A Human Cervical Mucosa Infection Model to Study HIV Pathogenesis in Ex Vivo Conditions

Place cervical uterine mucosa in a petri dish. Separate the ectocervix and endocervix mucosa from the submucosa. Slice the mucosal layer into smaller explants and transfer them into a conical tube.

Add the human immunodeficiency virus type 1, or HIV-1 into the explant tube. The HIV-1 virus particles approach the exposed mucosal layer and bind to the CD4 receptor and the CXCR4 co-receptor on the host cell.

Upon binding, the viral envelope fuses with the host cell membrane, releasing a viral capsid containing viral RNA and reverse transcriptase enzyme into the cytoplasm. Inside the cytoplasm, the reverse transcriptase converts the viral RNA into complementary DNA, or cDNA.

The cDNA moves into the host cell nucleus, where integrase incorporates viral DNA into the host genome. The viral DNA directs the synthesis of new viral RNA and proteins which assemble into new virus particles. These particles bud off from the host cell, acquiring a lipid envelope.

Rinse the explants with a suitable buffer to remove any unbound virus. Finally, place the explants on a gelatin sponge in a multiwell plate with a suitable medium and incubate.

The air-liquid interface maximizes oxygen exposure and nutrient accessibility through the sponge capillaries, enhancing model viability for downstream analysis.

Use the lid of the Petri dish as a cutting surface to dissect the tissue. Holding the tissue gently with forceps, separate the mucosa of the ectocervix and/or endocervix from the underneath stromal and submucosa with a scalpel and blade to obtain strips of mucosa. Cut the mucosa into 2-millimeter-wide strips. Remove and discard as much submucosa as possible, leaving only a 2-millimeter-thick layer of tissue. Then, cut the strips into 2-millimeter-thick blocks.

Transfer tissue explants into a new 100-millimeter Petri dish containing 10 to 20 milliliters of CMT to avoid tissue desiccation while proceeding with the dissection. At the end of the dissection, swirl the plate to randomize the explant distribution. With sterile forceps, transfer the explants into sterile 1.5-milliliter conical tubes. Remove any medium in the tube using a pipette.

For optimal results, explants of cervix should be processed and infected as soon as possible after surgery, ideally the day of the surgery. Alternatively, tissues can be stored submerged in CMT at 4 degrees overnight and infected immediately after dissection.

Thaw the HIV-1 preparation in a water bath at 37 degrees Celsius. Once thawed, transfer the virus into the tubes containing the explants. Transfer the tubes into a thermo-shaker and incubate for two hours at 37 degrees Celsius, rocking at 300 RPM. Gently invert the tubes a few times every 30 to 60 minutes.

Meanwhile, fill a six-well plate with 3 to 4 milliliters per well of sterile phosphate-buffered saline. At the end of the incubation with HIV-1, use a pipette to discard all virus preparation in the tubes containing the explants into a container with disinfectant solution. Then, use forceps and a pipette to transfer the explants into the six-well plate containing PBS.

After allowing the explants to rest in PBS for one minute at room temperature, wash them by gently pipetting the PBS up and down into the well two to three times. Then, discard the PBS into a container with disinfectant solution using a pipette. Add 3 to 4 milliliters of new PBS to each well. Repeat two more times for a total of three washes. Discard the PBS just before transferring the explants to the sponges to avoid tissue desiccation.

Now, use forceps to transfer five to eight explants on top of each gelatin sponge into a 12-well plate. Return the plate to the incubator.