Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the noninvasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.
Angiogenesis, which refers to the formation of new blood vessels from already existing vessels, is a promising therapeutic target and a complex multistep process involving many different factors. Pericytes (PCs) are attracting attention as they are considered to make significant contributions to the maturation and stabilisation of newly formed vessels, although not much is known about the precise mechanisms involved. Since there is no single specific marker for pericytes, in vivo models may complicate PC identification and the study of PCs in angiogenesis would benefit from in vitro models recapitulating the interactions between PCs and endothelial cells (ECs) in a three-dimensional (3D) configuration. In this study, a 3D in vitro co-culture microvessel model incorporating ECs and PCs was constructed by bottom-up tissue engineering. Angiogenesis was induced in the manner of sprout formation by the addition of a vascular endothelial cell growth factor. It was found that the incorporation of PCs prevented expansion of the parent vessel diameter and enhanced sprout formation and elongation. Physical interactions between ECs and PCs were visualised by immunostaining and it disclosed that PCs covered the EC monolayer from its basal side in the parent vessel as well as angiogenic sprouts. Furthermore, the microvessels were visualized in 3D by using a non-invasive optical coherence tomography (OCT) imaging system and sprout features were quantitatively assessed. It revealed that the sprouts in EC–PC co-culture vessels were longer and tighter than those in EC mono-culture vessels. The combination of the microvessel model and the OCT system analysis can be useful for the visualisation and demonstration of the multistep process of angiogenesis, which incorporates PCs.
For augmentation or reconstruction of urinary bladder after cystectomy, bladder urothelium derived from human induced pluripotent stem cells (hiPSCs) has recently received focus. However, previous studies have only shown the emergence of cells expressing some urothelial markers among derivatives of hiPSCs, and no report has demonstrated the stratified structure, which is a particularly important attribute of the barrier function of mature bladder urothelium. In present study, we developed a method for the directed differentiation of hiPSCs into mature stratified bladder urothelium. The caudal hindgut, from which the bladder urothelium develops, was predominantly induced via the high-dose administration of CHIR99021 during definitive endoderm induction, and this treatment subsequently increased the expressions of uroplakins. Terminal differentiation, characterized by the increased expression of uroplakins, CK13, and CK20, was induced with the combination of Troglitazone + PD153035. FGF10 enhanced the expression of uroplakins and the stratification of the epithelium, and the transwell culture system further enhanced such stratification. Furthermore, the barrier function of our urothelium was demonstrated by a permeability assay using FITC-dextran. According to an immunohistological analysis, the stratified uroplakin II-positive epithelium was observed in the transwells. This method might be useful in the field of regenerative medicine of the bladder.
Elucidating the mechanisms underlying sprouting angiogenesis and permeability should enable the developmentof more effective therapies for various diseases, including retinopathy, cancer, and other vascular disorders. Wefocused on epidermal growth factor-like domain 7 (EGFL7) which plays an important role in NOTCH signalingand in the organization of angiogenic sprouts. We developed anEGFL7-knockdownin vitromicrovessel modeland investigated the effect of EGFL7 at a tissue level. We foundEGFL7knockdown suppressed VEGF-A-inducedsprouting angiogenesis accompanied by an overproduction of endothelialfilopodia and reduced collagen IVdeposition at the basal side of endothelial cells. We also observed impaired barrier function which reflected aninflammatory condition. Furthermore, our results showed that proper formation of adherens junctions andphosphorylation of VE-cadherin was disturbed. In conclusion, by using a 3D microvessel model we identifiednovel roles for EGFL7 in endothelial function during sprouting angiogenesis.
Angiogenesis is the formation of new capillaries from pre-existing blood vessels and participates in proper vas-culature development. In pathological conditions such as cancer, abnormal angiogenesis takes place. Angiogen-esis is primarily carried out by endothelial cells, the innermost layer of blood vessels. The vascular endothelialgrowth factor-A (VEGF-A) and its receptor-2 (VEGFR-2) trigger most of the mechanisms activating and regulat-ing angiogenesis, and have been the targets for the development of drugs. However, most experimental assaysassessing angiogenesis rely on animal models. We report anin vitromodel using a microvessel-on-a-chip. Itmimics an effective endothelial sprouting angiogenesis event triggered from an initial microvessel using a singleangiogenic factor, VEGF-A. The angiogenic sprouting in this model is depends on the Notch signaling, as observedin vivo. This model enables the study of anti-angiogenic drugs which target a specific factor/receptor pathway, asdemonstrated by the use of the clinically approved sorafenib and sunitinib for targeting the VEGF-A/VEGFR-2pathway. Furthermore, this model allows testing simultaneously angiogenesis and permeability. It demonstratesthat sorafenib impairs the endothelial barrier function, while sunitinib does not. Suchin vitrohuman model pro-vides a significant complimentary approach to animal models for the development of effective therapies.
The intestine acts as a center for nutrient and water absorption at the epithelium and plays an important role in immunity. Considering the complexity of its function and roles in living systems, a physiologically relevant gut in vitro model is desirable in both basic biology and the analysis of effects of some substances on functions of the gut; these analyses include the screening of drug and food candidates with regard to intestinal disorder at an early stage of medical development. In the present study, we constructed a three-dimensional (3D) gut model using human absorptive enterocytes (CACO-2 cells) by reconstitution of the gut epithelial sheet restricted on a high-reproducible ductal scaffold of collagen gel. Moreover, using the 3D gut model, we evaluated the morphology at cellular and tissue levels and conducted a phenotypic analysis of the intestinal physiological functions, which involved a permeability assay mimicking barrier disruption inducing inflammation and an absorption assay reflecting ingestive effects. The ductal structure, in vivo-like 3D epithelial structures, epithelial barrier, and effective absorptive function characterized the 3D gut model. The epithelial cells formed a villus-like buckling epithelium, vertical microvilli of increased density on the cell surface, and a crypt-like localized cell proliferating region. The mature shape of the epithelium may contribute to mimicking barrier function and effective absorption compared with that in the 2D gut model. Furthermore, we successfully mimicked the dextran sodium sulfate-induced epithelial barrier dysfunction as a trigger phenomenon of gut inflammation in the 3D gut model. The integrity of the epithelium and phenotypic analysis of the intestinal physiological functions in the simple and reproducible 3D gut model will allow for a drug screening system for assessing the effects on the functions of the gut epithelium from the lumen side.
Three-dimensional (3D) in vitro microvasculature in a polydimethylsiloxane-based microdevice was developed as a physiologically relevant model of angiogenesis. The angiogenic process is monitored using stage-top optical coherence tomography (OCT). OCT allows non-invasive monitoring of the 3D structures of the prepared host microvasculature and sprouted neovasculature without fluorescence staining. OCT monitoring takes only a few minutes to scan through the several-millimetre scale range, which provides the advantage of rapid observation of living samples. The obtained OCT cross-sectional images capture 3D features of the angiogenic sprouting process and provide information on the dynamics of luminal formation. The stage-top system used in this study enables the observer to visualize the in vitro dynamics of 3D cultured cells simply and conveniently, offering an alternative monitoring method for studies on angiogenesis and providing quantitative information about vascular morphological changes.
As a skin model, sheet like epidermal tissues cultured on insert well are widely used in assays Thickness of the skin sheets is known as one of these phenotypes, and it is effective feature for capturing morphological changes As a method for measuring the thickness, it is common to prepare a tissue section by the paraffin embedding method and measure the length from the upper part to the lower part of the tissue with a microscope However, since this method is invasive evaluation method, subsequent detailed assay cannot be performed In addition, because the evaluation is based on a tissue section, it is a local evaluation, and it is difficult to capture features such as distribution of tissue thickness Furthermore, embedding with paraffin is difficult to maintain the original shape of the tissue Therefore, in order to perform more appropriate thickness evaluation, a new method for non invasively observing and quantifying the tissue on the insert well is required In recent years, optical coherence tomography (OCT) has become widespread as a three dimensional diagnostic method for the fund us. OCT is a technique for rendering a three dimensional tissue as a tomographic image, and the near infrared light, used by OCT as a light source, is suitable for biological permeabili ty and low in cytotoxicity. In this study, the OCT imaging device Cell3iMager Estier (SCREEN Holdings Co., Ltd.) was used to acquire 3D images of non invasively stacked cell tissues, and we aimed to establish a n ew method for measuring of tissue thickness distribution. The cell sheet on the insert well was observed using Cell3iMager Estier , and a three dimensional image was obtained. Based on the obtained three dimensional image, cell regions were extracted by image processing, and the thickness distribution of the entire sheet was ca lcu lated. We report that it is possible to measure non invasively the change of the thickness distribution of the whole cell sheet by the experimental condition.
3 D ex vivo platforms are being diligently evaluated as better predictive drug efficacy testing tools in preclinical as well as clinical space in the quest for profiling of novel anticancer entities (as single or two drug combinations) Within this context, there has been growing need in improved imaging and analysis of complex 3 D structures Optical Coherent tomography ( has been widely used as one of the most important test in ophthalmology It is a non invasive imaging technology that renders the high resolution and cross sectional images from retina Given its tremendous use in in vivo application, in recently, the technology has been applied in 3 D in vitro ex vivo applications for performing imaging of spheroids/ organoids and large tissues This technology allows to perform l arge tissue imagin g, non invasive monitoring of macro and sprouted neo vasculature without the need for fluorescent staining for providing quantitative information about the vascular morphological changes, thereby allowing for the evaluation of anti angiogenic drugs in real time Aim To develop a novel OCT based technology for imaging and analysis of 3 D structures, such as, spheroid / tissues for g rowth morphological evaluation, q uantification of internal cavities d rug sensitivity t esting to capture the events leading to tumor cell death and assessing effect of anti angiogenic drugs Material Methods and Results All cells were cultured in a suitable manner, and cells were imaged by spectrum domain OCT (SD OCT) system The SD OCT system is outlined below The samples were imaged from the bottom surface Original images obtained by the OCT system contained noise (such as from the collagen gel surrounding the sample) To reduce this noise, image processing was applied for the collected original OCT images using original our software ( and ImageJ image processing software ( as follows The images were subsequently processed with filters The images were then converted into binary images so that the cell area is white and all other areas are black Each feature values ( cavities etc were analyzed
While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggested that these 22 parameters can be potentially employed to evaluate the quality of bovine before ET.