The authors acknowledge the Microscopy core facility at the Faculty of Health Sciences, Link&#246;ping University for providing access to advanced imaging systems; Karl-Eric Magnusson (Emeritus Scientist) at the Dept. of Clinical and Experimental Medicine, Link&#246;ping University for providing access to Imaris 3D/4D image processing software (Bitplane, Switzerland); and S. Braian for his help with the lung model cartoon. This work was supported by funds from the Swedish Research Council (Alternatives to animal research, 2012-1951) and Swedish Research Council (2012-3349) to M.L. and Swedish Foundation for Strategic Research to S.B. S.B. receive grants from the Karolinska Institutet, Swedish Research Council, the Swedish International Development Cooperation Agency (Sida) and the Swedish Civil Contingencies Agency (MSB), and the Swedish Heart and Lung Foundation (HLF). M.S. received grants from the Karolinska Institutet and Stockholm County Council.

Note: Human peripheral blood from healthy anonymous blood donors purchased at the blood bank of Link&#246;ping University Hospital, Sweden was used as the source of immune cells for this study. This protocol is designed for 24 mm 6-well plate inserts. Direct adaptation to other well formats is not recommended since the tissue model contracts both vertically and horizontally during development.
1. Preparation of Materials, Media and Culture of Bacteria/ Cell Lines
<ol>
	<li>Culture of bacteria:
	<ol>
		<li>Grow the mycobacterial strain M. tuberculosis H37Rv carrying the pFPV2 plasmid to constitutively express green fluorescent protein (GFP), in Middlebrook 7H9 medium containing 0.05% Tween-80, 0.5% glycerol, kanamycin (20 &#181;g/ml), and supplemented with Middlebrook albumin, dextrose and catalase enrichment (Middlebrook ADC Enrichment), at 37 &#176;C with 5% CO2 for 7-10 days. 
		Note: All experimental steps involving live virulent M. tuberculosis strains should be performed in a BSL-3 facility.</li>
	</ol>
	</li>
	<li>Prepare 1x Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) complete medium (supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 10 mM HEPES, 0.1 mM non-essential amino acids and 10% heat-inactivated fetal bovine serum (FBS)). Prepare also antibiotic-free DMEM complete medium.</li>
	<li>Prepare 1x Minimum Essential Medium (MEM) complete medium (1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 10 mM HEPES, 0.1 mM non-essential amino acids and 10% heat-inactivated fetal bovine serum (FBS)).</li>
	<li>Preparation of fibronectin/collagen-coated flasks (total 10 ml):
	<ol>
		<li>Pipette 8.8 ml sterile 1x phosphate buffered saline (PBS) into a clean tube. Add 1 ml bovine serum albumin (1 mg/ml), 100 &#181;l type I bovine collagen (3 mg/ml) and 100 &#181;l recombinant human fibronectin (1 mg/ml).</li>
		<li>Mix the solution by turning the tube upside-down 5 times. Flasks are coated with a fibronectin/collagen solution (1 ml for a T-25 and 2 ml for a T-75 flask). Leave it O/N at 37 &#176;C. After incubation, remove the solution and store the coated flasks at RT. 
		Note: The collected solution can be stored at 4 &#176;C and reused for three times. Storage for more than 2 weeks may cause the liquid to turn brown (formation of crystals), where upon it must be discarded.</li>
	</ol>
	</li>
	<li>Culture of fibroblasts:
	<ol>
		<li>Grow and maintain MRC-5, (a human lung fibroblast cell line derived from normal lung tissue of a 14-week old male foetus), in complete DMEM in 5% CO2 at 37 &#176;C. Use the fibroblasts at passages 24-26 and grow until 70-80% confluent. 
		Note: MRC-5 cell line at passage &#62;30 tend to lose the morphology and is not recommended for use in the tissue model.</li>
	</ol>
	</li>
	<li>Culture of epithelial cells:
	<ol>
		<li>Obtain 16HBE14o- (16HBE), an immortalized human bronchial epithelial cell line that retains the differentiated morphology and function of normal human airway epithelia, (this was a gift from Dr. Dieter Gruenert, Mt. Zion Cancer Center, University of California, San Fransisco, USA 10. ). Culture 16HBE cells in fibronectin/collagen-coated flasks and maintain the cells in complete MEM in 5% CO2 at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of 5x DMEM
	<ol>
		<li>Prepare 5x DMEM by dissolving 13.4 g of DMEM powder and 3.7 g of sodium bicarbonate in 150 ml of sterile distilled water. Adjust the pH of the medium to 7.3, make up the volume to 200 ml and filter it using 0.22 &#181;m membrane filter. Collect the filtered medium in a sterile container and stored until use at RT.</li>
	</ol>
	</li>
</ol>
2. Preparation of Collagen-embedded Fibroblasts
<ol>
	<li>Thaw frozen aliquots of FBS and L-glutamine in a 37 &#176;C water bath. After thawing keep the samples on ice. Place Sodium bicarbonate (71.2 mg/ml) and Gentamicin (50 mg/ml) at 4 &#176;C. Pre-cool 50 ml centrifuge tubes and 10 ml sterile pipettes to 4 &#176;C. 
	Note: All materials used (except the 5x DMEM) are chilled on ice prior to use and all steps are performed on ice. Type I Bovine Collagen (1.1 mg/ml) must be kept cold, as this prevents solidification of collagen.</li>
	<li>Prepare the fibroblast cells:
	<ol>
		<li>Warm Trypsin in a 37 &#176;C water bath and incubate sufficient quantity with Lung fibroblasts (MRC-5) cells for 10 min at 5% CO2 at 37 &#176;C. Neutralize the trypsin by adding 1x DMEM complete. Aspirate the cell suspension and centrifuge at 300 x g for 5 min. Aspirate the supernatant and resuspend the cells at 2.3 x 105 cells/ml in DMEM complete. Place the cells on ice until ready for use.</li>
	</ol>
	</li>
	<li>Prepare the pre-mix:
	<ol>
		<li>Add the following to a tube labeled &#8220;pre-mix&#8221;; 395 &#181;l of 5x DMEM, 40 &#181;l L-Glutamine, 120 &#181;l NaHCO3 (71.2 mg/ml), 440 &#181;l FBS , 5 &#181;l Gentamicin (50 mg/ml), Total volume 1,000 &#181;l, then swirl the pre-mix well and put on ice. 
		Note: The volumes given are for one 24 mm 6-well culture insert. Calculate the specific amounts required for the total number of inserts but add one extra to be sure, enough pre-mix is prepared.</li>
	</ol>
	</li>
	<li>Prepare the acellular collagen mixture:
	<ol>
		<li>To each culture add 1 ml of acellular collagen mixture. Add the following to a 50-ml conical tube on ice in the given order; 686 &#181;l of 1.1 mg/ml collagen, 250 &#181;l Pre-mix and 64 &#181;l 1x complete DMEM to a total volume of 1,000 &#181;l. Mix the solution well ensuring no air bubbles. Work fast and add the collagen on the wall of the tube to avoid air bubbles.</li>
		<li>Add 1 ml of the acellular layer mixture to the insert placed in the 6 well plate. Do not add any medium to the well outside the insert. Incubate for 30 min in a 37 &#176;C incubator. Make sure the acellular mixture covers the entire insert without any air bubbles.</li>
	</ol>
	</li>
	<li>Prepare the cellular collagen mixture:
	<ol>
		<li>Mix components of the cellular layer in a 50 ml conical tube kept on ice in the following order; 2 ml Collagen, 615 &#181;l Pre-mix, 58 &#181;l of 1x complete DMEM and 327 &#181;l cell suspension Lung fibroblasts (MRC-5) to make up the total volume to 3,000 &#181;l. Each culture requires 3 ml of cellular collagen mixture. 
		CRITICAL STEP: Make sure to mix the collagen and premix carefully before addition of the cell suspension. This will neutralize the pH of the collagen to avoid toxic effects on the fibroblasts.</li>
		<li>Add the cellular layer (3 ml) on top of the acellular collagen layer and incubate for 2 hr in a 37 &#176;C incubator. Work fast and add the collagen on the wall of the tube to avoid air bubbles.</li>
		<li>Following polymerization, add 2 ml of DMEM complete to the bottom of the 6 well plate (under the insert) and incubate for 24 hr. 
		NOTE: If polymerization did not occur, discard the plate inserts containing the fibroblast-collagen matrix and start-over again. The most likely cause is an error in addition or incorrect volume of one of the above listed reagents.</li>
	</ol>
	</li>
</ol>
3. Continuous Culture of the Fibroblast-collagen Matrix
<ol>
	<li>Carefully lift the insert using a clean forceps and aspirate the culture media from the bottom of the well. Add 2 ml complete DMEM to the bottom of the well followed by 2 ml complete DMEM within the insert. Avoid introducing air bubbles under the insert, as this will prevent the diffusion of nutrients between the outer and inner chambers. Remove air bubbles with a micropipette tip.</li>
	<li>Change the culture media (inside and below the insert) every second day and culture for approximately 5-7 days. Take care in removing the media from the insert. To avoid contact with the fibroblast-collagen matrix, slightly tilt the insert using a clean forceps and aspirate the media from the insert walls. 
	CRITICAL STEP: The fibroblasts in the collagen matrix should get an elongated phenotype, and remodel the collagen, which then contracts. In about 5-7 days, the matrix has contracted to form a platform (10-14 mm in diameter) in the center of the insert. The contracted matrix is ready for use in the next step. To obtain a uniform contraction of the matrix it is critical that the fibroblasts are well mixed with the collagen prior to seeding (Step 2.6.1).</li>
</ol>
4. Seeding of Immune Cells (Infected/Uninfected Monocyte-macrophage Mixture)
Note: The following experimental steps involve virulent mycobacteria and therefore must be performed in a BSL-3 facility.
<ol>
	<li>Preparation of primary monocytes and macrophages:
	<ol>
		<li>Isolate peripheral blood monocytes from donor blood using an established protocol. Isolate monocytes on the same day as setting up the tissue model and culture and differentiate them into macrophages for approximately 7 days before infection with M. tuberculosis. 
		NOTE: This will ensure both the macrophages and the contracted fibroblast-collagen matrix are available after 7 days. Also isolate fresh monocytes that will be added together with the infected macrophages.</li>
	</ol>
	</li>
	<li>Preparation of M. tuberculosis -infected macrophages
	<ol>
		<li>Harvest the cultured bacteria, wash with 1x PBS containing 0.05% Tween-80, resuspend in antibiotic-free complete DMEM, pass through a truncated sterile 27 G needle to disperse bacterial clumps and measure the optical density. 
		Note: Pre-determine the M. tuberculosis colony forming unit equivalents of optical density in the laboratory. This will give an estimate of the number of bacteria to be used for infection.</li>
		<li>Incubate the macrophages for 4 hr with M. tuberculosis at the multiplicity of infection (MOI) 10). After infection, wash 3x with 1x PBS to remove the extracellular bacteria. Use uninfected macrophages cultured in the same way but without M. tuberculosis, as controls.</li>
		<li>Detach the macrophages from the culture plate by treatment with 2 mM EDTA for 10 min at 37 &#176;C and resuspended the cells in antibiotic-free complete DMEM.</li>
	</ol>
	</li>
	<li>Labelling of monocytes 
	Note: Isolation and labeling of monocytes can be performed in the BSL-2 bench and then taken into BSL-3 facility for further processing.
	<ol>
		<li>Stain freshly prepared monocytes (2 x 107 cells) with a final concentration of 2 &#181;M PKH26 red dye for 5 min, according to the manufacturer&#8217;s instructions. Wash 3x and resuspend the cells with antibiotic-free complete DMEM at a density of 1 x 107 cells/ml.</li>
	</ol>
	</li>
	<li>Addition of immune cells to fibroblast-collagen matrix
	<ol>
		<li>After 5-7 days of culture of the fibroblast-collagen matrix, aspirate the culture media from the outer and inner chambers and add 1.5 ml of fresh antibiotic-free DMEM complete to the outer chamber.</li>
		<li>Prepare a labeled monocyte-macrophage (uninfected/infected) mixture with a ratio of MO:MQ (5:1) in 50 &#181;l DMEM complete. For 50,000 macrophages, take 250,000 labeled monocytes.</li>
		<li>Add 50 &#181;l MO:MQ mixture to the fibroblast-collagen matrix and incubate for 1 hr in 5 % CO2 at 37 &#176;C. After incubation, gently add 2 ml of culture media into the insert and incubate for an additional 24 hr in 5% CO2 at 37 &#176;C. 
		Note: As cells added are loosely attached, addition of media should be slow by gently adding on the walls of the insert.</li>
	</ol>
	</li>
</ol>
5. Seeding of Lung Epithelial Cells (16HBE)
Note: The following steps must be performed in a BSL-3 facility.
<ol>
	<li>Seed lung epithelial cells (16HBE) on top of the MO:MQ-fibroblast-collagen layer. To perform this, first dissociate 16HBE cells from the flask by treating with trypsin (as in 2.2.1.) and resuspend at 4 x 106 cells/ml in antibiotic-free DMEM.</li>
	<li>Aspirate the culture media inside and outside of the insert. Then add 1.5 ml antibiotic-free DMEM complete in the bottom of the well outside the insert.</li>
	<li>Add 50 &#181;l of 16HBE on top of the immune cell-fibroblast collagen matrix. Leave for 2 min in the hood and incubate for 1 hr in 37 &#176;C incubator with 5% CO2. After incubation, add gently 2 ml of antibiotic-free DMEM complete within the insert and culture at 37 &#176;C for 3 days. The culture step facilitates proliferation of epithelial cells in the tissue model. 
	Note: As cells added are loosely attached, addition of media should be slow and gentle by sliding through the walls of the insert.</li>
</ol>
6. Air-exposure of the 3D Lung Model
Note: After day 5 post addition of infected macrophages, the tissue models are air-exposed and the following steps must be performed in a BSL-3 facility.
<ol>
	<li>Aspirate the culture media inside and outside of the insert. 
	Note: In this step supernatants can be collected for detection of secreted factors. Centrifuge, sterile-filter and store supernatants at -70 &#176;C.</li>
	<li>Add 1.8 ml antibiotic-free DMEM complete in the outer chamber and incubate in 5% CO2 at 37 &#176;C incubator for 2 days. Do not add culture media within the insert. 
	NOTE: Air-lifting of the tissue model facilitates the formation of stratified epithelia and mucus secretion, which provides the strength to the tissue and physiological resemblance to human lung tissue.</li>
</ol>
7. Harvesting and Mounting the 3D Lung Tissue Model
 
Note: The following steps must be performed in a BSL-3 facility.
<ol>
	<li>On day 7 post implantation of infected macrophages, the tissue models are ready for harvesting. Remove the culture media entirely from the tissue model. Fix the tissue model with 4% paraformaldehyde for 30 min in the dark at RT. This step kills the bacteria and fixes the tissue/ cell morphology for further processing.</li>
	<li>Using a scalpel, separate the membrane from the well insert. Transfer the membrane containing the tissue to a well containing 1x PBS.</li>
	<li>Cut and remove the sides of the tissue model using a clean scalpel. Then slice the tissue model into 4 approximately equal square pieces. Transfer a piece of tissue onto a superfrost glass slide. Store the tissue pieces in 1x PBS at 4 &#176;C.</li>
	<li>Dry the tissue for 5 min and mount using ProLong Gold antifade with DAPI and coverslip. Leave the slides without disturbing in the dark at RT until dry. 
	Note: The thickness of the tissue may vary between the center and periphery, causing slight tilting of the coverslip. To avoid this, a spacer (for instance parafilm) may be placed on the corner of the cover slip.</li>
	<li>Apply nail polish to the edges of the coverslip and allow it to dry. Immerse the slides in 70% ethanol to make them safe to bring out of the BSL-3 facility.</li>
</ol>
8. Visualization, Acquisition and Quantitative 3D Analysis
<ol>
	<li>Visualize the tissue slides using a confocal microscope system with lasers emitting at 488 nm for excitation of GFP (green channel), 420 nm for DAPI (blue) and 555 nm for the PKH26-labeled monocytes (red) respectively.</li>
	<li>Acquire 3D images at a 512x512 resolution with Z-stacks covering at a minimum of 20 &#181;m thickness and having 1 - 1.5 &#181;m separation between stacks. Acquire 5-10 different fields covering the entire piece of tissue. 
	Note: Use the configuration such as Nyqvist for optimal settings of optical resolution (wavelength, laser power/exposure, pixel size and zoom). Avoid under or over saturation of pixels.</li>
	<li>Analyze the confocal images with 3D image processing software. For 3D quantification of cell clusters, the following steps are recommended for optimal analysis.</li>
	<li>Open the 3D image processing software and load the image. Measure the dimensions of objects to be analysed in the image, for example, the size of a nucleus, individual monocyte and single bacteria. These observations are useful for defining or filtering the objects.</li>
	<li>Using a display adjustment tool, optimise volume rendering by adjusting each channel for image contrast, brightness and blend opacity. This step is to minimise the interference of noise in volume rendering. 
	Note: Gamma correction may lead to manipulation of images and thus should be avoided.</li>
	<li>Create surfaces by choosing the red channel (monocytes) and set the threshold (automatic or manual selection). If required, use filters to restrict the selection of red monocytes or to exclude the background. Similarly, create surfaces for green (M. tuberculosis) and blue (nuclei) channels as described above.</li>
	<li>Export the data into Ms-Excel files. It is possible to export a particular parameter or all the data. The parameters that are relevant for analysis of cell clustering are volume, intensity, number of objects, number of voxels and sphericity.</li>
	<li>Save and export the images in a suitable picture format preferably TIFF. 
	NOTE: Animations can also be made using the animation menu and saved as a media file.</li>
	<li>Save the analysis settings of each channel using the Add parameters option and can be retrieved later using the Rebuild function. Simultaneously analyse more files using the Batch processing tool. 
	Note: All the images to be compared must be acquired, processed and analyzed in the same manner. For instance a 8 bit image (256 integer) should not be compared with a 12 bit image (4096 integer).</li>
</ol>

<ol>
	<li>Sakamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+Mycobacterium+tuberculosis+infection.">The pathology of Mycobacterium tuberculosis infection.</a> Veterinary pathology. 49, 423-439 (2012).</li><li>Saunders, B. M., Britton, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+and+death+in+the+granuloma:+immunopathology+of+tuberculosis.">Life and death in the granuloma: immunopathology of tuberculosis.</a> Immunol Cell Biol. 85, 103-111 (2007).</li><li>Kaufmann, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+issues+in+tuberculosis.+Ann+Rheum+Dis.">New issues in tuberculosis. Ann Rheum Dis.</a> Ann Rheum Dis. 63, ii50-ii56 (2004).</li><li>Morrison, J., Pai, M., Hopewell, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis+and+latent+tuberculosis+infection+in+close+contacts+of+people+with+pulmonary+tuberculosis+in+low-income+and+middle-income+countries:+a+systematic+review+and+meta-analysis.">Tuberculosis and latent tuberculosis infection in close contacts of people with pulmonary tuberculosis in low-income and middle-income countries: a systematic review and meta-analysis.</a> Lancet Infect Dis. 8, 359-368 (2008).</li><li>Barry 3rd, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+latent+tuberculosis:+rethinking+the+biology+and+intervention+strategies.">The spectrum of latent tuberculosis: rethinking the biology and intervention strategies.</a> Nat Rev Microbiol. 7, 845-855 (2009).</li><li>Puissegur, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+dual+model+of+mycobacterial+granulomas+to+investigate+the+molecular+interactions+between+mycobacteria+and+human+host+cells.">An in vitro dual model of mycobacterial granulomas to investigate the molecular interactions between mycobacteria and human host cells.</a> Cell Microbiol. 6, 423-433 (2004).</li><li>Kapoor, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Granuloma+In+Vitro+Model,+for+TB+Dormancy+and+Resuscitation.">Human Granuloma In Vitro Model, for TB Dormancy and Resuscitation.</a> PLoS One. 8, e53657 (2013).</li><li>Nguyen Hoang, ., T, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+functional+properties+in+a+three-dimensional+tissue+model+of+human+lung+mucosa.">Dendritic cell functional properties in a three-dimensional tissue model of human lung mucosa.</a> Am J Physiol Lung Cell Mol Physiol. 302, L226-L237 (2012).</li><li>Parasa, V. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Mycobacterium+tuberculosis+early+granuloma+formation+in+experimental+human+lung+tissue.">Modeling Mycobacterium tuberculosis early granuloma formation in experimental human lung tissue.</a> Dis Model Mech. 7, 281-288 (2014).</li><li>Cozens, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+expression+and+chloride+secretion+in+polarized+immortal+human+bronchial+epithelial+cells.">CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.</a> Am J Respir Cell Mol Biol. 10, 38-47 (1994).</li><li>Brighenti, S., Andersson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+immune+responses+in+human+tuberculosis:+learning+from+the+site+of+infection.">Local immune responses in human tuberculosis: learning from the site of infection.</a> J Infect Dis. 205, S316-S324 (2012).</li><li>Davis, J. M., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+granuloma+in+expansion+and+dissemination+of+early+tuberculous+infection.">The role of the granuloma in expansion and dissemination of early tuberculous infection.</a> Cell. 136, 37-49 (2009).</li><li>Gupta, U. D., Katoch, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+tuberculosis.">Animal models of tuberculosis.</a> Tuberculosis (Edinb). 85, 277-293 (2005).</li><li>Kashino, S. S., Napolitano, D. R., Skobe, Z., Campos-Neto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guinea+pig+model+of+Mycobacterium+tuberculosis+latent/dormant+infection.">Guinea pig model of Mycobacterium tuberculosis latent/dormant infection.</a> Microbes Infect. 10, 1469-1476 (2008).</li><li>Singhal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+tuberculosis+in+the+Wistar+rat:+a+model+for+protective+immunity+and+control+of+infection.">Experimental tuberculosis in the Wistar rat: a model for protective immunity and control of infection.</a> PLoS One. 6, e18632 (2011).</li><li>Subbian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphodiesterase-4+inhibition+alters+gene+expression+and+improves+isoniazid-mediated+clearance+of+Mycobacterium+tuberculosis+in+rabbit+lungs.">Phosphodiesterase-4 inhibition alters gene expression and improves isoniazid-mediated clearance of Mycobacterium tuberculosis in rabbit lungs.</a> PLoS Pathog. 7, e1002262 (2011).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor+neutralization+results+in+disseminated+disease+in+acute+and+latent+Mycobacterium+tuberculosis+infection+with+normal+granuloma+structure+in+a+cynomolgus+macaque+model.">Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model.</a> Arthritis Rheum. 62, 340-350 (2010).</li><li>Rahman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+immune+responses+in+human+tuberculosis:+few+CD8++effector+T+cells+but+elevated+levels+of+FoxP3++regulatory+t+cells+in+the+granulomatous+lesions.">Compartmentalization of immune responses in human tuberculosis: few CD8+ effector T cells but elevated levels of FoxP3+ regulatory t cells in the granulomatous lesions.</a> Am J Pathol. 174, 2211-2224 (2009).</li></ol>

The authors declare no competing financial interests.

The ability to recruit and form organized cell clusters at the site of infection is the hallmark of human TB 11. These dynamic structures known as tubercle granulomas primarily consist of immune cells (macrophages, monocytes, T-cells and B-cells) and multi-nucleated giant cells surrounding M. tuberculosis. The role of the granuloma has long been considered to wall off the infection, preventing local spread of bacteria. However, more recent studies show that granuloma formation is critical for early bacterial survival, growth and dissemination 12. A strategy of new studies is to identify molecules or pathways that could efficiently be targeted to inhibit the cellular migration in granuloma formation and/or TB dissemination. 
A caveat for novel studies on TB is the lack of models that recapitulate human TB. The most widely used experimental animals do not form true granuloma upon M. tuberculosis infection, and are therefore not appropriate choices for studies of TB 13-16. Non-human primates have the closest resemblance to human TB 17, but are not the preferred choice owing to high operational costs and ethical issues. Human TB is a complex immunological process and is difficult to model in vitro. Cell cultures of monolayers or co-cultures lack the 3D environment and tissue responses. Therefore, we have developed an innovative lung tissue model based on human primary immune cells and human lung-specific cell lines 8,9. The model displays characteristic features of human lung tissue, including epithelia with evenly integrated macrophages, formation of extracellular matrix, stratified epithelia and mucus secretion 9. 
The 3D human lung tissue model has several benefits over the in vitro single or co-cultures seeded on tissue culture plates or transwell inserts. First, the human lung-specific cells (fibroblasts and epithelial cells) are not commonly included in the in vitro single or co-cultures. Second, the immune cells and lung-specific cells are embedded in a 3D physiological context (collagen rich extra-cellular matrix products). The response of cells to a stimulus/infection and the migratory behaviour of cells, for instance formation of a granuloma, differ significantly between a 2D and 3D environment. Furthermore, the described method enables the 3D visualization and robust 3D quantitative analysis that provides pivotal information on spatial distribution and intricate cellular interactions. 
Experimental infection in the model tissue with M. tuberculosis resulted in clustering of macrophages at the site of infection, reminiscent of early TB granuloma (Figure 2 and 3). We have recently demonstrated that mutant strains defective in the ability to secrete the virulence factor ESAT-6 or Mycobacterium bovis BCG that lacks ESAT-6 did not induce the clustering of monocytes (no early granuloma), in contrast to the virulent M. tuberculosis 9. These data are consistent with the observations made from Mycobacterium marinum-infected zebrafish embryos, whose transparency allows for elegant live imaging of granuloma formation 12. As there is no gold-standard model for TB, we took advantage of the surgically resected tissue biopsies from TB patients for validation of the method 9. Our in vitro tissue model shares several characteristics with the lung and lymph node biopsies from TB patients, including the aggregation of macrophages in granuloma, the presence of both intra- and extracellular bacteria 18 and induction of necrosis 11. 
Although the described model has physiological relevance to human TB and has several advantages over other in vitro models, it has some limitations. For instance, out of more than 20 collagen proteins identified in humans, only type I is included to the model to mimic the extra-cellular matrix. However, type I collagen is a complex mixture of extra-cellular matrix products and is the most abundant collagen in the human body. Further, we have demonstrated the presence of collagen IV and several extra-cellular matrix proteins such as tropoelastin, vimentin and laminin, which are produced by the epithelial cells and fibroblasts in the tissue model, indicating the synthesis of new collagen 8. Presently, the lung tissue model only has monocytes and macrophages, besides lung-specific cells. It lacks neutrophils and lymphocytes that are also known to be present in the granuloma. Remarkably the model is not limited to the introduction of additional immune cells and is of interest to explore how they contribute to the complex cellular interactions in human TB. Implantation of primary alveolar macrophages, skin-specific cells and lung carcinoma cells has already been tested in the model. Since our objective was to use a model that closely resembles human TB, introduction of mouse cells have not been attempted.
In summary, the lung tissue model has implications for both basic mechanistic and applied studies. Potential applications of the lung model include the study of innate immunity, investigating mechanistic aspects of host defences such as phagosomal maturation, autophagy, production of cytokines, chemokines and anti-microbial peptides, and functional characterization of individual cell types. Strikingly, the in vitro tissue model allows manipulation of one or more cells types and provides a relevant tissue micro-environment, not only for studies on TB, but for a variety of infectious and non-infectious diseases that affect the lungs.

In humans, responses to infection, tissue inflammation, cellular recruitment, tissue remodeling and the regulation of tissue homeostasis are complex events involving different cell types. Hence, these processes are best studied in the local tissue environment. Previously, this has mainly been possible using experimental animal models. However, the widely used experimental animals hold many limits as they often respond to pathogens in a different way than humans and also display a different course of disease 1. A human in vitro lung tissue model holds the possibilities to study specific immune responses in the human lung.
Human tuberculosis infection (TB) is mainly a disease affecting the lungs. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of TB, reaches the lung via aerosol droplets that are transported to the alveolar space, where the bacteria are engulfed by pulmonary dendritic cells and alveolar macrophages as part of the innate immune response to the infection 2,3. Phagocytosis of the pathogen leads to the compartmentalization of the bug within a phagosome and ideally results in the neutralization and killing of the pathogen by the phagocyte. Up to 50% of individuals exposed to M. tuberculosis are believed to be able to clear the infection through the innate immune response 4. Other outcomes of infection are clearance by the adaptive immune system at a later stage, latent infection or in worst cases chronic active disease 5.
Previously there have been no in vitro tissue models for studies of human TB. Single cell cultures of human macrophages or other peripheral blood cells have often been used 6,7. The disadvantage of this approach is that they cannot reflect the dynamics of different cell types operating together in a lung tissue exposed to M. tuberculosis. Thus, there is a need for an in vitro model to be able to perform functional and mechanistic studies on TB. The cell-based in vitro human lung tissue model described here was originally established by our group for studies on dendritic cell functions 8. We have adapted this method for the study of TB.
The human lung tissue model presented here consists of tissue-specific epithelial cells and fibroblasts 8. These cells are cultured in a matrix of collagen on top of a porous membrane in a transwell insert and form structures resembling normal human lung tissue (Figure 1). When exposed to air the cells start to secrete mucus at the apical side 8. By implanting human primary macrophages infected with M. tuberculosis to the model, we have observed how the immune cells migrate in the tissue and form early stages of TB granulomas 9. This is the first human tissue model described for TB and it poses a promising tool for studying innate immune responses to TB and other diseases of the lung. To date, we have used only monocytes and macrophages as immune cells in the model but the level of complexity can be increased by inclusion of additional relevant cell types.
<img alt="Figure 1" src="/files/ftp_upload/53084/53084fig1.jpg" /> 
Figure 1. Schematic outline of the lung tissue model.&#160;(A) The model is composed of human lung-specific epithelial cells, M. tuberculosis-infected primary macrophages and red dye labeled monocytes seeded onto collagen embedded fibroblasts prepared on a transwell filter. Exposure of the tissue model to air initiates production of extra-cellular matrix proteins, mucus secretion and stratification by the epithelium. The 3D tissue model thus developed is a useful tool to study M. tuberculosis infection in an environment that closely resembles a human lung. (B) Representative microscopic images of the different steps in the preparation of the tissue model. (C) Complete structure of the lung model tissue section. Scale - 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53084/53084fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell culture inserts</td>
			<td>BD Falcon</td>
			<td>353092</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>MRC-5 cells, lung fibroblasts</td>
			<td></td>
			<td></td>
			<td>ATCC#CCL-171</td>
		</tr>
		<tr>
			<td>16HBE cells, lung epithelial cells</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Dieter Gruenert, Mt. Zion Cancer Center, University of California, San Fransisco, USA&#160;</td>
		</tr>
		<tr>
			<td>5 x Dulbecco&#8217;s modified Eagle&#8217;s medium (5 x DMEM)</td>
			<td>Gibco</td>
			<td>12800-082</td>
			<td>Made from powder but add 5 times less water. Adjust pH to 7.3 and filter it using a 0.2 &#181;m filter.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium with glucose (DMEM) 1x</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM) 1x with Earle&#8217;s salts</td>
			<td>Sigma</td>
			<td>M4655</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids Solution, 100x</td>
			<td>Life Technologies</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3 (71.2 mg/ml)</td>
			<td></td>
			<td></td>
			<td>Prepared in house</td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Heat inactivated for 30 min, 56 &#176;C</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/ml)</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes buffer solution 1M</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (Pen Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Axis-Shield</td>
			<td>7801</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure 0.5 M EDTA</td>
			<td>Gibco</td>
			<td>15575</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Collagen PA treated (500 ml)</td>
			<td>Organogenesis</td>
			<td>200-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure col purified Bovine Collagen solution (100 ml)</td>
			<td>Advanced biomatrix</td>
			<td>5005-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular matrix protein, Fibronectin (1 mg)</td>
			<td>BD</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human monocytes/macrophages</td>
			<td></td>
			<td></td>
			<td>Isolated from human whole blood or buffy coats.</td>
		</tr>
		<tr>
			<td>PKH26 Red fluorescent cell linker</td>
			<td>Sigma</td>
			<td>MINI26</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis H37Rv expressing green fluorescent protein</td>
			<td></td>
			<td></td>
			<td>M. tuberculosis H37Rv wild type was transformed with the pFPV2 plasmid constitutively expressing GFP.</td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 medium&#160;</td>
			<td>Difco</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Middlebrook ADC Enrichment</td>
			<td>BBL</td>
			<td>211887</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin B sulfate (20 &#181;g/ml)</td>
			<td>Sigma</td>
			<td>B5264</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold anti=-fade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin -EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iMaris Scientific 3D/4D image processing software, version 7.6.8</td>
			<td>Bitplane AG</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a 3d human lung tissue model for functional studies on mycobacterium tuberculosis infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D lung tissue model for human TB can be effectively used to study the host-pathogen interactions in M. tuberculosis infection. The basic steps of this method, representative microscopic images of different steps and an overall microscopic structure of a tissue section are outlined in Figure 1. The model has several components of human lung tissue, including lung fibroblasts, bronchial epithelial cells and primary monocytes/macrophages embedded in the 3D tissue environment. Besides incorporating components of human lung tissue, the model resembles physiological conditions namely stratification of epithelia and mucus secretion.
An example for the use of the lung tissue model in monitoring a TB infection is presented in Figure 2. For visualizing the M. tuberculosis-immune cell migration and interaction, we introduced macrophages infected with M. tuberculosis that express GFP (green) together with the freshly isolated PKH26-labelled monocytes (red) into the tissue model (blue, DAPI stained for nuclei). On day 7 post addition of M. tuberculosis-infected cells to the tissue model, confocal microscopy reveals clustering of red monocytes at the site of infection (green) (Figure 2), which mimics the hallmark lesions of human TB 9.
A series of representative images for 3D visualization of M. tuberculosis-infected tissue model and quantification of cell clusters is shown in Figure 3. The 3D visualization gives the user flexibility to interact, examine and quantify several features in a 3D image. The spatial arrangement of green bacteria and red monocyte clusters can be seen from the apical, rotational and lateral view as illustrated in Figure 3B, which reveals clustering of monocytes at the site of M. tuberculosis. The clusters were not observed in uninfected tissues (Figure 3A). We quantified the size and number of monocyte cell clusters and found that the size (volume) of cell clusters is enhanced (p&#60;0.001), while the number of individual monocytes decreased (p&#60;0.01) in M. tuberculosis infected tissues as compared to uninfected tissue models (Figure 3C and 3D). This data validates our previous finding of early granuloma formation in M. tuberculosis infection observed in lung tissue models analyzed in 2D tissue sections 9.
Our data suggests that the tissue model provides a natural 3D habitat to investigate the complex host cell-M. tuberculosis communication network. We also found that 3D visualization and quantitative analysis are better tools for studying the features in the tissue model (Figure 3). Quantification of a cell cluster (granuloma for instance) often stretches to several cell layers and can be completely captured by a 3D quantitative analysis. Moreover, visualization of exact spatial and temporal features of individual cells or bacteria in the model allow live-imaging, migration and tracking studies in a designated laboratory.

<img alt="Figure 2" src="/files/ftp_upload/53084/53084fig2.jpg" /> 
Figure 2. Monocyte in the tissue model cluster around virulent M. tuberculosis.&#160;Representative confocal images of uninfected and M. tuberculosis infected tissue model is presented. Panels from green (M. tuberculosis-GFP), red (PKH26-labelled monocytes), blue (DAPI-stained nuclei) and merged channels show the recruitment of monocytes in the infected tissue as compared to uninfected tissues. Scale - 100 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/53084/53084fig2large.jpg" style="font-size: 14px; line-height: 28px;" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53084/53084fig3.jpg" /> 
Figure 3. 3D visualization and quantitative analysis of tissue model provide useful information.&#160;Representative images of 3D visualization of the entire tissue model (A) uninfected tissue, (B) infected with M. tuberculosis, through optical sectioning using Zeiss LSM700 confocal microscope and quantitative analysis by Imaris image processing software (version 7.6.8). These images were acquired at 20X magnification, 14 z-stacks covering a tissue thickness of 19.5 &#181;m with 1.5 &#181;m interval, allowing the visualization from apical, rotational horizontal, rotational vertical and lateral view (A and B). (C) Quantitative analysis of monocyte cell clusters reveal enhanced (p&#60;0.0001) size of early granuloma clusters after M. tuberculosis infection when compared to absence of infection. (D) Quantification of number of monocytes showed a decline (p&#60;0.01) in infected tissue as compared to uninfected tissue, reiterating more clusters in the infected tissue. Green &#8211;M. tuberculosis-GFP, Red &#8211; PKH26-labelled monocytes, Blue &#8211; cell nuclei, Scale &#8211; 100 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/53084/53084fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection at JoVE.com

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

a-3d-human-lung-tissue-model-for-functional-studies-on-mycobacterium

Research

JoVE Journal

Immunology and Infection

18.7K Views.  Linköping University. Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Video: A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...