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Amino Acid Metabolism in Mamalian Cell Cultures
... It would also be advantageous to use proteins produced identically for both OT and NMR studies, eliminating any artifact related to construct and streamlining production pipelines. While mammalian cells, in general, are not tolerant to the high percentage of D 2 O in media, they do have an advantage in that certain amino acid synthetic pathways are absent (Eagle, 1959), facilitating the supplementing with specifically labeled amino acids for incorporation into highly expressed heterologous proteins. This is advantageous in implementing site-specific labeling strategies for NMR of high molecular mass proteins (Henot et al., 2021;Höfurthner et al., 2023). ...
... To standardize the procedure of adding amino acids to ILVFY À media, the small-scale culture of the Expi293F cells was initiated. The simplest procedure was to add back the deficient amino acids at levels equivalent to the standard formulation of Dulbecco's modified Eagle medium (DMEM) (Dulbecco & Freeman, 1959;Eagle, 1959) and referred to herein as 1X (see Methods for exact formulation). For this experiment, cells were plated at 2 Â 10 6 cells/mL at 3 mL/well in six-well flat bottom plates, transfected with N30βc1, and cultured for 4 days in standard conditions. ...
... This third method resulted in excess toxicity for the cultured cells and is not recommended as a purification when working with mammalian cells. (Dulbecco & Freeman, 1959;Eagle, 1959): 0.104 mg/mL Ile, 0.104 mg/mL Leu, 0.094 mg/mL Val, 0.066 mg/mL Phe, and 0.103 mg/mL Tyr as this appeared to enhance cell survival. These levels (1X) and multiples thereof (2X, 4X) were used as the basis for the small-scale titration experiments (Figure 4). ...
Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with ¹⁵N and ¹³C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium‐substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli‐based expression systems. One such class of proteins includes T‐cell receptors and their related preT‐cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the β subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid‐specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 μM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.
... But both the concentration of leachate and the killing ability are strongly affected by the presence of other soluble species. In the presence of Dulbecco's Modified Eagle Medium (DMEM, a common medium for mammalian cell growth [72]), there was strong killing and a high concentration. Additionally, we found that the generation of ROSs are not involved in the mechanism of Cu 2 O surfaces against P. aeruginosa. ...
... In contrast, we found a very large concentration of copper, 4,200 µM, leached into DMEM. DMEM is a widely used growth medium for mammalian cells [72], and therefore is also used as a medium for testing pathogens against mammalian cells [23]. Therefore, our first conclusion is that the concentration of dissolved copper that is presented to bacterial cells in Table 1 Total copper concentration as measured by ICP-MS. ...
Some very effective antimicrobial coatings exploit copper or cuprous oxide (Cu2O) as the active agent. The aim of this study is to determine which species is the active antimicrobial - dissolved ions, the Cu2O solid, or reactive oxygen species. Copper ions were leached from Cu2O into various solutions and the leachate tested for both dissolved copper and the efficacy in killing Pseudomonas aeruginosa. The concentration of copper species leached from Cu2O into aqueous solution varied greatly with the composition of the aqueous solution. For a range of solution buffers, killing of P. aeruginosa was highly correlated with the concentration of copper in the leachate. Further, 10 µL bacterial suspension droplets were placed on Cu2O coatings, with or without a polymer barrier layer, and tested for bacterial kill. Killing occurred without contact between bacterium and solid, demonstrating that contact with Cu2O is not necessary. We therefore conclude that soluble copper species are the antimicrobial agent, and that the most potent species is Cu+. The solid quickly raises and sustains the concentration of soluble copper species near the bacterium. Killing via soluble copper ions rather than contact should allow copper coatings to kill bacteria even when fouled, which is an important practical consideration.
... Tumor growth is influenced by both cell intrinsic and cell extrinsic factors, and nutrient availability is emerging as a critical environmental factor that can shape the metabolic fitness and proliferative capacity of tumors (1)(2)(3). Concurrent with these discoveries has been a renewed realization that standard cell culture media were not designed to mimic the nutrient environment found in vivo, but were rather designed to provide excess amounts of the minimal nutrients required to sustain cancer cell growth in vitro (4)(5)(6)(7)(8)(9). As a result, the nutrients present in these traditional culture media do not accurately recapitulate the complexity or abundance of nutrients found in vivo. ...
... To determine whether a unique component of HPLM is responsible for the reduced sensitivity to rigosertib, we combined HPLM stocks 8-18 and added them to RPMI and performed dose-curve analyses, where we found that stocks 8-18 were able to recapitulate the effect of HPLM on rigosertib sensitivity ( Figures 3A and 3B). We then analyzed stocks [8][9][10][11][12][13][14][15][16][17][18] . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism waste product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. Structural modelling studies suggest that uric acid interacts with the tubulin-rigosertib complex and may act as an uncompetitive inhibitor of rigosertib. These results offer a possible explanation for the failure of rigosertib in clinical trials and demonstrate the utility of physiological media to achieve in vitro results that better represent human therapeutic responses.
... quantity of amino acids that are available for the animal [28], giving valuable information on both the animal's requirements and nutritional balance of the diet. Animals synthetize specific proteins depending on their requirements [29]. Protein synthesis mainly depends on animal requirements, determined by DNA transcription, and amino acids provided by the diet [30]. ...
... The growth pattern of an animal also determines its protein requirements. The growth of all body components can be investigated by slaughtering and analysing animals Animals synthetize specific proteins depending on their requirements [29]. Protein synthesis mainly depends on animal requirements, determined by DNA transcription, and amino acids provided by the diet [30]. ...
The ideal protein concept refers to dietary protein with an amino acid profile that exactly meets animal’s requirement. Low quality protein levels in the diet have neg-ative implications on productive and reproductive traits, and a protein oversupply is energy costly and leads to an excessive N excretion, with potentially negative en-vironmental impact. Urea Nitrogen (UN), which corresponds to the amount of ni-trogen in the form of urea circulating in the bloodstream, is a metabolite that has been widely used to detect amino acid imbalances and deficiencies and protein re-quirements. This review aims to critically analyse how UN can contribute to accu-rately implement the ideal protein concept in monogastric animals, particularly in pigs, poultry, and rabbit nutrition (14.000 animals from 76 published trials). About 59, 37 and 4% of trials have been conducted in pigs, poultry, and rabbits, respec-tively. UN level was negatively correlated to main performance traits (PCC of -0.98 and -0.76, for average daily gain and feed conversion ratio, respectively), and lower UN level was related to higher milk yield and concentration. High level of UN was positively correlated to N excretion (PCC=0.99) and negatively correlated to protein retention (PCC=-0.99). Therefore, UN in blood seems to be a proper indicator of amino acid imbalance in monogastric animals. Great variability in the use of UN was observed in the literature, as determination medium (blood, plasma, or serum), units, feeding system used (ad libitum or restricted), among others. A standardiza-tion of the methods in each of the species, with the aim to harmonize comparison among works, is suggested. After review, it is recommended UN measurement in plasma and, whenever possible, the utilization of the same nutritional methodology (ad libitum conditions or restriction with blood sampling after refeeding at stand-ardised time). More studies are necessary to know the potential of UN and other bi-oindicators on amino acid deficiencies evaluation to get closer to ideal protein con-cept.
... Tumor growth is influenced by both cell intrinsic and cell extrinsic factors, and nutrient availability is emerging as a critical environmental factor that can shape the metabolic fitness and proliferative capacity of tumors (1)(2)(3). Concurrent with these discoveries has been a renewed realization that standard cell culture media were not designed to mimic the nutrient environment found in vivo, but were rather designed to provide excess amounts of the minimal nutrients required to sustain cancer cell growth in vitro (4)(5)(6)(7)(8)(9). As a result, the nutrients present in these traditional culture media do not accurately recapitulate the complexity or abundance of nutrients found in vivo. ...
In-Press Preview The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans. ABSTRACT The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic affects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans. 3
... The nanoformulations were screened for their anticancer activity using the human liver carcinoma cell line HepG2 (ATCC: HB-8065™) [36] and for cytotoxic evaluation against the normal human cell line HEK 293 as follows. The cells were grown in DMEM media supplemented with 10 % fetal bovine serum, 1 % penicillin-streptomycin, and 5 mM sodium pyruvate at 37 • C in a CO 2 incubator (5 % CO 2 , 95 % ERH) [37]. The cells were harvested at the sub-confluent stage using trypsin, washed with PBS, and resuspended in the growth media. ...
Cisplatin is a versatile drug used to treat various types of cancer, but it is associated with high toxicity and resistance problems. Several approaches, including nanotechnology, have been adopted to minimize the toxic effects and to overcome the resistance of cisplatin. Most of the nanoformulations involve the use of synthetic or semisynthetic polymers as drug carriers. In this study arabinoxylan nanoparticles have been investigated as drug reservoirs for intestinal drug delivery. The drug-loaded arabinoxylan nanoparticles (size: ∼1.8 nm, polydispersity index: 0.3 ± 0.04) were prepared and nanoformulation was characterized by various analytical techniques. The nanoformulation was found to be stable (zeta potential: 31.6 ± 1.1 mV). An in vitro cytotoxicity against HepG2 and HEK 293 cell lines was studied. The cell viability analysis showed greater efficacy than the standard cisplatin (IC50: cisplatin 2.4, arabinoxylan nanoformulation 1.3 μg mL⁻¹). The expression profile of carcinogenic markers revealed a six-fold upregulation of MLKL and 0.9-fold down regulation of KRAS, suggesting the activation of the necroptotic pathway by the drug-loaded nanoparticles. The nanoformulation exhibited a sustained release of cisplatin with a cumulative release of ∼40 % (at pH 7.4) and ∼30 % (at pH 5.5) over a period of 12 h with very low initial burst. The study suggests that the use of the new nanoformulation can significantly reduce the required dose of cisplatin without compromising efficacy and more efficient release at basic pH.
... Glutamic acid is considered an essential amino acid crucial for the survival and growth of cells in cell culture, including fibroblasts and human cells. It is synthesized from glucose and plays a vital role in protein synthesis, contributing to the growth and maintenance of cells (31)(32)(33). Cysteine serves a protective role against oxidative stress and possesses strong antioxi dant effects, contributing to cell stability and facilitating growth and function in suitable environments. Consequently, it enhances cellular resilience. ...
It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.
IMPORTANCE
The choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract’s abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.
... In cultured cells, amino acids from culture media are assimilated and synthesised to form the necessary pools crucial for sustaining normal metabolism, biosynthetic activities and overall cellular homeostasis, and they also play a pivotal role during viral infection [50][51][52]. Accordingly, our results showed that valine, phenylalanine and proline were increased during CHIKV infection. These findings demonstrate broad alterations in glycolysis and TCA cycle metabolism to actively support and facilitate the infection process [53] (Figures 6a and 8). ...
The arbovirus Chikungunya (CHIKV) is transmitted by Aedes mosquitoes in urban environments, and in humans, it triggers debilitating symptoms involving long-term complications, including arthritis and Guillain-Barré syndrome. The development of antiviral therapies is relevant, as no efficacious vaccine or drug has yet been approved for clinical application. As a detailed map of molecules underlying the viral infection can be obtained from the metabolome, we validated the metabolic signatures of Vero E6 cells prior to infection (CC), following CHIKV infection (CV) and also upon the inclusion of the nsP2 protease inhibitor wedelolactone (CWV), a coumestan which inhibits viral replication processes. The metabolome groups evidenced significant changes in the levels of lactate, myo-inositol, phosphocholine, glucose, betaine and a few specific amino acids. This study forms a preliminary basis for identifying metabolites through HR-MAS NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Ressonance Spectroscopy) and proposing the affected metabolic pathways of cells following viral infection and upon incorporation of putative antiviral molecules.
... The earliest evidence is from Harry Eagle, showing that supplementation with GSH can rescue cells grown without cystine. 119 At the time, it was not clear how this rescue occurred. Although GSH can be exported from a cell, 120 the import of GSH is more complicated. ...
Scientists in this field often joke, “If you don’t have a mechanism, say it’s ROS.” Seemingly connected to every biological process ever described, reactive oxygen species (ROS) have numerous pleiotropic roles in physiology and disease. In some contexts, ROS act as secondary messengers, controlling a variety of signaling cascades. In other scenarios, they initiate damage to macromolecules. Finally, in their worst form, ROS are deadly to cells and surrounding tissues. A set of molecules with detoxifying abilities, termed antioxidants, is the direct counterpart to ROS. Notably, antioxidants exist in the public domain, touted as a “cure-all” for diseases. Research has disproved many of these claims and, in some cases, shown the opposite. Of all the diseases, cancer stands out in its paradoxical relationship with antioxidants. Although the field has made numerous strides in understanding the roles of antioxidants in cancer, many questions remain.
... Amino acids, vitamins that are required for the growth and proliferation of mammalian cells in culture and found that cystine (Cys2) which is the oxidized thiol-containing form of cysteine (Cys) one of the most essential amino acids. In absence of Cys2, cells fail to grow as there will be rapid depletion of Cys-containing antioxidant tripeptide GSH (c-L-glutamyl-L-cysteinglycine) and subsequently the cells die due to the accumulation of L-ROS (Banni et al., 1977;Eagle et al., 1961;Eagle, 1959;Eagle, 1955). System X -C is a Cystine/Glutamate (Cys/Glu) antiporter which play an important role in ferroptosis. ...
Ferroptosis is a recent form of non-apoptotic cell death, which occurs due to imbalance of iron homeostasis (iron overload). Oxidative stress due to the impairment of the antioxidant system is a major pathophysiology during ferroptosis, which eventually results in lipid peroxidation. The cellular and physiological biomarker of ferroptotic cell death includes major alteration in the glutathione peroxidase 4 (GPX4) antioxidant system and lipid peroxidation induced loss of plasma membrane integrity. This review elaborates the mechanism of oxidative stress which triggers ferroptosis.
... Classical media have traditional compositions and are widely used in research studies. TK-ST cells were cultured in six classical media of different compositions with 10% FBS: three types of medium based on DMEM (Dulbecco and Freeman 1959), EMEM (Eagle 1959), RPMI-1640 medium (Moore et al. 1966), and Ham's F-12 medium (Ham 1965). We found that the three DMEM and RPMI-1640 cell lines showed almost the same growth rate in the TK-ST culture. ...
Common bottlenose dolphin (Tursiops truncatus) is a well-known cetacean species that inhabits temperate and tropical seas worldwide. Limited supply and poor quality of samples hinder the investigation of the effects of various pathogens and environmental pollutants on this cetacean species. Cultured cells are useful for experimental studies; however, no cell lines derived from cetaceans are generally available. Therefore, in this study, we established a novel kidney cell line, TK-ST, derived from T. truncatus. Primary cells exhibited the morphological characteristics of epithelial and fibroblast cells, but their immortalization and passaging resulted in a predominantly epithelial cell morphology. TK-ST was immortalized using the large T SV40 antigen and human telomerase reverse transcriptase and exhibited long-term stable cell growth. TK-ST cells are generally cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37°C and 5% CO2 but can also be cultured in 5–20% fetal bovine serum and several other classical media commonly used for common animal cell culture. TK-ST cells were found to be susceptible to several viruses, including the dolphin morbillivirus (most important virus in cetaceans), and exhibited cytopathic effects, facilitating the replication of the dolphin morbillivirus. Furthermore, mRNA expression levels of cytokine genes were increased in TK-ST cells after stimulation with lipopolysaccharides and poly(I:C). Therefore, the novel TK-ST cell line derived in this study can potentially be used for further in vitro studies on cetaceans.
... Despite being categorized as a non-essential amino acid, L-serine has repeatedly demonstrated beneficial effects by acting as a precursor to crucial molecules required for protein synthesis, cell proliferation, and development. Its role in the formation of sphingolipids in the central nervous system is essential for neural differentiation and survival [1,2]. L-serine is produced via the phosphorylated pathway in the mammalian brain and is derived from glucose from glycolysis rather than phosphenolpyruvate from gluconeogenesis [3]. ...
L-serine is a non-essential amino acid that plays a vital role in protein synthesis, cell proliferation, development, and sphingolipid formation in the central nervous system. It exerts its effects through the activation of glycine receptors and upregulation of PPAR-γ, resulting in neurotransmitter synthesis, neuroprotection, and anti-inflammatory effects. L-serine shows potential as a protective agent in various neurological diseases and neurodegenerative disorders. Deficiency of L-serine and its downstream products has been linked to severe neurological deficits. Despite its crucial role, there is limited understanding of its mechanistic production and impact on glial and neuronal cells. Most of the focus has been on D-serine, the downstream product of L-serine, which has been implicated in a wide range of neurological diseases. However, L-serine is approved by FDA for supplemental use, while D-serine is not. Hence, it is imperative that we investigate the wider effects of L-serine, particularly in relation to the pathogenesis of several neurological deficits that, in turn, lead to diseases. This review aims to explore current knowledge surrounding L-serine and its potential as a treatment for various neurological diseases and neurodegenerative disorders.
... Therefore, the tumor could not consume oxygen for OxPhos because glucose was the only carbon source metabolized into lactate. Later, in 1959, Minimal Essential Medium (MEM), developed by Harry Eagle [13], was introduced as a synthetic cell culture medium containing amino acids and vitamins. In 1958, a culture medium supplemented with FBS was developed by Arthur Robinson [14]. ...
In previous work, we showed that cancer cells do not depend on glycolysis for ATP production, but they do on fatty acid oxidation. However, we found some cancer cells induced cell death after glucose deprivation along with a decrease of ATP production. We investigated the different response of glucose deprivation with two types of cancer cells including glucose insensitive cancer cells (GIC) which do not change ATP levels, and glucose sensitive cancer cells (GSC) which decrease ATP production in 24 h. Glucose deprivation-induced cell death in GSC by more than twofold after 12 h and by up to tenfold after 24 h accompanied by decreased ATP production to compare to the control (cultured in glucose). Glucose deprivation decreased the levels of metabolic intermediates of the pentose phosphate pathway (PPP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in both GSC and GIC. However, glucose deprivation increased reactive oxygen species (ROS) only in GSC, suggesting that GIC have a higher tolerance for decreased NADPH than GSC. The twofold higher ratio of reduced/oxidized glutathione (GSH/GSSG) in GIS than in GSC correlates closely with the twofold lower ROS levels under glucose starvation conditions. Treatment with N-acetylcysteine (NAC) as a precursor to the biologic antioxidant glutathione restored ATP production by 70% and reversed cell death caused by glucose deprivation in GSC. The present findings suggest that glucose deprivation-induced cancer cell death is not caused by decreased ATP levels, but rather triggered by a failure of ROS regulation by the antioxidant system. Conclusion is clear that glucose deprivation-induced cell death is independent from ATP depletion-induced cell death.
... DMEM medium is formulated from various components, such as amino acids, vitamins, inorganic salts, sugars, and pH indicators. 43,44 RO and DMEM were independently supplemented with a 10 % v/v addition of fetal bovine serum (FBS, Thermo Fisher Scientific Inc, USA; cat. no. ...
Wave mixed single-use bioreactors (also called rocking bioreactors) are a group of devices in which mixing is caused by characteristic wave-like movement of liquid inside a pillow-shaped bag mounted on a continuously oscillating platform. This study aimed to investigate the influence of liquid phase viscosity and the presence of an antifoaming agent on the mass transfer in a 2 liter single-use bag used in a ReadyToProcess WAVE 25 bioreactor system. The mass transfer rate was quantified by measuring the volumetric liquid-side mass transfer coefficient k L a using a gassing-out method. Two liquid phase mixture types were independently composed of Dulbecco's modified Eagle medium or water and supplemented with fetal bovine serum (FBS). The viscosity of the liquid phases was modified with glycerin. The measurements were performed with and without the addition of a Pluronic F-68 antifoaming agent in a viscosity range of 0.9−4.2 mPa·s. It was shown that the viscosity of the liquid phase significantly impacts the values of the k L a coefficient, ranging from 0.8 to 3.7 h −1 at α = 7°and ω = 2 min −1. The addition of the antifoaming agent caused a decrease in the mass transfer rate. A correlation has been proposed for estimating k L a values as a function of the modified Reynolds number.
... Glutathione peroxidase 4 (GPX4) utilizes reduced glutathione (GSH) to reduce lipid hydroperoxides to lipid alcos, to protect cells against membrane lipid peroxidation and inhibit ferroptosis [37]. At that time, researchers found that cystine deprivation causes cell death with a unique microscopic morphology and that restoration of GSH promotes cell growth [38]. Insufficient cystine supply induces metabolic stress and provokes ferroptosis cell death in tumor cells [36]. ...
Osteosarcoma (OS) is the most frequent primary malignant bone tumor. Ferroptosis, a form of regulated cell death, is a key tumor suppression mechanism. Although methionine adenosyltransferase II alpha (MAT2A) has been reported to inhibit several tumor cells, it is unclear whether inhibition of MAT2A in OS cells can reduce ferroptosis. CCK-8, flow cytometry, and Transwell assays were performed to evaluate cell viability, cell apoptosis/cycle, and cell migration, respectively. The levels of ferrous iron and glutathione (GSH) levels in cells were measured to evaluate the degree of cell ferroptosis. Western blot analysis was performed to detect protein levels of MAT2A, p-STAT3 (Ser727)/STAT3, and solute carrier family 7 member 11 (SLC7A11) in OS cells. MAT2A was significantly upregulated in OS specimens and high MAT2A expression was associated with a poorer prognosis in OS patients. shRNA targeting MAT2A significantly increased OS cell apoptosis, triggered cell cycle arrest in the G2 phase, and attenuated migration ability in vitro. MAT2A depletion dramatically inhibited tumor progression of OS in vivo. Overexpression of MAT2A rescued the tumor inhibition caused by miR-26b-5p. MAT2A knockdown promoted OS cell ferroptosis. miR-26b-5p/MAT2A regulates tumor malignant progression and OS cell ferroptosis by controlling p-STAT3 and SLC7A11 expressions. Taken together, our study displayed that miR-26b-5p/MAT2A triggers ferroptosis in OS cells by increasing intracellular ferrous iron levels and inhibiting the STAT3/SLC7A11 axis. Our results reveal a MAT2A-mediated ferroptosis defense mechanism used by OS cells and propose a potential ferroptosis-inducing strategy for the treatment of OS patients.
... In contrast, the body can make sufficient amounts of the non-essential amino acids. Thus, they are not needed but may become temporarily necessary if their demand exceeds their supply [59,60]. ...
Phytochemicals from plant extracts are becoming increasingly popular in the world of food science and technology because they have positive effects on human health. In particular, several bioactive foods and dietary supplements are being investigated as potential treatments for chronic COVID. Hydroxytyrosol (HXT) is a natural antioxidant, found in olive oil, with antioxidant anti-inflammatory properties that has been consumed by humans for centuries without reported adverse effects. Its use was approved by the European Food Safety Authority as a protective agent for the car-diovascular system. Similarly, arginine is a natural amino acid with anti-inflammatory properties that can modulate the activity of immune cells, reducing the production of pro-inflammatory cytokines such as IL-6 and TNF-α. The properties of both substances may be particularly beneficial in the context of COVID-19 and long COVID, which are characterised by inflammation and oxidative stress. While L-arginine promotes the formation of • NO, HXT prevents oxidative stress and inflammation in infected cells. This combination could prevent the formation of harmful peroxynitrite, a potent pro-inflammatory substance implicated in pneumonia and COVID-19-associated organ dysfunction, as well as reduce inflammation, improve immune function, protect against free radical damage and prevent blood vessel injury. Further research is needed to fully understand the potential benefits of HXT and arginine in the context of COVID-19.
... Enders, J.F., Weller, T.H., and Robbins, F.C. won the Nobel Prize in Physiology in 1954 for their "Tissue Culture of Poliovirus". With the development and production of poliovirus vaccines [17][18][19], synthetic culture media such as 199 medium [20], Eagle's medium [21], and Dulbecco's medium [22] have been developed, and stable two-dimensional cultures have become possible. ...
Organ transplantation is the first and most effective treatment for missing or damaged tissues or organs. However, there is a need to establish an alternative treatment method for organ transplantation due to the shortage of donors and viral infections. Rheinwald and Green et al. established epidermal cell culture technology and successfully transplanted human-cultured skin into severely diseased patients. Eventually, artificial cell sheets of cultured skin were created, targeting various tissues and organs, including epithelial sheets, chondrocyte sheets, and myoblast cell sheets. These sheets have been successfully used for clinical applications. Extracellular matrix hydrogels (collagen, elastin, fibronectin, and laminin), thermoresponsive polymers, and vitrified hydrogel membranes have been used as scaffold materials to prepare cell sheets. Collagen is a major structural component of basement membranes and tissue scaffold proteins. Collagen hydrogel membranes (collagen vitrigel), created from collagen hydrogels through a vitrification process, are composed of high-density collagen fibers and are expected to be used as carriers for transplantation. In this review, the essential technologies for cell sheet implantation are described, including cell sheets, vitrified hydrogel membranes, and their cryopreservation applications in regenerative medicine.
... Basal media formulations are sufficient to keep the cell alive for a limited period; however, various media are used to proliferate for extended periods. Minimal essential medium (MEM) is frequently used to maintain cells in tissue culture comprising amino acids, vitamins, glucose, and salts (Eagle, 1959). Minute variations in MEM have created a new media commonly used for mammalian cell cultures, Dulbecco's modified Eagle's medium (DMEM) (van der Valk et al., 2010) In cell culture, 70% of the glucose is converted into lactate by highly proliferating cells; however, 20%-30% of the remaining glucose is available for tricarboxylic acid (Ryan et al., 1987). ...
Cellular agriculture is one of the evolving fields of translational biotechnology. The emerging science aims to improve the issues related to sustainable food products and food security, reduce greenhouse gas emissions and provide animal wellbeing by circumventing livestock farming through cell-based meat (CBM) production. CBM exploits cell culture techniques and biomanufacturing methods by manipulating mammalian, avian, and fish cell lines. The cell-based products ought to successfully meet the demand for nutritional protein products for human consumption and pet animals. However, substantial advancement and modification are required for manufacturing CBM and related products in terms of cost, palatability, consumer acceptance, and safety. In order to achieve high-quality CBM and its production with high yield, the molecular aspect needs a thorough inspection to achieve good laboratory practices for commercial production. The current review discusses various aspects of molecular biology involved in establishing cell lines, myogenesis, regulation, scaffold, and bioreactor-related approaches to achieve the target of CBM.
... To date, the Chinese hamster ovary (CHO) cells are the predominant host organism for recombinant therapeutic proteins such as monoclonal antibodies due to various advantages including their high adaptability to suspension culture conditions and capacity to achieve proper folding and disulfide bond formation of complex proteins as well as desirable post-translational modifications (Baycin-Hizal et al., 2012;Lim et al., 2010;Liu et al., 2015). However, unlike bacteria culture, successful culture of CHO cells requires a much more complex list of nutrients to be present in the culture media, including carbohydrates, amino acids, vitamins, etc. (Eagle, 1959;Ritacco et al., 2018). The very early culture media for CHO cells also required supplementation with serum but serum-free, chemically defined medium was developed later and is predominantly used now to avoid unwanted contamination and to achieve consistent, high productivities (Kim et al., 2020;Ritacco et al., 2018). ...
Cysteine is a critically important amino acid necessary for mammalian cell culture, playing key roles in nutrient supply, disulfide bond formation, and as a precursor to antioxidant molecules controlling cellular redox. Unfortunately, its low stability and solubility in solution make it especially problematic as an essential medium component that must be added to Chinese hamster ovary and other mammalian cell cultures. Therefore, CHO cells have been engineered to include the capacity endogenously synthesize cysteine by overexpressing multiple enzymes, including cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and glycine N-methyltransferase (GNMT) to reconstruct the reverse transsulfuration pathway and overcome a key metabolic bottleneck. Some limited cysteine biosynthesis was obtained by overexpressing CBS and CTH for converting homocysteine to cysteine but robust metabolic synthesis from methionine was only possibly after incorporating GNMT which likely represents a key bottleneck step in the cysteine biosynthesis pathway. CHO cells with the reconstructed pathway exhibit the strong capability to proliferate in cysteine-limited and cysteine-free batch and fed-batch cultures at levels comparable to wildtype cells with ample cysteine supplementation, providing a selectable marker for CHO cell engineering. GNMT overexpression led to the accumulation of sarcosine byproduct, but its accumulation did not affect cell growth. Furthermore, pathway reconstruction enhanced CHO cells' reduced and glutathione levels in cysteine-limited conditions compared to unmodified cells, and greatly enhanced survivability and maintenance of redox homeostasis under oxidative stress induced by addition of menadione in cysteine-deficient conditions. Such engineered CHO cell lines can potentially reduce or even eliminate the need to include cysteine in culture medium, which not only reduces the cost of mammalian media but also promises to transform media design by solving the challenges posed by low stability and solubility of cysteine and cystine in future mammalian biomanufacturing processes.
... pH value and osmolality were 7.0-7.6 and 250-340 mOsmol/Kg for all cultures, respectively (Supporting Information: Figure S4). (Eagle, 1959). Therefore, to avoid limitations of nonquantified metabolites, cell culture medium was additionally exchanged with a high flow-rate (5 RV/d) 2 h before infection. ...
We present a proof‐of‐concept study for production of a recombinant vesicular stomatitis virus (rVSV)‐based fusogenic oncolytic virus (OV), rVSV‐Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi‐perfusion in small‐scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process‐related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK‐21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV‐NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID50/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell‐specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large‐scale manufacturing of fusogenic rVSV‐NDV at HCD for all three candidate cell lines.
... Before the concept of 'ferroptosis' was proposed in 2012, the observation of similar phenomena had been much discussed within the literature. In the mid-twentieth century, Eagle et al. [1][2][3] pioneeringly discovered that cysteine/ cystine deprivation led to cell death while this type of cell death could be resisted by endogenous synthesis of cysteine utilizing methionine and glucose. In 2003, in the study of Dolma et al. [4], erastin was initially identified in a synthetic lethal high-throughput screening, which induced a form of cell death lacking features of apoptosis. ...
As an essential trace element in the body, iron is critical for the maintenance of organismal metabolism. Excessive iron facilitates reactive oxygen species generation and inflicts damage on cells and tissues. Ferroptosis, a newly identified iron-dependent type of programmed cell death, has been implicated in a broad set of metabolic disorders. Ferroptosis is mainly characterized by excess iron accumulation, elevated lipid peroxides and reactive oxygen species, and reduced levels of glutathione and glutathione peroxidase 4. The vast emerging literature on ferroptosis has shown that numerous diseases, such as cancers, neurodegeneration, and autoimmune diseases, are associated with ferroptosis. Meanwhile, recent studies have confirmed the relationship between ferroptosis and eye diseases including keratopathy, cataract, glaucoma, retinal ischemia-reperfusion injury, age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and retinoblastoma, indicating the critical role of ferroptosis in ocular diseases. In this article, we introduce the primary signaling pathways of ferroptosis and review current advances in research on ocular diseases involving iron overload and ferroptosis. Furthermore, several unanswered questions in the area are raised. Addressing these unanswered questions promises to provide new insights into preventing, controlling, and treating not only ocular diseases but also a variety of other diseases in the near future.
... Bioscience or biotechnology scientists artificially grow cells and use them as research materials [1][2][3][4][5]. Because, among in vitro cultured cells, several types of cells such as embryonic stem cells (ESCs) are hard to culture without feeder cells or a specific extracellular matrix, natural proteins are needed for the culturing and attachment [6][7][8]. ...
... For most of the commercially available cell culturing media, the nutrient compositions are not as close as the in vivo environment for cells because they were designed to provide a minimal amount of nutrients for continuous cultivation of cells, instead of reproducing a physiological cellular environment like human plasma. For example, Eagle's Minimal Essential Medium (EMEM) is a synthetic cell culture medium developed by Harry Eagle, which includes 6 salts, 8 vitamins, and essential amino acids [7]. Dulbecco's Modified Eagle's Medium (DMEM) was introduced with higher concentrations of amino acids and vitamins based on EMEM to avoid nutrient exhaustion during the cultivation process without attendance for a longer period of time [8]. ...
Aiming to reduce the gap between in vitro and in vivo environment, a complex culture medium, Plasmax, was introduced recently, which includes nutrients and metabolites with concentrations normally found in human plasma. Herein, to study the influence of this medium on cellular behaviors, we utilized Plasmax to cultivate two cancer cell lines, including one breast cancer cell line, MDA-MB-231BR, and one brain cancer cell line, CRL-1620. Cancer cells were harvested and prepared for transcriptomics and proteomics analyses to assess the discrepancies caused by the different nutritional environments of Plasmax and two commercial media: DMEM, and EMEM. Total RNAs of cells were extracted using mammalian total RNA extract kits and analyzed by next-generation RNA sequencing; proteomics analyses were performed using LC-MS/MS. Gene oncology and pathway analysis were employed to study the affected functions. The cellular invasion and cell death were inhibited in MDA-MB-231BR cell line when cultured in Plasmax compared to DMEM and EMEM, whereas the invasion, migration and protein synthesis of CRL-1620 cell line were activated in Plasmax in relative to both commercial media. The expression changes of some proteins were more significant compared to their corresponding transcripts, indicating that Plasmax has more influence upon regulatory processes of proteins after translation. This work provides complementary information to the original study of Plasmax, aiming to facilitate the selection of appropriate media for in vitro cancer cell studies.
... Its main features include iron overload, ROS accumulation, and elevated lipid peroxidation (Bertrand 2017). Although ferroptosis has been known since the 1950s and 1960s (Eagle 1959;Wang et al. 2004;Yonezawa et al. 1996), the first theory was only proposed in 2012 . Ferroptosis is mainly characterized by appreciable mitochondrial morphological changes, including reduced volume, increased membrane density, and the absence or reduction of mitochondrial crests (Dixon et al. 2012Friedmann Angeli et al. 2014). ...
Ferroptosis is a recently discovered modulated cell death mechanism caused by the accumulation of iron-dependent lipid peroxides to toxic levels and plays an important role in tumor immunology and neurology. Recent studies have shown that ferroptosis may play a crucial role in bacterial infection pathogenesis, which may be useful in anti-infection therapies. However, how bacteria enter cells to induce ferroptosis after invading the host immune system remains largely unknown. In addition, the current studies only focus on the relationship between a single bacterial species or genus and host cell ferroptosis, and there is no systematic summary of its regulatory mechanism. Therefore, our review firstly sums up the role of ferroptosis in bacterial infection and its regulatory mechanism, and innovatively speculates on the function and potential mechanism of extracellular vesicles (EVs) in bacterial-induced ferroptosis, in order to provide possible novel directions and ideas for future anti-infection research.
Key points
• Ferroptosis presents a novel mechanism for bacterial host interaction
• EVs provide the potential mechanism for bacterial-induced ferroptosis
• The relationship of EVs with ferroptosis provides possible directions for future treatment of bacterial infection
... As early as the last century, some pioneering studies have shown that cystine deprivation induces cell death, and dead cells show a unique microscopic morphology [18]. Further research found that the recovery of GSH can promote cell growth in cystinefree medium [19]. Since then, some early studies have only focused on the importance of amino acids for cell culture. ...
Ferroptosis is a new type of programmed cell death with unique morphological, biochemical, and genetic features. From the initial study of histomorphology to the exploration of subcellular organelles and even molecular mechanisms, a net connecting ferroptosis and fibrosis is being woven and formed. Inflammation may be the bridge between both processes. In this review, we will discuss the ferroptosis theory and process and the physiological functions of ferroptosis, followed by a description of the pathological effects and the underlying mechanisms of ferroptosis in the pathogenesis of tumorigenesis, ischemic damage, degenerative lesions, autoimmune diseases, and necroinflammation. We then focus on the role of ferroptosis in the fibrosis process in the liver, lung, kidney, heart, and other organs. Although the molecular mechanism of ferroptosis has been explored extensively in the past few years, many challenges remain to be resolved to translate this information into antifibrotic practice, which is becoming a promising new direction in the field of fibrotic disease prevention and treatment.
... Glutamic acid is one of the amino acids with the highest concentrations in female reproductive fluids, and its concentration has been shown to be in the range of 0.05-5.5 mM in the oviductal fluid of various mammalian species, including humans [4][5][6][7][8][9][10][11]. Glutamic acid (L-Glu) is a "nonessential" amino acid (classified according to nutritional requirements of several cell lines [12]) and exists in its ionic form under physiological conditions. Since glutamate salts (including monosodium glutamate, a widely used food additive) dissociate in aqueous solutions, the term glutamate can be used for both glutamate salts and glutamic acid [13]. ...
Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using RT-PCR, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected expression of two AMPA receptor subunits, three kainate receptor subunits and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from GRIA3 and/or GRIA4 subunits), kainate receptors (formed from GRIK 3 and GRIK 4 or GRIK 5 subunits) and GRM5 glutamate receptor were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pre-treatment of blastocysts with AMPA, kainate and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through activation of cell membrane receptors.
... Several metals are also considered essential components in media as well as the human body [16,17]. Eagle's minimal essential medium (EMEM) is one of the most traditional and widely used synthetic medium containing 5 to 10% serum [18]. Today, there are wide variety media that are modified versions of the traditional medium; which are provided metals from raw materials such as bovine serum [19]. ...
... The same measurement was performed on cell culture medium in a regular 5 mm NMR glass tube in order to obtain a reference value without cells and scaffold. Considering an extracellular sodium concentration of 144.4 mM of standard cell culture minimal essential medium (MEM) [17,18] and an intracellular human fibroblasts sodium concentration of 10 mM [19], the percentage cell volume was calculated according to [20]. The analysis revealed that 3D collagen-based embedded cells occupy 1.43% of the total sample volume within the sensitive NMR coil. ...
NMR flow devices provide longitudinal real-time quantitative metabolome characterisation of living cells. However, discrimination of intra- and extracellular contributions to the spectra represents a major challenge in metabolomic NMR studies. The present NMR study demonstrates the possibility to quantitatively measure both metabolic intracellular fingerprints and extracellular footprints on human control fibroblasts by using a commercially available flow tube system with a standard 5 mm NMR probe. We performed a comprehensive 3D cell culture system characterisation. Diffusion NMR was employed for intra- and extracellular metabolites separation. In addition, complementary extracellular footprints were determined. The implemented perfused NMR bioreactor system allowed the determination of 35 metabolites and intra- and extracellular separation of 19 metabolites based on diffusion rate differences. We show the reliability and sensitivity of NMR diffusion measurements to detect metabolite concentration changes in both intra- and extracellular compartments during perfusion with different selective culture media, and upon complex I inhibition with rotenone. We also demonstrate the sensitivity of extracellular footprints to determine metabolic variations at different flow rates. The current method is of potential use for the metabolomic characterisation of defect fibroblasts and for improving physiological comprehension.
Animal cell culture has marked a significant revolution in science, allowing different fields to benefit from its use. The aim of this chapter is to introduce animal cell culture, covering its main advances over the years, its main applications, such as the use of animal cell culture for vaccine production, and to address the main challenges facing the technology. Several challenges still need to be addressed, such as the development of serum-free media, the optimization of culture due to shear sensitivity in bioreactors, cost implications, among others. The field is constantly evolving, and the outlook is positive for the emergence of new applications and the achievement of new production scales.
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Los cultivos celulares son una herramienta que permite el mantenimiento de células in vitro conservando al máximo sus características fisiológicas, bioquímicas y genéticas, los cuales pueden presentar propiedades muy diversas dependiendo de la especie, tejido de origen y el medio de cultivo empleado. El uso de cultivos celulares de dípteros constituye una metodología valiosa en diferentes disciplinas, tales como fisiología, genética, bioquímica y patología; así, por ejemplo, se han podido estudiar diversas enfermedades tanto de interés en medicina humana como en veterinaria. En la presente revisión se presenta información de múltiples líneas celulares del orden Diptera, las cuales se han empleado para llevar a cabo estudios de interacción célula a célula, flujo intracelular de metabolitos, producción de bioinsecticidas, amplificación viral y producción de péptidos antimicrobiano. En Colombia, se han establecido 12 líneas celulares de dípteros las cuales fueron caracterizadas y estandarizadas, algunas de ellas se han empleado como sustratos en estudios del ciclo biológico de parásitos del género Leishmania, también, en la evaluación a la susceptibilidad de arbovirus. En esta tendencia, se analiza y discute la información de las líneas celulares derivadas de dípteros con el fin de realizar el presente trabajo de revisión sobre sus características y aplicaciones.
In biomedical research, germ-free and gnotobiotic mouse models enable the mechanistic investigation of microbiota-host interactions and their role on (patho)physiology. Throughout any gnotobiotic experiment, standardized and periodic microbiological testing of defined gnotobiotic housing conditions is a key requirement. Here, we review basic principles of germ-free isolator technology, the suitability of various sterilization methods, and the use of sterility testing methods to monitor germ-free mouse colonies. We also discuss their effectiveness and limitations, and share the experience with protocols used in our facility. In addition, possible sources of isolator contamination are discussed and an overview of reported contaminants is provided.
A surface plasmon resonance (SPR) optical fiber sensor with multimode-coreless-multimode (MNM) structure was developed, which modified by L-glutamine-binding protein (QBP) for detection of L-glutamine (Gln). The QBP was immobilized on the surface of gold films by chemical cross-linking and exhibited a binding affinity for L-glutamine. The conformation of QBP can be changed from the "open" to the "closed", which led to a red-shift of the SPR peak when QBP bounded to L-glutamine. There was a good linear correlation between is a dependence of the SPR peak on and the concentration of L-glutamine concentration in the range 10-100 μM, with a sensitivity of 10.797nm/log10[Gln] for L-glutamine in the in vitro embryo culture (IVC) medium environment, and the limit of detection (LOD) is 1.187 μM. This QBP-modified MNM structure optical fiber SPR sensor provides a new idea for the developmental potential assessment of embryos in the process of in vitro embryo culture.
The basic techniques for cell culture are introduced in this chapter. Primarily, the requirements for cell culture are an air-conditioned room of cleanroom class 10,000 (FS209E) equipped with lights and ventilation, a laminar flow hood, a CO2 incubator, sterilizers with both saturated steam and dry heat, a low speed centrifuge with refrigerator, upright and inverted phase contrast microscopes, a freezer at −20, a deep freezer at − 80 ̊C, a refrigerator at 4 ̊C, a sink, disposable sterile plasticware (flasks, dishes, tubes, and pipettes), balance, ultrapure water, and a supply of media, and other reagents needed for the cell environment. Further helpful apparatuses are a pH meter, a cell counter (hemocytometer), a vacuum pump, a pipette-aid, micropipettes, a liquid nitrogen tank, a fluorescent microscope, and so on. Attention to safety and the maintenance of equipments is essential to understand the significance, the reasons, and the mechanisms. Contamination of microbials such as bacteria, yeast, staphylococcus, fungus, and mycoplasma should be strictly avoided in cell culture. Simultaneously careful attention not to overgrow but grow with sufficient cell density and to avoid passages for a long time because the phenotype of the cell may subject to change. This knowledge will help researchers with even a little prior experience to set up a suitable laboratory for basic cell culture.KeywordsCell cultureTechniquesCellsPrinciples
Amino acids (AAs) and ammonia are metabolic markers essential for nitrogen metabolism and cell regulation in both plants and humans. NMR provides interesting opportunities to investigate these metabolic pathways, yet lacks sensitivity, especially in case of ¹⁵N. In this study, spin order embedded in p‐H2 is used to produce on‐demand reversible hyperpolarization in ¹⁵N of pristine alanine and ammonia under ambient protic conditions directly in the NMR spectrometer. This is made possible by designing a mixed‐ligand Ir‐catalyst, selectively ligating the amino group of AA by exploiting ammonia as a strongly competitive co‐ligand and preventing deactivation of Ir by bidentate ligation of AA. The stereoisomerism of the catalyst complexes is determined by hydride fingerprinting using ¹H/D scrambling of the associated N‐functional groups on the catalyst (i.e., isotopological fingerprinting), and unravelled by 2D‐ZQ‐NMR. Monitoring the transfer of spin order from p‐H2 to ¹⁵N nuclei of ligated and free alanine and ammonia targets using SABRE‐INEPT with variable exchange delays pinpoints the monodentate elucidated catalyst complexes to be most SABRE active. Also RF‐spin locking (SABRE‐SLIC) enables transfer of hyperpolarization to ¹⁵N. The presented high‐field approach can be a valuable alternative to SABRE‐SHEATH techniques since the obtained catalytic insights (stereochemistry and kinetics) will remain valid at ultra‐low magnetic fields.
A novel fluorogenic sensor N-benzo[b]thiophen-2-yl-methylene-4,5-dimethyl-benzene-1,2-diamine (BTMPD) was synthesized and characterized by using spectroscopic methods including UV-visible, FT-IR, 1H NMR, 13C NMR, and mass spectrometry. The designed fluorescent probe, owing to its remarkable properties, behaves as an efficient turn-on sensor for the sensing of amino acid Serine (Ser). Also, the strength of the probe enhances upon the addition of Ser via charge transfer, and the renowned properties of the fluorophore were duly found. The sensor BTMPD shows incredible execution potential with respect to key performance indicators such as high selectivity, sensitivity, and low detection limit. The concentration change was linear ranging from 5 × 10-8 M to 3 × 10-7 M, which is an indication of the low detection limit of 1.74 ± 0.02 nM under optimal reaction conditions. Interestingly, the Ser addition leads to an increased intensity of the probe at λ = 393 nm which other co-existing species did not. The information about the arrangement and the features of the system and the HOMO-LUMO energy levels was found out theoretically using DFT calculations which is fairly in good agreement with the experimental cyclic voltammetry results. The fluorescence sensing using the synthesized compound BTMPD reveals the practical applicability and its application in real sample analysis.
This chapter overviews numerous application studies on spray-dried vaccines, from laboratory-scale research to clinical trials. The main focus is on vaccines against influenza, measles, hepatitis B (HepB), tuberculosis (TB), human papillomavirus (HPV), cholera, anthrax, typhoid, diphtheria, tetanus, pertussis, rotavirus, poliovirus, vesicular stomatitis virus (VSV), herpes simplex virus (HSV), norovirus, human immunodeficiency virus (HIV), Newcastle disease virus, tularemia, pneumococcal disease, meningococcal disease, and mRNA vaccines. This high number of applications illustrates the wide variety of spray-dried vaccines. The development status of the respective application is presented and summarized by vaccine type and route of administration. The focus is on the spray drying process parameters, formulation strategies, particle design properties, storage stability results, and efficacy. To begin with, an excursus is given on commercialized spray-dried pharmaceutical products that have already been approved, which may pave the way for spray-dried vaccines. In particular, a brief overview of spray-dried inhalable drugs and single-use dry powder inhalers for pulmonary delivery is provided. In addition, highly recommended review articles and books on (bio)pharmaceutical spray drying are presented.
Human trophoblast cultures provide powerful tools to model key processes of placental development. In vitro trophoblast studies to date have relied on commercial media which contains non-physiological levels of nutrients, and the impact of these conditions on trophoblast metabolism and function is unknown. Here we show that the physiological medium (Plasmaxä) with nutrient and metabolite concentrations recapitulating human plasma improves human trophoblast stem cell (hTSC) proliferation and differentiation compared to standard medium (DMEM-F12). hTSCs cultured in Plasmax-based medium also show altered glycolytic and mitochondrial metabolism, as well as reduced S-adenosylmethionine/S-adenosyl-homosysteine ratio compared to DMEM-F12-based medium. These findings demonstrate the importance of the nutritional environment for phenotyping cultured human trophoblasts.
This work presents the nature of N-benzyloxycarbonyl-l-serine through quantum chemical calculations and spectral analysis. The optimized geometry and vibrational frequencies were computed by density functional theory. The geometric parameters were theoretically obtained and those values were compared with experimental data. The calculated vibrational wavenumbers of the heading compound display exact agreement with the recorded spectrum. The frontier molecular orbital band gap energy confirmed that the heading compound has a good reactivity and stability of the molecule. The theoretical UV-Vis spectrum turned into analysis in gas and with different solvents. The nuclear magnetic resonance spectrum was calculated. Each evaluation was in comparison with the experimental ones. The molecular electrostatic potential energy map is an effective model to identify electrophilic and nucleophilic sites. Further, natural bond orbital and Mulliken charge analysis had been additionally calculated. Drug-likeness studies were carried out. This result shows that the heading compound has a good biological activity. Anticancer activity was tested based on the molecular docking evaluation and it was diagnosed that the heading compound can act as a potential inhibitor of liver cancer.
Cancer cells need a steady supply of nutrients to evade cell death and proliferate. Depriving cancer cells of the amino acid cystine can trigger the non-apoptotic cell death process of ferroptosis. Here, we report that cancer cells can evade cystine deprivation-induced ferroptosis by uptake and catabolism of the cysteine-rich extracellular protein albumin. This protective mechanism is enhanced by mTORC1 inhibition and involves albumin degradation in the lysosome, predominantly by cathepsin B (CTSB). CTSB-dependent albumin breakdown followed by export of cystine from the lysosome via the transporter cystinosin fuels the synthesis of glutathione, which suppresses lethal lipid peroxidation. When cancer cells are grown under non-adherent conditions as spheroids, mTORC1 pathway activity is reduced, and albumin supplementation alone affords considerable protection against ferroptosis. These results identify the catabolism of extracellular protein within the lysosome as a mechanism that can inhibit ferroptosis in cancer cells.
Two-dimensional cell cultures are established models in research for studying and perturbing cell-type specific functions. However, many limitations apply to the cell growth in a monolayer using standard cell culture media. Although they have been used for decades, their formulations do not mimic the composition of the human cell environment. In this study, we analyzed the impact of a newly formulated human plasma-like media (HPLM) on cell proliferation, mitochondrial bioenergetics, and alterations of drug efficacies using three distinct cancer cell lines. Using high-resolution respirometry, we observed that cells grown in HPLM displayed significantly altered mitochondrial bioenergetic profiles, particularly related to mitochondrial density and mild uncoupling of respiration. Furthermore, in contrast to standard media, the growth of cells in HPLM unveiled mitochondrial dysfunction upon exposure to the FDA-approved kinase inhibitor sunitinib. This seemingly context-dependent side effect of this drug highlights that the selection of the cell culture medium influences the assessment of cancer drug sensitivities. Thus, we suggest to prioritize media with a more physiological composition for analyzing bioenergetic profiles and to take it into account for assigning drug efficacies in the cell culture model of choice.
Modern day ethics and laws call for more safety and use of fewer animals in biomedical research. It became crucial to develop novel in vitro devices of higher relevance. Since the end of the twentieth century, several systems have been proposed by researchers in attempts to palliate the shortcomings of current systems. Notably, organs-on-chip systems are specifically tailored to recapitulate tissue functions in a manner that remains easily accessible for the experimenter. Despite the significant improvements that were brought during the last century to in vitro cell and tissue culture systems, the field of bioengineering is still young and much progress remains to be done. The work presented here details the development of an organ-on-chip that includes a biocompatible and actuatable hydrogel membrane, with controlled physico-chemical properties. Such chip is relevant when hosting barrier tissues, which are composed of several cell types, disposed on each side of a barrier, as well as within its bulk, and are often submitted to mechanical stimuli. During this PhD, several objectives have been attained. Notably, we: - Designed and produced an organ-on-chip including a biocompatible and actuatable hydrogel layer, as well as a microfluidic system allowing the independent control of both flow and actuation. - Characterized the deformation of the hydrogel layer. - Cultured intestinal cells within the chip, which formed a three dimensionally structure epithelium, and characterized its apparent permeability to molecules of varying sizes
Mammalian cell culture is a fundamental tool used to study living cells. Presently, the standard protocol for performing cell culture involves the use of commercial media that contain an excess of nutrients. While this reduces the likelihood of cell starvation, it creates non-physiologic culture conditions that have been shown to 're-wire' cellular metabolism. Recently, researchers have developed new media like Plasmax, formulated to approximate the nutrient composition of human blood plasma. Although this represents an improvement in cell culture practice, physiologic media may be vulnerable to nutrient depletion. In this study we directly addressed this concern by measuring the rates of glucose and amino acid depletion from Plasmax in several cancer cell lines (PC-3, LNCaP, MCF-7, SH-SY5Y) over 48 hours. In all cell lines, depletion of glucose from Plasmax was rapid such that, by 48h, cells were hypoglycemic (<2mM glucose). Most amino acids were similarly rapidly depleted to sub-physiological levels by 48h. In contrast, glucose and most amino acids remained within the physiological range at 24h. When the experiment was done at physiological oxygen (5%) versus standard (18%) with LNCaP cells, no effect on glucose or amino acid consumption was observed. Using RNA sequencing, we show that this nutrient depletion is associated with enrichment of starvation responses, apoptotic signalling, and endoplasmic reticulum stress. A shift from glycolytic metabolism to mitochondrial respiration at 5% O 2 was also measured using Seahorse analysis. Taken together, these results exemplify the metabolic considerations for Plasmax, highlighting that cell culture in Plasmax requires daily media exchange.
The field of proteomics is continually improving, requiring the development of new quantitative methods. Stable isotope labeling in cell culture (SILAC) is a metabolic labeling technique originating in the early 2000s. By incorporating isotopically labeled amino acids into the media used for cell culture, unlabeled versus labeled cells can be differentiated by the mass spectrometer. Traditional SILAC labeling has been expanded to pulsed applications allowing for a new quantitative dimension of proteomics - temporal analysis. The complete introduction of Heavy SILAC labeling chased with surplus unlabeled medium mimics traditional pulse-chase experiments and allows for the loss of heavy signal to track proteomic changes over time. In a similar fashion, pulsed SILAC (pSILAC) monitors the initial incorporation of a heavy label across a period of time, which allows for the rate of protein label integration to be assessed. These innovative techniques have aided in inspiring numerous SILAC-based temporal and spatial labeling applications, including super SILAC, spike-in SILAC, spatial SILAC, and a revival in label multiplexing. This review reflects upon the evolution of SILAC and the pulsed SILAC application, introduces advances in SILAC labeling, and proposes future perspectives for this novel and exciting field.
The parental HeLa cell population, a morphologically uniform, human cancer cell strain, grown for several years in tissue culture by procedures always involving massive inocula, has been shown to contain different mutant cell types.
Two clonal lines have been isolated and established as reliable stock cultures. Both strains exhibit 100 per cent plating efficiency in high or low serum concentrations in the presence of a feeder system. In the absence of a feeder system and in low serum concentrations, the two strains are quantitatively differentiable: S3 still exhibits 100 per cent plating efficiency, while that of S1 lies in the neighborhood of zero.
These differences have remained stable throughout 100 successive generations of growth of each strain including 2 single cell isolations.
Application of these techniques to studies in the genetics of mammalian somatic cells and to specific cell-cell interactions has been indicated.
Only minimal amounts of poliomyelitis virus were formed by HeLa cells placed in a medium free from glucose and glutamine, even if the medium contained an otherwise full complement of essential and non-essential amino acids, purines, pyrimidines, NB4+, and serum protein. Conversely, within one log of the optimal yield of virus was formed by HeLa cells in a medium containing only glucose, glutamine, and salts, even if the cells had been starved in this medium for 12 hours prior to their inoculation.
The presence of glucose alone caused an average 170-fold increase in viral output beyond the amounts formed by the glucose- and glutamine-depleted cells. The addition of glutamine alone caused an average 2000-fold increase; and the addition of both increased the viral formation 40,000-fold. Qualitatively similar results were obtained with unstarved cells, not previously depleted of glucose and glutamine. It follows that only a small proportion of HeLa cells are capable of forming virus unless either glucose or glutamine, or both, are present in the medium.
The elaboration of virus was significantly delayed in media containing glucose but no glutamine.
The absence of glucose and glutamine did not prevent the fixation of poliomyelitis virus by the cell. When these compounds were added to previously depleted cells even 6 hours after inoculation, and after the excess free virus had been removed by washing and by the addition of specific antiserum, normal amounts of virus were formed despite the degenerative changes caused by the previous glucose and glutamine deprivation. Possible functions of glucose and glutamine in the elaboration of virus are discussed in the text.
Such factors other than glucose, glutamine, or salts (e.g. amino acids, purines, pyrimidines, vitamins, protein, or NH4+) as may be needed by HeLa cells for the propagation of poliomyelitis virus, need not be present in the medium and cannot be easily washed out of the cell. Even 12 hours' total deprivation of the cells in salt solution prior to inoculation only slightly decreased their virus-synthesizing capacity in a similarly deficient medium, provided only that adequate amounts of glucose and glutamine were retained.
Monkey kidney cells tested in their first culture passage, 24 hours after their isolation from the animal host, required the same 13 amino acids for survival and growth as cell lines serially propagated in culture for years. Under the conditions of the present experiments, arginine, cystine, glutamine, histidine, and tyrosine proved necessary, over and above the 8 amino acids required for nitrogen balance in man.
With the serially propagated lines, glutamic acid substituted for glutamine only at extremely high and non-physiological levels. In the monkey kidney cell cultures, however, glutamic acid and glutamine were interchangeable, mole for mole; and aspartic acid and asparagine were also effective as glutamine substitutes.
Glycine was growth-stimulatory for monkey kidney cells in primary culture, and the cells grown in a glycine-deficient medium usually failed to survive subculture.
It has previously been shown that in full grown monolayer cultures of HeLa cells infected with poliovirus the only components of the medium necessary for maximum virus output are glucose, glutamine, and salts (1, 2). Rappaport (3) has similarly reported maximum poliovirus production in monkey kidney cultures in the absence of a number of growth-essential amino acids; and a number of growth-essential amino acids were found by Bader and Morgan (4) to be non-essential in the production of psittacosis virus by the L cell. The question remains as to whether, in a medium lacking nutritionally essential amino acids, poliovirus protein is synthesized from the pre-existing free amino acids of the pool, or whether instead there is a breakdown of cell protein to products which are then used for viral synthesis. An approach to this problem has been provided by a study of the effect of cell population density on the nutritional requirements for poliovirus synthesis by HeLa cells. In two other virus cell systems it has already been shown that dilution of the infected cell suspension in saline greatly reduces the virus production per cell. Chicken fibroblasts infected with Western equine encephalomyelitis and sus- pended in Earle's saline at a low population density require embryo extract in order to produce virus normally (5). Pereira (6) has shown that when chick fibroblasts infected with fowl plaque virus are suspended in saline the virus yield per cell is higher at high population densities. These findings suggest that cellular components essential for virus synthesis may be lost when cells are suspended at low concentrations in a deficient medium.
The amino acid requirements of a human uterine carcinoma cell (HeLa strain) have been defined. The 12 compounds previously found to be essential for the growth of a mouse fibroblast proved similarly essential for this human epithelial cell. They included arginine, cyst(e)ine, histidine, and tyrosine, in addition to the eight amino acids required for nitrogen balance in man (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine). Only the L-amino acids were active; the D-enantiomorphs had no demonstrable effect at physiologic concentrations.
The minimum concentrations required for survival and limited growth varied from 0.003 µM per ml. for L-tryptophan, to 0.1 µM per ml. for L-lysine. The concentrations permitting optimum growth similarly varied from 0.01 µM per ml. for tryptophan, to 0.1 µM per ml. for leucine, isoleucine, threonine, lysine, and valine. The latter optimum concentrations of the individual amino acids were closely correlated with their serum levels. With at least six of the amino acids, high concentrations, in the range 1 to 10 µM per ml., caused a definite growth inhibition.
In the absence of a single essential amino acid, degenerative changes occurred in the cells, culminating in their death and dissolution. In the early stages, however, these degenerative changes could be reversed by the restoration of the missing component.
Strain L cells, of mouse fibroblastic origin, have been cultivated in vitro in completely synthetic medium M 150 and in various modifications of this medium. The amino acid changes in the nutrient medium during cell cultivation have been studied by paper chromatography. A characteristic pattern of amino acid uptake and accumulation in the medium has been found. No change in the alanine concentration was observed but omission of alanine from the culture medium resulted in its accumulation in appreciable amounts. Omission of glutamic acid did not alter the pattern of amino acid changes by the cells. Omission of glutamine increased the uptake of amino acids and prevented amino acid accumulation. Omission of both glutamic acid and glutamine resulted in a virtual cessation of amino acid changes in the culture medium. Strain L cells decreased the adenine content of the medium and produced small amounts of hypoxanthine. These changes were not affected by alterations in the amino acid content of the medium. Omission of glutamic acid and glutamine from the culture medium did not cause an appreciable decrease in cell population or apparent degeneration of the cultures over a 30-day period.
A minimal growth medium supplemented with dialyzed serum, which sufficed for the propagation of a wide variety of human cell strains in heavily inoculated monolayer and suspension cultures, did not permit the regular or optimal growth of small numbers of HeLa, HeLa S3, conjunctiva, or KB cells deriving from suspension cultures. At threshold concentrations of serum, the plating efficiency of single cells was greatly reduced as compared with their plating efficiency in a medium containing dialyzed serum instead of whole serum, and the clones which did develop grew at a slower rate. The nutritional deficiency could be overcome by adding the seven amino acids which are ordinarily not nutritionally essential. In most of the experiments serine alone sufficed.
In monolayers of HeLa cells, glucose, glutamine, and balanced salts promoted essentially maximal yields of poliovirus.Fructose substituted for glucose in this respect, but not galactose, ribose, ribose plus pyruvate, or ribose-5PO4. The effective concentrations of glucose and fructose for viral synthesis were the same as those required for cell growth.Glutamic acid at high concentrations (10–20 mM) substituted for glutamine in poliovirus production. The effective concentrations of glutamic acid and glutamine were approximately the same as those required for cell growth.Adaptation of the cells to glutamic acid reduced the concentration of glutamic acid necessary for virus production.The need for added glutamine in order to obtain maximal virus production is probably related to its rapid disappearance from the free amino acid pool.
A number of the co-factors here tested (FMN, DPN, cocarboxylase) had essentially the same activity as the corresponding vitamin (riboflavin, nicotinamide and thiamine, respectively) in promoting the growth of the mouse fibroblast, in terms of either the effective concentration or the amount of growth obtained. Several vitamin conjugates (FAD, TPN, coenzyme A and pyridoxal phosphate) proved significantly less active than the parent vitamin (riboflavin, nicotinamide, pantothenate, and pyridoxal, respectively). The present experiments provide no information as to the degree to which these differences may reflect only variations in cell permeability, rather than partial blocks in the utilization of these specific co-factors, or in their conversion to a more active form. Natural citrovorum factor, however, was somewhat more active than folic acid. Of the vitamin congeners or precursors here tested, pyridoxine and pyridoxal were essentially equivalent in activity; nicotinic acid was significantly less active than either nicotinamide or DPN in terms of the amount of growth obtained, although the effective concentrations were of the same order of magnitude; while PABA was wholly inactive as a substitute for folic acid. As with the co-factors, the present experiments do not exclude differential cell permeability as the basis of these differences.
1. A strain of Sarcoma 180 maintained by routine trocar implant passage in CFW mice has been isolated and serially propagated in vitro from biopsy specimens in a medium containing only the growth factors demonstrably essential for the HeLa cell and the L strain of mouse fibroblasts. The generation time in these media is circa 30 hrs and this cell line is now in the 32 nd transplant, having undergone more than 100 generations during 230 days in vitro. 2. Inocula prepared from these cultures after 215 days in vitro have produced characteristic sarcomata within 3-5 days in 100% of the CFW mice implanted subcutaneously with 1.0 × 105 or more cells. The ED50 in CFW mice is circa 4.5-5.0 × 103 cells, and the latent period between injection and the development of palpable tumors increases with decreasing inocula.
The histories of 8 strains of human epithelial-like cells derived from bone marrow, ascitic and pleural fluids have been described. Preliminary observations on their morphology have been reported and the significance of these observations has been discussed.
Three tissue culture cell lines deriving from normal human tissue (liver, conjunctiva, and intestine) and two lines deriving from human cancer [KB (nasopharynx), and J-111 (monocytic leukemia)] have been examined with respect to their amino acid requirements in comparison with those of a HeLa cell and a mouse fibroblast. With the possible exception of tryptophan, all seven cell lines required the same amino acids (arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, valine); and in the absence of any one of these 12, cytopathogenic changes developed which culminated in the death of the cell. The provision of non-essential amino acids, purines, pyrimidines, and NH4+ had a glutamine-sparing effect, but did not eliminate the need for this amino acid. Extremely high and nonphysiological concentrations of glutamic acid (20 mM) did, however, substitute for glutamine.It is not yet clear whether tryptophan is similarly essential for survival and growth, or whether it is merely growth stimulatory for some of the five cell lines here studied.The concentration of the individual amino acids necessary for optimum growth varied somewhat among the six strains; but there were not significant or consistent differences in this respect between the lines deriving from normal and from malignant tissues.
A strain of rabbit fibroblasts designated strain RM3–73 requires folic acid, nicotinamide, pantothenic acid, pyridoxal, riboflavin, and thiamine for continuous proliferation in a medium containing 2% (v/v) dialyzed horse serum. The concentration of each vitamin which permits optimal proliferation was determined by propagating the cells serially in media containing graded concentrations of the compound. A group of eight amino acids which are not required for serial propagation in the presence of pyridoxal replaced the requirement for this vitamin under experimental conditions employed.
A spinner culture system for the growth of tissue cells in fluid suspension has been described. Actively proliferating mammalian cell suspensions, in volumes of 25 to 400 ml have been established utilizing the prototype “L” cell. Similar, although less extensive, investigations have been conducted with strain HeLa. A 300 to 400 ml “L” cell spinner culture has been continuously maintained for the last 5 months. It is suggested that the described submerged culture system provides a more quantitative tool for investigations relating to the response of the mammalian cell to infectious or pharmacologic agents.
In the absence of exogenous energy and nitrogen, resting yeast cells are capable of replenishing both their nucleotide and free amino acid pools. This replenishment phenomenon is the result of intracellular protein and nucleic acid breakdown rather than of cell lysis. Isotopically labeled cells showed turnover rates of 6.6 · 10−3 h−1 and 1.5 · 10−3 h−1 for protein and nucleic acid respectively. Protein and nucleic acid degradation, as well as their synthesis, are energy-requiring reactions.When exogenous energy is available, the degradation products are reutilized for protein and nucleic acid synthesis. Employing 15N-purine-grown cells, it was found that the amino groups of nucleic acid purines can serve as nitrogen reservoirs for limited protein synthesis.
Strain L cells, of mouse fibroblastic origin, have been cultivated in vitro in completely synthetic medium M 150 and in various modifications of this medium. The amino acid changes in the nutrient medium during cell cultivation have been studied by paper chromatography. A characteristic pattern of amino acid uptake and accumulation in the medium has been found. No change in the alanine concentration was observed but omission of alanine from the culture medium resulted in its accumulation in appreciable amounts. Omission of glutamic acid did not alter the pattern of amino acid changes by the cells. Omission of glutamine increased the uptake of amino acids and prevented amino acid accumulation. Omission of both glutamic acid and glutamine resulted in a virtual cessation of amino acid changes in the culture medium. Strain L cells decreased the adenine content of the medium and produced small amounts of hypoxanthine. These changes were not affected by alterations in the amino acid content of the medium. Omission of glutamic acid and glutamine from the culture medium did not cause an appreciable decrease in cell population or apparent degeneration of the cultures over a 30-day period.
Twelve amino acids had proved essential for the survival and growth of both the mouse fibroblast (strain L) and a human carcinoma cell (strain HeLa). In media lacking a single essential amino acid; in which the cells would otherwise degenerate and die, normal growth and multiplication were obtained on the addition of a dipeptide containing the essential amino acid. The effective concentrations of the dipeptide were of the same order of magnitude as those required of the simple amino acid. It remains to be seen whether the dipeptide is incorporated into cellular protein as such, or whether it is first split into its component amino acids(3).