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

The overall goal of this procedure is to construct an in vitro lung tissue model for studies of M tuberculosis infection. This is accomplished by first layering a fibroblast collagen matrix onto the filter of a cell culture plate insert. In the second step, infected immune and lung epithelial cells are seated onto the fibroblast collagen layer.

The resulting 3D tissue model is then air exposed to facilitate the mucus secretion and stratification of the epithelium. In the final step, the tissue is harvested and mounted. Ultimately, immunofluorescence microscopy is used to analyze the formation of M tuberculosis induced early granuloma.

The main advantage of this technique or existing methods, such as in vitro mono layer of cells, is that the cells in this system are present in a 3D microenvironment, allowing the formation of granulomas upon infection with virulent microbacteria resembling human TB To prepare collagen embedded fibroblasts begin by treating lung fibroblast cells with tripsin for 10 minutes at 37 degrees Celsius when the cells have detached, stop the reaction with eight milliliters of complete dmem and then spin down the cells. Resuspend the pellets at 230, 000 cells per milliliter in fresh dmm and place the cells on ice. Next, add one insert per well into a six.

Well plate and dispense one milliliter of freshly prepared a cellular layer mixture into each insert, making sure that the mixture covers the entire insert without any air bubbles. Then place the plate in a 37 degrees Celsius incubator for 30 minutes. At the end of the incubation mix two milliliters of collagen with 615 microliters of premix in a sterile conical tube on ice to neutralize the pH of the collagen per insert.

Then add 58 microliters of complete dmem and 327 microliters of lung fibroblasts per insert to the collagen mixture. Pipetting up and down to distribute the cells evenly throughout the mixture. Now quickly add three milliliters of the cellular layer down the side of each insert onto each acellular collagen layer.

Taking care to avoid air bubbles and return the plate to the cell culture incubator for another two hours. Following the polymerization of the collagen, add two milliliters of complete dmem to the bottom of each well, and return the plate to the incubator for another 24 hours. The next day, use a pair of clean forceps to carefully lift the inserts one at a time and aspirate the culture medium from the bottom of each.

Well then add two milliliters of complete DMM to the bottom of each well, and two milliliters of complete DMM to each insert. Changing both volumes of medium every second day for five to seven days. To prepare the infected macrophages, begin by incubating a macrophage culture for four hours with virulent M tuberculosis at a multiplicity of infection of 10.

At the end of the incubation, wash the cells three times with PBS to remove the extracellular bacteria and treat the macrophages with two millimolar, A DTA for 10 minutes at 37 degrees Celsius. When the cells have detached resus, suspend them in antibiotic free complete dmm. Then aspirate all of the medium from the fibroblast collagen matrix plate and add 1.5 milliliters of fresh antibiotic free complete DMM to the outer chambers.

Next, mix fluorescently labeled monocytes with the infected macrophages at a five to one ratio in 50 microliters of the antibiotic-free complete DM EM per insert. Then add 50 microliters of the monocyte macrophage mixture to the inside of each insert and return the plate to the incubator. After one hour, slowly add two milliliters of culture, medium down the walls of the inserts to avoid dislodging the cells and incubate the cultures for another 24 hours.

To seed the cultures with lung epithelial cells, add 4 million epithelial cells per milliliter in 50 microliters of complete dmm on top of the immune cell fibroblast collagen matrices. Incubate the cells for two minutes at room temperature, followed by one hour at 37 degrees Celsius. Then gently add two milliliters of antibiotic-free, complete DMM to the inserts as just demonstrated, and culture the cells for another three days.

Five days after seeding the infected macrophages aspirate all of the culture medium from the plate and add 1.8 milliliters of antibiotic free, complete DMM to the outer chambers. Then air exposed the 3D tissue models in the incubator for two days without adding medium to the insert on day seven. Post seeding.

Remove the medium entirely from the plate and mix the tissue models with 4%paraldehyde for 30 minutes at room temperature in the dark. Then using a scalpel, separate the membranes from the well inserts. Next, use a clean scalpel to carefully remove the sides of the tissue models and slice each tissue into four approximately equal sized square pieces.

Transfer the pieces onto super frosts glass slides and let them dry for five minutes. Then using fluorescent mounting, medium containing anti fade and dpi, mount the tissues and cover them with cover slips. Leave the slides at room temperature in the dark until they are dry, and then seal the edges of the cover slip with nail polish.

When the lacquer is dried, immerse the slides in 70%ethanol for a transfer out of the BSL three facility. To visualize the tissues, acquire 3D images of five to 10 different fields covering the entire piece of tissue at a 512 by 512 resolution with Zack covering at a minimum of 20 micron thickness with a one to 1.5 micron separation between the stacks on a fluorescent confocal microscope for 3D quantification of the cell clusters. Open the 3D image processing software and load the image of interest.

Next, using the display adjustment tool, adjust each channel for image, contrast, brightness, and blend opacity to optimize the volume rendering and to minimize the noise interference. Then to visualize the tissue surface, select the red monocyte channel and set the appropriate thresholds using the filters to restrict the selection of the red monocytes or to exclude the background as necessary. Finally, export the data into the appropriate spreadsheet program for further downstream analysis.

On day seven, post M tuberculosis infected cell seeding confocal microscopy reveals a clustering of red monocytes at the site of infection, as noted by the presence of the green bacteria mimicking the lesions of human tb known as granuloma 3D. Visualization provides the flexibility of interacting, examining, and quantifying several features. A 3D image, for example, in these images depicting a cluster of monocytes at the site of em tuberculosis.

The spatial arrangement of green bacteria and red monocyte clusters can be observed from the apical, rotational and lateral views. As expected, these clusters were not observed in the uninfected tissues. Quantification of the size and number of monocyte, cell clusters and M tuberculosis infected tissues revealed that the size of the cell clusters is enhanced while the number of individual monocytes is decreased compared to the uninfected tissue models.

Potential users of this lung tissue model include a study of role of different immune cells and tissue immunity in tb, the study of mycobacteria phenotypes and testing of drug candidates that kill mycobacteria within the granuloma.