Recently, the understanding of dynamic cellular changes that occur in vivo has advanced significantly, both at the extracellular and intracellular levels. These changes might fluctuate with daily, circadian, weekly, or monthly intervals, and the approaches used to understand these changing conditions in vitro should parallel in vivo studies. In addition, the in vitro milieu should be optimized and better defined, so that artefacts due to in vitro culture systems would not pose dangers for the proper interpretation of results. In this article, we discuss some of these issues and propose solutions.

Culture media are generally supplemented with glucose, amino acids, and lipids. Looking at the general compositions of the most commonly used media (i.e. RPMI, DMEM, IMDM), the glucose concentration ranges from 2- to 4-fold higher than the physiologic plasma concentration: in RPMI it is 200mg/dl, DMEM is 450mg/dl, and IMDM is 450mg/dl, compared with a physiological range in plasma of 60–100mg/dl [1, 3, 4]. Amino acid concentrations differ even more dramatically, ranging from 200- to 500-fold higher than normal plasma levels in humans and mice [1, 3, 4]^*. Such is also the case with lipids, in particular the levels of cholesterol, phospholipids, and triglycerides [1, 3, 4]^*. Supra-physiologic concentrations will profoundly impact the results and cellular responses when compared with normal physiology. Glucose, amino acids, and lipids activate a series of intracellular responses such as the mammalian target of rapamycin (mTOR) pathway, the nutrient energy sensing systems (ie. AMPK, SIRT1), and nuclear receptors such as the PPARs [5]. Given a series of reports showing that these systems can profoundly affect cell growth and survival as well as having dynamic and oscillatory activities [6], it must be considered that constant supra-physiologic concentrations of these nutrients in culture media will influence the experimental results and their interpretation in light of normal physiology.

The addition of serum to culture media is often crucial for both normal and transformed or immortalized cells. The enormous value of this supplement is undisputed, yet it carries implications that are rarely pondered or questioned. Bovine serum derived from foetal calves (FBS) has a very high concentration of placental hormones including prolactin, chorionic gonadotropin, growth hormone, leptin, oestrogen, progesterone, and cortisol, among others [1, 2]. All of these factors, even when diluted to 10% of their original concentrations into the culture media, remain at such high levels to practically reproduce an in vitro environment of pregnancy rather than a condition of normalcy. Obviously, these conditions affect cells at multiple levels. Consequently, this key aspect has sometimes been dealt with by using autologous sera, which may nonetheless still be used at supra-physiologic concentrations. Finally, little consideration has been given to the possibility that culture media components themselves may have hormonal activity. For example, phenol red, which bears a structural resemblance to some nonsteroidal estrogens and is used ubiquitously as a pH indicator in tissue culture media, has significant estrogenic activity at the concentrations (15–45 mM) typically found in tissue culture media [7].

Commonly, cell incubators have an internally controlled and static atmosphere that is supplemented with gases such as carbon dioxide (CO₂; 5%) and atmospheric air. CO₂ supplementation is necessary to maintain a physiologic pH in the culture media because of its buffering capacity (carbonate/bicarbonate buffering system; HCO3⁻/H₂CO₃) [1, 2, 8]^*. The pressure of O₂ is basically at equilibrium within the atmosphere and is dissolved to approximately 160 mm/Hg in culture media (about 20% in the air). However, both CO₂ and O₂ concentrations in the incubator chambers do not change dynamically, as they do in the blood stream (i.e., the arterial versus venous blood), but remain static. Although O₂ pressure in the atmosphere is at 160 mm/Hg, when at equilibrium in the culture media the concentration of O₂ in cultures becomes non physiologic [1, 2, 8]^*. Indeed, in normal blood and peripheral tissues, the concentration of O₂ is approximately 100 mm/Hg (about 13%) in the arterial blood and approximately 40 mm/Hg (about 8%) in venous blood [8]. These substantial differences in the concentration of O₂ between incubators and normal blood (160 mm/Hg versus 40–100 mm/Hg, respectively) are capable of modifying the respiratory and glucose/fatty acid oxidative processes and mitochondrial respiration in a very powerful manner [5]. Although CO₂ may be less influential on in vitro studies because the arterial concentration of this gas is approximately 40 mm/Hg (as in the incubators at 5%), and its level in the venous blood averages 46 mm/Hg (about 8%), the above considerations suggest that what is observed in cells kept in culture can differ significantly from what occurs in the whole organism.

Culture plates do not provide the dynamic changes in temperature, nutrients, hormones, and growth factors that are present in vivo. Because it is generally assumed that the internal human body temperature is 37°C, tissue culture incubators are set to this constant temperature. However, although the core body temperature is maintained constant, dynamic changes still occur due to the external temperature (of the environment), and owing to variations between vital organs (truncular tissues) and the periphery (i.e. limbs), where the temperature is generally 2–3°C lower [9]. Another critical variation in the circadian body temperature takes place during the sleep–wake cycle, with significantly lower temperatures during the period of sleep compared with the period of wakefulness. These subtle differences in body temperature have a profound impact on cellular and tissue metabolism and functions [9]. It must be reiterated that these dynamic changes cannot be excluded as possible factors affecting in vitro cellular responses. Like body temperature, variations in nutrients exist in the whole body, as do hormonal fluctuations that can occur hourly, intradiurnally, or through circadian cycles (e.g., cortisol, leptin, prolactin, GH). These dynamic changes are not present within in vitro cultures and cannot be reproduced in these systems.

The use of immortalized or transformed cells has provided unique possibilities in the molecular dissection of intracellular mechanisms that determine the different cellular responses. Non-immortalized cell lines from normal tissues and primary cultures provide a more physiologic condition to study the processes leading to intracellular signalling. Immortalized cell lines or transformed cells have high basal levels of nutrient sensing factors such as mTOR and AMPK, which do not reflect normal physiology [5]. Conversely, non-immortalized cells have several “fuel sensing systems” that are sensitive to changes in the extracellular and intracellular milieu [5]. The switch in fuel utilization is common and can profoundly affect metabolic responses depending on the intracellular milieu (i.e., glycolysis to fatty-acid oxidation). For example, in both hypothalamic and immune responses, the cyclic and dynamic changes in intracellular activities of multiple enzymes and kinases can differentially affect outcomes and experimental results.

In vitro approaches have limitations that are often underestimated or not considered. The scientific community should be well aware of these aspects when interpreting data and should place a greater emphasis on developing in vitro new approaches that take into consideration the dynamic environmental and intracellular processes that are typical characteristics of normal physiology.