France

Mammalian cell culture is widely used for discovery and development. Recently, increasing
attention has been paid to the importance of maintaining physiologically-relevant conditions in cell
culture. Although oxygen level is a particularly important consideration, it is rarely regulated by
experimentalists. The atmospheric O2
levels commonly used in cell culture are significantly higher
than those experienced by most mammalian cells in vivo, leaving cells susceptible to oxidative damage, senescence, transformation, and otherwise aberrant physiology. A barrier to incorporating O2
regulation into most cell culture workflows has been the expense of investing in new equipment, as
the vast majority of laboratory CO2
incubators do not regulate O2
. Here, we describe an inexpensive
(&lt;CAD 1000), portable and user-friendly O2/CO2
incubator that can establish and maintain physiological O2
, CO2
, and temperature values within their physiological ranges. We used an Arduino-based
approach to add O2 and CO2 control to a temperature-regulating egg incubator. Our incubator
was tested against a commercial laboratory O2/CO2
incubator. Using Presens OxoDish technology, we demonstrate that at a setpoint value of 5% gas-phase incubator O2
, media O2 averaged
5.03 (SD = 0.03) with a range of 4.98–5.09%. MCF7, LNCaP and C2C12 cell lines cultured in the
incubator displayed normal morphology, proliferation, and viability. Culture for up to one week
produced no contamination. Thus, our incubator provides an inexpensive means of maintaining
physioxia in routine mammalian cell culture.

SEB Annual Conference 2022

An Inexpensive Incubator for Mammalian Cell Culture Capable of Regulating O2, CO2, and Temperature

Discover the art of creating extraordinary science at SEB’s Annual Conference at Le Corum Conference Center     in Montpellier, France from 5 to  8 July 2022.

SEB’s Annual Conference is celebrated for creating the perfect composition of research, new discoveries and collaborative connections. Be creative and innovative by exchanging ideas and knowledge with masters of biology from all over the world.

2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years.
	<br><br><br>


Please register for the conference by clicking on this link:

https://www.sebiology.org/events/seb-annual-conference-2022/registration.html

The site will be open for registered participants closer to the event.

2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France

Mammalian cell culture is widely used for discovery and development. Recently, increasing
attention has been paid to the importance of maintaining physiologically-relevant conditions in cell
culture. Although oxygen level is a particularly important consideration, it is rarely regulated by
experimentalists. The atmospheric O2
levels commonly used in cell culture are significantly higher
than those experienced by most mammalian cells in vivo, leaving cells susceptible to oxidative damage, senescence, transformation, and otherwise aberrant physiology. A barrier to incorporating O2
regulation into most cell culture workflows has been the expense of investing in new equipment, as
the vast majority of laboratory CO2
incubators do not regulate O2
. Here, we describe an inexpensive
(<CAD 1000), portable and user-friendly O2/CO2
incubator that can establish and maintain physiological O2
, CO2
, and temperature values within their physiological ranges. We used an Arduino-based
approach to add O2 and CO2 control to a temperature-regulating egg incubator. Our incubator
was tested against a commercial laboratory O2/CO2
incubator. Using Presens OxoDish technology, we demonstrate that at a setpoint value of 5% gas-phase incubator O2
, media O2 averaged
5.03 (SD = 0.03) with a range of 4.98–5.09%. MCF7, LNCaP and C2C12 cell lines cultured in the
incubator displayed normal morphology, proliferation, and viability. Culture for up to one week
produced no contamination. Thus, our incubator provides an inexpensive means of maintaining
physioxia in routine mammalian cell culture.

poster

Many animals experience periods of reduction in food quality and quantity. Metabolic responses of animals to periods of reduction in crucial nutrient and food deprivation may have positive and negative impacts on the animal performance. I will present results on the mitochondrial responses to reduction in omega-3 dietary availability in a marine fish, golden grey mullet (Chelon auratus) and naturally-occurring periods of food deprivation in brown trout (Salmo trutta). 
We show that mitochondria of fish fed on the low omega 3 highly unsaturated fatty acids (n-3 HUFA) diet had higher mitochondrial efficiency than those of fish maintained on the high n-3 HUFA diet. Yet, mitochondrial efficiency varied up about 2-fold among individuals on the same dietary treatment, resulting in some fish consuming half the oxygen and energy substrate to produce the similar amount of ATP than conspecific on similar diet.
In another study, after a 2-week period of fasting, brown trout increased their oxidative phosphorylation (OXPHOS) respiration and decreased proton leakage (LEAK) respiration, suggesting a selective increase in the capacity to produce ATP without a concomitant increase in energy dissipated through proton leakage. However, this was associated with an almost two-fold increase in mitochondrial H2O2 levels.
Our results emphasize that (i) shift in energy metabolism in response to food change may help preserve limited resources but potentially come at a cost of increased oxidative stress; (ii) individual variation in mitochondrial efficiency may explain intraspecific variation in response to food changes.







Mitochondrial responses to varying diet quality and quantity: shift in metabolic efficiency and capacity.

Thermal acclimation is a mechanism that ectotherms use to maintain performance across a range of temperatures. In variable environments, acclimating quickly may be as important as acclimation capacity. The phenotypic changes comprising acclimation require energy and nutrients that ectotherms obtain from their diet. Thus, diet may be a critical factor determining acclimation rate and capacity. Using a temperate omnivorous fish, opaleye (Girella nigricans), we tested the hypotheses that diet can impact thermal acclimation rate and capacity for cardiac performance. We offered juvenile opaleye an ad-lib herbivorous (algae), carnivorous (shrimp), or omnivorous (algae and shrimp) diet for two weeks at 12°C and measured the thermal performance curve (TPC) for maximum heart rate (fHmax) and cardiac thermal limits. Next, we raised the temperature in the holding tanks from 12 to 20ºC, mimicking an abrupt heatwave, and independently sampled fish on days 1, 3, 7, and 14 post-temperature change using the same cardiac test. Acclimation capacity for fHmax was sensitive to diet, with the herbivorous treatment displaying the smallest change in fHmax. However, acclimation rate and all cardiac thermal limits were insensitive to diet, with &gt;60% of acclimation capacity occurring in the first 3 days at 20°C. Our results indicate that for generalist species, like opaleye, variation in diet is an important determinant of thermal performance on environmentally relevant timescales. Our findings also add to growing evidence that it is essential to integrate acclimation time into our predictions of species responses to temperature change.

Diet effects on acclimation rate and capacity for maximum heart rate in a temperate fish (Girella nigricans)

All animals must acquire food for energy, but there is a vast diversity in how different species and even different individuals approach this task. Individuals within a species appear to fall along a bold-shy continuum, whereby some fish acquire food aggressively and with seemingly high risk, while others appear more opportunistic and cautious. Greater food consumption generally results in faster growth, but only if the energy acquired through food is more than enough to compensate for heightened metabolism associated with a more active lifestyle. Fast-growing phenotypes also tend to have elevated baseline metabolism – at least when food is plentiful – which further complicates the link between food consumption and growth. The net energy gained from a meal (as calculated from the specific dynamic action (SDA) coefficient) is optimised with larger meal sizes, but the digestion of large meals can erode the aerobic metabolic scope available for other critical activities such as predator avoidance. Thus, it is expected that fish in predator-rich habitats should regulate meal sizes to maximise growth efficiency without dangerously compromising aerobic capacity, and this balance may differ between individual metabolic phenotypes. This presentation will discuss these complicated interplays with a goal to better understand what drives intraspecific differences in growth performance.

Why some fish grow faster than others: examining the interplay between metabolic phenotype, predator threat and growth performance

In several organisms including fish, lipids are the most efficient source of energy compared to other nutrients. Some fatty acids (e.g. oleic acid, palmitic acid) are known to be preferentially used by organisms to create or to store energy. Here, we aimed to understand the relationship between FA profile, individual growth performance and well-being of European sea bass (Dicentrarchus labrax). For that, we coupled the measurements of growth rate and Fulton’s condition factor (K), both widely used to estimate growth and the well-being of a fish, to an innovative non-lethal sampling method for FA analysis that allows us to measure the evolution of the fatty acid profile individually. Eighteen tagged sea bass juveniles were reared during 130 days in the same food and temperature conditions, in triplicate. White muscle subcutaneous biopsy was carried out on each fish at day 0 and day 130 for FA analysis. Fish were weighted and measured to calculate growth rate (Thermal Growth Coefficient, TGC) and K at d0 and  d130. The fatty acid profiles of the three best and lowest performing fish (TGC related) were compared. The two biopsies performed over the 130 days induced no mortality. All the fish had positive TGC and showed an increase in K (1.01 ±0.15 at d0; 1.20 ±0.12 at d130). The results will be discussed trying to establish a link between FA, TGC and K, on an individual scale.

Relation between fatty acid profile, growth rate and condition factor using a non-lethal sampling method in European sea bass.

The cold of temperate winters slows, and may constrain, ectotherm physiology. Yet many fish species remain active in the winter, with continued performance facilitated by physiological compensation. Conversely, certain species (e.g., cunner, Tautogolabrus adspersus) enter winter dormancy – an energy-savings strategy characterized by inactivity, fasting, and slow metabolism – which allows them to endure frigid, food-poor winters and persist at poleward latitudes. We hypothesized that winter dormancy is a consequence of severe constraints on digestion and growth in the cold, which make it inefficient to remain active and feeding. Cunner become dormant below ~7.5°C, so we predicted negative growth and zero feeding below ~7.5°C even when provided food. In voluntarily feeding fish acclimated to 15, 9, or 3°C, daily food consumption and specific growth rates decreased significantly with declining temperature, and dormant fish at 3°C had near-zero feeding rates with negative growth. We then repeated this experiment with force-feeding 1% body weight every third day of acclimation to determine if fasting and weight loss results from physiological constraints of cold on digestion and growth processes. Contrary to our hypothesis, force-fed cunner showed positive growth at 3°C, indicating that the fasting, inactive, and non-foraging phenotype characteristic of dormancy is a behavioural response rather than a consequence of intrinsic physiological constraints. We are currently analyzing specific dynamic action. Our findings are consistent with an overarching conclusion emerging from our work on cunner: physiological performance is not severely constrained by cold in winter-dormant fishes, and ecological factors are more likely to be driving winter dormancy. 

Fasting in winter-dormant fish is a behavioural response rather than a consequence of constraints of cold on digestion and growth

Torpor is a state of active metabolic depression associated with a decrease of body temperature that enables individuals to save energy to cope with environmental fluctuations. Heterothermic species can use short, daily bouts of torpor during the active season, e.g., for young individuals to sustain processes of growth, development and pre-hibernation fattening. Hibernators can also employ torpor bouts lasting several days, weeks or months, called hibernation, to survive long winters with no or little food available. Feeding can affect the use of torpor and the seasonal expression of hibernation. For instance, reduced food availability facilitates the use of torpor, which in turn can increase nutrient assimilation during euthermic phases. Also, various proportions of omega fatty acids modulate hibernation onset and impacts hibernation performances, notably the duration and depth of torpor bouts, during the winter. Saturated fatty acids ensure the optimal fuelling of hibernation, while some unsaturated fatty acids regulate oxidative and metabolic functions including the proper functioning of the heart, a key organ to maintain the activity during deep torpor. Further, contrasted torpor and hibernating patterns, resulting from feeding regimes that differ in terms of food quantity and/or quality, can lead to contrasted life-history strategies and impacts on somatic maintenance, survival and longevity. In this talk, I will present evidence for effects of feeding on the seasonal regulations of the expression of torpor and hibernation as strategies affecting the survival and life-history traits of individuals from hibernating species.

Effects of feeding on the energetics of torpor in hibernating species

Migratory flight requires birds to sustain high levels of energy expenditure, fueled almost exclusively by fat, for prolonged periods of time. Beyond fueling flight, the types of dietary fatty acids may influence performance. The natural doping hypothesis proposes that shorebirds feed on diets rich in essential n-3 long chain polyunsaturated fatty acids (LCPUFA), which help prime the flight muscles for endurance flight by enhancing membrane fludity, promoting rapid fatty acid transport, and activating cellular signalling pathways to increase aerobic and fatty acid oxidation capacity. We fed western sandpipers (Calidris mauri) formulated diets low or high in n-3 LCPUFA (EPA and DHA) and flew them for up to 8 hours in a wind tunnel to assess endurance flight performance. We found that a diet high in n-3 LCPUFA decreased flight energy expenditure by 12%. Activities of key oxidative enzymes, citrate synthase and carnitine palmitoyl transferase were significantly greater in the flight muscles of birds fed the n-3 LCPUFA enriched diet, and we are investigating further effects on the regulation of lipid metabolism and oxidative capacity. In addition, we present evidence for limited capacity for endogenous synthesis of n-3 LCPUFA from precursor fatty acids, suggesting an essential dietary requirement for n-3 LCPUFA. These findings support the natural doping hypothesis by demonstrating performance-enhancing effects at the biochemical and whole animal level, and they indicate a critical role of dietary n-3 LCPUFA during migration for shorebirds.

Flight testing natural doping: Influence of dietary n-3 long chain polyunsaturated fatty acids on migratory performance in western sandpipers

Changes in the early-life microbiota of hosts might affect infectious disease risk throughout life, if such disruptions during formative times alter immune system development. For this talk, I present empirical evidence that an early-life disruption of gut microbiota has immediate and long-term consequences on parasite resistance across taxa, including Cuban tree frogs and passerine birds. In these studies, we experimentally manipulated the gut microbiota of hosts, during the immune developmental period, with mild antibiotics then quantified their ability to resist parasitic nematodes (frogs) or nest flies (birds). An early-life microbiota disruption had varying effects on host resistance to parasitism. A microbiota disruption in tapoles led to a decrease in parasite resistance later in life, whereas a disruption in nestling birds lead to either an increase or decrease in resistance to nest flies, depending on the host species. These results support the idea that preventing early-life disruption of host-associated microbiota might confer protection against diseases, but this idea depends on the host taxa.

Early-life disruption of gut microbiota affects resistance to parasites

Phosphate sensing SPX proteins control arbuscule life-time in Medicago

Contagious cancers are somatic cell lineages that are transmitted between individuals via the transfer of living tumour cells, meaning that they can survive beyond the host that spawned them. In other words: they are tumours that metastasize across different hosts. For this reason, they represent an interesting and unique model to illuminate insights into the general mechanisms of cancer development and metastasis. Since 2016, we have collected 6,300 common cockles from 33 locations covering all the Atlantic Coast of Europe, from Morocco to Russia. 342 cockles (5.4%, 350/6,300) from 5 different countries were diagnosed with cancer by cytological and histological methods. The contagious nature of these cancer samples was assessed with sequencing data as it has been previously done in dogs and Tasmanian devils that also suffer from contagious cancers. Phylogenetic analyses based on variants from mitochondrial DNA revealed nine paraphyletic clonal cancer lineages, some with a distribution extending over thousands of miles. The analysis of nuclear structural variants, however, does not reveal nine cancer lineages, seeming to suggest a maximum of two independent origins of the disease. Multiple captures of the mitochondria from the host cells by the cancer lineages, throughout the long-term evolution of these two cancer lineages, may explain these contradicting observations. This work represents the most comprehensive analysis of a marine contagious cancer performed to date and reveals that mitochondrial and nuclear DNA of cockle transmissible cancers tell slightly different evolutionary stories that are part of the same book.

Premium content

An Inexpensive Incubator for Mammalian Cell Culture Capable of Regulating O2, CO2, and Temperature

Next from SEB Annual Conference 2022

Mitochondrial responses to varying diet quality and quantity: shift in metabolic efficiency and capacity.

Similar lecture

The Determinator: How does respiratory complex I control metabolic flux, fertility and longevity?

Stay up to date with the latest Underline news!

PRESENTATIONS

CONFERENCES

COMPANY

RESOURCES