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

Overview

This video demonstrates ex vivo infection of human uterine mucosal tissue with human immunodeficiency virus type 1, HIV-1, and their maintenance on top of gelatin sponges at the air-liquid interface. This method allows monitoring of HIV-1 replication and pathogenesis within human tissues.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. 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 on the same day of surgery. Alternatively, specimens can be stored submerged in CMT (Culture Media supplemented with antibiotics) 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 a medium to avoid tissue desiccation.
4. Cut the mucosa into 2 mm-wide strips, and 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, and 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 the dissection, swirl the plate to randomize the explant distribution.
6. With sterile forceps, transfer the explants into the 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 a minimum of 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 a 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 a 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 opposite containers according to the institute's regulations on handling hazardous biologicals and the specific risk assessment.

Materials

Name	Company	Catalog Number	Comments
Gelfoam 12-7 mm Absorbable gelatin sponge	Pfizer	NDC: 0009-0315-08	Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue.
SPONGOSTAN Standard 70 × 50 × 10 mm Absorbable hemostatic gelatin sponge	Ferrosan Medical Devices	MS0002	Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue.
RPMI 1640 with phenol red and glutamine	ThermoFisher Scientific	21875034	To RPMI1640 add MEM nonessential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).
Sterile phosphate-buffered saline (PBS) pH 7.4 w/o calcium, magnesium and phenol red			To use for storage and transportation of surgical specimens. PBS can be replaced with another isotonic solution with physiologic pH. Culture medium is best for overnight storage.
Sterile cell culture-grade water
70% ethanol solution
Disinfectant solution for biological waste disposal
Sterile metal forceps or tweezers
Sterile metal scissors
Sterile flat weighing metal spatula
Sterile scalpels and blades
6-well cell culture plates
Sterile Petri dish, 100 mm × 20 mm
Cell-free HIV-1 viral preparation HIV-1BaL	NIH AIDS Reagent Program	510	The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 °C. Avoid repeated freezing and thawing.
Cell-free HIV-1 viral preparation HIV-1LAI.04	NIH AIDS Reagent Program	2522	The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 °C. Avoid repeated freezing and thawing.
Sterile 1.5 and 2mL screw cap tubes
Water bath set at 37 °C
Biological safety cabinet
Dry bath shaker with heating block for 1.5 mL tubes, set at 37 °C, 300 rpm