Functional, patient-derived 3D tri-culture models of the uterine wall in a microfluidic array

Busch, Caroline and Hill, Christopher J. and Paterson, Karla and Mellin, Ronan and Zagnoni, Michele and Hapangama, Dharani K. and Sandison, Mairi E. (2024) Functional, patient-derived 3D tri-culture models of the uterine wall in a microfluidic array. Human Reproduction. ISSN 1460-2350 (In Press) (https://doi.org/10.1093/humrep/deae214)

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Abstract

Study question: Can a functional in vitro model, containing the main cellular components of the uterine wall be generated from cells derived from patient tissues? Summary answer: We present a three-dimensional physiologically-relevant, organ-on-a-chip model of the uterine wall containing primary endometrial and myometrial cellular participants, generated from human uterine tissue. What is already known: A highly dynamic reproductive organ, the human uterus plays fundamental physiological roles in menstruation and childbirth. The endometrial-myometrial junction (EMJ) defines the interface between the inner mucosal layer (endometrium) and outer smooth muscle zone (myometrium) that comprises the uterine wall. The EMJ is implicit in several uterine pathologies of unknown aetiology, including adenomyosis and abnormally invasive placenta; however, despite this, no patient-derived in vitro models of the uterine wall containing all EMJ participants currently exist. Study design, size, duration: We employed microfluidic technology to characterise multiple miniaturised models of the uterine wall. Protocols were tested that included variations in the seeding order of endometrial and myometrial fractions, and the addition of a low viscosity extracellular matrix to influence cell behaviour. Ultimately, functional hormone responses of patient-derived uterine wall models were then assessed. Participants/materials, setting, methods: Endometrial (n=9) and myometrial biopsies (n=4) were enzymatically dissociated to create epithelial, stromal and myometrial cellular fractions. Cell suspensions were seeded into non-adhesive poly(dimethylsiloxane) microfluidic devices containing 5x5 microwell arrays. The fate of individual cell types was monitored in real-time using fluorescent tracers, and cell phenotype was characterised by immunocytochemistry. Model functionality was assessed by measuring Ca2+ responses to agonist stimulation, and both IGFBP-1 and Osteopontin secretion in response to hormone stimulation. Main results and the role of chance: When subjected to microfluidic culture in isolation, endometrial stromal cells and smooth muscle myocytes formed compact spheroids, whilst epithelial cells produced diffuse aggregates. Tri-cultures were established by sequential seeding of individual or combined cell fractions at various ratios. Regardless of protocol, epithelial cells localised to the outer periphery of tri-culture spheroids, which varied in morphology across the protocols. Incorporation of 5% [v/v] Matrigel® improved the reproducibility of 3D aggregates, which exhibited robust self-assembly of a stromal/smooth muscle core encased in epithelium. Exposure of tri-cultures to oestradiol, medroxyprogesterone acetate and cyclic adenosine monophosphate increased secretion of insulin-like growth factor binding protein-1, which indicates stromal decidualisation, as well as enhancing epithelial cell osteopontin secretion. Stimulation with endothelin-1 induced Ca2+ signalling in myocytes. Limitations, reasons for caution: Endometrial and myometrial tissue was collected from relatively few donors. Myometrial tissue was collected from pregnant donors, which may influence myocyte phenotype. Furthermore, endometrial tissue sampling was from women not having hysterectomy, thus, may not include the deeper basalis region, which may limit the physiological mimicry of the final models. Wider implications of the findings: Our novel approach to modelling the uterine wall in 3D captures all relevant main cell types in a medium-throughput system, enabling the screening of hundreds of cultures in parallel from a single biopsy. This system shows great promise for examining the cellular interplay between physiological cues and EMJ pathologies, such as the impact of uterine peristalsis and cyclical hormones on the pathogenesis of adenomyosis.

ORCID iDs

Busch, Caroline, Hill, Christopher J., Paterson, Karla, Mellin, Ronan, Zagnoni, Michele ORCID logoORCID: https://orcid.org/0000-0003-3198-9491, Hapangama, Dharani K. and Sandison, Mairi E. ORCID logoORCID: https://orcid.org/0000-0003-1021-1461;
CORE (COnnecting REpositories)