Macrophage Polarization Related to Crystal Phases of Calcium Phosphate Biomaterials.

Calcium phosphate (CaP) materials influence macrophage polarization during bone healing. However, the effect of the crystal phase of CaP materials on the immune response of bone remains unclear. In this study, the effect of the crystal phases of CaP materials on the regulation of macrophage polarization was investigated. Human THP-1 cells and mouse RAW 264 cells were cultured with octacalcium phosphate (OCP) and its hydrolyzed form Ca-deficient hydroxyapatite to assess the expression of pro-inflammatory M1 and anti-inflammatory M2 macrophage-related genes. OCP inhibited the excessive inflammatory response and switched macrophages to the anti-inflammatory M2 phenotype, which promoted the expression of the interleukin 10 (IL10) gene. In contrast, HL stimulated an excessive inflammatory response by promoting the expression of pro-inflammatory M1 macrophage-related genes. To observe changes in the microenvironment induced by OCP and HL, inorganic phosphate (Pi) and calcium ion (Ca2+) concentrations and pH value in the medium were measured. The expression of the pro-inflammatory M1 macrophage-related genes (tumor necrosis factor alpha (TNFα) and interlukin 1beta (IL1ß)) was closely related to the increase in ion concentration caused by the increase in the CaP dose. Together, these results suggest that the microenvironment caused by the crystal phase of CaP materials may be involved in the immune-regulation capacity of CaP materials.

Induction of spontaneous phenotype conversion in Ralstonia solanacearum by addition of iron compounds in liquid medium.

Ralstonia solanacearum is a soil-borne pathogen that causes bacterial wilt in plants. The wild-type strain of R. solanacearum undergoes spontaneous phenotype conversion (PC), from a fluidal to non-fluidal colony morphology. PC mutants are non-pathogenic due to reduced virulence factors, and can control wilt diseases as biological control agents. The induction factors of PC in R. solanacearum are currently unclear. Here, we investigated the effect of iron treatment on bacterial growth of wild-type strain and PC mutant, and PC of the wild-type strain in liquid medium. Interestingly, PC was frequently induced in the single cultured wild-type strain by iron treatment; however, PC was not induced in the co-culture. In a co-culture of both strains, the PC mutant showed increased growth compared to the wild-type strain by iron treatment. Furthermore, we investigated the effects of iron treatment on the bacterial growth and PC of the wild-type strain under different culture conditions of medium type (MM broth, BG broth, and water medium), iron compounds, and pH. In BG broth, PC occurred frequently regardless of iron treatment. In MM broth, the optimal conditions for high frequency induction of PC by iron treatments were treatment of iron (III) EDTA, and under pH 7-8. Conversely, PC was not induced by iron treatment in water medium and in MM broth under pH 5 conditions. Common to the culture conditions wherein PC was not induced by iron treatment, the bacterial density of the wild-type strain was as low as 106 CFU mL-1 or less. Finally, we investigated the effects on bacterial growth and PC of the wild-type strain by the iron treatment and addition of culture filtrate after cultivation of the wild-type strain at high concentration. In medium containing only the culture filtrate, PC did not occur. However, in medium containing the culture filtrate and iron, PC occurred frequently. Our results thus suggest that high-density growth of the wild-type strain as well as the presence of iron are involved in inducing PC in R. solanacearum.

Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions.

Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.

Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains.

The bacterium that causes syphilis, Treponema pallidum subsp. pallidum, has now been cultured in vitro continuously for periods exceeding 3 years using a system consisting of coculture with Sf1Ep rabbit epithelial cells in TpCM-2 medium and a low-oxygen environment. In addition, long-term culture of several other syphilis isolates (SS14, Mexico A, UW231B, and UW249B) and the T. pallidum subsp. endemicum Bosnia A strain has been achieved. During in vitro passage, T. pallidum subsp. pallidum exhibited a typical bacterial growth curve with logarithmic and stationary phases. Sf1Ep cells are required for sustained growth and motility; however, high initial Sf1Ep cell numbers resulted in reduced multiplication and survival. Use of Eagle's minimal essential medium as the basal medium was not effective in sustaining growth of T. pallidum subsp. pallidum beyond the first passage, whereas CMRL 1066 or M199 supported long-term culture, confirming that additional nutrients present in these more complex basal media are required for long-term culture. T. pallidum subsp. pallidum growth was dependent upon the presence of fetal bovine serum, with 20% (vol/vol) being the optimal concentration. Omission of reactive oxygen species scavengers dithiothreitol, d-mannitol, or l-histidine did not dramatically affect survival or growth. Additionally, T. pallidum subsp. pallidum can be successfully cultured in a Brewer jar instead of a specialized low-oxygen incubator. Phosphomycin or amphotericin B can be added to the medium to aid in the prevention of bacterial or fungal contamination, respectively. These results help define the parameters of the T. pallidum subsp. pallidum culture system that are required for sustained, long-term survival and multiplication.IMPORTANCE Syphilis is caused by the bacterium Treponema pallidum subsp. pallidum Until recently, this pathogen could only be maintained through infection of rabbits or other animals, making study of this important human pathogen challenging and costly. T. pallidum subsp. pallidum has now been successfully cultured for over 3 years in a tissue culture system using a medium called TpCM-2. Here, we further define the growth requirements of this important human pathogen, promoting a better understanding of the biology of this fastidious organism.

Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy.

This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells were collected from each blastocyst after thawing, and the embryos were cultured in vitro for up to 10 days. The outgrowths after culturing beyond implantation were sampled and subjected to chromosome analysis using next-generation sequencing. Chromosomal concordance rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-positive rate (FPR), and false-negative rate (FNR) of niPGT-A and PGT-A against each outgrowth were analyzed. The concordance rate between the niPGT-A and outgrowth samples was 9/16 (56.3%), and the concordance rate between the PGT-A and outgrowth samples was 7/16 (43.8%). NiPGT-A exhibited 100% sensitivity, 87.5% specificity, 88.9% PPV, 100% NPV, 12.5% FPR, and 0% FNR. PGT-A exhibited 87.5% sensitivity, 77.8% specificity, 87.5% PPV, 75% NPV, 14.3% FPR, and 22.2% FNR. NiPGT-A may be more accurate than PGT-A in terms of ploidy diagnostic accuracy in outgrowths.

Improved Non-Invasive Preimplantation Genetic Testing for Beta-Thalassemia Using Spent Embryo Culture Medium Containing Blastocoelic Fluid.

Objectives: To compare successful beta-thalassemia (ß-thalassemia) detection rates obtained using spent culture medium and spent culture medium containing blastocoelic fluid (BF). Method: This study involved data from 10 couples who underwent preimplantation genetic testing (PGT) for ß-thalassemia. A total of 26 samples of spent culture medium containing BF (group A) and 33 samples without BF (group B) were collected and analyzed. The DNA concentration and ß-thalassemia detection rates were evaluated. Results: The HBB mutation analysis results of 34 samples were concordant with the biopsy results (34/59, 57.6%). In group A, the HBB mutation analysis results of 19 of 26 samples (73.1%) were concordant with the biopsy results. The concordance rate in group A was higher than that in group B (15/33, 45.5%; P < 0.05). The haplotyping results of 38 samples were concordant with the biopsy results (38/59, 64.4%). The concordance rate in group B was 17/33 (51.5%), which was significantly lower than that in group A (21/26, 80.8%) (P < 0.05). In group A, the mean DNA concentration of samples with <10% fragmentation was 107.3 ± 70.1 ng/µL, which was lower than that of samples with ≥10% fragmentation (194.6 ± 28.0 ng/µL) (P < 0.05). However, the detection rates of <10% and ≥10% fragmentation were not significantly different (P > 0.05). Conclusion: The ß-thalassemia detection rate with non-invasive PGT using the spent culture medium containing BF was higher than that using the spent culture medium alone. Fragmentation is associated with DNA concentration in the spent culture medium containing BF.

Extraction and characterization of fungal chitin nanofibers from Mucor indicus cultured in optimized medium conditions.

Chitin nanofibers (ChNFs) were extracted from Mucor indicus by a purification method followed by a mechanical treatment, cultivated under obtained optimum culture medium conditions to improve fungal chitin production rate. A semi synthetic media containing 50 g/l glucose was used for cultivation of the fungus in shake flasks. The cell wall analysis showed that N-acetyl glucosamine (GlcNAc) content, which is an indication of chitin content, was maximum in presence of 2.5 g/l H3PO4, 2.5 g/l of NaOH, 1 g/l of yeast extract, 1 mg/l of plant hormones, and 1 ml/l of trace metals. The chemical characterizations demonstrated that the isolated fibers had a degree of deacetylation lower than of 10%, and were phosphate free. The FTIR results revealed successful removal of different materials during the purification step. The FE-SEM of fibrillated chitin illustrated an average diameter of 28 nm. Finally, X-ray diffraction results showed that the crystallinity index of nanofibers was 82%.

Comparison of Transforming Growth Factor 1 (TGF-β 1) Expression in Various Lysate Platelet-Rich Fibrin (L-PRF) Concentrations on Human Dental Pulp Stem Cell Differentiation

ABSTRACT Objective: To compare Transforming growth factor-β1 (TGF-β1) expression in various L-PRF concentrations on the hDPSC differentiation process. Material and Methods: hDPSC cell cultures were subjected to serum starvation by reducing FBS levels in the hDPSC culture media. Lysate PRF was obtained from the PRF gel, which was then incubated at 4°C for 24 h. The supernatant was dried, transferred to a 2-ml Eppendorf tube, and stored at −20°C. The evaluation of TGF-β1 expression in 1%, 5%, 10%, and 25% L-PRF samples and 10% FBS (control) during the process of hDPSC differentiation was quantified using an ELISA reader on day 7. The expression of TGF-β1 was subjected to a one-way ANOVA test, followed by Bonferroni's post hoc test with significant values (p<0.05). Results: Significant differences were noted in TGF-β1 expression between 1%, 5%, 10%, and 25% L-PRF and the control group (10% FBS). The highest TGF-β1 expression occurred with 25% L-PRF (0.645 ± 0.048), followed by 10% L-PRF (0.461 ± 0.035), 10% FBS (0.374 ± 0.013), 5% L-PRF (0.275 ± 0.045), and the lowest expression was with 1% L-PRF (0.160 ± 0.045). Conclusion: The best result of TGF-B1 expression in hDPSC differentiation was in the 25% L-PRF group.

Serial Cell-free DNA Assessments in Preclinical Models.

Studying circulating cell-free DNA (cfDNA) release within preclinical model systems provides opportunities to investigate the mechanisms and kinetics underlying this process under various conditions. We present a detailed protocol for longitudinal evaluation of cfDNA release through (1) seeding of cancer cell lines and establishment of xenograft tumors, (2) treatment of cancer cells and xenograft tumors, (3) serial collection of cell line media and xenograft blood, and (4) processing and isolation of cfDNA for (5) quantification of cfDNA by quantitative PCR. For complete details on the use and execution of this protocol please refer to Rostami et al. (2020).

Characterization of the secretory profile and exosomes of limbal stem cells in the canine species.

Limbal stem cells (LSCs) are a quiescent cell population responsible for the renewal of the corneal epithelium. Their deficiency is responsible for the conjunctivization of the cornea that is seen in different ocular pathologies, both in humans and in the canine species. The canine species represents an interesting preclinical animal model in ocular surface pathologies. However, the role of LSCs in physiological and pathological conditions in canine species is not well understood. Our objective was to characterize for the first time the soluble factors and the proteomic profile of the secretome and exosomes of canine LSCs (cLSCs). In addition, given the important role that fibroblasts play in the repair of the ocular surface, we evaluated the influence of the secretome and exosomes of cLSCs on their proliferation in vitro. Our results demonstrated a secretory profile of cLSCs with high concentrations of MCP-1, IL-8, VEGF-A, and IL-10, as well as significant production of exosomes. Regarding the proteomic profile, 646 total proteins in the secretome and 356 in exosomes were involved in different biological processes. Functionally, the cLSC secretome showed an inhibitory effect on the proliferation of fibroblasts in vitro, which the exosomes did not. These results open the door to new studies on the possible use of the cLSC secretome or some of its components to treat certain pathologies of the ocular surface in canine species.

LC/MS analysis of three-dimensional model cells exposed to cigarette smoke or aerosol from a novel tobacco vapor product.

A novel tobacco vapor product (NTV) contains tobacco leaves and generates nicotine-containing aerosols using heating elements. Subchronic biological effects have been evaluated previously using three-dimensional bronchial epithelial model cells by repeated exposure to cigarette smoke (CS) and the NTV aerosols; however, the intracellular exposure characteristics have not been studied in detail. In this study, cells were initially exposed to an aqueous extract (AqE) of cigarette smoke (CS) at two concentration levels, and the cell lysate underwent untargeted analysis by LC-high resolution mass spectrometry to determine the exogenous compounds present in the cells. Among the thousands of peaks detected, four peaks showed a CS-dependency, which were reproducibly detected. Two of the peaks were nicotine and nicotine N-oxide, and the other two putative compounds were myosmine and norharman. The cells were then exposed to an AqE of CS in various combinations of exposure and post-exposure culture durations. Post-exposure culturing of cells with fresh medium markedly decreased the peak areas of the four compounds. The in-vitro switching study of CS to NTV aerosols was investigated by intermittently exposing cells to an AqE of CS four times, followed by exposure to either an AqE of CS, NTV aerosol or medium another four times. Switching to NTV reduced myosmine and norharman levels, which are known CS constituents. The results indicate that extracellular compounds inside cells reflect the exposure state outside cells. Thus, monitoring functional changes to cells in these exposure experiments is feasible.

Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs.

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (Kp,uu) at the steady-state.

ß-Adrenoceptor Activation in Breast MCF-10A Cells Induces a Pattern of Catecholamine Production Similar to that of Tumorigenic MCF-7 Cells.

Adrenaline, which participates in the neuroendocrine response that occurs during stress and perimenopause, may be tumorigenic. This exploratory study aimed at investigating whether non-tumorigenic and tumorigenic human breast epithelial cell lines are able to synthesize adrenaline. The study was carried out in non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) human breast cell lines. Expression of enzymes involved in adrenaline synthesis was characterized by RT-qPCR, immunocytochemistry and western blot. Catecholamines and analogue compounds were quantified by HPLC-ECD. Functional assessment of the impact of drugs on cells' tumorigenic potential was assessed by determination of cell viability and clonogenic ability. Both MCF-10A and MCF-7 cells produce catecholamines, but the capacity to produce adrenaline is lower in MCF-10A cells. ß-adrenoceptor activation increases the capacity of MCF-10A cells to produce adrenaline and favor both cell viability and colony formation. It is concluded that exposure of human breast epithelial cells to ß-adrenoceptor agonists increases cell proliferation and the capacity to produce adrenaline, creating an autocrine potential to spread these adrenergic effects in a feed-forward loop. It is conceivable that these effects are related to tumorigenesis, bringing a new perspective to understand the claimed anticancer effects of propranolol and the increase in breast cancer incidence caused by stress or during perimenopause.

Comparative Analysis of the Different Dyes' Potential to Assess Human Normal and Cancer Cell Viability In Vitro under Different D/H Ratios in a Culture Medium.

In this study, using new approach (laser diffraction + biological dyes), we have demonstrated the decrease of cells viability in vitro in the deuterated growth medium, whereas in the deuterium-depleted medium, there was an increase of cell viability. We have also found that not all dyes are equally sensitive to the D/H ratios in the culture medium (system) as well as to the different cell types (cancer vs normal cells).

Higher nisin yield is reached with glutathione and pyruvate compared with heme in Lactococcus lactis N8.

There are different studies that aim to enhance the production of nisin by Lactococcus lactis since its chemical synthesis is not possible. In this study, glutathione (GSH) and pyruvate, which are known to reduce the oxidative stress of cells, have been shown to trigger the production of nisin at both transcriptional and translational levels in L. lactis cells grown under aerobic condition. Presence of GSH and pyruvate caused more nisin yield than the heme-supplemented medium. Moreover, the expression of genes that encode stress-related enzymes were apparently upregulated in the presence of GSH and pyruvate. It can be concluded that GSH and pyruvate contribute to the defense system of L. lactis cells and so that higher biomass was obtained which in turn enhance nisin production. Antioxidant effect of GSH and pyruvate was known; however, their stimulating effect on nisin production was shown for the first time in this study.

Site-specific N-glycosylation of HeLa cell glycoproteins.

We have characterized site-specific N-glycosylation of the HeLa cell line glycoproteins, using a complex workflow based on high and low energy tandem mass spectrometry of glycopeptides. The objective was to obtain highly reliable data on common glycoforms, so rigorous data evaluation was performed. The analysis revealed the presence of a high amount of bovine serum contaminants originating from the cell culture media - nearly 50% of all glycans were of bovine origin. Unaccounted, the presence of bovine serum components causes major bias in the human cellular glycosylation pattern; as is shown when literature results using released glycan analysis are compared. We have reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites, and 178 glycoforms. HeLa glycoproteins were found to be highly (68.7%) fucosylated. A medium degree of sialylation was observed, on average 46.8% of possible sialylation sites were occupied. High-mannose sugars were expressed in large amounts, as expected in the case of a cancer cell line. Glycosylation in HeLa cells is highly variable. It is markedly different not only on various proteins but also at the different glycosylation sites of the same protein. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa cell line.

A new method to confirm the absence of human and animal serum in mesenchymal stem cell culture media.

Mesenchymal stem cells are an ideal source for regenerative medicine. For clinical use, cell culture should be done at stable conditions, thus the use of serum should be avoided because of the batch-to-batch variations of serum. Although several kinds of serum-free media are available, a method to confirm whether they contain serum has not been established yet. During studies on effect of adipocyte mesenchymal stem cells (Ad-MSCs) on pain using a human pain gene array, we noticed that BDKRB1 gene was constantly upregulated when serum was used in the culture medium. In this study, we attempted to establish further the potential of this gene as a new marker indicative of the presence of serum in media. Using a real-time quantitative PCR gene array screening containing 84 functional genes, we verified BDKRB1 as a specific gene upregulated in the presence of serum. The expression of BDKRB1 in Ad-MSCs was induced not only by bovine serum but also by human serum. The BDKRB1 expression was induced even when Ad-MSCs was cultured with 0.1% serum in the medium. We concluded that BDKRB1 is a valuable marker to detect traces of both human and animal serum in Ad-MSCs cultures. Our study provides a new method to confirm the absence of serum in media and ensure a stable cell culture condition.

Silicate-substituted strontium apatite nano coating improves osteogenesis around artificial ligament.

BACKGROUND: Treatment of anterior cruciate ligament injuries commonly involves the use of polyethylene terephthalate (PET) artificial ligaments for reconstruction. However, the currently available methods require long fixation periods, thereby necessitating the development of alternative methods to accelerate the healing process between tendons and bones. Thus, we developed and evaluated a novel technique that utilizes silicate-substituted strontium (SrSiP). METHODS: PET films, nano-coated with SrSiP, were prepared. Bone marrow mesenchymal cells (BMSCs) from femurs of male rats were cultured and seeded at a density of 1.0 × 104/cm2 onto the SrSiP-coated and non-coated PET film, and subsequently placed in an osteogenic medium. The osteocalcin concentration secreted into the medium was compared in each case. Next, PET artificial ligament, nano-coated with SrSiP, were prepared. BMSCs were seeded at a density of 4.5 × 105/cm2 onto the SrSiP-coated, and non-coated artificial ligament, and then placed in osteogenic medium. The osteocalcin and calcium concentrations in the culture medium were measured on the 8th, 10th, 12th, and 14th day of culture. Furthermore, mRNA expression of osteocalcin, alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor 2 (Runx2) was evaluated by qPCR. We transplanted the SrSiP-coated and non-coated artificial ligament to the tibiae of mature New Zealand white rabbits. Two months later, we sacrificed them and histologically evaluated them. RESULTS: The secretory osteocalcin concentration in the medium on the film was significantly higher for the SrSiP group than for the non-coated group. Secretory osteocalcin concentration in the medium on the artificial ligament was also significantly higher in the SrSiP group than in the non-coated group on the 14th day. Calcium concentration on the artificial ligament was significantly lower in the SrSiP group than in the non-coated group on the 8th, 10th, 12th, and 14th day. In qPCR as well, OC, ALP, BMP2, and Runx2 mRNA expression were significantly higher in the SrSiP group than in the non-coated group. Newly formed bone was histologically found around the artificial ligament in the SrSiP group. CONCLUSIONS: Our findings demonstrate that artificial ligaments using SrSiP display high osteogenic potential and thus may be efficiently used in future clinical applications.

Application of the optimized and validated LC-MS method for simultaneous quantification of tryptophan metabolites in culture medium from cancer cells.

Kynurenine pathway is the main route of tryptophan degradation generating a number of immunoregulatory compounds. Some conditions like oxidative stress, inflammatory factors might enhance tryptophan degradation. Process is active in several cells including fibroblasts, cancer cells, and immune cells, therefore it is intensively studied in context of cancer microenvironment. The validated and standardized methodology for kynurenine quantification is crucial for reliable comparison of results obtained in different studies. This paper concerns an approach for simultaneous quantification of four major tryptophan metabolites of the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in cell culture supernatants by liquid chromatography coupled with single quadrupole mass spectrometer. During development of the novel method, the principal component analysis was used to select the best mobile phase and to ensure the optimal conditions for simultaneous quantification of metabolites. The analysis involves simple protein precipitation with acidified methanol and 3-nitrotyrosine as an internal standard. The obtained limits of detection and quantification in cell culture medium were in the range of 3.31-10.80 nmol/L and 9.60-19.50 nmol/L, respectively. At the validation step, other method parameters (linearity, precision, accuracy, recovery, matrix effects) were also evaluated and satisfactory results were obtained for all target compounds. The method was applied to study tryptophan metabolites by determination of kynurenines in cell culture medium from two different human cancer cell lines (MDA-MD-231 and SK-OV-3) in context of exposure to glycation products.