A Whale in a Dish: Recognizing the Benefits and Limitations of Cell Culture
By: Natalie Kubicki (Trinity '23)
November 2022
How Science Adapts
Marine mammals are not a typical “model organism.” What we can do with fruit flies and mice, we must come up with unique methods of study for marine mammals. In a utopian scientific world, we would get biopsies from deep-diving marine mammals at multiple time points in their dives, have technologies to immediately analyze the genome, and be able to modify and observe physiological responses in the organism in real time. However, we do not have access to these animals throughout their diving (nor would we want to repeatedly biopsy an animal) and very few parts of scientific analysis are immediate. Thus, science adapts and in vitro methods have proven to provide valuable insights to whole organism studies.
Cell culture is an incredible, accessible model to examine cellular adaptations. The ability to identify a target gene, manipulate that gene to achieve a desired response, and observe cellular and molecular changes is an extraordinary scientific tool. However, scientific processes and large-scale applications are improved by recognizing the limitations of a method. Our typical incubator cell-culture conditions maintain fibroblast cells at 20% oxygen, 5% CO2, 37C, and constant pressure. Typical deep-dives expose these skin cells to much lower levels of oxygen, lower temperatures, and higher pressures.
Oxygen Levels
20% oxygen was established as the baseline oxygen level because it is the atmospheric oxygen level. However, human lung alveolar cells are really the only cells that we know are briefly exposed to 20% oxygen. Therefore, human fibroblasts and deep-diving marine mammal fibroblasts are more likely being exposed to hyperoxic, or too-high of oxygen conditions. As defined by Donnelly et al. (2022), oxygen conditions must be distinguished in “the ambient environment, biological compartments, and control of mitochondrial oxygen consumption and signaling.”
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The blood oxygen volume for deep diving marine mammals typically is 20% at the beginning of their dives and then reduces to approximately 2% after 15 minutes of diving (Ramirez et al. 2017). While this is not necessarily indicative of the partial pressure of oxygen in fibroblast tissues, there is a basis for maintaining the cell culture at 20% and making our hypoxic exposure conditions 2%.
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In my experiences with exposing cells to hypoxia, I have not seen that the cells in the hypoxic conditions grow at a much faster rate than the cells growing at 20% oxygen. However, I have not grown cells at hypoxia for long periods of time. Interesting future experimentation could be to measure levels of ROS production or just cell doubling time in hypoxia vs. normoxia in the incubators to see if 20% oxygen is truly an optimal growth condition.
Oxygen levels outside of normoxia.
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Φ is % air/O2 at 100 kPa and 25C
Arterial blood while diving from gray seals (Halichoerus) and hooded seals (Cystophora)
Pressure and Temperature
We are most interested in these species' hypoxia adaptations in comparison to humans. However, it is important to recognize that when studying baseline comparisons across species without manipulating other factors such as oxygen levels, some of the seen differences may also be due to thermoregulation or pressure adaptations, not just hypoxia tolerance adaptation.
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37C is the standard temperature for incubation because that is the body temperature of warm blooded animals. Deep-diving marine mammals are no different, as they are endotherms. However, these mammals have many thermoregulatory strategies to maintain the 37C when diving to depths with water temperatures of -2C. The animal is maintaining this 37C body temperature, but skin cells (which we are culturing), are exposed to extreme temperatures.
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The pressure of the incubator is assumed to be atmospheric pressure, or 1 atm. The deepest diving Cuvier’s beaked whale can experience pressures up to 300 atm. Future experimentation could include the Duke Hyperbaric Center, where we could run experiments ± hypoxia and in high/low pressure conditions.
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Since these cold temperature, high pressure, and low oxygen conditions frequently coincide, the differences that we see in hypoxia may also be the organism preparing for an expected change in other conditions.
Conclusions
While there are limitations to cell culture, controls and comparative studies can help us to see differences that are related to the variables we are manipulating. For example, the 20% normoxia to 2% hypoxia conditions may not actually be physiologically normoxia and hypoxia to these cells in the whole organism, but we can still examine cellular responses to decreased oxygen. Temperature, pressure, and CO2 levels are held constant in the incubator, so by only manipulating the oxygen levels, we can hypothesize that any cellular differences we are seeing between cells of the same species are due to the oxygen variation.
Studying protected, deep-diving, elusive marine mammals is a challenge, but with scientific adaptations such as cell culture we can learn so much about their unique adaptations. The limitations of cell culture must be remembered when applying this information to a whole organism. The benefits of cell culture must be recognized when we can make cross-species comparisons of cellular adaptations and make mechanistic hypotheses for future scientific study.
Citations
Donnelly C, Schmitt S, Cecatto C, Cardoso L, Komlódi T, Place N, Kayser B, Gnaiger E (2022) The ABC of hypoxia – what is the norm? MitoFit Preprints 2022.25.v2. https://doi.org/10.26124/mitofi t:2022-0025.v2
Ramirez, J. M., Folkow, L. P., & Blix, A. S. (2007). Hypoxia Tolerance in Mammals and Birds: From the Wilderness to the Clinic. Annual Review of Physiology, 69(1), 113–143. https://doi.org/10.1146/annurev.physiol.69.031905.163111
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Pain, S. (2022, May 31). Call of the deep. Knowledgable Magazine. https://knowablemagazine.org/article/living-world/2022/deep-diving-animals-ocean-twilight-zone
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University of Hawai’i. (2022). Compare-Contrast-Connect: The Deep Divers. Exploring Our Fluid Earth.
