Improved filtration method to isolate pure populations of primary bovine endometrial epithelial and stromal cells for immunological studies

Paul Kelly,Amy Brewer,Cliona O’Farrelly,Kieran G Meade,Anne Barry-Reidy

doi:10.1007/s11259-020-09770-3

Abstract

ObjectivesIsolation and culture of distinct primary endometrial cells are key to reliable in-vitro models to investigate the uterine immune response and optimse new disease interventions. Details on the isolation method and purity of distinct cell populations is lacking in currently available protocols leading to inconsistent results across laboratories.MethodsBovine endometrial tissue from non-pregnant bovine uteri were collected immediately post-mortem and separated using differential size filtering. Isolations (n = 15) yielded an average of 3.1 × 105 ± 0.7 × 105 epithelial cells and 1.88 × 106 ± 5.44 × 105 stromal fibroblasts per uterine horn. Following expansion in culture, the purity of cell populations was confirmed using morphology and positive staining for cytokeratin and vimentin which identifies epithelial and stromal fibroblast populations, respectively. Using PCR, cDNA from both cell populations was negative for CD45, a marker of immune cells.ResultsOn challenge with a bacterial PAMP (LPS), epithelial and stromal fibroblasts showed a marked increase in the expression of the inflammatory mediators IL8, IL6, S100A8 and S100A9, with both cell populations displaying distinct expression profiles. Here we provide a detailed methodology on the culture of primary bovine endometrial epithelial and stromal cells and demonstrate these cells provide a physiologically relevant model for studies of endometrial inflammation and its regulation.

Highlights

The endometrium, the mucosal lining of the uterus, plays a complex role maintaining a homeostatic environment to allow for successful implantation and pregnancy while tolerating the local microbiome and protecting against invading pathogens
Vet Res Commun (2020) 44:29–39 role in defence against viral infection (Donofrio et al 2007). Possession of such a wide repertoire of protective mechanisms demonstrates the pivotal roles epithelial and stromal fibroblast cells play in the early stages of the innate immune response before immune cell recruitment (MacKintosh et al 2013)
The forceps was used to hold the edge of the endometrial lining while the curved scissors passed underneath the endometrial lining, cutting away the fibres that tie it to the lower functional layer composed of mainly stromal fibroblasts

Summary

Introduction

The endometrium, the mucosal lining of the uterus, plays a complex role maintaining a homeostatic environment to allow for successful implantation and pregnancy while tolerating the local microbiome and protecting against invading pathogens. In addition to their barrier function, epithelial cells are potent producers of a wide selection of host defence peptides (HDPs) including defensins, cathelicidins and whey acid proteins, as well as pro-inflammatory cytokines and chemokines such as IL-6 and IL-8 (Cronin et al 2012; Davies et al 2008; Narciandi et al 2011; Whelehan et al 2014). Vet Res Commun (2020) 44:29–39 role in defence against viral infection (Donofrio et al 2007) Possession of such a wide repertoire of protective mechanisms demonstrates the pivotal roles epithelial and stromal fibroblast cells play in the early stages of the innate immune response before immune cell recruitment (MacKintosh et al 2013). Detailed investigation into the innate immune properties of endometrial cells is hindered by difficulty in isolating and culturing these distinct cell populations

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