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Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface

View Article: PubMed Central - PubMed

ABSTRACT

The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions.

No MeSH data available.


Culture design comparison. (A) liquid-liquid interface (LLI); (B) air-liquid interface (ALI).
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viruses-08-00241-f001: Culture design comparison. (A) liquid-liquid interface (LLI); (B) air-liquid interface (ALI).

Mentions: To generate the Vk2/E6E7 (Vk2) culture, 60,000 cells were seeded on 0.4 µm pore-sized transwell polystyrene inserts (BD Falcon, Mississauga, ON, Canada), and keratinocyte serum-free medium (Life Technologies, Carlsbad, CA, USA) supplemented with 0.1 ng/mL human recombinant epidermal growth factor (Life Technologies), 0.05 mg/mL of bovine pituitary extract (Life Technologies), CaCl2, and 100 units/mL penicillin/streptomycin (Sigma Aldrich, Oakville, ON, Canada) were added to the apical and basolateral sides of the culture, as described in [41]. LLI culture was generated by keeping the media on both the apical (300 µL) and basolateral sides (500 µL) (Figure 1A). For ALI culture, media from the apical side was removed after 24 h of seeding the cells and media was only present in the basolateral side (1000 µL) (Figure 1B). Media was replenished every 24 h for 10 days of culture.


Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface
Culture design comparison. (A) liquid-liquid interface (LLI); (B) air-liquid interface (ALI).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035955&req=5

viruses-08-00241-f001: Culture design comparison. (A) liquid-liquid interface (LLI); (B) air-liquid interface (ALI).
Mentions: To generate the Vk2/E6E7 (Vk2) culture, 60,000 cells were seeded on 0.4 µm pore-sized transwell polystyrene inserts (BD Falcon, Mississauga, ON, Canada), and keratinocyte serum-free medium (Life Technologies, Carlsbad, CA, USA) supplemented with 0.1 ng/mL human recombinant epidermal growth factor (Life Technologies), 0.05 mg/mL of bovine pituitary extract (Life Technologies), CaCl2, and 100 units/mL penicillin/streptomycin (Sigma Aldrich, Oakville, ON, Canada) were added to the apical and basolateral sides of the culture, as described in [41]. LLI culture was generated by keeping the media on both the apical (300 µL) and basolateral sides (500 µL) (Figure 1A). For ALI culture, media from the apical side was removed after 24 h of seeding the cells and media was only present in the basolateral side (1000 µL) (Figure 1B). Media was replenished every 24 h for 10 days of culture.

View Article: PubMed Central - PubMed

ABSTRACT

The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions.

No MeSH data available.