A microphysiological model of human trophoblast invasion during implantation

Successful establishment of pregnancy requires adhesion of an embryo to the endometrium and subsequent invasion into the maternal tissue. Abnormalities in this critical process of implantation and placentation lead to many pregnancy complications. Here we present a microenigneered system to model a complex sequence of orchestrated multicellular events that plays an essential role in early pregnancy. Our implantation-on-a-chip is capable of reconstructing the three-dimensional structural organization of the maternal-fetal interface to model the invasion of specialized fetal extravillous trophoblasts into the maternal uterus. Using primary human cells isolated from clinical specimens, we demonstrate in vivo-like directional migration of extravillous trophoblasts towards a microengineered maternal vessel and their interactions with the endothelium necessary for vascular remodeling. Through parametric variation of the cellular microenvironment and proteomic analysis of microengineered tissues, we show the important role of decidualized stromal cells as a regulator of extravillous trophoblast migration. Furthermore, our study reveals previously unknown effects of pre-implantation maternal immune cells on extravillous trophoblast invasion. This work represents a significant advance in our ability to model early human pregnancy, and may enable the development of advanced in vitro platforms for basic and clinical research of human reproduction.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Sample size was not predetermined. We performed at least 3 independent experiments for each tested condition,each containing 3 technical replicates or more. Sample sizes were determined using widely accepted guidelines for 3D cell culture in engineered microphysiological devices, as well as our extensive experience in the development and engineering of physiological in vitro tissue and organ models. In addition, all independent experiments and replication attempts in this study were successful and showed very consistent biological phenomena, which were sufficient to ensure statistical significance. Considering the experimental unit being observed as defined by Lazic et al as extent of invasion by extravillous trophoblasts, and the experiments performed on our device being essentially cell culture experiments, we considered biological replicates as (to the extent possible) independent experiments performed on different days, different passage numbers and cells from different patients. Since no prior information on these variables were available, we made sure at least 3 replicates were performed for each experiment attempted.
No data were excluded from the study.
To avoid confounding effects, comparisons were made with technical replicates concurrently established in an identical manner using similar numbers of cells, same solutions of culture media and same commercial lots of primary human cells when applicable. Fetal EVTs from different patients, but similar gestational ages were utilized, and studies using maternal endometrial cells isolated cells from independent patients for each experiment to ensure reproducibility of experimental findings. All human subjects showed similar baseline demographic characteristics with no known pathology. All experiments contained biological (at least 3) and technical replicates (at least 3) to confirm reliable reproducibility of experimental findings, and were successful. No data was excluded.
All replicates on organ-on-a-chip-based platform were randomly distributed between experimental groups.
Studies utilized well-definted quantitative metrics and did not involve subjective measurements or qualitative rating schema. Researchers were not blinded to allocation of experimental groups, data collection or analysis. Validation data for each antibody used can be found on the manufacturers' websites and datasheets as summarized below. In addition, for imaging, specificity of primary antibodies was validated by negative control staining in cultured cells that are not known to express the target antigen, and for flow cytometry, fluorescence minus one controls were carried out. CD31 (Abcam # ab24590) is a mouse monoclonal antibody for CD31 of endothelial cells. It was tested for applications in immunohistochemistry stainings for human species. VE-Cadherin (Cell Signaling #2500) is a rabbit monoclonal antibody for VE-cadherin. It has been validated for western blotting, immunoprecipitation, immunocytochemistry, and flow cytometry for human, bovine, and pig. Cleaved Caspase-3 (Cell Signaling #9661) is a rabbit polyclonal antibody to human Cleaved-Caspase-3. It has been validated for western blotting, immunoprecipitation, and immunocytochemistry stainings for human, mouse, rabbit, and monkey. ki67 (Abcam #ab245113) is a mouse monoclonal antibody to KI67. It has been validated for application in flow cytometry and immunocytochemistry stainings for only human species. Cytokeratin-7 (Abcam # ab181598) is rabbit monoclonal antibody for Cytokeratin 7. It has been validated for western blotting, immunocytochemistry stainings, and flow cytometry for human, mouse, and rat. VCAM-1(Novus Biologicals[6G9] NBP1-47491) is a mouse monoclonal antibody to VCAM-1. It has been validated for western blotting, immunohistochemistry stainings for human, canine, and primate species. Complement C4(Thermo Fisher # MA5-32856) is a rabbit monoclonal antibody for Complement C4. It has been validated for use in western blotting, immunohistochemistry stainings for only human species. Prolactin (Thermo Fisher # MA5-11998) is a mouse monoclonal antibody for prolactin. It has been validated for use in immunohistochemistry stainings and ELISA for only human species. Fibroblast surface protein (Abcam # ab11333) is a mouse monoclonal antibody for fibroblast surface protein. It has been validated for use in immunohistochemistry stainings and western blotting for only human species.. HLAG (Biorad #MCA2044): As stated on the BioRad website, this product was tested for application in immunofluoresence experiments. No other validation data was listed on website, but we tested validity of antibody by co-staining with cytokeratin 7 and human placental lactogen in first trimester formalin fixed paraffin embedded tissue and found specific expression in extravillous trophoblasts only. In addition, this antibody was tested along with other HLA-G antibody clones to ensure specificity of staining. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.