C2C12: Manufacturing life in a laboratory
Macdonald Hall is the building on campus hosting the University of Ottawa’s physics department. It is down a set of stairs, in the basement, that resides the Center for Interdisciplinary Nanophysics: a state-of-the-art research facility renovated in 2011 (Biological Physics, 2012). A large door marks the entrance and has to be electronically unlocked to access the facilities. As you pass the door, a long hallway stretches on: shoes rest on your right, under coat hooks. Surrounded by key locked doors, a dozen at most, I make my way to the cell culture room. I began my adventure here, in the cell culture room of the Pelling Laboratory for Biophysical Manipulation (www.pellinglab.net). It is led by curiosity that physicists, biologists, engineers and artists explore possible physical manipulation and re-purposing of life. I thank them for allowing me to share their passion by spending time in the lab and for teaching me the ways of biophysical research.
Unfrozen into existence, it is inside a round 10cm petri dish that my C2C12 started growing. They arrived to me after their 10th passage: this means they had been split 10 times from the initial cell line. It is quite youthful, I have been told. A passage constitutes of cell splitting: cells grow continuously and, when they become too confluent (cells touching each other), they can fuse. In cell culture, this is too be avoided because it may lead to unwanted cell differentiation (Fisher-Aylor & Williams, 2011). In need of a passing every 2-4 days, I will see these cells grow older very fast.
The cell line I am cultivating, C2C12, is an immortal line of mouse myoblasts (a precursor of myocyte or muscle fiber) which comes from the thigh muscle of a 2 month old C3H mouse (Fisher-Aylor et Williams, 2011). This work was done by Yaffe and Saxel (1977). To stay alive, the cells need to be in a regulated environment. This is the role the incubator plays: it is a warm humid box kept at 37°C with 5% of CO2. Maintaining the cell also means providing them with the same basic essential matter as other living organisms require: energy.
The cells grow, stuck to the bottom of a petri dish, into a carefully concocted growth media. 89% of this mixture is Dulbecco’s modified Eagle’s medium (DMEM). Originally, Eagles’ minimal essential medium (EMEM) contained amino acids, salts, glucose and vitamins. Dulbecco’s variant contains more amino acids, vitamins and glucose (HiMedia, 2011). It is suitable for the growth of most cells, including mammalian cells. To DMEM is added 1% Penicillin Streptomycin, or Pen Strep for short (Life Technologies, 2015a). Toxic at a high dose, this antibiotic carries out the role of protecting the cell cultures from contamination. The final 10% of the mixture is Foetal Bovine Serum (FBS).
What is FBS?
Sold at around 400$ for a 500 mL bottle (Life Technologies, 2015b), FBS is a serum extracted from the blood of foetus found in the womb at slaughterhouses. Serum is blood without cells and clotting agents and a great source of nutrients and growth factors (Humane Research Australia, 2013; Jochems et al., 2002). Its production being tied to the meat industry, the price of FBS fluctuates given livestock numbers, weather, food costs and milk and beef prices (Jochems et al., 2002). The initial step in acquisition of the serum is through brutal extraction of the blood directly from the living calf’s heart. These calves are taken from the carcass of their mother when cows are found pregnant at slaughter (Humane Research Australia, 2013; Jochems et al., 2002). No anesthesia is given to the foetus’ who may experience pain and discomfort as blood is drained from the heart, by a needle stuck between the ribs, into a sterile bag (Jochems et al., 2002). The use of this product and it’s methods of production are generally justified by the end product: it is a purer serum, containing more growth hormones then would adult blood and the extraction method reduces the risk of contamination (Rauch et al., 2011). It has been shown the foetuses are still alive at the time of extraction. They are capable of experiencing discomfort, and their under-developed nervous system may lead to a more intense experience of pain (Jochems et al., 2002). Thus, many ethical concerns have been raised about the production and use of FBS in terms of animal welfare. Cell culture is often seen as an alternative to cruelty against animals in animal testing (Jochems et al., 2002): but can this alternative really be sustainable if it itself produces more suffering? Given these facts, the thought of more than 1 million bovine fetuses being harvested to support the 500 000 litres per year market seems debilitating (Gstraunthaler, 2003).
Amongst concerns tied to the use of FBS, are technical ones. Batch-to-batch variation is a significant concern that requires pre-test for every batch sold (Jochems et al., 2002; Gstraunthaler, 2003). The exact composition of FBS remains unknown (Rauch et al., 2011) and it can have effects on cell culture. Jochems et al. (2002) have found in the literature that FBS can interfere with genotypic and phenotypic cell stability as well as supressing cell spreading, attachment and differentiation. All of these effects can influence the results of a study. Another way in which results could be affected is through contamination of the serum (Jochems et al., 2002; Gstraunthaler, 2003; Rauch et al., 2011).
FBS, with its richness in growth factor needed for cell proliferation, can be used almost unversally as a supplement for mammalian and insect cell culture (Gstraunthaler, 2003). Despite this great utility, the ethical and practical issues raised about the use of FBS has led to a search for alternatives (Jochems et al., 2002; Gstraunthaler, 2003, Rauch et al., 2011). Serum-free media exist as an alternative. Consisting mainly of DMEM for essential nutrients and another highly enriched nutrient mixture, factors like hormones, growth factors, lipids and vitamins are specifically added (Gstraunthaler, 2003). Unlike FBS, these media need to be highly specific to different cell lines, but more and more recipes have been published and databases established (Gstraunthaler, 2003). Using serum-free media can lower the use of FBS (and therefore plays in favor of animal welfare) and address certain technical concerns such as variation between batches and elimination of possible contamination. Other alternatives are also in the works. In the lab, an alternative serum obtained from expired donated human blood is currently being tested in the hope of eliminating use of FBS. The use of human platelet lysates, obtained from expired blood, seems to show high potential as growth media for multiple cell lines and it’s use addresses the ethical and technical issues associated to FBS (Rauch et al., 2011). Testing of this alternative in the lab in still pending analyses.
So far, I have been able to keep my cell culture alive and going healthily. Visiting my culture a few times a week. It is not only the cells themselves that are at the center of my experience but the protocols at hand, the use of laboratory material, biological and non-biological waste, the laboratory members, the science behind it all and, most importantly, the way in which all of these elements coexist in the basement of Macdonald Hall.
As this adventure begins, I look forward to not only cultivating cells, but manipulating life. The way in which humans arrive to grasp life, encapsulate it in a petri dish and play with it leads me to question the nature of our relationship with these microscopic living entities. Such as shown with FBS, the lives involved in cell culture go beyond that of the researcher and the researched cells. The how and why of these manipulations is intriguing and the Pelling Lab provides answers: within logics of do-it-yourself, repurposing and curiosity, the aim is to achieve better understanding of life itself.
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Rauch, C., Feifel, E., Amann, E-M., Spötl, HP., Schennach, H., Pfaller, W., & Gstraunthaler, G. (2011). Alternatives to the Use of Fetal Bovine Serum: Human Platelet Lysates as a Serum Subsitute in Cell Culture Media. Altex(28), 305-316.
Yaffe, D. & Saxel, O. (1977). Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature, 270, 725-727.