which process the clotted substance is seperated from the serum by refrigerated centrifugation. Afterwards, the 'empty' fetus is destructed to assure it will not be used for human consumption.
Bovine fetuses can feel pain before birth.
The heart of the fetus must function in order to obtain an adequate (read: commercially satisfactorily) amount of fetal blood for FBS production. If the heart functions, the fetus is - by definition - alive. But it is not receiving any form of anaesthesia prior to being exposed to a cardiac puncture, which represents a problem because it is an extremely painful procedure in animals after birth. The last ten to fifteen years more and more scientific data is piling up showing that the fetuses of mammals (in particular those of the species whose newborns are relatively well-developed at birth, like bovines, horses, guinea pigs, sheep, goats, pigs) can experience pain or discomfort well before birth. In a recent guideline on the humane euhanasia of experimental animals, it is said that such animals could experience pain or discomfort as early as 30% gestation time. For a bovine fetus this is as from 3 months as the total gestation period is 9 months. Bovine fetuses used for FBS harvest must at least be 3 months of age (otherwise they are simply too small), but commonly they are of 6 months of age or older. So, all bovine fetuses used for FBS production are capable of sensing pain, yet they are never anaesthesized! What makes it even worse, is the finding that mammal fetuses are not just able to feel pain from a certain timing in their development, they are even more susceptible to pain than adults! After birth, this 'over'-susceptibility decreases to adult values as the newborn grows older. Apart from the difficulties in themselves of assessing possible pain and suffering in a fetus, in this case this is also hampered by the lack of oxygen in the fetus resulting from the few minutes between killing of the mothercow and the performance of a cardiac puncture. Lack of oxygen is argued to cause some degree of unconsciousness in the fetus, but exactly 'how much' unconsciouss a bovine fetus is at the moment of puncture has never been scientifically investigated. To make it even more complicated, mammal fetuses are highly resistant to lack of oxygen. But how does this all add up in terms of animal suffering? No one knows!
In normal laboratory settings, this would imply that a scientist wishing to perform a cardiac puncture on a bovine fetus for serum harvest using the procedure described above, must consider anaesthesizing the fetus somehow before the puncture. This a applied ethics, as it is normal that scientists err on the side of caution wherever animal suffering may be involved, not on the side ignorance or desinterest. This is apart of the question, whether man has the right to use animals at all for human interests.
This represents an ethical problem, which sheds another light on its use in so-called 'alternative' methods replacing 'direct' animal experiments. Most people oppose to hurting or using animals for the purpose of e.g. make-up, tobacco and toiletry research. Blood sampling is considered a procedure in most Western countries. The application of sera in cell culture is never ethically weighed, so a lot of serum is used - if not "thrown away" - without any restrictions from an Animals Ethics Committee of the relevant institution, in research dealing with eg make up substances. So, current make up and tobacco and toiletry research still hurts animals! Apparently, there is something as a contradictio in vitro when it comes to the use of FBS in the culturing of cells as "alternative" to animal experiments. According to the current animal experimentation legislation of the UK, the procedure described above would be considered an animal experiment (all fetuses are covered by that law as from 50% gestation period). A more open FBS-seller says that bovine fetuses would be or have been bred specifically for the purpose of harvesting their blood in some central European countries. Per year around 500,000 liters of FBS are obtained from > 1,000,000 bovine fetuses worldwide. With the exception of a few, commercial suppliers are almost all very reserved to give information on any aspect of FBS production: now you know why!
Alternatives to FBS.
Concerns over the periodic fluctuations in cost and availability of fetal bovine serum, its quality (contaminations, support of cell growth, smuggling), imprecise composition (batch-to-batch variation, presence of substances with unknown function) and the ethical issue stimulate the creative quest for fetal bovine serum alternatives. Substitutes include 100% synthetic media (cell type specific = expensive to develop), reduced-serum media (broader applicability then synthetic media), alternative post-natal mammalian serum substitutes like new-born calf, horse, adult bovine and donor bovine serum (more endotoxins and antibodies present, in particular in new-born calf serum), fetal horse / pig serum (other composition than FBS, same ethical problem). It seems fair to say that 100% synthetic medium is the best alternative currently at hand. The cell type cultured determines the optimum serum (substitute) and its concentration.
Animal welfare in cell culture.
Altogether, what should be kept in mind is (1) fetuses of many mammal species can feel pain already long before the moment of birth, (2) the use of fetal bovine serum represents an ethical problem because it is not clear to what extent the unborn cow feels pain from the heart puncture, apart from the question of using animals for human interests at all, (3) the use of other animal sera (like donor sera) also implies certain animal welfare aspects becoming related to cell culture work, as does (4) the killing of animals for the purpose of getting cells to be cultured. The overall conclusion is that cell culture work does not free us from animal welfare considerations as taken for granted by many!
For more information on the issue of fetal bovine serum and its alternatives, you may wish to visit the following site: http://prex.las.vet.uu.nl/nca , option <Documents>, which contains the summary of a report on livestock blood sera production methods and related ethical implications, as well as a downloadable version of the report. If accepted, a publication on this issue will see the light (it has been conceived and is now in its fetal stage) probably late 2000. The NCA (Netherlands Centre Alternatives to animal use) is considering to organise a workshop on this issue in cooperation with ECVAM (European Centre for the Validation of Alternative Methods). As this is the first time this issue has been touched upon in a scientific manner, a workshop would be the proper event to come to conclusions regarding the animal welfare aspects of this form of unborn animal use.
Carlo E.A. Jochems, Wageningen Agricultural University
NL-6708 PG Wageningen