Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

The protocol for collection of human tissues may require ethical approval by the local competent authorities. In case of indicated surgeries such as tonsillectomy and hysterectomy, the specimens are not collected specifically for research purpose and the study is not considered human subjects research (National Institutes of Health. Research on Human Subjects. https://humansubjects.nih.gov/walkthrough-investigator#tabpanel11). However, obtaining tissue donors' personal and medical data (e.g., sex, age, current drug use, history of infections, etc.) that may help interpret experimental results, poses concerns about informed consent and privacy that must be addressed in the protocol for tissue collection.

Caution: The entire procedure should be carried out in a biological safety cabinet. All human specimens, including those from 'healthy' individuals, may harbor infectious agents. The operator should receive training to work with blood-borne pathogens to safely handle tissue specimens, even if experiments do not involve the use of HIV. A risk assessment of the procedure of tissue dissection and infection with HIV should be carried out by trained staff to ensure the safety of the operator and coworkers. The use of infectious HIV requires dedicated biosafety level 2 or 3 environments depending on the country and institute-specific regulations on hazardous biologicals and blood-borne pathogens.

1. Preparation of Gelatin Sponges

NOTE: Although it is not strictly necessary to prepare gelatin sponges before tissue dissection, it is more convenient to follow the order outlined here, especially when handling a large amount of tissue and/or from many donors.

1. Prepare culture medium (CM) and supplement it with antibiotic solution (Tircacillin-clavulanate mix) before use to make CMT (Table of Materials). Discard any leftover antibiotic solution.
   NOTE: Ticarcillin and clavulanate in CMT are poorly stable. If needed, re-supplement CMT with antibiotic solution after about 24 h from preparation. It is not required to add antibiotic solution to explant medium every 24 h during culture.
2. Fill a 100 mm x 20 mm Petri dish with about 20–30 mL of CMT.
   NOTE: This setting is optimal to prepare enough gelatin sponges for two 6-well plates or one 12-well plate at the time. If many plates are required, use a wider Petri dish and larger CMT volume to speed up the preparation.
3. Transfer the gelatin sponge(s) into the Petri dish using sterile forceps. Wet the sponges on both sides and allow the sponges to soak up medium for 1 min. Press the sponges against the bottom of the Petri dish for about 2 min using a sterile spatula.
   NOTE: This step is of crucial importance because the presence of air bubbles in the sponge will block the capillaries through which culture medium nutrients reach the tissue. Gelatin sponges are extremely brittle when dehydrated. Gently push down on the sponge with the spatula, especially at the beginning, to avoid breaking the sponge.
4. Use sterile scissors to cut the rehydrated gelatin sponges into pieces of equal size of about 1 cm². Transfer 1 sponge piece using forceps into each well of a culture plate. Add 3–4 mL of CMT into each well of a 6-well plate (tonsils) and 1 mL per well into a 12-well plate (cervix). Place the plates in the incubator, set at 37 °C, 5% CO₂, ≥ 90% humidity, until the tissue explants are ready to be loaded on the sponges.
   Note: The volume of CMT to add into the plate should be adjusted to the thickness of the sponge to keep the explants at the liquid-air interface.

2. Dissection of Human Tonsillar Tissue

NOTE: Tonsils should be processed as soon as possible after surgery, ideally the same day of surgery. Alternatively, specimens can be left overnight submerged in CMT at 4 °C.

1. Allow the CM enough time to reach room temperature (RT) or put it in a water bath pre-warmed at 37 °C. Supplement the CM with antibiotic solution (CMT) before use. Discard any antibiotic solution left over. Pour 70% ethanol solution into a clean container to soak forceps and scalpel whenever needed during the dissection procedure. Clean the tools and change the scalpel blade between dissections of specimens from different donors.
2. Transfer the tonsils from the transportation container into a 100 mm-Petri dish containing 10-20 mL of CMT using sterile forceps.
   NOTE: Specimens with widespread areas of cauterized, bloody, or necrotic tissue should be discarded.
3. Use the lid of the Petri dish as a cutting surface to dissect tissue. Holding the tissue gently with forceps, cut each tonsil into several big pieces using sterile scalpel and blade.
   Caution: Handling sharp tools to dissect human specimens increases the risk of injury and contamination. The operator should consider wearing metal, mesh, cut-resistant gloves as additional protection.
4. Remove cauterized, bloody, and fibroid tissue, and any parts containing tonsilloliths (calcified material) and/or with green-brownish color using forceps and scalpel.
   NOTE: While working on one piece of tissue, it is important to keep the rest of the tissue submerged in medium to avoid tissue desiccation.
5. Cut each tissue piece into slices of about 2 mm in thickness. Remove any unwanted part as in the previous step. Cut the tissue slices into 2 mm-thick strips. Cut the tissue strips into 2 mm-thick blocks. This should result in tissue explants of roughly 8 mm³.
6. Transfer the explants into a new 100 mm Petri dish containing 10–20 mL of CMT to avoid tissue desiccation while proceeding with the dissection. At the end of dissection, swirl the plate to randomize the explant distribution. Place 9 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.
   NOTE: Typically, explants are cultured overnight and inoculation of cultures with HIV-1 is performed the next day. This is to make sure that no bacterial or fungal contamination develops in the culture. In addition, cells tend to egress tonsillar tissue explants after dissection. This process is largely completed within the first 24 h of culture¹.
7. Dispose of all biological waste in apposite containers according to the institute's regulations on handling hazardous biologicals and the specific risk assessment.

3. Infection of Tonsillar Tissue Explants with HIV-1

NOTE: To reduce result variability between independent experiments, the same virus preparation should be used for an entire study. Virus can be propagated in exogenously activated peripheral blood mononuclear cells and then the culture supernatant can be aliquoted for storage at -80 °C to avoid repeated freezing and thawing (Table of Materials).

1. Put the CM in a water bath pre-warmed at 37 °C. Supplement the CM with antibiotic solution (CMT) before use. Discard any leftover antibiotic solution.
2. Aspirate the medium in the 6-well plate(s) 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, and aspirate and discard it. Add 3-4 mL of CMT to each well.
3. Thaw the HIV-1 virus preparation in the water bath at 37 °C. If necessary, dilute the virus stock with CM (Table 1).
   NOTE: The virus stock should be diluted so that the selected inoculum is contained in a volume of 5–8 µL (Table 1). For an efficient infection, it is critical to use the minimum time required between thawing the HIV-1 preparation and infecting tissue explants.
4. Pipette the virus inoculum directly on top of each of the 9 explants on a gelatin sponge. Return the plate(s) to the incubator as soon as the infection is completed. Discard any residual virus preparation. Continue to Section 5.
   NOTE: To accurately deliver the virus inoculum the operator should consider using a reverse pipetting technique and changing the tip for every well. This involves pushing the pipette piston down to the purge position (the second stop) to draw the virus inoculum up, and pushing to the first stop to deliver the inoculum, which helps minimizing bubble formation and the error associated with repeated sampling of small volumes, i.e., 5–8 µL.

4. Dissection and Infection of Uterine Cervical Mucosa

NOTE: For optimal results, explants of cervical mucosae should be processed and infected as soon as possible after surgery, ideally the same day of surgery. Alternatively, specimens can be stored submerged in CMT at 4 °C overnight, and infected immediately after dissection.

1. Allow the CM enough time to reach RT or put it in a water bath pre-warmed at 37 °C. Supplement the CM with antibiotic solution (CMT) before use. Discard any leftover antibiotic solution. Pour 70% ethanol solution into a clean container to soak the forceps and scalpel whenever needed during the dissection procedure. Clean the tools and change the scalpel blade between dissections of specimens from multiple donors.
2. Using sterile forceps, transfer the sample from the transportation container into a 100 mm Petri dish containing 10–20 mL of CMT.
3. 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 muscular tissue (submucosa) with a scalpel and blade to obtain strips of mucosa.
   NOTE: While working on one piece of tissue, it is important to keep the rest of the tissue submerged in medium to avoid tissue desiccation.
4. Cut the mucosa into 2 mm-wide strips, remove and discard as much submucosa as possible, leaving only a 2 mm-thick layer of tissue. Cut the strips into 2 mm-thick blocks. This should result in tissue explants of roughly 8 mm³.
   Caution: Handling sharp tools to dissect human specimens increases the risk of injury and contamination. The operator should consider wearing metal, mesh, cut-resistant gloves as additional protection.
5. Transfer tissue explants into a new 100 mm Petri dish containing 10–20 mL of CMT to avoid tissue desiccation while proceeding with the dissection. At the end of dissection, swirl the plate to randomize the explant distribution.
6. With sterile forceps, transfer the explants into sterile 1.5-mL conical tube(s) (maximum 16 explants per tube). Remove any medium in the tube using a pipette.
7. Thaw the HIV-1 preparation in a water bath at 37 °C. If necessary, dilute the virus stock with CM. Transfer the virus into the tube(s) containing the explants. Discard any residual virus preparation.
   NOTE: The virus stock should be diluted so that the selected inoculum is contained in a volume of minimum 0.5 mL (Table 1). To reduce result variability between independent experiments, the same virus preparation should be used for an entire study (Table of Materials).
8. Transfer the tubes into a thermo-shaker and incubate for 2 h at 37 °C rocking at 300 rpm. Gently invert the tubes a few times every 30–60 min.
   Note: For an efficient infection, it is critical to use the minimum time required between thawing the HIV-1 preparation and transferring the tubes to 37 °C.
9. Fill a 6-well plate with 3–4 mL per well of sterile phosphate buffer saline (PBS). At the of the incubation with HIV-1, discard all virus preparation in the tube(s) into a container with disinfectant solution using a pipette. Transfer the explants into the 6-well plate containing PBS using forceps.
10. Allow the explants to rest in PBS for 1 min at RT, and then wash them by gently pipetting up-and-down the PBS into the well 2-3 times. Discard the PBS into a container with disinfectant solution using a pipette. Add 3–4 mL 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.
    NOTE: Explants of cervical mucosa, and in particular endocervix, release great amounts of mucus during infection. It is critical to wash and remove as much mucus as possible because unspecific retention on explants and subsequent release of virus into culture supernatant can mask virus replication.
11. Using forceps, transfer 5-8 explants on top of each gelatin sponge in a 12-well plate.Return the plate to the incubator.
12. Dispose of all biological waste in apposite containers according to the institute's regulations on handling hazardous biologicals and the specific risk assessment.

5. Histoculture

1. Change the CM every 3 days starting from the time of infection until day 12–15 post-infection. The CM and/or explants can be sampled at regular intervals throughout the culture time to assess HIV-1 replication, among other parameters.
2. Put the CM in a water bath pre-warmed at 37 °C.
   NOTE: It is not required to supplement the CM with antibiotic solution at this stage.
3. Collect a sample of CM with a pipette and transfer it into sterile 1.5 or 2 mL screw cap tube(s) for storage at -80 °C.
   NOTE: The CM from replicate wells are pooled at collection.
4. Harvest the tissue explants with sterile forceps, remove the excess medium on explants by placing them on a piece of paper (optional), and transfer explants into sterile 1.5 or 2 mL screw cap tube(s). Process or store the tissue samples as required by the experiment.
   NOTE: For flow cytometry, explants are immediately digested with an enzymatic cocktail to obtain a single-cell suspension (for more details see references 1, 9, 16, and 17). For RNA extraction, explants are kept in a RNA stabilization solution at 4 °C overnight before storing them at -80 °C. For DNA extraction or in situ staining (cervix), explants can be snap-frozen in liquid nitrogen or dry ice/ethanol slurry and stored at -80 °C. Alternatively, tonsillar explants can be fixed by submersion in a solution of PBS and 4% paraformaldehyde for in situ staining.
5. Aspirate the residual CM 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.
6. Add 3–4 mL of CM to each well. Using sterile forceps, return any tissue explants that dropped into the medium to the gelatin sponge. Return the plate to the incubator.
7. At the end of culture, dispose of all biological waste in apposite containers according to the institute's regulations on handling hazardous biologicals and the specific risk assessment.