Three-dimensional microengineered vascularised endometrium-on-a-chip

Abstract

Study question: Can we reconstitute physiologically relevant 3-dimensional (3D) microengineered endometrium in-vitro model?

Summary answer: Our representative microengineered vascularised endometrium on-a-chip closely recapitulates the endometrial microenvironment that consists of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D extracellular matrix in a spatiotemporal manner.

What is known already: Organ-on-a-chip, a multi-channel 3D microfluidic cell culture system, is widely used to investigate physiologically relevant responses of organ systems.

Study design, size, duration: The device consists of five microchannels that are arrayed in parallel and partitioned by array of micropost. Two central channels are for 3D culture and morphogenesis of stromal fibroblast and endothelial cells. In addition, the outermost channel is for the culture of additional endometrial stromal fibroblasts that secrete biochemical cues to induce directional pro-angiogenic responses of endothelial cells. To seed endometrial epithelial cells, on Day 8, Ishikawa cells were introduced to one of the two medium channels to adhere on the gel surface. After that, the microengineered endometrium was cultured for an additional 5-6 days (total ∼ 14 days) for the purpose of each experiment.

Participants/materials, setting, methods: Microfluidic 3D cultures were maintained in endothelial growth Medium 2 with or without oestradiol and progesterone. Some cultures additionally received exogenous pro-angiogenic factors. For the three distinct layers of microengineered endometrium-on-a-chip, the epithelium, stroma and blood vessel characteristics and drug response of each distinct layer in the microfluidic model were assessed morphologically and biochemically. The quantitative measurement of endometrial drug delivery was evaluated by the permeability coefficients.

Main results and the role of chance: We established microengineered vascularised endometrium-on-chip, which consists of three distinct layers: epithelium, stroma and blood vessels. Our endometrium model faithfully recapitulates in-vivo endometrial vasculo-angiogenesis and hormonal responses displaying key features of the proliferative and secretory phases of the menstrual cycle. Furthermore, the effect of the emergency contraception drug levonorgestrel was evaluated in our model demonstrating increased endometrial permeability and blood vessel regression in a dose-dependent manner. We finally provided a proof of concept of the multi-layered endometrium model for embryo implantation, which aids a better understanding of the molecular and cellular mechanisms underlying this process.

Large scale data: N/A.

Limitations, reasons for caution: This report is largely an in-vitro study and it would be beneficial to validate our findings using human primary endometrial cells.

Wider implications of the findings: Our 3D microengineered vascularised endometrium-on-a-chip provides a new in-vitro approach to drug screening and drug discovery by mimicking the complicated behaviours of human endometrium. Thus, we suggest our model as a tool for addressing critical challenges and unsolved problems in female diseases, such as endometriosis, uterine cancer and female infertility, in a personalised manner.

Study funding/competing interest(s): This work is supported by funding from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) to Y.J.K. (No. 2018R1C1B6003), to J.A. (No. 2020R1I1A1A01074136) and to H.S.K. (No. 2020R1C1C100787212). The authors report no conflicts of interest.

Keywords: 3D culture; drug screening; endometrial angiogenesis; endometrium; microfluidic.

Figure 1.

Microfluidic device to reconstitute 3D vascularised endometrial microenvironment. (A) Schematic overview and confocal images to mimic natural endometrial microenvironment using a microfluidic 3D tri-culture model, endometrial stromal fibroblasts, endometrial epithelial cells and endothelial cells. Confocal image shows natural endometrial sprouting morphogenesis from the pre-formed blood vessel network. Scale bar, 400 μm. (B) Representative confocal images show different angiogenic sprouts response under S1P and VEGF-A. Scale bar, 400 μm.

Figure 2.

Endometrial epithelial cell responses under proliferative and secretory phase conditions. (A) Schematic of the female menstrual cycle showing contribution of the different hormones (oestrogen (E2) and progesterone (P4)). (B) Schematic of the experimental process. (C) Schematic illustration shows endometrial epithelial cell growth within microfluidic platform. (D) Representative confocal microscopic images of endometrial epithelial cells. Scale bar, 200 μm. (E) Quantification of endometrial epithelial cell thickness difference in response to E2 and E2+P4 treatment. One-way ANOVA, plotted as mean ± SEM; ** P < 0.01, **** P < 0.0001.

Figure 3.

Oestradiol and progesterone response of endometrial stromal fibroblasts and endothelial cells within 3D endometrium on a chip. (A) Schematic illustration and representative confocal images of F-actin cytoskeleton of endometrial stromal fibroblasts. Scale bar, 400 μm. (B) F-actin cytoskeleton area of endometrial stromal fibroblasts. (C) F-actin intensity of endometrial stromal fibroblasts. (D) Schematic illustration and representative confocal image of angiogenic sprouts and vascular network. Scale bar, 400 μm. (E) Angiogenic sprouts area in stroma-angiogenic sprout channel. (F) Vascular network area in vascular network channel. (G) CD31-positive plot profile of arbitrary transverse line one-way ANOVA, plotted as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 4.

Levonorgestrel response in 3D endometrium-on-a-chip. (A) Schematic illustration of levonorgestrel treatment on 3D endometrium-on-a-chip. (B and C) Propidium iodide area of the epithelium layer and stromal layer, respectively. (D) Representative confocal images represent live/dead signals. (E) Schematic illustration of levonorgestrel treatment on 3D endometrium-on-a-chip and blood vessel regression monitoring. (F) Representative confocal image of angiogenic sprouts and vascular network before levonorgestrel treatment (Day 10) and after levonorgestrel treatment (Day 13). Scale bar, 600 μm. (G) Blood vessel regression index was calculated as the ratio of final vascular network area to initial vascular network area. (H) Blood vessel regression index in response to different levonorgestrel dosages from 0 to 10 000 ng/ml. One-way ANOVA, plotted as mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 5.

Endometrial permeability measurement in response to levonorgestrel treatment. (A) Schematic of the experimental process of permeability measurement with levonorgestrel treatment. Endometrial permeability was measured after 48 h treatment of levonorgestrel. (B) The levonorgestrel-treated condition showed significantly higher permeability than that of the control. (C) Time series fluorescence micrographs were captured and analysed for endometrial permeability. Non-paired Student’s t-test, plotted as mean ± SD; **** P < 0.0001.

Figure 6.

3D microengineered implantation model. (A and B) Schematic of the experimental process of testing the 3D microengineered implantation model. (C) Bright field image of stably attached protein-coated microbeads. Scale bar: 200 μm. (D) Graph shows that beads carrying HB-EGF and IGF-1 exhibited significantly higher attachment than BSA-coated microbeads. One-way ANOVA, plotted as mean ± SEM; ** P < 0.01.