Center for Infectious Diseases and Vaccinology, The Biodesign Institute at Arizona State University, 1001 South McAllister Avenue, Tempe, AZ 85287-5401, USA.
Biol Reprod. 2010 Mar;82(3):617-27. doi: 10.1095/biolreprod.109.080408. Epub 2009 Dec 9.
We have developed an in vitro human vaginal epithelial cell (EC) model using the innovative rotating wall vessel (RWV) bioreactor technology that recapitulates in vivo structural and functional properties, including a stratified squamous epithelium with microvilli, tight junctions, microfolds, and mucus. This three-dimensional (3-D) vaginal model provides a platform for high-throughput toxicity testing of candidate microbicides targeted to combat sexually transmitted infections, effectively complementing and extending existing testing systems such as surgical explants or animal models. Vaginal ECs were grown on porous, collagen-coated microcarrier beads in a rotating, low fluid-shear environment; use of RWV bioreactor technology generated 3-D vaginal EC aggregates. Immunofluorescence and scanning and transmission electron microscopy confirmed differentiation and polarization of the 3-D EC aggregates among multiple cell layers and identified ultrastructural features important for nutrient absorption, cell-cell interactions, and pathogen defense. After treatment with a variety of toll-like receptor (TLR) agonists, cytokine production was quantified by cytometric bead array, confirming that TLRs 2, 3, 5, and 6 were expressed and functional. The 3-D vaginal aggregates were more resistant to nonoxynol-9 (N-9), a contraceptive and previous microbicide candidate, when compared to two-dimensional monolayers of the same cell line. A dose-dependent production of tumor necrosis factor-related apoptosis-inducing ligand and interleukin-1 receptor antagonist, biomarkers of cervicovaginal inflammation, correlated to microbicide toxicity in the 3-D model following N-9 treatment. These results indicate that this 3-D vaginal model could be used as a complementary tool for screening microbicide compounds for safety and efficacy, thus improving success in clinical trials.
我们使用创新的旋转壁式生物反应器（RWV）技术开发了一种体外人阴道上皮细胞（EC）模型，该模型重现了体内的结构和功能特性，包括具有微绒毛、紧密连接、微褶皱和粘液的分层鳞状上皮。这种三维（3-D）阴道模型为候选杀微生物剂的高通量毒性测试提供了一个平台，旨在对抗性传播感染，有效补充和扩展了现有的测试系统，如外科外植体或动物模型。阴道 EC 细胞在旋转、低流体剪切环境中生长在多孔、胶原涂层的微载体珠上；使用 RWV 生物反应器技术生成 3-D 阴道 EC 聚集物。免疫荧光和扫描及透射电子显微镜确认了 3-D EC 聚集物在多个细胞层中的分化和极化，并鉴定了对营养吸收、细胞-细胞相互作用和病原体防御很重要的超微结构特征。用各种 Toll 样受体（TLR）激动剂处理后，通过细胞计数珠阵列定量细胞因子的产生，证实 TLR2、3、5 和 6 被表达和功能化。与相同细胞系的二维单层相比，3-D 阴道聚集物对壬苯醇醚-9（N-9）的抵抗力更强，N-9 是一种避孕和先前的杀微生物候选物。N-9 处理后，与宫颈阴道炎症相关的肿瘤坏死因子相关凋亡诱导配体和白细胞介素 1 受体拮抗剂的产生呈剂量依赖性，与 3-D 模型中的杀微生物毒性相关。这些结果表明，这种 3-D 阴道模型可作为筛选杀微生物化合物的安全性和有效性的补充工具，从而提高临床试验的成功率。
