Isolation of MDCK cells with low expression of mdr1 gene and their use in membrane permeability screening

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P--glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.

Comparative Susceptibility of Madin–Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens

Madin–Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75–31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5–7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-β, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

Transcriptomic Characterization Reveals Attributes of High Influenza Virus Productivity in MDCK Cells

The Madin–Darby Canine Kidney (MDCK) cell line is among the most commonly used cell lines for the production of influenza virus vaccines. As cell culture-based manufacturing is poised to replace egg-based processes, increasing virus production is of paramount importance. To shed light on factors affecting virus productivity, we isolated a subline, H1, which had twice the influenza virus A (IAV) productivity of the parent (P) through cell cloning, and characterized H1 and P in detail on both physical and molecular levels. Transcriptome analysis revealed that within a few hours after IAV infection, viral mRNAs constituted over one fifth of total mRNA, with several viral genes more highly expressed in H1 than P. Functional analysis of the transcriptome dynamics showed that H1 and P responded similarly to IAV infection, and were both subjected to host shutoff and inflammatory responses. Importantly, H1 was more active in translation and RNA processing intrinsically and after infection. Furthermore, H1 had more subdued inflammatory and antiviral responses. Taken together, we postulate that the high productivity of IAV hinges on the balance between suppression of host functions to divert cellular resources and the sustaining of sufficient activities for virus replication. Mechanistic insights into virus productivity can facilitate the process optimization and cell line engineering for advancing influenza vaccine manufacturing.

Physical basis for the determination of lumen shape in a simple epithelium

The formation of a hollow lumen in a formerly solid mass of cells is a key developmental process whose dysregulation leads to diseases of the kidney and other organs. Hydrostatic pressure has been proposed to drive lumen expansion, a view that is supported by experiments in the mouse blastocyst. However, lumens formed in other tissues adopt irregular shapes with cell apical faces that are bowed inward, suggesting that pressure may not be the dominant contributor to lumen shape in all cases. Here we use live-cell imaging to study the physical mechanism of lumen formation in Madin-Darby Canine Kidney cell spheroids, a canonical cell-culture model for lumenogenesis. We find that in this system, lumen shape reflects basic geometrical considerations tied to the establishment of apico-basal polarity. A physical model incorporating both cell geometry and intraluminal pressure can account for our observations as well as cases in which pressure plays a dominant role.

Mechanism of Antiviral Activity of Nitazoxanide against the Influenza Virus: Effect of Tizoxanide on AdenosineTriphosphate in Influenza-virus Infected Madin Darby Canine Kidney Cells

Background: Nitazoxanide (NTZ) is a broad-spectrum antiviral undergoing clinical development for treating influenza and other viral respiratory infections such as those caused by rhinovirus/enterovirus and coronavirus including the emerging SARS-CoV-2. Methods: Nitazoxanide is a mild uncoupler of oxidative phosphorylation, which is modulating the ATP production in cells. ATP is an essential component of viral replication, and we have evaluated the effect of tizoxanide (TIZ), the active circulating metabolite of NTZ, on ATP in Madin-Darby canine kidney (MDCK) cells and in MDCK cells infected with influenza A and B viruses. Results: TIZ decreased cellular ATP in a dose-dependent manner in MDCK cells and in MDCK cells infected with influenza A and B viruses. Maximum inhibition of ATP in influenza infected or uninfected MDCK cells reached up to 45% after 6 and 24 hours of exposure to 100 micrometer TIZ. The decrease in cellular ATP did not affect cell viability and was reversible after eliminating TIZ from the culture. Conclusion: The concentrations of TIZ required to decrease cellular ATP levels were similar to those reported to inhibit replication of influenza A and B viruses in our laboratory. A decrease in ATP triggers activation of AMP-activated protein kinase, which is known to suppress the secretion of pro-inflammatory cytokines. Additional studies are warranted to evaluate the effect of TIZ on mitochondrial function.

Apical Actin-myosin Network Regulates the Tight Junction of Polarized Madin-Darby Canine Kidney Cells

The tight junction outlines the apicolateral border of epithelial cells like a belt, sealing the paracellular space when cells form contacts with each other. The permeability and morphology of tight junction are regulated by actomyosin contractility, which has been conventionally thought from the purse-string-like circumferential actomyosin belt along tight junction. Spatially, the tight junction is close to the apical actin network, which exerts inward contractions orthogonal to the tight junction. To test the contributions from apical actin network, we laser-ablated spots on the apical surface of polarized Madin-Darby Canine Kidney (MDCK) epithelial cells. Laser ablation severed the apical cytoskeleton network, decreased in-plane tension, increased the apical surface area, and rendered the tight junction less tortuous in shape. Consistent with these observations, changes in MDCK cell sheet morphology due to cell proliferation, or perturbation with the ROCK inhibitor Y27632 increased the density of the apical actin network and decreased tight junction tortuosity. The morphological analysis revealed scutoids in flat MDCK cell sheets, contrary to predictions from a previous model that only considered cell-cell interactions as line tension. Additional cell-cell interactions from apical in-plane tension provides probable cause for the occurrence of scutoids on flat geometry. Taken together, our findings identify the importance of the apical actin network exerting in-plane apical tension to regulate tight-junction mechanobiology and epithelial cell shape.

On the mechanical regulation of epithelial tissue homeostasis

Despite recent efforts to understand homeostasis in epithelial tissues, there are many unknowns surrounding this steady state. It is considered to be regulated by mechanoresponse, but unlike for single cells, this remains heavily debated for tissues. Here, we show that changes in matrix stiffness induce a non-equilibrium transition from tubular to squamous Madin-Darby Canine Kidney II tissues. Nonetheless, despite different cell morphologies and densities, all homeostatic tissues display equivalent topologies, which, hence, must be actively targeted and regulated. On the contrary, the mechanoresponse induces dramatic changes in the large-scale organization of the colonies. On stiff gels, this yields an unreported cooperative state of motile cells displaying higher densities than in the arrested homeostatic state. This suggests a more complex relation between cell density and motility than previously anticipated. Our results unequivocally relate the mechanosensitive properties of individual cells to the evolving macroscopic structures, an effect that could be important for understanding the emergent pathologies of living tissues.