Organ-on-chip of the cervical epithelial layer: A platform to study normal and pathological cellular remodeling of the cervix

Abstract

Damage to the cervical epithelial layer due to infection and inflammation is associated with preterm birth. However, the individual and/or collective roles of cervical epithelial layers in maintaining cervical integrity remain unclear during infection/inflammation. To determine the intercellular interactions, we developed an organ-on-chip of the cervical epithelial layer (CE-OOC) composed of two co-culture chambers connected by microchannels, recapitulating the ectocervical and endocervical epithelial layers. Further, we tested the interactions between cells from each distinct region and their contributions in maintaining cervical integrity in response to LPS and TNFα stimulations. The co-culture of ectocervical and endocervical cells facilitated cellular migration of both epithelial cells inside the microchannels. Compared to untreated controls, both LPS and TNFα increased apoptosis, necrosis, and senescence as well as increased pro-inflammatory cytokine productions by cervical epithelial cells. In summary, the CE-OOC established an in vitro model that can recapitulate the ectocervical and the endocervical epithelial regions of the cervix. The established CE-OOC may become a powerful tool in obstetrics and gynecology research such as in studying cervical remodeling during pregnancy and parturition and the dynamics of cervical epithelial cells in benign and malignant pathology in the cervix.

Keywords: cervical ripening; cervix; epithelial-to-mesenchymal transition; organ-on-a-chip; pregnancy; preterm birth.

FIGURE 1

CE-OOC design and cell culture within. The CE-OOC is designed to recapitulate the cervical epithelial layer in vitro by co-culturing ectocervical and endocervical epithelial cells separated by type IV collagen-filled microfluidic channels through which cells can migrate to form the transformation zone epithelium of the cervix. A, Schematic representation of the anatomy of the cervical epithelial layer. Left: gross morphology view; right: cross-sectional view. B, The CE-OOC design shown in 3D, where the two cell culture chambers separated by 24 shallow microchannels. C, Cross-sectional view showing the height difference between the cell culture chambers (500 μm height) and the microchannels (5 μm height). D, An image of the CE-OOC device showing the outer ectocervical epithelial cell culture chamber filled with blue color dye and the inner endocervical epithelial cell culture chamber filled with yellow color dye. E, Bright-field and fluorescence microscopy images of the ectocervical epithelial and endocervical epithelial cell cultures in the CE-OOC, showing cell morphology as well as CK-18 (red) and vimentin (green) expression. Nuclei are stained blue (DAPI: 4′,6-diamidino-2-phenylindole). Scale bar = 500 μm

FIGURE 2

Monocultured ectocervical and endocervical epithelial cell morphology in the CE-OOC under LPS and TNFα treatments for 96 hours. Bright-field (BF) microscopy showing the morphology of A, ectocervical and B, endocervical epithelial cells in the CE-OOC monoculture under control, LPS, and TNFα treatment for 96 hours. Magnified images were shown to highlight the morphology of the cervical epithelial cells under normal, LPS-, and TNFα-treated conditions. Scale bar = 500 μm (top) and 100 μm (bottom)

FIGURE 3

Monocultured ectocervical and endocervical epithelial cell migration in the CE-OOC under control, LPS, and TNFα treatment conditions. Representative bright-field images and quantification of migrated monocultured ectocervical epithelial cells (A-B) and endocervical epithelial cells (C-D) in the CE-OOC device under control, LPS, and TNFα treatment conditions. Error bars represent mean ± SEM. n = 5 biological replicates. *P < .05; **P < .01

FIGURE 4

Effects of LPS and TNFα treatment on cell transition and migration in the CE-OOC monoculture condition. Fluorescence microscopy images showing cell morphology, CK-18 (red), and vimentin (green) in the inner chamber, collagen-filled microchannels, and outer chamber of untreated (control), LPS-treated, and TNFα-treated ectocervical epithelial cells A, and endocervical epithelial cells B, Quantification of the ratio of vimentin and CK-18 relative fluorescence unit (RFU) in ectocervical C, and endocervical epithelial cells D, in the chambers and microchannels of the CE-OOC. Nuclei are stained blue (DAPI: 4′,6-diamidino-2-phenylindole). Scale bar = 50 μm. Error bars represent mean ± SEM. n = 9 technical replicates. A total of 5–10 cells per replicate were analyzed to calculate the average RFU of vimentin and CK-18 . *P < .05

FIGURE 5

Effect of LPS and TNFα on the cell fate in the CE-OOC co-culture condition. Annexin V (green) and propidium iodide (red) staining of untreated (control), LPS- and TNFα-treated co-culture of ectocervical epithelial cells A, and endocervical epithelial cells B, Nuclei are stained blue (DAPI: 4′,6-diamidino-2-phenylindole). Scale bar, 100 μm. Quantification of the percentage of cells that are viable, early apoptotic, late apoptotic, and necrotic in ectocervical epithelial cells (A) and endocervical epithelial cells (B). Error bars represent mean ± SEM. n = 5 technical replicates. C, Senescence-associated β-galactosidase (SA β-gal) staining in control and LPS- and TNFα-treated ectocervical epithelial cells and endocervical epithelial cells in CE-OOC co-culture. Arrows show the SA β-gal-positive cells. Scale bar, 100 μm. Quantification of senescent cells in control and LPS- and TNFα-treated ectocervical epithelial cells and endocervical epithelial cells in CE-OOC monoculture. Error bars represent mean ± SEM. n = 9 technical replicates. A total of 400–600 cells were analyzed per replicate of the apoptosis, necrosis, and senescence assays. *P < .05; **P < .01; and ***P < .001

FIGURE 6

Effect of LPS and TNFα on cell migration in the CE-OOC co-culture condition. A, Fluorescence microscopy of co-culture experiments showed that both ectocervical epithelial cells (blue) and endocervical epithelial cells (yellow) migrate through the collagen-filled microchannels. The bottom panel highlights the migrated ectocervical epithelial cells (blue) and endocervical epithelial cells (yellow) that have integrated into the microchannels. Scale bar, 200 μm. The right panel is a schematic representing the migration of cervical epithelial cells inside the microchannels. B, Quantification of migrating ectocervical epithelial cells that enter through collagen-filled microchannels. n = 3 biological replicates. C, Quantification of migrating endocervical epithelial cells that enter through the collagen-filled microchannels. Error bars represent mean ± SEM. n = 3 biological replicates

FIGURE 7

Pro-inflammatory cytokine production and propagation in the CE-OOC co-culture systems. Luminex assay measured the baseline media concentrations of IL-6 and IL-8 from ectocervical (outer chamber) A, and endocervical epithelial cell media (inner chamber) B, For IL-6, ectocervical epithelial cells: 58.98 ± 18.21 pg/mL and endocervical epithelial cells: 470.6 ± 173.2 pg/mL. For IL-8, ectocervical epithelial cells: 556.1 ± 246 pg/mL and endocervical epithelial cells: 9515 ± 2048 pg/mL. C-D, IL-6 levels from ectocervical (C) and endocervical epithelial cell media (D) under control (untreated), LPS, and TNFα treatments. E-F, IL-8 levels from ectocervical (outer chamber) (E) and endocervical epithelial cell media (inner chamber) (F) under control (untreated), LPS, and TNFα treatment. G, Graphical summary of inflammatory cytokine production in the CE-OOC culture systems. The treatment of both chambers with TNFα promoted inflammation in both cervical epithelial cells, as supported by increased IL-6 and IL-8 production. Treatment in only one chamber also induced increased IL-8 production in the adjacent chamber indicating the propagation of inflammatory mediators in the CE-OOC. n = 5 biological replicates. The IL-6 and IL-8 levels were considered to be increased (↑) if there was a twofold change from the baseline (control/control) IL-6 and IL-8 levels. Otherwise, the result was classified as no change from baseline. *P < .05