scholarly journals A 3D endometrial organotypic model simulating the acute inflammatory decidualisation initiation phase with epithelial induction of the key endometrial receptivity marker, integrin αVβ3

2021 ◽  
Author(s):  
R Fraser ◽  
R Smith ◽  
C-J Lin
Keyword(s):  
Integrin Αvβ3 ◽  
Reproductive Technologies ◽  
Endometrial Receptivity ◽  
Model Systems ◽  
Receptor Protein ◽  
Phenotypic Change ◽  
Hormonal Stimulation ◽  
Culture Model ◽  
Epithelial Phenotype

Abstract STUDY QUESTION Is it possible to develop a simplified physiological in vitro system representing the key cell-types associated with a receptive endometrial phenotype? SUMMARY ANSWER We present a new concept to investigate endometrial receptivity, with a 3D organotypic co-culture model to simulate an early and transient acute autoinflammatory decidual status that resolves in the induction of a receptive endometrial phenotype. WHAT IS KNOWN ALREADY Embryo implantation is dependent on a receptive uterine environment. Ovarian steroids drive post-ovulation structural and functional changes in the endometrium, which becomes transiently receptive for an implanting conceptus, termed the ‘window of implantation’, and dysregulation of endometrial receptivity is implicated in a range of reproductive, obstetric, and gynaecological disorders and malignancies. The interactions that take place within the uterine microenvironment during this time are not fully understood, and human studies are constrained by a lack of access to uterine tissue from specific time-points during the menstrual cycle. Physiologically relevant in vitro model systems are therefore fundamental for conducting investigations to better understand the cellular and molecular mechanisms controlling endometrial receptivity. PARTICIPANTS/MATERIALS, SETTING, METHODS An endometrial stromal cell (ESC) line, and endometrial epithelial cells (EECs) isolated from uterine biopsy tissue and expanded in vitro by conditional reprogramming, were used throughout the study. Immunocytochemical and flow cytometric analyses were used to confirm epithelial phenotype following conditional reprogramming of EECs. To construct an endometrial organotypic co-culture model, ESCs were embedded within a 3D growth factor-reduced Matrigel structure, with a single layer of conditionally reprogrammed EECs seeded on top. Cells were stimulated with increasing doses of medroxyprogesterone acetate, cAMP and estradiol, in order to induce ESC decidual transformation and endometrial receptivity. Decidual response and the induction of a receptive epithelial phenotype were assessed by immunocytochemical detection and quantitative in-cell western analyses, respectively. MAIN RESULTS AND THE ROLE OF CHANCE A transient upregulation of the IL-33 receptor protein, ST2L, was observed in ESCs, indicating a transient autoinflammatory decidual response to the hormonal stimulation, known to induce receptivity gene expression in the overlying epithelium. Hormonal stimulation increased the EEC protein levels of the key marker of endometrial receptivity, integrin αVβ3 (n = 8; *P <0.05; ***P < 0.0001). To our knowledge, this is the first demonstration of a dedicated endometrial organotypic model, that has been developed to investigate endometrial receptivity, via the recapitulation of an early decidual transitory acute autoinflammatory phase and induction of an epithelial phenotypic change, to represent a receptive endometrial status. LIMITATIONS, REASONS FOR CAUTION This simplified in vitro ESC-EEC co-culture system may be only partly representative of more complex in vivo conditions. WIDER IMPLICATIONS OF THE FINDINGS The 3D endometrial organotypic model presented here may offer a valuable tool for investigating a range of reproductive, obstetric, and gynaecological disorders, to improve outcomes for assisted reproductive technologies, and for the development of advances in contraceptive methods. STUDY FUNDING/COMPETING INTEREST(S) This work was supported in part by an MRC Centre Grant (project reference MR/N022556/1). RF was the recipient of a Moray Endowment award and a Barbour Watson Trust award. C-JL is a Royal Society of Edinburgh Personal Research Fellow, funded by the Scottish Government. The authors have no conflicts of interest to declare.

scholarly journals High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

2017 ◽  
Vol 22 (8) ◽  
pp. 974-984 ◽  
Author(s):  
Tijmen H. Booij ◽  
Hester Bange ◽  
Wouter N. Leonhard ◽  
Kuan Yan ◽  
Michiel Fokkelman ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Signaling Pathways ◽  
Polycystic Kidney Disease ◽  
Kinase Inhibitors ◽  
Renal Cysts ◽  
In Vitro Assays ◽  
Phenotypic Change ◽  
Polycystic Kidney ◽  
Culture Model

Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD.

The influence of in vitro fertilization and embryo culture on the embryo epigenetic constituents and the possible consequences in the bovine model

2017 ◽  
Vol 8 (4) ◽  
pp. 411-417 ◽  
Author(s):  
M.-A. Sirard
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Genetic Model ◽  
Culture Media ◽  
Reproductive Technologies ◽  
The Body ◽  
Model Systems ◽  
Gene Perturbations

Medically assisted reproductive technologies, such as in vitro embryo production, are increasingly being used to palliate infertility. Eggs are produced following a hormonal regimen that stimulates the ovaries to produce a large number of oocytes. Collected oocytes are then fertilized in vitro and allowed to develop in vitro until they are either frozen or transferred to mothers. There are controversial reports on the adverse impacts of these technologies on early embryos and their potential long-term effects. Using newly developed technological platforms that enable global gene expression and global DNA methylation profiling, we evaluated gene perturbations caused by such artificial procedures. We know that cells in the early embryo produce all cells in the body and are able to respond to their in vitro environment. However, it is not known whether gene perturbations are part of a normal response to the environment or are due to distress and will have long-term impacts. While the mouse is an established genetic model used for quality control of culture media in clinics, the bovine is a large mono-ovulating mammal with similar embryonic kinetics as humans during the studied developmental window. These model systems are critical to understand the effects of assisted reproduction without the confounding impact of infertility and without the limitations imposed by the scarcity of donated human samples and ethical issues. The data presented in this review come mostly from our own experimentation, publications, and collaborations. Together they demonstrate that the in vitro environment has a significant impact on embryos at the transcriptomic level and at the DNA methylation level.

3D printing novel in vitro cancer cell culture model systems for lung cancer stem cell study

2021 ◽  
Vol 122 ◽  
pp. 111914
Author(s):  
Alejandro Herreros-Pomares ◽  
Xuan Zhou ◽  
Silvia Calabuig-Fariñas ◽  
Se-Jun Lee ◽  
Susana Torres ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Culture ◽  
Stem Cell ◽  
3D Printing ◽  
Cancer Stem Cell ◽  
Model Systems ◽  
Cell Culture Model ◽  
Culture Model ◽  
Cell Study

Hyperphosphorylation of MEF2A in primary adipocytes correlates with downregulation of human GLUT4 gene promoter activity

2007 ◽  
Vol 292 (4) ◽  
pp. E1149-E1156 ◽  
Author(s):  
David P. Sparling ◽  
Beth A. Griesel ◽  
Ann Louise Olson
Keyword(s):  
Cell Culture ◽  
Transcription Factor ◽  
Promoter Activity ◽  
Model Systems ◽  
Regulatory Control ◽  
Cell Culture Model ◽  
In Vitro System ◽  
Culture Model ◽  
Enhancer Factor

GLUT4 promoter activity is regulated by hormonal, metabolic, and tissue-specific controls. This complicates the study of GLUT4 gene transcription, as no cell culture model adequately recapitulates these extracellular regulators. While investigating cultured primary adipocytes as a model system for GLUT4 transcription, we observed that GLUT4 mRNA was specifically and rapidly downregulated upon tissue dispersal. Downregulation of GLUT4 mRNA was mediated in part by loss of regulatory control by the trans-acting factors that control GLUT4 transcriptional activity [the myocyte enhancer factor 2 (MEF2) transcription factor family and the GLUT4 enhancer factor] and their cognate DNA binding sites in transgenic mice. The differences in GLUT4 transcription when whole adipose tissue and cell culture model systems are compared can be correlated to a posttranslational phosphorylation of the transcription factor MEF2A. The difference in the MEF2A phosphorylation state in whole tissue vs. isolated cells may provide a further basis for the development of an in vitro system that could recapitulate fully regulated GLUT4 promoter activity. Development of an in vitro system to reconstitute GLUT4 transcriptional regulation will further efforts to discern the molecular mechanisms that underlie GLUT4 expression.

scholarly journals In vitro Approaches to Model Breast Tumor Complexity

2021 ◽  
Author(s):  
Heizel Rosado-Galindo ◽  
Lyanne Suarez ◽  
Maribella Domenech
Keyword(s):  
Breast Cancer ◽  
Cell Culture ◽  
Tumor Microenvironment ◽  
Breast Tumor ◽  
Model Systems ◽  
Biological Complexity ◽  
Culture Model ◽  
Wide Range ◽  
Key Aspects

Cell culture technologies have provided biomedical researchers with fast and accessible tools to probe the breast tumor microenvironment. Exponential progress in fabrication methods combined with multiparametric approaches have enabled the development of cell culture model systems with enhanced biological complexity to identify key aspects that regulate breast cancer (BC) progression and therapeutic response. Yet, the culture parameters and conditions employed influence the behavior of tumor cells, thereby affecting its tissue biomimetic capabilities. In this chapter we review the wide range of culture platforms employed for the generation of breast tumor models and summarize their biomimetic capabilities, advantages, disadvantages and specific applications.

1120: Citrate and Vitamin E Blunt the SWL-Induced Free Radical Surge in an In-Vitro MDCK Cell Culture Model

2004 ◽  
Vol 171 (4S) ◽  
pp. 295-295
Author(s):  
Fernando C. Delvecchio ◽  
Ricardo M. Brizuela ◽  
Karen J. Byer ◽  
W. Patrick Springhart ◽  
Saeed R. Khan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Free Radical ◽  
Vitamin E ◽  
Mdck Cell ◽  
Cell Culture Model ◽  
Culture Model

Human 3-dimensional liver organoids: in vitro model systems to study liver diseases

2020 ◽  
Author(s):  
H Gaitantzi ◽  
C Cai ◽  
S Asawa ◽  
K Böttcher ◽  
M Ebert ◽  
...  
Keyword(s):  
Liver Diseases ◽  
In Vitro Model ◽  
Model Systems ◽  
3 Dimensional ◽  
Vitro Model ◽  
Liver Organoids

The experience of holding the cycles of assisted reproductive technology with defrost, a biopsy, genetic study and refreezing of embryos in patients with multiple unsuccessful implantations

2016 ◽  
pp. 166-170
Author(s):  
Y.V. Masliy ◽  
◽  
I.O. Sudoma ◽  
P.S. Mazur ◽  
D.A. Mykytenko ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Genetic Diagnosis ◽  
Morphological Characteristics ◽  
Implantation Rate ◽  
Ovarian Response ◽  
Reproductive Technologies ◽  
Comparative Genome Hybridization ◽  
Comparative Genome ◽  
Vitro Fertilization

The objective: to study the possibility of using frozen blastocysts for biopsy and genetic testing and performance measurement transfer euploeded 5–7-day-old embryos after thawing, biopsies, refreezing and thawing in patients with unsuccessful implantation. Patients and methods. The object of the study was the group of patients with repeated failure of implantation (4) in programs of auxiliary reproductive technologies (ART), subject to transfer to the uterus in total (i.e. in all the programs) for at least 6 good quality embryos based on morphological characteristics). All women had sufficient ovarian reserve. The patient was treated for infertility within the ART programs of the clinic of reproductive medicine "Nadiya" in the period from 2006 to 2016. The sample included couples who were not carriers of chromosomal rearrangements, without anomalies of the uterus (congenital and acquired: a doubling of the uterus, one-horned uterus, intrauterine membrane, synechia, submucous myoma of the uterus). All women had a positive ovarian response to controlled stimulation with gonadotropins (at least 7 oocytes) and a sufficient number of cryopreserved embryos. The first group (G1) included 64 women who trophectodermal a biopsy was performed on fresh blastocysts (in a loop controlled ovarian hyperstimulation). The second group (G2) were included 31 women who underwent thawing previously cryopreserved blastocysts trophectodermal re-biopsy and vitrification of blastocysts. Results. It was found that the performance of transfers euploid embryos that were vitrified, bioptrone and revitriphted, a little lower than those that were bioptrone fresh and vitrified only once. At the same time computationa genetic diagnosis previously vitrified blastocysts using comparative genome hybridization in patients with recurrent failed implantation allows to obtain a reasonable pregnancy rate (58%), implantation rate (33.3 %) and the birth of living children (45.1 %). Conclusion. Reprising biopropane embryos does not cause significant destructive impact and allows you to achieve pregnancy and birth of the alive child. Key words: in vitro fertilization, reusable unsuccessful implantation, a method of comparative genome hybridization, refreezing.

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