scholarly journals Generation of Miniaturized Ovaries by in Vitro Culture From Mouse Gonads

2021 ◽  
Author(s):  
Si Won Jang ◽  
Hoon Jang ◽  
Hyun Woo Choi
Keyword(s):  
Ovarian Development ◽  
Primordial Germ Cells ◽  
Nutrient Deficiency ◽  
Culture Conditions ◽  
The Body ◽  
Reproductive Age ◽  
Considerable Research ◽  
Complex Process ◽  
Genetic And Environmental Factors

Abstract The incidence of infertility among individuals of reproductive age has been growing due to genetic and environmental factors, and considerable research efforts are focused on solving this issue. Ovarian development is an overly complex process in the body, involving the interaction between primordial germ cells and gonad somatic cells. However, follicles located in the center of the in vitro ovary are poorly formed or die owing to ovarian complexity, nutrient deficiency, and signaling deficiency. In the present study, we optimized methods for dissociating gonads and culture conditions for the in vitro generation of miniaturized ovaries. The gonads from embryos were dissociated into cell masses and cultured on a Transwell-COL membrane for 3~5 weeks. Approximately 12 follicles were present per in vitro ovary. We observed that miniaturized ovaries successfully matured to MII oocytes in vitro from 150 to 100 µm gonad masses. This method will be useful for investigating follicle development and oocyte production.

Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro

Development ◽  
1978 ◽  
Vol 46 (1) ◽  
pp. 119-133
Author(s):  
Janet Heasman ◽  
C. C. Wylie
Keyword(s):  
Xenopus Laevis ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Structural Features ◽  
Culture Conditions ◽  
Structural Basis ◽  
Electron Microscopic ◽  
Microscopic Studies

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.

scholarly journals Direct repression of Nanog and Oct4 by OTX2 modulates contribution of epiblast-derived cells to germline and somatic lineage

Development ◽  
2021 ◽  
Author(s):  
Luca Giovanni Di Giovannantonio ◽  
Dario Acampora ◽  
Daniela Omodei ◽  
Vincenzo Nigro ◽  
Pasquale Barba ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Regulatory Network ◽  
Primordial Germ Cells ◽  
Culture Conditions ◽  
Specific Cell ◽  
Temporal Window ◽  
Pluripotency Gene ◽  
Gene Regulatory ◽  
Cell Culture Conditions

In mammals the pre-gastrula proximal epiblast gives rise to Primordial Germ Cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work showed that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here we show that OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs occurs also in mutant embryos. We propose that the OTX2 repressive control of Oct4 and Nanog is at the basis of the mechanism determining epiblast contribution to germline and somatic lineage.

Transport of Metal Oxide Nanoparticles Across Calu-3 Cell Monolayers Modelling the Air-Blood Barrier

2011 ◽  
Vol 3 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Christine Schulze ◽  
Ulrich F. Schaefer ◽  
Matthias Voetz ◽  
Wendel Wohlleben ◽  
Cornel Venzago ◽  
...  
Keyword(s):  
Metal Oxide ◽  
Pore Size ◽  
Metal Oxide Nanoparticles ◽  
Culture Conditions ◽  
The Body ◽  
Epithelial Barrier ◽  
Nanoparticle Transport ◽  
Small Pore ◽  
Barrier Model

Abstract As inhalation is the major exposure route for nanoparticles, the question if inhaled particles can overcome the respiratory epithelial barrier and hence enter the body is of great interest. Here, we adapted the for soluble substances well established Calu-3 in vitro air-blood barrier model to the use of nanoparticle transport testing. As the usually used filter supports hindered particle transport due to their small pore size, supports with a pore size of 3 μm had to be used. On those filters, barrier and transport characteristics of the cells were tested and culture conditions changed to obtain optimal conditions. Functionality was confirmed with transport experiments with polystyrene model particles prior to testing of industrially relevant engineered metal oxide particles. Except for CeO2 nanoparticles, no transport across the epithelial barrier model could be detected. Paracellular permeability and barrier function was not affected by any of the nanoparticles, except for ZrO2.

Factors influencing the efficiency of isolation and culture of human embryonic germ cells

2004 ◽  
Vol 1 (3) ◽  
pp. 191-196
Author(s):  
Hua Jin-Lian ◽  
Dou Zhong-Ying ◽  
Xu Xiao-Ming ◽  
Li Song ◽  
Yang Yu-Ai ◽  
...  
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Essential Amino Acids ◽  
The Body ◽  
Leukaemia Inhibitory Factor ◽  
Basic Medium ◽  
Feeder Cells ◽  
Embryonic Fibroblasts ◽  
Factors Influencing

AbstractEmbryonic germ (EG) cells are pluripotent cells derived from the primordial germ cells of gonads, gonadal ridges and mesenteries, and analogies of foetuses, with the ability to undergo both self-renewal and multiple differentiation. These cells can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cell of the body. The present paper investigates some factors influencing the efficiency of isolation and culture of human EG cells, such as foetus age, culture serum, added cytokines and feeder cells. The results demonstrate that foetuses of 7–12 weeks are optimal for in vitro culture of human EG cells. The basic medium consisted of DMEM, 1×non-essential amino acids, 2 mM l-glutamine and 1 mM sodium pyruvate. Supplementation with 15% foetal bovine serum, 4 ng/ml human recombinant leukaemia inhibitory factor, 4 ng/ml basic fibroblast growth factor and 20 ng/ml stem cell factor clearly improved the efficiency of isolation and culture of human EG cells. Murine embryonic fibroblasts were better feeder cells than human embryonic fibroblasts, bovine embryonic fibroblasts or STO cell line.

scholarly journals Osteocyte Characterization on Polydimethylsiloxane Substrates for Microsystems Applications

2012 ◽  
Vol 16 ◽  
pp. 27-42 ◽  
Author(s):  
Spencer L. York ◽  
Ahmad R. Arida ◽  
Karan S. Shah ◽  
Palaniappan Sethu ◽  
Marnie M. Saunders
Keyword(s):  
Mechanical Load ◽  
Collagen Type I ◽  
Bone Cell ◽  
Culture Conditions ◽  
Substrate Material ◽  
Research Field ◽  
The Body ◽  
Type I

In the body, osteocytes reside in lacunae, lenticular shaped cavities within mineralized bone. These cells are linked to each other and surface-residing osteoblasts via physical channels known as gap junctions. It has been suggested that osteocytes sense mechanical load applied to bone and relay that signal to osteoclasts and osteoblasts. Currentin vitroandin vivomodels of mechanotransduction face temporal and spatial barriers. Recent advances in polydimethylsiloxane (PDMS) based microfabrication techniques may be able to overcome some of these hurdles. However, before the bone research field can effectively utilize microsystems techniques, fundamental groundwork must be completed. This study characterized the behaviour of osteocytes on PDMS coated with collagen type I (CTI) and provides the framework for bone cell mechanotransduction studies using microsystems. The goal was to determine whether osteocytes were adversely affected by the substrate material by comparing their behaviour to a standard glass substrate. In addition, optimal culture conditions and time points for growing osteocytes on PDMS substrates were determined. Results of this study suggested that use of PDMS does not adversely affect osteocyte behaviour. Furthermore, the results demonstrated that osteocytes should be cultured for no less than 72 hours prior to experimentation to allow the establishment and maintenance of phenotypic characteristics. These results completed essential groundwork necessary for further studies regarding osteocytes in microsystems modelling utilizing PDMS.

scholarly journals Epigenetic Manipulation Facilitates the Generation of Skeletal Muscle Cells from Pluripotent Stem Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Tomohiko Akiyama ◽  
Shunichi Wakabayashi ◽  
Atsumi Soma ◽  
Saeko Sato ◽  
Yuhki Nakatake ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Myogenic Differentiation ◽  
Ectopic Expression ◽  
Cell Types ◽  
Culture Conditions ◽  
The Body ◽  
Specific Cell ◽  
Modifying Factors

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation.

Tissue and cell interactions in mammalian PGC development

Development ◽  
2021 ◽  
Vol 148 (23) ◽  
Author(s):  
Christopher B. Cooke ◽  
Naomi Moris
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Cell Types ◽  
Supporting Cell ◽  
Tissue Level ◽  
Neighbouring Cell ◽  
Tissue Interactions ◽  
Considerable Research ◽  
Robust Regulation

ABSTRACT Primordial germ cells (PGCs) form early in embryo development and are crucial precursors to functioning gamete cells. Considerable research has focussed on identifying the transcriptional characteristics and signalling pathway requirements that confer PGC specification and development, enabling the derivation of PGC-like cells (PGCLCs) in vitro using specific signalling cocktails. However, full maturation to germ cells still relies on co-culture with supporting cell types, implicating an additional requirement for cellular- and tissue-level regulation. Here, we discuss the experimental evidence that highlights the nature of intercellular interactions between PGCs and neighbouring cell populations during mouse PGC development. We posit that the role that tissue interactions play on PGCs is not limited solely to signalling-based induction but extends to coordination of development by robust regulation of the proportions and position of the cells and tissues within the embryo, which is crucial for functional germ cell maturation. Such tissue co-development provides a dynamic, contextual niche for PGC development. We argue that there is evidence for a clear role for inter-tissue dependence of mouse PGCs, with potential implications for generating mammalian PGCLCs in vitro.

scholarly journals Development of Microfluidic, Serum-Free Bronchial Epithelial Cells-on-a-Chip to Facilitate a More Realistic In vitro Testing of Nanoplastics

2021 ◽  
Vol 3 ◽  
Author(s):  
Govind Gupta ◽  
Srikanth Vallabani ◽  
Romain Bordes ◽  
Kunal Bhattacharya ◽  
Bengt Fadeel
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Cellular Uptake ◽  
Culture Conditions ◽  
Bronchial Epithelial Cells ◽  
The Body ◽  
Bronchial Epithelial ◽  
Serum Free ◽  
Static Conditions

Most cell culture models are static, but the cellular microenvironment in the body is dynamic. Here, we established a microfluidic-based in vitro model of human bronchial epithelial cells in which cells are stationary, but nutrient supply is dynamic, and we used this system to evaluate cellular uptake of nanoparticles. The cells were maintained in fetal calf serum-free and bovine pituitary extract-free cell culture medium. BEAS-2B, an immortalized, non-tumorigenic human cell line, was used as a model and the cells were grown in a chip within a microfluidic device and were briefly infused with amorphous silica (SiO2) nanoparticles or polystyrene (PS) nanoparticles of similar primary sizes but with different densities. For comparison, tests were also performed using static, multi-well cultures. Cellular uptake of the fluorescently labeled particles was investigated by flow cytometry and confocal microscopy. Exposure under dynamic culture conditions resulted in higher cellular uptake of the PS nanoparticles when compared to static conditions, while uptake of SiO2 nanoparticles was similar in both settings. The present study has shown that it is feasible to grow human lung cells under completely animal-free conditions using a microfluidic-based device, and we have also found that cellular uptake of PS nanoparticles aka nanoplastics is highly dependent on culture conditions. Hence, traditional cell cultures may not accurately reflect the uptake of low-density particles, potentially leading to an underestimation of their cellular impact.

scholarly journals The use of chitosan-based biomaterials for the treatment of hard-healing wounds

2019 ◽  
Vol 73 ◽  
pp. 768-781
Author(s):  
Marta Kędzierska ◽  
Katarzyna Miłowska
Keyword(s):  
Wound Healing ◽  
Chronic Wounds ◽  
Healing Process ◽  
Medical Engineering ◽  
Fibrin Clot ◽  
The Body ◽  
Complex Process ◽  
Other Substances

Wound healing is a complex process that engages skin cells, the blood, the immune system and a number of circulating substances in the body. Infections, contamination of the wound or a vast area of damage complicate and delay the natural process of skin regeneration. The incidence of hard-to-heal wounds is an increasingly common problem, because they can significantly impair the quality of life of the patient. For this reason, it is extremely important to look for factors (drugs, dressings or other substances) that could accelerate and relieve wound healing. Among many compounds in the area of medical engineering interest, attention should be paid to natural polysaccharides, e.g. chitosan and alginate. This article is devoted to biomaterials that play an important role in the treatment of chronic wounds. These include the following: hydrogels, non-wovens, membranes and chitosan sponges as well as chitosan-alginate composites or chitosan composites combined with zinc oxide and nanosilver. The material, which has chitosan as a base, works on all stages of the healing process. Many in vitro, in vivo and clinical studies that provide the basis for using chitosan materials as a substitute for conventional bandages and dressings have been carried out. At the stage of hemostasis, it accelerates platelet aggregation and the formation of a fibrin clot. In the inflamed stage, they cause the proliferation of neutrophils and macrophages that cleanse the wound, releasing cytokines at the wound site. Studies have shown that chitosan mimics the native extracellular matrix, providing the optimal microenvironment for the wound.

scholarly journals Improvement of developmental competence of aged porcine oocytes by means of the synergistic effect of insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF)

2011 ◽  
Vol 50 (No. 7) ◽  
pp. 300-310 ◽  
Author(s):  
I. Petrová ◽  
R. Rajmon ◽  
M. Sedmíková ◽  
Z. Kuthanová ◽  
F. Jílek ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Insulin Like Growth Factor ◽  
Large Numbers ◽  
Complex Process ◽  
Epidermal Growth ◽  
The Many

There is a constant increase in demand for large numbers of high-quality oocytes of domestic mammals for various reproductive biotechnologies. Prolonged in vitro culture represents one of the many possible ways of meeting this demand. However, oocytes further cultured in vitro after reaching the stage of metaphase II undergo the complex process called “ageing”, which decreases the quality of oocytes. The aim of the present study was to improve the culture conditions for in vitro matured porcine oocytes by supplement of the epidermal growth factor (EGF) or the insulin-like growth factor 1 (IGF-1) in order to reduce the adverse effects of ageing. Supplement of either EGF or IGF-1 had no significant effect on the ageing of porcine oocytes. Significant effects were demonstrated when the oocytes were cultured with both EGF and IGF-1. A combination of 10 µg EGF/ml with 25 ng IGF-1/ml was the most effective. Oocytes aged under these conditions retained very good developmental competence. We observed development to the morula (21%) or blastocyst (25%) stage in oocytes aged for 1 day with EGF and IGF-1. Porcine oocytes matured in vitro are more resistant to the ageing when cultured in the presence of both EGF and IGF-1 and these conditions retain an elevated developmental competence for a certain time.  

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