key: cord-283339-pbgeoxdu authors: Jonsdottir, Hulda R.; Dijkman, Ronald title: Characterization of Human Coronaviruses on Well-Differentiated Human Airway Epithelial Cell Cultures date: 2014-12-18 journal: Coronaviruses DOI: 10.1007/978-1-4939-2438-7_8 sha: doc_id: 283339 cord_uid: pbgeoxdu The human airway serves as the entry point of human respiratory viruses, including human coronaviruses. In this chapter we outline the methods by which we establish fully differentiated airway epithelium and its use for human coronavirus propagation. Additionally, we outline methods for immunofluorescence staining of these cultures for virus detection, characterization of cell tropism, and how to perform antiviral assays and quantify viral replication. The human airway serves as the entry point of human respiratory viruses, including human coronaviruses (HCoVs). In order to properly recapitulate the complex anatomy of the human lung specialized cell culture models have been developed to resemble both the upper and lower airways [ 1 -3 ] . Primary human bronchial epithelial cells cultured in an air-liquid interface (ALI) system serve as a universal platform to study human respiratory viruses [ 4 -6 ] . These human airway epithelial (HAE) cultures morphologically and functionally resemble the upper conducting airways in vivo. In these cultures, the epithelial layer is pseudostratifi ed and after differentiation they contain many different cell types such as basal, ciliated, and goblet cells and furthermore, generate protective mucus equivalent to that of in vivo epithelium [ 7 ] . Establishment of HAE cultures requires time and patience but the differentiated cultures allow for a number of advantageous analyses in respiratory virus research. We have adapted and optimized our methods based on previously published work [ 8 -10 ] . Moreover, we have standardized methods for the propagation of human coronaviruses and evaluation of the effects of antiviral compounds on both viral replication and cell viability. We are able to propagate all known human coronaviruses in this system and can easily evaluate their tropism by immunohistochemistry [ 5 , 11 ] . In this chapter we outline the methods by which we establish fully differentiated airway epithelium and use it for human coronavirus propagation. Additionally, we outline methods for immunofl uorescence staining of these cultures for virus detection, characterization of cell tropism and how to perform antiviral assays and quantify viral replication. Carry out all procedures in a biosafety cabinet according to local biosafety regulations. Cell culture fl asks are coated for 2 h with a mixture of Type I and III collagen that is necessary to effi ciently expand the number of primary airway epithelial cells. 5. Culture fl asks can be directly used. Optional : Store coated fl asks at +4 °C for a maximum of 6 weeks. The inserts need to be coated overnight with collagen type IV, necessary for development and long-term maintenance of differentiated primary airway epithelial cell cultures. 1. Mix 7.2 ml of fi lter-sterile dH 2 O with 800 µl of Collagen Type IV solution (0.5 mg/ml). 2. Apply 150 µl per 12-well inserts, or 50 µl per 24-well inserts. After completing one plate, make sure that the entire surface of each well is covered with the 1:10 collagen solution. 3. Air-dry the inserts overnight in a laminar fl owhood, and afterwards expose them to UV-light (type C) for 30 min. 4. To remove traces of acetic acid wash inserts twice with at least 500 µl of PBS. 5. After these steps, coated inserts can be used directly. Optional : Store at +4 °C (wrapped in foil) for a maximum of 6 weeks. Repeat UV-exposure and washing steps before use. Primary epithelial cells can be isolated from whole lung tissue resections of tracheal and/or bronchial origin according to the following protocol. Smaller lung tissue resections can be processed with the same protocol. All procedures are performed at room temperature unless stated otherwise. 1. Trim the bronchial tissue free of connective tissue and fat using forceps and scissors or a scalpel. If needed, cut the bronchial tissue into 2 cm segments. 2. Wash the cleaned tissue three times in washing solution. 3. Fill the desired number of 50 ml tubes with 30 ml of wash solution and transfer as many tissue segments as possible into a single tube, until the volume reaches 36 ml. Then add 4 ml of 10× digestion solution to each tube, to end volume 40 ml (40 mg Protease/0.4 mg DNase). 4. Place tubes on a rocking platform/tube roller at +4 °C and incubate for 48 h. 5. Place the 50 ml tube containing the digested tissue on ice and add 4 ml of heat-inactivated FBS to each tube (to a fi nal concentration of 10 % ( v / v )), to neutralize protease activity. Invert tubes three times. 6. Pour solution along with the tissue onto a large petri dish, and gently scrape off the epithelium from the collagen-cartilage surface, using a scalpel in the reverted angle. Pool solutions containing dissociated cells into a 50 ml conical tube and wash the petri dish once with PBS. 7. Centrifuge for 5 min at 500 × g . Wash cells once with HBSS and resuspend cells in BEGM to a concentration of, approximately, 5 × 10 6 cells/ml. 8. Count cells using a hemocytometer and seed into collagen coated fl asks with 20 ml of pre-warmed BEGM. An appropriate amount of cells for T75 fl asks ranges between 0.5 and 1.0 × 10 6 cells. 9. Change medium the next day to remove red blood cells and any unattached epithelial cells. 10. To prevent acidifi cation of the medium change it every 2-3 days, until 80-90 % confl uence. When the primary cells have reached 80-90 % confl uence in the expansion phase one can dissociate and seed the dedifferentiated primary cells on collagen type IV coated inserts, according to the following protocol. All procedures are performed at room temperature unless stated otherwise. 1. Remove BEGM and transfer it into a 50 ml tube and wash the cell monolayer twice with 12 ml of HBSS. 2. Dissociate the bronchial cells for 3 min at 37 °C in a humidifi ed 5 % CO 2 incubator with the appropriate amount of trypsin (25 cm 2 : 1 ml, 75 cm 2 : 3 ml). If needed tap the fl ask to dissociate the cells ( see Note 7 ). 3. Collect the cells in the previously collected BEGM and centrifuge for 5 min at 500 × g . Carefully discard the supernatant and resuspend cells in HBSS and centrifuge the suspension for 5 min at 500 × g . Discard the supernatant and resuspend cells in pre-warmed ALI medium and count using a hemocytometer. 8. The next day, medium in the apical compartment must be changed to remove any unattached cells. Discard the old medium and wash the apical surface with 500 µl HBSS and apply 500 µl of pre-warmed ALI medium to the apical side. Adjust volume to 200 µl for 24-well inserts. All incubation steps are performed at room temperature on a gyrorocker (20-30 rpm), unless stated otherwise 1. After the apical washing has been collected the apical surface is washed twice with 500 µl of PBS before cells are fi xed with formalin-solution for later immunofl uorescence analysis. 10. Apply primary antibodies ( see Table 2 ) diluted in 250 µl CB solution dropwise to the apical surface and incubate for 120 min. 11. Wash the apical surface three times with 500 µl of CB solution for 5 min ( see Note 11 ). 12. Apply the appropriately diluted conjugated secondary antibodies in 250 µl CB solution dropwise to the apical surface and incubate for 60 min. 13. Wash the apical surface twice with 500 µl of CB solution for 5 min. 14. Incubate cells with nucleic acid counter stain solution diluted in 250 µl of CB solution for 5 min. 15. Wash the apical surface once with 500 µl of CB solution for 5 min. 16. Lastly, wash the apical surface twice with 500 µl of PBS for 5 min to remove residual saponin and restore cell membrane integrity. 17. Before removing the washing solution, apply mounting medium on a glass slide (use 1-2 drops). Remove any air bubbles. 18. Excise the membrane from the plastic holder and carefully place the basolateral side of the membrane on top of the mounting medium, without generating air bubbles. 19. Then slowly add one drop of mounting medium on top of each membrane. 20. Slowly place the coverslip, in a tilted fashion, on top of the membrane without generating air bubbles. 21. Allow the mounting medium to polymerize for 30 min, after which the slide can directly be analyzed. 1. Pre-warm ALI medium to 37 °C. 2. Mix antiviral compounds (e.g. K22, recombinant interferons) in various concentrations or by serial dilution in ALI medium. Include non-treated controls. Also, to exclude viral inhibition by solvents (e.g. DMSO) include solvent controls. 3. For evaluation of either prophylactic or therapeutic effects of antivirals, the HAE cultures can be incubated with the compounds diluted in the basolateral medium prior to, during or after infection. 4. Infect cultures apically with human coronaviruses as described in Subheading 3.2 . 5. Collect apical washings in HBSS as described in Subheading 3.2 for viral quantifi cation by plaque assay and cells for viral quantifi cation by Renilla Luciferase Assay or qRT-PCR. CellTiter-Glo substrate to room temperature. 2. Transfer the buffer to the amber bottle containing the substrate to reconstitute the enzyme. Mix by gently swirling the bottle. 3. Wash the apical side of the HAE cultures three times with 500 µl HBSS to remove excess mucus. 4. Apply 50 µl of HBSS to the apical side and mix with equal volume of reconstituted CellTiter-Glo enzyme solution (optimized for 24-well inserts, for other insert sizes adjust buffer amount accordingly) and incubate for 5 min at room temperature on a gyro-rocker to induce cell lysis. 5. Next incubate the plate for 10 min at room temperature to allow for stabilization of the luminescence signal. 6. Transfer 20 µl of cell lysate to a white, non-transparent 96-well plate for analysis. 7. Record luminescence ( see Note 12 ). To account for background signal include empty wells in your analysis. 8. Program your luminometer settings with 10 s measure time followed by a 2 s delay. 100 µl of assay buffer should be dispensed into each well. If the luminometer is not equipped with an injector the assay buffer can be added manually using a multichannel pipette. 9. To adjust samples for background include empty wells in your analysis. The current protocol is optimized for HCoV-229E, but can easily be adapted to any other cell line and coronavirus strain. 1. Seed 150,000 target cells in a 12-well cluster plates with 1 ml of complete medium per well and incubate overnight at 37 °C in a humidifi ed 5 % CO 2 -incubator. 2. Make 6 tenfold serial dilutions of the harvested virus supernatants in 1 ml and inoculate the cells. 3. Incubate inoculum for 2 h at 33 °C in a humidifi ed 5 % CO 2incubator before removing the serial diluted virus inoculums from the cells and replace with 1 ml of overlay medium. 4. Incubate titration plates for 3-4 days at 33 °C in a humidifi ed 5 % CO 2 -incubator. 5 . Remove overlay and wash wells twice with water to remove residual Avicel. 6. Subsequently add approximately 0.5-1 ml of crystal violet solution to each well and incubate for 10 min. 7. Remove crystal violet solution and wash the cells once with water and allow the plates to air-dry before counting the number of plaques. 10. The fi xed HAE cultures can be kept for 1-3 months at 4 °C if the CB is fi lter-sterilized (0.2 µM) and all the procedure were performed under sterile conditions. After cold storage it is preferential to acclimatize the fi xed cultures for 15 min to room temperature on a gyro-rocker (20-30 rpm) prior to continuation of the staining protocol. 11. To prevent bleaching of the fl uorophores one should cover the inserts from daylight exposure during each incubation step. 12. Luminometer settings depend on the manufacturer. However, a measurement time of 1-2 s per well has proved effective. 13. For this assay cultures must be infected with coronaviruses expressing a Renilla Luciferase reporter gene. 14. If your luminometer is equipped with an injector you must remember to account for priming by increasing the volume of required Renilla Assay buffer by 2-3 ml. 15. Primers targeting HCoV-NL63, HCoV-HKU1, HCoV-229E, and HCoV-OC43 have been characterized and described [ 4 , 14 , 15 ] . Add the volume up to 34 ml, mix gently by inverting the tube three times. Filter-sterilize the solution through a 0.22 µm fi lter, and store at −20 °C in aliquots of 3.5 ml in 15 ml tubes To prepare the 1,000× stock, fi rst confi rm the RA concentration of the ethanol stock by diluting it 1:100 in absolute EtOH. Measure the absorbance at 350 nm using a spectrophotometer and a 1 cm light path quartz cuvette (or NanoDrop with 0.1 cm light path), blanked on 100 % EtOH. The absorbance of the diluted stock should equal 0.45 (0.045 on a NanoDrop) Dissolve 42 mg ferrous sulfate, 12.2 g magnesium chloride, and 1.62 g calcium chloride-dihydrate in 80 ml H 2 O Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface Air-liquid interface (ALI) culture of human bronchial epithelial cell monolayers as an in vitro model for airway drug transport studies Differentiation of human alveolar epithelial cells in primary culture: morphological characterization and synthesis of caveolin-1 and surfactant protein-C Culturing the unculturable: human coronavirus HKU1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures Effi cient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential Infection of human airway epithelium by human and avian strains of infl uenza a virus Mucin gene expression during differentiation of human airway epithelia in vitro. Muc4 and muc5b are strongly induced Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells A serum-free method for culturing normal human bronchial epithelial cells at clonal density Well-differentiated human airway epithelial cell cultures Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus Interferon lambda 4 signals via the IFNlambda receptor to regulate antiviral activity against HCV and coronaviruses Identifi cation of cell lines permissive for human coronavirus NL63 Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E This work was supported by the 3R Research Foundation Switzerland (project 128-11).