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

Abstract

Source: Introini, A., et al., Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture. J. Vis. Exp. (2018).

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 ....

Materials

List of materials used in this article

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