428. Normal follicle growth and maturation in a three-dimensional in vitro culture system: follicular environment manipulation and assessment of oocyte outcomes

2008 ◽  
Vol 20 (9) ◽  
pp. 108
Author(s):  
K. R. Dunning ◽  
L. K. Akison ◽  
D. L. Russell ◽  
R. J. Norman ◽  
R. L. Robker
Keyword(s):  
Culture System ◽  
Cumulus Cell ◽  
Three Dimensional ◽  
Oocyte Quality ◽  
Metabolic Effects ◽  
Follicle Growth ◽  
Cumulus Expansion ◽  
Follicle Diameter

In vivo, the oocyte matures in a niche environment surrounded by somatic cells, and later in ovarian follicular development, by follicular fluid. Maternal diet influences the environment in which an oocyte matures but the mechanisms by which an altered metabolic profile, such as hyperinsulinemia, affects oocyte quality are not known. We investigated the use of a three dimensional follicle culture system allowing direct manipulation of the follicular environment thus circumventing systemic hormonal and metabolic effects. Secondary follicles (113.4 ± 1.02µm, n = 54) were isolated from mice at d12, encapsulated individually in 2µl of alginate matrix, and cultured in aMEM/5%FCS/10 mIU/mL LH/100 mIU FSH at 37°C/5%CO2, with media sampling and replacement every second day. Following 12 days of culture there was a significant 3-fold increase in follicle diameter (320 ± 10.1µm, n = 51). Histological analysis showed normal follicular morphology and antrum formation. Analysis of oestradiol (15.0ng/mL), androstenedione (7.8ng/mL) and progesterone (23.7ng/mL) in the media at d12 confirmed normal steroidogenesis and differentiation. Treatment of follicles with an ovulatory stimulus (1.5IU/mL hCG/5ng/mL Egf), resulted in cumulus expansion and hyaluronan localising to the cumulus oocyte complex (COC) and follicular basement membrane. These analyses were consistent with follicle growth and induction of ovulation in vivo. Further, COCs isolated from follicles and matured in vitro (IVM) in the presence of Egf and FSH, underwent cumulus expansion (CEI 2.8 ± 0.2) and were capable of fertilisation and blastocyst development. LH did not induce IVM COC expansion (CEI 1.36 ± 0.2), reflecting the normal in vivo differentiation process. However, culturing follicles in high insulin (5ug/mL) led to a significant increase in the degree of IVM cumulus expansion in response to LH (CEI 2.1 ± 0.3) indicating inappropriate cumulus cell differentiation, which may lead to poorer oocyte quality. These results demonstrate that this technique recapitulates normal in vivo folliculogenesis and is useful for manipulation of the follicular environment and assessment of oocyte outcomes.

scholarly journals Cumulus Cell Expansion, Its Role in Oocyte Biology and Perspectives of Measurement: A Review

2015 ◽  
Vol 45 (4) ◽  
pp. 212-225 ◽  
Author(s):  
J. Nevoral ◽  
M. Orsák ◽  
P. Klein ◽  
J. Petr ◽  
M. Dvořáková ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hyaluronic Acid ◽  
High Performance ◽  
Uv Light ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cumulus Expansion

Abstract Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA), spectrophotometry, and high-performance liquid chromatography (HPLC) in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.

scholarly journals Sex steroids influence organizational but not functional decidualization of feline endometrial cells in a 3D culture system†

2019 ◽  
Vol 101 (5) ◽  
pp. 906-915 ◽  
Author(s):  
Kathryn Wilsterman ◽  
Xinmiao Bao ◽  
Allegra D Estrada ◽  
Pierre Comizzoli ◽  
George E Bentley
Keyword(s):  
Sex Steroids ◽  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
In Vitro Models ◽  
Sex Steroid ◽  
Endometrial Cells ◽  
Organizational Patterns

Abstract Successful implantation requires complex signaling between the uterine endometrium and the blastocyst. Prior to the blastocyst reaching the uterus, the endometrium is remodeled by sex steroids and other signals to render the endometrium receptive. In vitro models have facilitated major advances in our understanding of endometrium preparation and endometrial–blastocyst communication in mice and humans, but these systems have not been widely adapted for use in other models which might generate a deeper understanding of these processes. The objective of our study was to use a recently developed, three-dimensional culture system to identify specific roles of female sex steroids in remodeling the organization and function of feline endometrial cells. We treated endometrial cells with physiologically relevant concentrations of estradiol and progesterone, either in isolation or in combination, for 1 week. We then examined size and density of three-dimensional structures, and quantified expression of candidate genes known to vary in response to sex steroid treatments and that have functional relevance to the decidualization process. Combined sex steroid treatments recapitulated organizational patterns seen in vivo; however, sex steroid manipulations did not induce expected changes to expression of decidualization-related genes. Our results demonstrate that sex steroids may not be sufficient for complete decidualization and preparation of the feline endometrium, thereby highlighting key areas of opportunity for further study and suggesting some unique functions of felid uterine tissues.

scholarly journals Bacterial Infection-Mimicking Three-Dimensional Phagocytosis and Chemotaxis in Electrospun Poly(ε-caprolactone) Nanofibrous Membrane

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 569
Author(s):  
Seung-Jun Lee ◽  
Perry Ayn Mayson A Maza ◽  
Gyu-Min Sun ◽  
Petr Slama ◽  
In-Jeong Lee ◽  
...  
Keyword(s):  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
Culture Conditions ◽  
Lung Epithelial Cells ◽  
Nanofibrous Membrane ◽  
Infection Model ◽  
Layer System

In this study, we developed a three-dimensional (3D) in vitro infection model to investigate the crosstalk between phagocytes and microbes in inflammation using a nanofibrous membrane (NM). Poly(ε-caprolactone) (PCL)-NMs (PCL-NMs) were generated via electrospinning of PCL in chloroform. Staphylococcus aureus and phagocytes were able to adhere to the nanofibers and phagocytes engulfed S. aureus in the PCL-NM. The migration of phagocytes to S. aureus was evaluated in a two-layer co-culture system using PCL-NM. Neutrophils, macrophages and dendritic cells (DCs) cultured in the upper PCL-NM layer migrated to the lower PCL-NM layer containing bacteria. DCs migrated to neutrophils that cultured with bacteria and then engulfed neutrophils in two-layer system. In addition, phagocytes in the upper PCL-NM layer migrated to bacteria-infected MLE-12 lung epithelial cells in the lower PCL-NM layer. S. aureus-infected MLE-12 cells stimulated the secretion of tumor necrosis factor-α and IL-1α in 3D culture conditions, but not in 2D culture conditions. Therefore, the PCL-NM-based 3D culture system with phagocytes and bacteria mimics the inflammatory response to microbes in vivo and is applicable to the biomimetic study of various microbe infections.

In Vitro Follicle Growth, Maturation, and Gene Expression: Three-Dimensional (3D) Matrigel Culture versus Two-Dimensional Culture (2D)

2021 ◽  
Vol 11 (12) ◽  
pp. 2512-2515
Author(s):  
Byoung-San Moon ◽  
Seungki Lee ◽  
Jung Kyu Choi
Keyword(s):  
Gene Expression ◽  
Three Dimensional ◽  
Ovarian Follicles ◽  
Dimensional System ◽  
Two Dimensional ◽  
Follicle Growth ◽  
Adult Mice ◽  
Three Dimensional Culture

This research aimed to compare the In Vitro growth, maturation, and gene expression in ovarian follicles collected from adult mice (6–8-week-old) between two-dimensional and three-dimensional cultures. First, we confirmed In Vitro follicle growth and maturation using adult mice with outbred characteristics and analyzed the expression of genes related to follicular development. We found that the three-dimensional culture system utilizing a Matrigel drop to create an in vivo-like ovarian microenvironment was more efficient in terms of In Vitro follicle growth, maturation, and gene expression than the two-dimensional system (non-physical environment). The in vivo-like three-dimensional culture of ovarian follicles provides new insights into the physiology and development of ovarian follicle in vivo, thereby contributing to new strategies to improve female fertility.

scholarly journals Three-Dimensional Culture System of Cancer Cells Combined with Biomaterials for Drug Screening

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2754 ◽  
Author(s):  
Teruki Nii ◽  
Kimiko Makino ◽  
Yasuhiko Tabata
Keyword(s):  
Cell Culture ◽  
Cancer Cells ◽  
Drug Screening ◽  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
New Drugs ◽  
Biological Research

Anticancer drug screening is one of the most important research and development processes to develop new drugs for cancer treatment. However, there is a problem resulting in gaps between the in vitro drug screening and preclinical or clinical study. This is mainly because the condition of cancer cell culture is quite different from that in vivo. As a trial to mimic the in vivo cancer environment, there has been some research on a three-dimensional (3D) culture system by making use of biomaterials. The 3D culture technologies enable us to give cancer cells an in vitro environment close to the in vivo condition. Cancer cells modified to replicate the in vivo cancer environment will promote the biological research or drug discovery of cancers. This review introduces the in vitro research of 3D cell culture systems with biomaterials in addition to a brief summary of the cancer environment.

scholarly journals Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation

2017 ◽  
Vol 114 (29) ◽  
pp. E5796-E5804 ◽  
Author(s):  
Ye Yuan ◽  
Lee D. Spate ◽  
Bethany K. Redel ◽  
Yuchen Tian ◽  
Jie Zhou ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Fourfold Increase

Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called “FLI medium,” improves nuclear maturation of oocytes in cumulus–oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus–oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.

270 MITOCHONDRIA AND REACTIVE OXYGEN SPECIES COLOCALIZATION IN IN VIVO AND IN VITRO MATURED OOCYTES FROM SUPEROVULATED ADULT EWES

2011 ◽  
Vol 23 (1) ◽  
pp. 233
Author(s):  
B. Ambruosi ◽  
N. A. Martino ◽  
M. Filioli Uranio ◽  
F. Silvestre ◽  
F. Binetti ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cumulus Cell ◽  
Reactive Oxygen ◽  
Oocyte Quality ◽  
Nuclear Chromatin ◽  
Oxygen Species ◽  
Fluorescent Intensity ◽  
Intracellular Ros

Analyses of energy and redox status parameters are emerging technologies to improve oocyte quality assessment. Mitochondria (mt) play a vital role in the oocyte to support maturation, fertilization, and pre-implantation development. They are the major source of reactive oxygen species (ROS) produced during oxidative phosphorylation, which are not only by-products of cell metabolism but also important molecules for regulation of intracellular cell signaling. The aim of the present study was to test for mt/ROS colocalization in oocytes recovered from superovulated adult ewes and examined after in vivo or in vitro maturation (IVM). Cumulus–oocyte complexes of 8 superovulated (fluorogestone acetate + D-cloprostenol for oestrus synchronization, pFSH/pLH and eCG for superovulation) adult (2 to 8 years of age) ewes were recovered (ovariohysterectomy by midventral laparotomy performed 54 h after vaginal sponge removal) either from flushing oviducts (oviducal oocytes) or from ovarian growing follicles (1–5 mm in diameter; follicular oocytes). Follicular oocytes were analysed after IVM (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent nuclear chromatin, mt, and ROS evaluation. Hoechst 33258 and Mitotracker Orange CMTM Ros were used to label nuclear chromatin and mt (Ambruosi et al. 2009) and 2′,7′-dichloro-dihydro-fluorescein diacetate was used for ROS labelling (Hashimoto et al. 2000 Mol. Reprod. Dev. 57, 353–360). Oocytes at the metaphase II (MII) stage showing regular ooplasmic size (>130 μm in diameter) and morphology were selected for confocal analysis of mt/ROS fluorescence distribution, intensity, and colocalization. Forty oviducal MII oocytes recovered from 8 ewes were analysed. Thirty-two oocytes recovered from the ovaries of 4 ewes underwent IVM, and 23 out of 32 (72%) reached nuclear maturation and were analysed. The rate of oocytes showing perinuclear mt distribution pattern did not differ between oviducal and IVM oocytes (33%, 13/40 v. 43%, 10/23; not significant). In these oocytes, fluorescent intensity of mt labelling and intracellular ROS levels did not differ between oviducal and IVM ooocytes (996.27 ± 363.57 v. 798.13 ± 275.91; not significant; and 1808.11 ± 442.78 v. 1473.29 ± 662.49, for mt and ROS, respectively; not significant), whereas mt/ROS colocalization was significantly higher in ovulated oocytes than in IVM oocytes (Pearson coefficient 0.67 ± 0.11 v. 0.39 ± 0.19, respectively; P < 0.001). In conclusion, in oocytes of adult ewes, mt aggregation, apparent energy status, and intracellular ROS levels do not differ between ovulated and IVM oocytes, but mt/ROS colocalization differs between the 2 groups. As it was reported for other cell systems that such a difference can be indicative of healthy status of ovulated oocytes, we suggest that mt/ROS colocalization could be considered as a suitable marker of oocyte quality. Financial support was provided by Fondazione Cassa di Risparmio di Puglia 2008. Project: Salvaguardia di razze ovine autoctone pugliesi (R.U. DPA Resp. Sci. Prof. M. E. DellAquila).

scholarly journals Design of biomimetic cellular scaffolds for co-culture system and their application

2017 ◽  
Vol 8 ◽  
pp. 204173141772464 ◽  
Author(s):  
Yun-Min Kook ◽  
Yoon Jeong ◽  
Kangwon Lee ◽  
Won-Gun Koh
Keyword(s):  
Extracellular Matrix ◽  
Culture System ◽  
Three Dimensional ◽  
Biochemical Properties ◽  
Culture Systems ◽  
Specific Tissue ◽  
Multicellular Interactions ◽  
Cell Functionality

The extracellular matrix of most natural tissues comprises various types of cells, including fibroblasts, stem cells, and endothelial cells, which communicate with each other directly or indirectly to regulate matrix production and cell functionality. To engineer multicellular interactions in vitro, co-culture systems have achieved tremendous success achieving a more realistic microenvironment of in vivo metabolism than monoculture system in the past several decades. Recently, the fields of tissue engineering and regenerative medicine have primarily focused on three-dimensional co-culture systems using cellular scaffolds, because of their physical and biological relevance to the extracellular matrix of actual tissues. This review discusses several materials and methods to create co-culture systems, including hydrogels, electrospun fibers, microfluidic devices, and patterning for biomimetic co-culture system and their applications for specific tissue regeneration. Consequently, we believe that culture systems with appropriate physical and biochemical properties should be developed, and direct or indirect cell–cell interactions in the remodeled tissue must be considered to obtain an optimal tissue-specific microenvironment.

Embryonic Stem Cell-Derived Embryoid Bodies in Three-Dimensional Culture System Form Hepatocyte-Like Cells in Vitro and in Vivo

2004 ◽  
Vol 10 (11-12) ◽  
pp. 1716-1724 ◽  
Author(s):  
Tetsuya Imamura ◽  
Li Cui ◽  
Ruifeng Teng ◽  
Kohei Johkura ◽  
Yasumitsu Okouchi ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Culture System ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
System Form ◽  
Three Dimensional Culture
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