Primary Blood Cells Research Articles

A Novel Method for Primary Blood Cell Culturing and Selection in Drosophila melanogaster.

The blood cells of the fruit fly Drosophila melanogaster show many similarities to their vertebrate counterparts, both in their functions and their differentiation. In the past decades, a wide palette of immunological and transgenic tools and methods have been developed to study hematopoiesis in the Drosophila larva. However, the in vivo observation of blood cells is technically restricted by the limited transparency of the body and the difficulty in keeping the organism alive during imaging. Here we describe an improved ex vivo culturing method that allows effective visualization and selection of live blood cells in primary cultures derived from Drosophila larvae. Our results show that cultured hemocytes accurately represent morphological and functional changes following immune challenges and in case of genetic alterations. Since cell culturing has hugely contributed to the understanding of the physiological properties of vertebrate blood cells, this method provides a versatile tool for studying Drosophila hemocyte differentiation and functions ex vivo.

Development of SFC-MS Method for Quantification of Eicosanoids Biosynthesized in Primary Human Blood Cells.

Eicosanoids are lipid mediators generated from arachidonic acid with pro- and anti-inflammatory properties. Despite these lipid mediators being known for decades, quantitative determination in biological samples is still challenging due to low abundance, instability, the existence of regio- and stereoisomers, and a wide polarity range that hampers chromatographic separation. In this study, we developed a supercritical fluid chromatography mass spectrometry (SFC-MS) platform for the quantification of relevant eicosanoids. Application of a chiral amylose-based column and modifier combination of 2-propanol/acetonitrile offered separation and sufficient resolution of 11 eicosanoids (5-, 12-, 15-HETE, PGB1, LTB4, t-LTB4, 20-OH-LTB4, PGE2, PGD2, PGF2α, TxB2) with baseline separation of isobaric analytes within 12 min. The method was validated in terms of range (78-2500 ng/mL), linearity, accuracy, precision, and recovery according to EMA guidelines. Finally, we confirmed the method's applicability by quantifying eicosanoid levels in human primary blood cells. In conclusion, we present a validated SFC-MS method for the determination of relevant eicosanoids in biological samples with a wide range of polarity while maintaining baseline separation of isobars, which allows coupling to a single quadrupole mass detector.

Development of l-Dopa-containing diketopiperazines as blood-brain barrier shuttle

In our overall goal to develop anti-Parkinson drugs, we designed novel diketopiperazines (DKP1-6) aiming to both reach the blood-brain barrier and counteract the oxidative stress related to Parkinson's Disease (PD).The anti-Parkinson properties of DKP 1–6 were evaluated using neurotoxin-treated PC12 cells, as in vitro model of PD, while their cytotoxicity and genotoxicity potentials were investigated in newborn rat cerebral cortex (RCC) and primary human whole blood (PHWB) cell cultures. The response against free radicals was evaluated by the total antioxidant capacity (TAC) assay. Comet assay was used to detect DNA damage while the content of 8-hydroxyl-2′-deoxyguanosine (8-OH-dG) was determined as a marker of oxidative DNA damage. PAMPA-BBB and Caco-2 assays were employed to evaluate the capability of DKP1-6 to cross the membranes. Stability studies were conducted in simulated gastric and intestinal fluids and human plasma.Results showed that DKP5-6 attenuate the MPP + -induced cell death on a nanomolar scale, but a remarkable effect was observed for DKP6 on Nrf2 activation that leads to the expression of genes involved in oxidative stress response thus increasing glutathione biosynthesis and ROS buffering. DKP5-6 resulted in no toxicity for RCC neurons and PHWB cells exposed to 10–500 nM concentrations during 24 h as determined by MTT and LDH assays and TAC levels were not altered in both cultured cell types. No significant difference in the induction of DNA damage was observed for DKP5-6. Both DKPs resulted stable in simulated gastric fluids (t1/2 > 22h). In simulated intestinal fluids, DKP5 underwent immediate hydrolysis while DKP6 showed a half-life higher than 3 h. In human plasma, DKP6 resulted quite stable. DKP6 displayed both high BBB and Caco-2 permeability confirming that the DKP scaffold represents a useful tool to improve the crossing of drugs through the biological membranes.

Vaccine mRNA Can Be Detected in Blood at 15 Days Post-Vaccination.

COVID-19 mRNA vaccines effectively reduce incidence of severe disease, hospitalisation and death. The biodistribution and pharmacokinetics of the mRNA-containing lipid nanoparticles (LNPs) in these vaccines are unknown in humans. In this study, we used qPCR to track circulating mRNA in blood at different time-points after BNT162b2 vaccination in a small cohort of healthy individuals. We found that vaccine-associated synthetic mRNA persists in systemic circulation for at least 2 weeks. Furthermore, we used transmission electron microscopy (TEM) to investigate SARS-CoV-2 spike protein expression in human leukemic cells and in primary mononuclear blood cells treated in vitro with the BNT162b2 vaccine. TEM revealed morphological changes suggestive of LNP uptake, but only a small fraction of K562 leukemic cells presented spike-like structures at the cell surface, suggesting reduced levels of expression for these specific phenotypes.

A Comparative Study on the Viability of Normal and Cancerous Cells upon Irradiation with a Steady Beam of THz Rays

Terahertz (THz) electromagnetic radiation is commonly used in astronomy, security screening, imaging, and biomedicine, among other applications. Such approach has raised the question of the influence of THz irradiation on biological objects, especially the human body. However, the results obtained to date are quite controversial. Therefore, we performed a comparative study on the viability of normal cells and cancer cells upon irradiation with a steady beam of THz rays. We used human peripheral blood mononuclear cells and cancer cell lines. Primary human mononuclear blood cells (monocytes, and B-, and T-cells) showed an increased death rate, determined by cell counting and fluorescence microscopy, upon 0.14 THz irradiation. The effect of THz radiation was different among malignant cells of B- and T-cell origin (Ramos and Jurkat cells) and epithelial cancer cells (MCF7 and LNCaP). This was demonstrated by cell counting and by the alamarBlue assay. In conclusion, THz radiation can result in the death of human primary and malignant cells. However, the mechanism of this phenomenon is largely unknown. Hence, more work should be done to shed some light on the mechanism of action of THz irradiation in living organisms to enhance technologic developments.

TAS‐116 (pimitespib), a heat shock protein 90 inhibitor, shows efficacy in preclinical models of adult T‐cell leukemia

Adult T‐cell leukemia/lymphoma (ATL) is a highly chemoresistant malignancy of peripheral T lymphocytes caused by human T‐cell leukemia virus type 1 infection, for which there is an urgent need for more effective therapeutic options. The molecular chaperone heat shock protein 90 (HSP90) plays a crucial role in nuclear factor‐κB (NF‐κB)‐mediated antiapoptosis in ATL cells, and HSP90 inhibitors are new candidate therapeutics for ATL. Accordingly, we investigated the anti‐ATL effects of a novel oral HSP90 inhibitor, TAS‐116 (pimitespib), and the mechanisms involved in ex vivo and in vivo preclinical models. TAS‐116 achieved IC50 values of less than 0.5 μmol/L in 10 ATL‐related cell lines and less than 1 μmol/L in primary peripheral blood cells of nine ATL patients; no toxicity was observed toward CD4+ lymphocytes from healthy donors, indicating the safety of this agent. Given orally, TAS‐116 also showed significant inhibitory effects against tumor cell growth in ATL cell‐xenografted mice. Furthermore, gene expression profiling of TAS‐116‐treated Tax‐positive or ‐negative cell lines and primary ATL cells using DNA microarray and multiple pathway analysis revealed the significant downregulation of the NF‐κB pathway in Tax‐positive cells and cell‐cycle arrest in Tax‐negative cells and primary ATL cells. TAS‐116 suppressed the activator protein‐1 and tumor necrosis factor pathways in all examined cells. These findings strongly indicate the efficacy of TAS‐116, regardless of the stage of ATL progression, and its potential application as a novel clinical anti‐ATL therapeutic agent.

Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9

To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34+ progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2mut) cells, whereas ASXL1mut as well as DNMT3Amut cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells.

Dynamic CTCF binding directly mediates interactions among cis-regulatory elements essential for hematopoiesis

While constitutive CCCTC-binding factor (CTCF)–binding sites are needed to maintain relatively invariant chromatin structures, such as topologically associating domains, the precise roles of CTCF to control cell-type–specific transcriptional regulation remain poorly explored. We examined CTCF occupancy in different types of primary blood cells derived from the same donor to elucidate a new role for CTCF in gene regulation during blood cell development. We identified dynamic, cell-type–specific binding sites for CTCF that colocalize with lineage-specific transcription factors. These dynamic sites are enriched for single-nucleotide polymorphisms that are associated with blood cell traits in different linages, and they coincide with the key regulatory elements governing hematopoiesis. CRISPR-Cas9–based perturbation experiments demonstrated that these dynamic CTCF-binding sites play a critical role in red blood cell development. Furthermore, precise deletion of CTCF-binding motifs in dynamic sites abolished interactions of erythroid genes, such as RBM38, with their associated enhancers and led to abnormal erythropoiesis. These results suggest a novel, cell-type–specific function for CTCF in which it may serve to facilitate interaction of distal regulatory emblements with target promoters. Our study of the dynamic, cell-type–specific binding and function of CTCF provides new insights into transcriptional regulation during hematopoiesis.

A red-letter day for RBC research

Hematology The study of primary human red blood cell (huRBC) disorders such as sickle cell disease (SCD) and infectious diseases such as malaria has been hampered by a lack of in vivo models of human erythropoiesis. Song et al. transferred human fetal liver cells into MISTRG mice, which are immunodeficient and are genetically engineered with several human genes involved in hematopoiesis. This approach was unsuccessful because mature huRBCs are rapidly destroyed in the mouse liver. They then used CRISPR-Cas9 to mutate these mice into a fumarylacetoacetate hydrolase–deficient strain, allowing them to replace the mouse liver with engrafted human hepatocytes. These mice exhibited enhanced human erythropoiesis and circulating huRBC survival and could recapitulate SCD pathology when reconstituted with SCD-derived HSCs. Science , this issue p. [1019][1] [1]: /lookup/doi/10.1126/science.abe2485

Insight into the Biological Activity of Hennosides-Glucosides Isolated from Lawsonia inermis (henna): Could They Be Regarded as Active Constituents Instead.

Henna is the current name of the dye prepared from the dry leaf powder of Lawsonia inermis (Lythraceae). Several studies have focused on the chemistry and pharmacology of the henna dyeing active compound, lawsone, obtained from the main constituents of leaves, hennosides, during the processing of plant material. However, knowledge regarding the biological activity of hennosides is largely lacking. In this paper, the redox activity of three hennoside isomers is reported. The pro-oxidative activity was confirmed by their ability to induce mild lysis of erythrocytes and to increase the level of methemoglobin at the concentration ≥ 500 μg/mL. The antioxidant activity of hennosides (concentration ≥100 μg/mL) was determined by FRAP and ABTS assays. At concentration of 500 μg/mL, antioxidant activity of hennoside isomers was equivalent to 0.46 ± 0.08, 0.62 ± 0.28 and 0.35 ± 0.03 mM FeSO4 × 7H2O, and 0.15 ± 0.01, 0.30 ± 0.01 and 0.09 ± 0.01 mM Trolox. Hennosides at 100 μg/mL concentration did not influence viability of human breast cancer cell lines MDA231 and MCF-7 and primary human peripheral blood and periodontal ligament-mesenchymal stem cells, but produced a modest increase in concentration of antioxidants in the cell culture supernatants. The evidenced antioxidant and pro-oxidant activities indicate their potential to act as redox balance regulator, which opens up the possibility of using hennosides in commercial phytomedicines.

Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.

Gene expression profiling of SARS-CoV-2 infections reveal distinct primary lung cell and systemic immune infection responses that identify pathways relevant in COVID-19 disease.

To identify key gene expression pathways altered with infection of the novel coronavirus SARS-CoV-2, we performed the largest comparative genomic and transcriptomic analysis to date. We compared the novel pandemic coronavirus SARS-CoV-2 with SARS-CoV and MERS-CoV, as well as influenza A strains H1N1, H3N2 and H5N1. Phylogenetic analysis confirms that SARS-CoV-2 is closely related to SARS-CoV at the level of the viral genome. RNAseq analyses demonstrate that human lung epithelial cell responses to SARS-CoV-2 infection are distinct. Extensive Gene Expression Omnibus literature screening and drug predictive analyses show that SARS-CoV-2 infection response pathways are closely related to those of SARS-CoV and respiratory syncytial virus infections. We validated SARS-CoV-2 infection response genes as disease-associated using Kaplan–Meier survival estimates in lung disease patient data. We also analysed COVID-19 patient peripheral blood samples, which identified signalling pathway concordance between the primary lung cell and blood cell infection responses.

Modulators of histone demethylase JMJD1C selectively target leukemic stem cells.

Leukemic stem cells (LSCs) comprise a very rare cell population that results in the development of acute myeloid leukemia. The selective targeting of drivers in LSCs with small molecule inhibitors holds promise for treatment of acute myeloid leukemia. Recently, we reported the identification of inhibitors of the histone lysine demethylase JMJD1C that preferentially kill MLL rearranged acute leukemia cells. Here, we report the identification of jumonji domain modulator #7 (JDM-7). Surface plasmon resonance analysis showed that JDM-7 binds to JMJD1C and its family homolog JMJD1B. JDM-7 did not significantly suppress cell proliferation in liquid cell culture at higher doses, although it led to a significant decrease in semi-solid colony formation experiments at lower concentrations. Moreover, low doses of JDM-7 did not suppress the proliferation of erythroid progenitor cells. We identified that JDM-7 downregulates the LSC self-renewal gene HOXA9 in leukemia cells. We further found that the structure of JDM-7 is similar to that of tadalafil, a drug approved by the US Food and Drug Administration. Molecular docking and surface plasmon resonance analysis showed that tadalafil binds to JMJD1C. Moreover, similar to JDM-7, tadalafil suppressed colony formation of leukemia cells in semi-solid cell culture at a concentration that did not affect primary umbilical cord blood cells. In summary, we have identified JDM-7 and tadalafil as potential JMJD1C modulators that selectively inhibit the growth of LSCs.

RNA Targeting in Acute Myeloid Leukemia.

Nucleosides and their analogues constitute an essential family of anticancer drugs. DNA has been the presumptive target of the front-line prodrug for acute myeloid leukemia (AML), cytarabine (ara-C), since the 1980s. Here, the biomolecular targeting of ara-C was evaluated in primary white blood cells using the ara-C mimic "AzC" and azide-alkyne "click" reactions. Fluorescent staining and microscopy revealed that metabolic incorporation of AzC into primary white blood cells was unexpectedly enhanced by the DNA polymerase inhibitor aphidicholine. According to RNaseH digestion and pull-down-and-release experiments, AzC was incorporated into short RNA fragments bound to DNA in peripheral blood monocytes (PBMCs) collected from all six healthy human donors tested. Samples from 22 AML patients (French-American-British classes M4 and M5) exhibited much more heterogeneity, with 27% incorporating AzC into RNA and 55% into DNA. The overall survival of AML patients whose samples incorporated AzC into RNA was approximately 3-fold higher as compared to that of the DNA cohort (p ≤ 0.056, χ2 = 3.65). These results suggest that the RNA primers of DNA synthesis are clinically favorable targets of ara-C, and that variable incorporation of nucleoside drugs into DNA versus RNA may enable future patient stratification into treatment-specific subgroups.

Predictive Value of Risk Factors for Chronic Idiopathic Thrombocytopenic Purpura in Patients with Acute Type of Disease

BACKGROUND: Immune thrombocytopenic purpura (ITP) is an autoimmune disease in which autoantibodies react with platelet surface antigens and results in mild to severe thrombocytopenia due to decreased platelet count or inhibition of platelet production. Given the relatively high prevalence of ITP among children and the lack of standard diagnostic testing for the diagnosis of chronic disease, this study evaluated the predictive value of risk factors for chronic ITP in hospitalized patients.
 METHODS: This prospective cohort study was performed on 65 children with ITP who referred to Ali Asghar and Rasool Akram Hospitals in Tehran, Iran, during the years 2017 and 2018. Relationships between different risk factors, including age of diagnosis, gender, white cell count, primary platelet count, mean platelet volume (MPV), history and type of the previous patient infection, FCG gene mutation, and type of FCG mutation with a chronic disease incidence were investigated using multiple logistic regression model.
 RESULTS: Of 65 patients, 31 (47.69%) were male and 34 (52.31%) were female included in the study. Twenty-eight patients (43.08%) had acute ITP and 37 (56.92%) had chronic ITP. Frequency of FCG gene mutation in patients with chronic and acute type ITP was 16.36% and 7.27%, respectively (p = 0.51). No association was found between the history of the previous infection and its type with the chronic incidence of ITP. The multiple logistic regression model showed that three factors, including the absolute number of lymphocytes, age of diagnosis, and primary white blood cells (WBC) count were directly linked to chronic ITP. Furthermore, three factors of platelet, sex, and MPV were indirectly related to chronic ITP. In addition, the absolute number of lymphocytes, age of diagnosis and primary WBC count were significantly associated with chronic ITP. The receiver operating characteristic analysis showed that the cutoff rate of these factors was 0.31. Further analysis of these risk factors in comparison with the gold standard demonstrated that the diagnostic sensitivity and specificity of these risk factors for chronic ITP were 73.08% and their specificity was 88.57%, indicating the high importance and predictive power of these risk factors.
 CONCLUSIONS: According to the results of this study, for the first time in Iran, six risk factors, including the absolute number of lymphocytes, age at diagnosis, sex, MPV level, platelet level at time of diagnosis, and primary WBC count were considered as the most important risk factors affecting the incidence of chronic ITP. Of course, more comprehensive studies can definitely lead to more comprehensive models.

Spatial patterns of CTCF sites define the anatomy of TADs and their boundaries

BackgroundTopologically associating domains (TADs) are genomic regions of self-interaction. Additionally, it is known that TAD boundaries are enriched in CTCF binding sites. In turn, CTCF sites are known to be asymmetric, whereby the convergent configuration of a pair of CTCF sites leads to the formation of a chromatin loop in vivo. However, to date, it has been unclear how to reconcile TAD structure with CTCF-based chromatin loops.ResultsWe approach this problem by analysing CTCF binding site strengths and classifying clusters of CTCF sites along the genome on the basis of their relative orientation. Analysis of CTCF site orientation classes as a function of their spatial distribution along the human genome reveals that convergent CTCF site clusters are depleted while divergent CTCF clusters are enriched in the 5- to 100-kb range. We then analyse the distribution of CTCF binding sites as a function of TAD boundary conservation across seven primary human blood cell types. This reveals divergent CTCF site enrichment at TAD boundaries. Furthermore, convergent arrays of CTCF sites separate the left and right sections of TADs that harbour internal CTCF sites, resulting in unequal TAD ‘halves’.ConclusionsThe orientation-based CTCF binding site cluster classification that we present reconciles TAD boundaries and CTCF site clusters in a mechanistically elegant fashion. This model suggests that the emergent structure of nuclear chromatin in the form of TADs relies on the obligate alternation of divergent and convergent CTCF site clusters that occur at different length scales along the genome.Graphical abstract

Parallel bimodal single-cell sequencing of transcriptome and chromatin accessibility.

Joint profiling of transcriptome and chromatin accessibility within single cells allows for the deconstruction of the complex relationship between transcriptional states and upstream regulatory programs determining different cell fates. Here, we developed an automated method with high sensitivity, assay for single-cell transcriptome and accessibility regions (ASTAR-seq), for simultaneous measurement of whole-cell transcriptome and chromatin accessibility within the same single cell. To show the utility of ASTAR-seq, we profiled 384 mESCs under naive and primed pluripotent states as well as a two-cell like state, 424 human cells of various lineage origins (BJ, K562, JK1, and Jurkat), and 480 primary cord blood cells undergoing erythroblast differentiation. With the joint profiles, we configured the transcriptional and chromatin accessibility landscapes of discrete cell states, uncovered linked sets of cis-regulatory elements and target genes unique to each state, and constructed interactome and transcription factor (TF)–centered upstream regulatory networks for various cell states.

Successful delivery of large-size CRISPR/Cas9 vectors in hard-to-transfect human cells using small plasmids

With the rise of new powerful genome engineering technologies, such as CRISPR/Cas9, cell models can be engineered effectively to accelerate basic and disease research. The most critical step in this procedure is the efficient delivery of foreign nucleic acids into cells by cellular transfection. Since the vectors encoding the components necessary for CRISPR/Cas genome engineering are always large (9–19 kb), they result in low transfection efficiency and cell viability, and thus subsequent selection or purification of positive cells is required. To overcome those obstacles, we here show a non-toxic and non-viral delivery method that increases transfection efficiency (up to 40-fold) and cell viability (up to 6-fold) in a number of hard-to-transfect human cancer cell lines and primary blood cells. At its core, the technique is based on adding exogenous small plasmids of a defined size to the transfection mixture.

MO068A KIDNEY-ON-THE-CHIP APPROACH USING PRIMARY HUMAN TUBULAR CELLS IN A 3D CO-CULTURE SYSTEM

Abstract Background and Aims Conventional 2D mono-culture in vitro models using immortalized cell lines are still widely used in experimental nephrology, although their value is limited by poor translatability and predictive value for the in vivo or even human situation. The implementation of more sophisticated in vitro assays as routine cell culture systems is often limited by complex protocols and long lasting procedures. We aimed to establish and validate a relatively easy-to-use but yet (patho-) physiologically relevant cell culture assay that mimics key aspects of the in vivo situation of renal tubules, including a leak-thight epithelium with a luminal and baso-lateral side, interstitial matrix, a peri-tubular capillary and circulating blood cells inside its lumen. Method We utilized the 3-lane OrganoPlate® system (Mimetas, Leiden, Netherlands) as a scaffold. After infusing a collagen I matrix in the middle channel (C2), primary human renal progenitor cells are seeded into the upper channel (C1), adhering to the C2-matrix. The plate is put on a perfusion rocker (Mimetas), that facilitates continuous gravity-triggered bi-directional perfusion of C1. Within 48h the cells form a leak-tight tubular structure with a continuous lumen. Next, human endothelial cells are seeded into the bottom channel (C3), which adhered to the opposite site of the C2-matrix and – upon continuous perfusion – formed a vessel-like structure with a continuous lumen, as well. Finally, primary human white blood cells were isolated and seeded into C3 (see figure). Results Establishing the whole tubule-on-the-chip as described above takes on average three days. We investigated its operational life span by monitoring the barrier integrity of the tubular structure in C1 using a fluorescence-labeled dextran (150 kDa). Over a course of 5 days the tubular integrity did not decline, suggesting that the co-culture system remains stable and functional for at least 5 days. In accordance with other studies, the primary human tubular cells constituting the 3D tubule-on-the-chip expressed higher levels of functionally relevant proteins, e..g the tight-junction protein ZO-1 and the sodium-potassium-pump Na-K-ATPase, compared to standard 2D settings without perfusion. This emphasizes, that even primary cells show a physiologically reduced phenotype in standard 2D settings, which possibly impedes the identification and representative quantification of physiologically and hence also patho-physiologically relevant mechanisms in vitro. To study the interaction of cells in the tubule-on-the-chip, we investigated the recruitment of immune cells from C3 (vessel) across C2 (interstitium) to C1 (renal tubule), which - in vivo - represents a detrimental mechanism of action in intrarenal forms of AKI. Under baseline conditions the immune cells inserted into C3 did not leave their compartment. Upon damaging the tubular cells in C1 with extracellular histones, neutrophils and monocytes left C3 (extravasation), migrated through C2 and could be found in close contact with epithelial cells of C1. This serves as a proof of principle, that the tubule-on-the-chip is applicable to study complex cell-cell and cell-substrate interactions, such as chemokine-mediate immune cell homing. Measuring lactate dehydrogenase release for a number of known nephrotoxic agents revealed, that tubular cells forming a 3D-structure while kept under perfusion show significantly different responses to the same dose compared to standard 2D conditions, suggesting that dose-response studies using target cells out of their tissues context can be misleading when extrapolating results from in vitro to in vivo. Conclusion The results of this study suggest, that sophisticated 3D co-culture models of a renal tubule including an interstitial compartment, a peri-tubluar capillary and circulating immune cells are feasible and potentially suited to allow for in depth mechanistic studies in vitro.

The in vitro cytotoxic, genotoxic, and oxidative damage potentials of the oral artificial sweetener aspartame on cultured human blood cells

Background/aimAspartame (APM, L-aspartyl-L-phenylalanine methylester) is a low-calorie, nonsaccharide artificial sweetener widely used in foods and beverages. When metabolized by the body, APM is broken down into aspartic acid, phenylalanine amino acids, and a third substance, methanol. Since the amino acid phenylalanine serves as a neurotransmitter building block affecting the brain, and methanol is converted into toxic formaldehyde, APM has deleterious effects on the body and brain. Thus, its safety and, toxicity have been the subjects of concern ever since it was first discovered. Although many studies have been performed on it, due to the presence of conflicting data in the literature, there are still numerous question marks concerning APM. Therefore, the safety of aspartame was tested using in vitro methods.Materials and methodsWe aimed to evaluate the in vitro cytotoxic effects by using 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release tests, genotoxic damage potential by using chromosome aberration (CA) assay, and antioxidant/oxidant activity by using total antioxidant capacity (TAC) and total oxidative stress (TOS) analysis in primary human whole blood cell cultures.ResultsThe results of the MTT test showed that APM led to significant decreases in cell viability in a clear concentration-dependent manner. Moreover, an increase in CA frequency was found in the cells treated with APM. However, APM treatments did not cause any significant changes in TAC and TOS levels in whole blood cultures.ConclusionOverall, the obtained results showed that APM had genotoxicity potential and a concentration-dependent cytotoxic activity in human blood cells.