This method can help answer key questions in the HIV field, such as the identification of the determinants of virus transmission and pathogenesis within two of the most effected organs by the infection. In fact, the key advanced driving disease progression take place in tissues. The features and complexity of infection are poorly reflected by cells in circulation or isolated and infected in vitro.
The main advantage of the ex vivo tissue culture to study HIV pathogenesis is that it retains its original spacial and functional relationships between cells although for two to three weeks. To begin, fill a 100 millmeter by 20 millimeter Petri dish with 20 to 30 milliliters of CMT. Transfer gelatin sponges into the Petri dish using sterile forceps.
Wet the sponges on both sides and allow the sponges to soak up medium for one minute. Press the sponges against the bottom of the Petri dish for about two minutes using a sterile spatula. Use sterile scissors to cut the rehydrated gelatin sponges into pieces of equal size of about one square centimeter.
Then use forceps to transfer one sponge piece into each well of a culture plate. Add three to four milliliters of CMT into each well of a 6-well plate for the tonsils and one milliliter per well into a 12-well plate for the cervix. Place the plates in the incubator, set at 37 degrees Celsius, 5%CO2, and greater than or equal to 90%humidity until the tissue explants are ready to be loaded on the sponges.
Transfer the tonsils from the transportation container into a 100 millimeter Petri dish containing 10 to 20 milliliters of CMT using sterile forceps. Use the lid of the Petri dish as a cutting surface to dissect the tissue. Holding the tissue gently with forceps, cut each tonsil into different, big pieces using a sterile scalpel and blade.
Use forceps and a scalpel to remove cauterized, bloody, and fibroid tissue, as well as any parts containing tonsilloliths or greenish-brown color. Cut each tissue piece into slices of about two millimeters in thickness. Remove any unwanted part as in the previous step.
Then cut the tissue slices into two-millimeter-thick strips. Finally, cut the tissue strips into two-millimeter-thick blocks. Transfer the 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. Place nine tissue explants on top of each gelatin sponge in a 6-well plate using forceps, allowing space between them. Return the plates to the incubator.
Typically, explants are cultured overnight. Inoculation with HIV is performed the next day. This is to make sure that no bacterial or fungal contamination develops in the culture.
Also, cells tend to regress tissue explants immediately after dissection. Aspirate the medium in the 6-well plate with a pipette, and discard it in a container with disinfectant solution. Tilt the plate and gently push the gelatin sponges to the upper part of the well to allow the medium to gather at the bottom.
Aspirate and discard the medium. Then add three to four milliliters of CMT to each well. Now thaw the HIV-1 virus preparation in the water bath at 37 degrees Celsius.
Pipette the virus inoculum directly on top of each of nine explants on a gelatin sponge. Return the plate to the incubator. 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 two-millimeter-wide strips. Remove and discard as much submucosa as possible, leaving only a two-millimeter-thick layer of tissue.
Then cut the strips into two-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 optimum 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 thermoshaker, 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 6-well plate with three to four 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 6-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 three to four 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.
Collect a sample of culture medium with a pipette, and transfer it into sterile 1.5 or two milliliter screw cap tubes for storage at minus 80 degrees Celsius. Harvest the tissue explants with sterile forceps, and transfer the explants into sterile 1.5 or two milliliter screw cap tubes. Process or store the tissue samples as required by the experiment.
Aspirate the residual culture medium in the well with a pipette, and discard it into a container with disinfectant solution. Tilt the plate and gently push the gelatin sponges to the upper part of the well to allow the medium to gather at the bottom, and then aspirate and discard it. Now add three to four milliliters of culture medium to each well.
Using sterile forceps, return any tissue explants that dropped into the medium to the gelatin sponge before returning the plate to the incubator. At the end of the culture, dispose of all biological waste in apposite containers according to the institute's regulations on handling hazardous biologicals as well as the specific risk assessment. Inter-donor variability can affect HIV-1 replication as measured by the concentration of the HIV core protein p24 gag in tissue explant culture supernatant, as demonstrated for cervical tissue from three different donors infected with the same virus inoculum.
For the first donor, the accumulation of p24 gag in culture medium of explants infected with HIV-1 BaL alone clearly indicates a product infection. The antiretroviral drug 3TC that completely abrogates HIV replication was used as a negative control. The amount of p24 gag measured in culture medium of 3TC-treated explants reveals the fraction of virus inoculum aspecifically absorbed and released by tissue explants during culture.
In tissue explants from the second donor, HIV-1 bowel infection results in a steady decrease in the concentration of p24 gag over time, indicating an abort of infection or undetectable by p24 gag quantification. This is confirmed by 3TC-treated control explants that show a similar HIV replication kinetic. Infection of tissue explants from the third donor yields little amount of virus.
In this case, the inclusion of the 3TC treatment as a negative control is necessary to ascertain the presence of infection with de novo virus production. Following this procedure, analytical methods like flow cytometry or immunostaining can be performed at different time points to answer questions about phenotype, proportion, or tissue location of the cell population of interest. When designing the experiment, it is important to estimate the impact of inter-donor variability on the experimental readout of choice.
Inter-donor variability can be partially controlled by setting stringent clinical and, possibly, experimental inclusion and exclusion criteria. Ex vivo human tissue cultures can be used to study the pathogenesis of microbes other than HIV, such as vaccinia virus or herpes viruses. They have also been used as a preclinical platform for antimicrobial toxicity and ethicacy studies.
Please be aware that all human specimens, even from healthy individuals, can harbor infectious agents. Appropriate education, training, and protections are required to handle human tissues to ensure the safety of you and your coworkers.
