Dataset Information

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium.

ABSTRACT: The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-?m porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal vs. disease-related endometrial microenvironments.

SUBMITTER: Gnecco JS

PROVIDER: S-EPMC5489603 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium.

Gnecco Juan S JS Pensabene Virginia V Li David J DJ Ding Tianbing T Hui Elliot E EE Bruner-Tran Kaylon L KL Osteen Kevin G KG

Annals of biomedical engineering 20170120 7

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells ...[more]

PMID: 28108942

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Project description:Study questionDoes the uterine vasculature play a localized role in promoting stromal cell decidualization in the human endometrium?Summary answerOur study demonstrated that hemodynamic forces induced secretion of specific endothelial cell-derived prostanoids that enhanced endometrial perivascular decidualization via a paracrine mechanism.What is known alreadyDifferentiation of stromal cell fibroblasts into the specialized decidua of the placenta is a progesterone-dependent process; however, histologically, it has long been noted that the first morphological signs of decidualization appear in the perivascular stroma. These observations suggest that the human endometrial vasculature plays an active role in promoting stromal differentiation.Study design, size, durationPrimary human endometrial stromal cells were co-cultured for 14 days with primary uterine microvascular endothelial cells within a microfluidic Organ-on-Chip model of the endometrium.Participants/materials, setting, methodsCultures were maintained with estradiol and a progestin, with or without continuous laminar perfusion to mimic hemodynamic forces derived from the blood flow. Some cultures additionally received exogenous agonist-mediated challenges. Decidualization in the microfluidic model was assessed morphologically and biochemically. ELISA was used to examine the culture effluent for expression of decidualization markers and prostaglandins. Immunofluorescence was used to monitor cyclooxygenase-2 expression in association with decidualization.Main results and the role of chanceA significantly enhanced stromal decidualization response was observed in the co-cultures when the endothelial cells were stimulated with hemodynamic forces (e.g. laminar shear stress) derived from controlled microfluidic perfusion (<0.001). Furthermore, the enhanced progestin-driven stromal differentiation was mediated via cyclooxygenase-2 and the paracrine action of prostaglandin E2 and prostacyclin. Altogether, these translational findings indicate that the vascular endothelium plays a key physiologic role during the early events of perivascular decidualization in the human endometrium.Large scale dataN/A.Limitations, reasons for cautionThis report is largely an in vitro study. Although we were able to experimentally mimic hemodynamic forces in our microfluidic model, we have not yet determined the contribution of additional cell types to the decidualization process or determined the precise physiological rates of shear stress that the microvasculature of the endometrium undergoes in vivo.Wider implications of the findingsIdentification of specific endothelial-derived prostaglandins and their role during endometrial reproductive processes may have clinical utility as therapeutic targets for reproductive disorders such as infertility, endometriosis, adenomyosis, pre-eclampsia and poor pregnancy outcomes.Study funding/competing interest(s)This work was supported by the Veterans Affairs (I01 BX002853), the Bill and Melinda Gates Foundation Grand Challenges Exploration (OPP1159411), the Environmental Toxicology Training Grant (NIH T32 ES007028) and the Environmental Protection Agency STAR Center Grant (83573601).Conflict of interestThe authors report no conflicts of interest.Trial registration numberN/A.

Project description:BACKGROUND:Pancreatic stellate cells (PSCs), a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. Culturing various cells in microfluidic (microchannel) devices has proven to be a useful in studying cellular interactions and drug sensitivity. Here we present a microchannel plate-based co-culture model that integrates tumor spheroids with PSCs in a three-dimensional (3D) collagen matrix to mimic the tumor microenvironment in vivo by recapitulating epithelial-mesenchymal transition and chemoresistance. METHODS:A 7-channel microchannel plate was prepared using poly-dimethylsiloxane (PDMS) via soft lithography. PANC-1, a human pancreatic cancer cell line, and PSCs, each within a designated channel of the microchannel plate, were cultured embedded in type I collagen. Expression of EMT-related markers and factors was analyzed using immunofluorescent staining or Proteome analysis. Changes in viability following exposure to gemcitabine and paclitaxel were measured using Live/Dead assay. RESULTS:PANC-1 cells formed 3D tumor spheroids within 5 days and the number of spheroids increased when co-cultured with PSCs. Culture conditions were optimized for PANC-1 cells and PSCs, and their appropriate interaction was confirmed by reciprocal activation shown as increased cell motility. PSCs under co-culture showed an increased expression of ?-SMA. Expression of EMT-related markers, such as vimentin and TGF-?, was higher in co-cultured PANC-1 spheroids compared to that in mono-cultured spheroids; as was the expression of many other EMT-related factors including TIMP1 and IL-8. Following gemcitabine exposure, no significant changes in survival were observed. When paclitaxel was combined with gemcitabine, a growth inhibitory advantage was prominent in tumor spheroids, which was accompanied by significant cytotoxicity in PSCs. CONCLUSIONS:We demonstrated that cancer cells grown as tumor spheroids in a 3D collagen matrix and PSCs co-cultured in sub-millimeter proximity participate in mutual interactions that induce EMT and drug resistance in a microchannel plate. Microfluidic co-culture of pancreatic tumor spheroids with PSCs may serve as a useful model for studying EMT and drug resistance in a clinically relevant manner.

Dataset Information

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium.

Publications

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium.

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