{"paper_id":"c6332193-7f0d-4095-b9c5-badd30360535","body_text":"Hormone-responsive, patient-derived models of the uterine wall in a microfluidic array \n \n \nCaroline Busch1,2, Christopher J. Hill3,4, Karla Paterson5, Michele Zagnoni2,5, Dharani K. Hapangama3,4, Mairi E. \nSandison1* \n1Department of Biomedical Engineering, University of Strathclyde, 2Department of Electronic and Electrical Engineering, \nUniversity of Strathclyde, 3Department of Women's and Children's Health, University of Liverpool, 4Liverpool Women's \nHospital NHS Foundation Trust, 5 ScreenIn3D Limited, Glasgow \n*Corresponding author: mairi.sandison@strath.ac.uk \n \n \nAbstract theme: Bioengineered Models \n \n \nIntroduction \nDespite their prevalence and their profound impact on patient’s lives, chronic gynaecological disorders (such \nas endometriosis and adenomyosis)  remain under -researched. Both to advance our understanding of the \ncellular behaviours underlying t hese conditions and to provide new tools for screening of potential therapies, \nadvanced in vitro models are required. 3D patient-derived cultures that mimic the uterine wall, comprising both \nendometrial and myometrial cell types,  are an attractive and unex plored route. Here, we describe an organ -\non-a-chip approach to creating such cultures, demonstrating multiplexed, hormone-responsive uterine models \nwithin a microfluidic array. \n \n \nMaterials and Methods \nEndometrial (obtained from pipelle biopsies)  and myometrial (from patients undergoing elective C-sections) \ntissue samples were enzymatically dissociated to produce epithelial, stromal and myometrial cell fractions [1]. \nThese cell suspensions were sequentially seeded into a microfluidic device with m ultiple, individually \naddressable channels , each containing a 5x5  microwell arra y. The devices w ere fabricated in PDMS and \nfunctionalised to create a non -adherent surface as previously published [ 2]. The resultant 3D cultures were \nmaintained in vitro for up to 15 days and were subjected to hormone stimulation with β-estradiol (E2, 10 nM), \nmedroxyprogesterone acetate (MPA, 100 nM) and cAMP (500 µM). Cellular organisation was monitored by \nlive-cell imaging using fluorescent tracers  and by end -point immunocytochemistry  (pan-cytokeratin for \nepithelial cells, calponin for myocytes (SMCs), vimentin for stromal cells ), with model functionality assessed \nby measuring IGFBP-1 and osteopontin secretion (by ELISA) following hormone stimulation. \n \n \nResults and Discussion \nA series of cell injection protocols, sequentially seeding the three cellular fractions over 2 -4 days, were \ninvestigated. Regardless of protocol, epithelial cells ultimately localised to the outer periphery of the 3D \ncultures. Optimum results – where self-organisation resulted in robust 3D cultures with an outer epithelial layer \nencircling an inner myometrial core, thus mimicking the architecture of the uterine wall – were obtained when \nthe myometrial cells were seeded last  and when 5% (v/v) Matrigel® was incorporated into the culture media . \nConsistent cellular organisation was obtained when directly comparing patient-derived cultures produced from \ndifferent endometrial biopsies (n=3, all biopsies from secretory phase tissue).  Exposure of these cultures to \nE2, MPA and cAMP resulted in an increase in the secretion of both osteopontin and IGFBP -1, indicating \nstromal decidualisation [3].  \n \n \nConclusions \nThese functional, 3D multicellular cultures show promise for personali sed modelling of gynaecological \ndisorders, enabling screening of patient-derived cultures in a medium-throughput format. \n \n \nReferences  \n1.  Hapangama DK et al. Human Reproduction. 34(2): 56-68, 2019. \n2.  Mulholland T et al, Scientific Reports 8: 14672, 2018. \n3.  von Wolff M et al, Fertility & Sterility, 81(1): 741-748, 2004. \n \n \nAcknowledgements \nCB was supported by an Organ-on-a-Chip Technologies Network Pump Priming Project grant, CJH/DKH by a \nWellbeing of Women project grant RG 2137 and DKH by a MRC CTRF (MR/VOO7238/1).","source_license":"CC0","license_restricted":false}