The overall goal of this protocol is to predict drug binding to caseum from tuberculous lesions. This method can help determine how effectively antimicrobials penetrate the caseus core of necrotic lesions without the use of animal models and expensive imaging techniques. Although this method can provide insight into tuberculosis direct discovery, it can also be applied to other diseases that are characterized by the formation of lesions or abscesses where a drug penetration is limited.
Visual demonstration of this method is critical as consistency in the performance of the protocol will ensure reproducibility of data between experiments. Today, I will demonstrate the cell culture procedure and preparation of the surrogate matrix. Hsin-pin, a research associate from our lab, will demonstrate the right essay.
Grow THP-1 monocytes in T-175 cell culture flasks containing 80 mL of RPMI 1640 medium, with 2 millimolar of l-glutamine and 10 percent fetal bovine serum. Incubate the flasks at 37 degrees Celsius with five percent carbon dioxide for three to four days. Once the cells are about 70 percent confluent, split them into two 50 mL conical tubes, and centrifuge them at 150 times g for five minutes.
After the spin discard the supernatant and suspend the palate in 10 mL of RPMI 1640 medium. Pipe at five microliters of this culture into a 1.5 mL tube containing 45 microliters of trypan blue. Mix thoroughly by pipetting.
Tranfer 10 microliters to a hemocytometer. And using a light microscope at 10x magnification, count the number of viable THP-1 monocytes. After determining the viable cells per milliliter of the culture, dilute it with RPMI medium to a final density of 1.25 times 10 to the 6 cells per milliliter.
Add 40 microliters of 100 micromolar PMA to the tube. And then transfer the culture to a 140 times 20 millimeter cell culture plate. Allow the cells to adhere overnight in the incubator.
Next, dilute pure iliac acid in ethanol to a concentration of 0.1 molar. Here, 31.7 microliters of iliac acid is dissolved in 968.3 microliters of ethanol. Dilute the solution in fresh, pre-warmed RPMI medium to a concentration of 10 millimolar.
Dilute the iliac acid suspension to a final working concentration 0.4 millimolar in RPMI medium prewarmed to 37 degrees Celsius. The dilution of iliac acid in prewarmed RPMI media is critical to ensure sufficient dissolution of the fatty acid in an aqueous solution. Using a pipette, remove the existing medium and non-adherent cells from the cell culture plate containing THP-1 macrophages, and gently add 40 mL of 0.1 millimolar iliac acid.
Incubate at 37 degrees Celsius in the incubator overnight. The next day, use a light microscope at 40x magnification to visually confirm the presence of numerous lipid body inclusions in THP macrophage. Lipid bodies appear as small, clear, spherical structures in the cytoplasm.
Remove all the RPMI medium from the cell culture plate and gently wash the adherent cells twice with PBS, using a 50 mL serological pipette. Add 40 mL of 5 millimolar EDTA and PBS to the plate. Incubate for 15 minutes at 37 degrees Celsius.
Next, to detach the foamy macrophages, use a 10 mL serological pipette and repeatedly pipette up and down over the surface of the whole plate. Transer the cell suspension to a 50 mL conical tube. And centrifuge it at 150 times g for five minutes.
After the spin, resuspend the cell palette in 10 mL of PBS and transfer it to a pre-weighed 15 mL conical tube. Centrifuge again at 150 times g for five minutes. Then carefully aspirate the supernatant using a serological pipette and discard it.
Subject the foamy macrophage palettes to three freestyle cycles. Alternate between dry ice and 37 degree Celsius incubation on a thermal block to lyse the cells. Then incubate them at 75 degrees Celsius for 20 to 30 minutes to denature the proteins in the matrix.
Store the palettes at negative 20 degrees Celsius until they are needed. The rapid equilibrium devices, or RED essay shown in this section of the video may be carried out using either the surrogate matrix we just made or casing from TB infected rabbit lesions. Here, we'll show the essay as it's performed on the surrogate matrix.
Prepare 10 millimolar stock solutions of all the test compounds. Here, moxyfloxacin is prepared in DMSO. Dilute to 500 micromolar working solutions in DMSO prior to each essay.
Way the tube containing excised caseum or surrogate palette. Subtract the weight of the empty tube to derive the weight of the caseum or surrogate palette alone. Add two to three metal beads per tube and then add PBS at a ratio of one to nine W to V.Using a vortex, disrupt the caseum or the surrogate matrix for one minute.
Add 6.5 microliters of the prepared test compound to 643.5 microliters of the homogenate. To achieve the final concentration of five micromolar and vortex. Next, place the red inserts into the base plate.
Prepare the inserts for each test, each compound in triplicate. Add 200 microliters of the drug-spiked matrix into the red donor chamber of each red insert. Then add 350 microliters of PBS into each receiver chamber.
Place an adhesive seal on the plate and incubate it 37 degrees Celsius on the thermal mixer at 200 rotations per minute for four hours. After the incubation, gently mix the contents of the donor and receiver chambers by pipetting up and down two to three times. Transfer 20 microliters of homogenate from each of the donor chambers to 20 microliters of clean PBS in a 1.5 mL tube.
Similarly, transfer 20 microliter aliquots of PBS samples from the receiver chambers to 20 microliters of clean homogenate. Matrix matching eliminates the need for two separate color vision curves in homogenate and PBS to be made for quantitative analysis. Contents of the donor chamber make sediment over time.
Gently mix the contents by pipetting before removing aliquots. Add 160 microliters of one-to-one methanol to acetonitrile, containing 500 nanograms per milliliter of diclofenac or 10 nanograms per milliliter of verapamil to each tube as an internal standard. Vortex to precipitate the proteins.
Centrifuge at 10000 x g for five minutes to sediment the precipitate and then transfer the supernatants into 96-well deep well plates for analysis by liquid chromatography mass spectrometry, LCMS. Build calibration curves from one to 1000 nanomolars for each test compound by making dilutions of the compound stock solution while keeping the same matrix composition as the samples. Quantify the concentration of test compound in samples from the donor and receiver chambers using an LCMS method.
Calculate the unbound fraction, or fu, of the drug and diluted matrix, using equation one shown here. Then, calculate the fu in undiluted matrix using equation two, where D is equal to a dilution factor of 10. Finally, check the recovery or mass balance of each compound using equation three shown here.
To identify compounds with stability, metabolism or nonspecific binding issues. Note that recovery typically falls between 70 percent and 130 percent. The protocol described in this video has been used to test hundreds of tuberculosis drug development compounds for their predicted efficiency at penetrating caseum.
The dialysis membrane of the red inserts allows for unbound small molecules to diffuse from the donor well to the receiver well, finally achieving an equilibrium between both compartments. Small molecules that are bound to macromolecules such as proteins or lipids are trapped in the donor well. Quantitative analysis of the samples from both compartments allows for the calculation of the unbound fraction, fu, of each small molecule in the caseum or surrogate matrix, as shown here.
The higher the fu, the more extensively the drug diffuses into caseum. Matrix assisted laser desorption ionization, or MALDI mass spectrometry imaging, was performed for six drugs. Ion maps of representative lung legions are shown here where the signal intensity is indicated by the scale bar to the left.
Hematoxylin and ESN or HNE standing of adjacent sections is shown below them. The black-white contour lines highlight the caseous center of each lesion. These data are consistent with the hypothesis that is drug molecules diffuse into the outer room of caseum from its interface, with cellular layers of the lesion.
Binding to caseous macromolecules sequesters the drug, preventing it from further diffusing into the caseous core. As shown here, the matrix prepared from THP-1-derived foamy macrophages is an effective surrogate to true caseum. Binding in both matrices strongly correlates with each other.
Following this procedure, several compounds can be tested simultaneously to save time and resources. We call this cassette testing. Since its development, this essay has been used by multiple lead optimization programs for tuberculosis drug discovery.
Don't forget that working with microbacteria and tuberculosis-infected tissue specimens can be extremely hazardous and a validated tissue sterilization protocol, using gamma radiation should be used prior to handling infected casing samples in a BSL2 lab. Thanks for watching and good luck with your experiments.
