142 Mimicking the follicular environment: in vitro maturation of prepubertal ovine oocytes in a liquid marble bioreactor as a 3-dimensional culture system

2022 ◽  
Vol 34 (2) ◽  
pp. 309
Author(s):  
D. Bebbere ◽  
F. Ariu ◽  
S. Nieddu ◽  
S. Ledda
Keyword(s):  
Culture System ◽  
In Vitro Maturation ◽  
3 Dimensional ◽  
Liquid Marble

45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 135 ◽  
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
M. Anzar
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Time Intervals ◽  
Early Embryo Development ◽  
In Vitro Culture System ◽  
Bovine Oocytes

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

scholarly journals Effect of single-oocyte culture system on in vitro maturation and developmental competence in mice

2014 ◽  
Vol 13 (3) ◽  
pp. 153-159 ◽  
Author(s):  
Manami Nishio ◽  
Yumi Hoshino ◽  
Kentaro Tanemura ◽  
Eimei Sato
Keyword(s):  
Culture System ◽  
In Vitro Maturation ◽  
Developmental Competence

scholarly journals Cumulus co-culture system does not improve the in-vitro maturation (IVM) of oocytes in mice

2016 ◽  
Vol 21 (2) ◽  
pp. 109-113
Author(s):  
Tahereh Rahiminia ◽  
Azita Faramarzi ◽  
Arezoo Khoradmehr ◽  
Mohammad Ali Khalili
Keyword(s):  
Culture System ◽  
In Vitro Maturation

286 EFFECTS OF PORCINE FOLLICULAR FLUID DURING IN VITRO MATURATION OF OOCYTES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 258
Author(s):  
B. Agung ◽  
T. Otoi ◽  
D. Fuchimoto ◽  
S. Senbon ◽  
A. Onishi ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Follicular Fluid ◽  
Culture System ◽  
In Vitro Maturation ◽  
Control Group ◽  
Rotating System ◽  
Vitro Fertilization ◽  
Maturation Medium

When used as a solo maturation medium for oocytes, porcine follicular fluid (pFF) promoted male pronucleus formation (MPF) of oocytes after in vitro maturation (IVM), using a static system, and in vitro fertilization (IVF) in pigs (Naito et al. 1988 Gamete Res. 21, 289–295). However, the developmental competence of oocytes matured in pFF after IVM/IVF has not been reported. This study was conducted to assess the ability of pFF as a maturation medium to support IVM/IVF of porcine oocytes and their subsequent in vitro development. pFF, including cumulus–oocyte complexes (COCs), was aspirated from follicles (2–5 mm in diameter) of prepubertal crossbred gilt ovaries, and large clusters of follicular cells (FC) were removed from pFF by filtration through 212 �m of mesh. All of the COCs in filtered pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the filtered pFF, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. COCs were transferred to 3.5 mL (in a 15-mL test tube) of MpFF with FC (5.2 � 106 cells mL-1) and cultured for 44–48 h at 38.5�C in 5% O2 and 5% CO2 using the rotating culture system. As a control group, COCs were cultured in 2 mL of MpFF without FC in a 35-mm Petri dish by the standard static culture system. After maturation, culture oocytes were co-incubated (IVF) for 5 h with frozen–thawed sperm in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and then some of them were fixed 10 h after IVF to assess the fertilization status; the rest of them were cultured in PZM (Yoshioka et al. 2002 Biol. Reprod. 60, 112–119) for 7 days to assess their early embryonic development. All of the data were analyzed by ANOVA. Oocytes cultured with FC in the rotating system (R group) showed significantly higher sperm penetration (71.0%), MPF formation (70.5%), and normal fertilization (monospermic fertilization with female and male pronuclei; 31.5%) rates than those in the control group (56.0%, 56.9%, and 17.6%, respectively; P &lt; 0.05). Also, the R group showed significantly higher rates of 8-cell embryos at 2 days after IVF and blastocyst formation at 7 days after IVF than those of the control group (17.2% vs. 8.3% and 10.9% vs. 4.5%, respectively; P &lt; 0.05). These results indicate that porcine oocytes matured in pFF supplemented with FC using the rotating system have the ability to be penetrated by sperm and form MPF, and to develop to the blastocyst stage at higher rates, than oocytes cultured in the standard static maturation culture system. In conclusion, the pFF can be a sole and simple maturation culture medium useful for the in vitro production of blastocysts in pigs.

P–269 Effect of well-of-the-well culture as in vitro maturation system for human oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
B Dura. Lopez ◽  
I Moya ◽  
P Torres ◽  
M J Gomez-Torres ◽  
A Monzo ◽  
...  
Keyword(s):  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Study Groups ◽  
Culture Conditions ◽  
Human Oocytes ◽  
Nuclear Maturation ◽  
Secreted Factors ◽  
Experimental Group

Abstract Study question Can the Well-of-the-Well system (WOW), applied on denuded oocytes, improve germinal vesicle breakdown (GVBD) and maturation rate? Summary answer In vitro maturation (IVM) of denuded germinal vesicle (GV) oocyte using WOW culture system increases nuclear maturation competence when compared with droplet conventional culture What is known already Further research remains necessary to address the mechanism of oocyte maturation in order to refine culture conditions and improve the implantation rate of in vitro matured oocytes. Several studies on bovine oocytes have shown that oocyte-secreted factors (an uncharacterized mix of growth factors secreted by the oocyte) enhance oocyte developmental competence during in vitro maturation. These oocyte-secreted factors may accumulate at the bottom of the micro-well, as suggested for the WOW culture system. Previous reports suggested that diffusible factors secreted by individual oocytes probably accumulated in a micro-well WOW dish, may provide a suitable microenvironment for their in vitro maturation. Study design, size, duration A total of 879 GV collected between 2017 and 2019 were included in this study. They were randomly allocated into two experimental groups: (1) single-cultured oocytes (SC) that were cultured individually in micro-droplets, and (2) group-cultured oocytes (WOW) that were cultured in a microwell culture system using the WOW dish (culture dish for time lapse incubator). The nuclear maturation was assessed after 24 hours and 48 hours of IVM Participants/materials, setting, methods GV oocytes were obtained from 609 patients undergoing controlled ovarian stimulation cycles. Oocytes from the experimental group (1) were placed individually in conventional 25μl micro-droplets in a 35 mm dish. Oocytes from the experimental group (2) were placed in 80 μl droplet individually in each of 9 microwells of WOW dish. All GV oocytes were matured in a single step embryo culture medium, supplemented with human menopausal gonadotropin and synthetic serum substitute. Main results and the role of chance Mature oocyte (MII) was considered when we observed rupture of the GV and the presence of a first polar body in the perivitelline space during the first 24 or 48 hours of culture under inverted optical microscope. GVBD noted significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [GVBD: SC group; 70% (318/455) vs. WOW group; 83% (352/424)] and 48 hours [GVBD: SC group; 77% (319/416) vs. WOW group; 94% (398/424)]. The maturation rates (MR) showed significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [MR: SC group; 51% (233/455) vs. WOW group; 80% (338/424)] and 48 hours [MR: SC group; 71% (295/416) vs. WOW group; 91% (387/424)]. Limitations, reasons for caution There is no data on cleavage and blastocyst rates. There are no previous reports comparing the maturation rates in denuded human oocytes single-cultured in individually droplet or group-cultured in WOW dish. Wider implications of the findings: Our results must be taken into account in order to improve the culture conditions for the optimization of the in vitro maturation technique in human oocytes from stimulated cycles. We now provide evidence that group-cultured oocytes in WOW dish increase GVBD and maturation rates. Trial registration number Not applicable

scholarly journals In vitro growth of mouse primary and early preantral follicles in a 3-dimensional culture system

2003 ◽  
Vol 80 ◽  
pp. 79 ◽  
Author(s):  
Erkan Buyuk ◽  
Ozgur Oktem ◽  
Jeff Roberts ◽  
Zev Rosenwaks ◽  
Kutluk Oktay
Keyword(s):  
Culture System ◽  
In Vitro Growth ◽  
Preantral Follicles ◽  
3 Dimensional

scholarly journals A Novel Three-Dimensional Co-Culture System to Study Leukemia in the Bone Marrow Microenvironment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1864-1864 ◽  
Author(s):  
Zeena Salman ◽  
Juan Carlos Balandrán-Juárez ◽  
Rosana Pelayo ◽  
Monica L. Guzman
Keyword(s):  
Bone Marrow ◽  
Culture System ◽  
Lymphoblastic Leukemia ◽  
Cell Monolayer ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Bone Marrow Niche ◽  
3 Dimensional

Abstract The need for novel therapies in acute leukemia has been motivated by sub-optimal 5-year survival rates of 25.4% in acute myeloid leukemia (AML) and approximately 70% in acute lymphoblastic leukemia (ALL). While these rates are higher in the pediatric population, novel approaches are necessary in all age groups to improve outcomes. Pre-clinical studies of novel therapeutics using in vitro and in vivo methods remain suboptimal with frequent lack of correlation with clinical outcomes at the bedside. Recent evidence has shown that human leukemia xenografts into immunodeficient mice yield variable results, indicating that treatment using these methods is not replicable. When using in vitro cell culture methods, the well-documented protective effects of the bone marrow (BM) microenvironment (BMME) on leukemia are not mimicked. Furthermore, these techniques cannot be used to investigate the effects of novel agents on leukemia stem cells (LSC) and their mobilization, which is important in the ablation of leukemia. Thus, we explored a novel 3-dimensional co-culture system to study the effects of drugs on leukemia cells in the presence of stroma in an environment more similar to that of human leukemia in the BMME. We generated a 3-dimensional (3D) spheroid co-culture system using human stromal cell line (HS-5) cells or human mesenchymal stromal cells (hBMSC) from primary AML or ALL BM. To evaluate the dynamics of the 3D system, we labeled the stroma cells with GFP and the leukemia cells with mCherry. We observed rapid homing to the center of the 3D stroma. We evaluated ROS levels, proliferation status, hypoxia and expression of key niche proteins such as CXCL12 in leukemia cells found outside and inside the 3D system. These methods were compared to similar treatments in leukemia cell monolayer culture and 2-dimensional co-culture systems. We treated this system with various drugs such as cytarabine, doxorubicin, TG02 (a multi-kinase inhibitor with LSC mobilization effects), and plerixafor; we then harvested cells from the outer and inner layers and evaluated these separately by multi-parameter flow cytometry for viability and mobilization of LSCs in relation to the stroma and xenotransplant assays. Our studies reveal that the 3D culture system has lower ROS internally, suggesting a similarly hypoxic environment to BMME. Our studies also reveal that, when treated with cytarabine, AML cells closest to the stromal center of the spheroid remain protected, with higher viability compared to those farther from stroma, and even more so than leukemia cells in a 2-D bilayer with stroma or in a monolayer. A lower CXCL12 level was also observed in the stroma of leukemic BM compared to healthy BM within the co-culture system. This culture method possesses many of the characteristics of leukemia cells within the bone marrow niche and should be considered for future in vitro pre-clinical drug testing to model the tumor within its microenvironment. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Promise of Lung Organoids for Growth and Investigation of Pneumocystis Species

2021 ◽  
Vol 2 ◽  
Author(s):  
Nikeya Tisdale-Macioce ◽  
Jenna Green ◽  
Anne-Karina T. Perl ◽  
Alan Ashbaugh ◽  
Nathan P. Wiederhold ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Culture System ◽  
Ex Vivo ◽  
Alveolar Type ◽  
Proof Of Concept ◽  
3 Dimensional ◽  
Alveolar Epithelial

Pneumocystis species (spp.) are host-obligate fungal parasites that colonize and propagate almost exclusively in the alveolar lumen within the lungs of mammals where they can cause a lethal pneumonia. The emergence of this pneumonia in non-HIV infected persons caused by Pneumocystis jirovecii (PjP), illustrates the continued importance of and the need to understand its associated pathologies and to develop new therapies and preventative strategies. In the proposed life cycle, Pneumocystis spp. attach to alveolar type 1 epithelial cells (AEC1) and prevent gas exchange. This process among other mechanisms of Pneumocystis spp. pathogenesis is challenging to observe in real time due to the absence of a continuous ex vivo or in vitro culture system. The study presented here provides a proof-of-concept for the development of murine lung organoids that mimic the lung alveolar sacs expressing alveolar epithelial type 1 cells (AEC1) and alveolar type 2 epithelial cells (AEC2). Use of these 3-dimensional organoids should facilitate studies of a multitude of unanswered questions and serve as an improved means to screen new anti- PjP agents.

Sign in / Sign up

Export Citation Format

Share Document