scholarly journals Optimized culture systems for the preimplantation ICR mouse embryos with wide range of EDTA concentrations

2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Seema Thapa ◽  
Seung Hee Kang ◽  
Yun Seok Heo
Keyword(s):  
Large Volume ◽  
Culture System ◽  
Ethylenediaminetetraacetic Acid ◽  
Culture Media ◽  
Mouse Embryos ◽  
Blastocyst Development ◽  
Culture Systems ◽  
Development Rates ◽  
Icr Mouse ◽  
Wide Range

In this study, in vitro preimplantation embryo culture media especially for outbred stock mice (Institute of Cancer Research (ICR)) were optimized with different concentrations of ethylenediaminetetraacetic acid (EDTA). A plot with embryo development rates against EDTA concentrations ranging from 0 to 500 µM showed a unique pattern with two characteristic peaks. Two hundred micromolar was adopted as an optimal concentration of EDTA. The optimized media were also evaluated with two culture systems: conventional large volume culture system (1 ml) and micro-droplet culture system. In the conventional large volume culture system, the blastocyst development rates were compared among three different media (F-10, KSOM and KSOM with the optimized 200 µM EDTA). The rates were 0.4%, 16.7% and 57.6%, respectively. The development rates for the micro-droplet (10 µl) culture system were 73.9%. In conclusion, 200 µM EDTA concentration in 10 µl droplets in the KSOM medium was found as the most suitable culture conditions for ICR mouse embryos, as the blastocyst development rate was higher in the micro-droplet culture system than in the traditional conventional large volume culture system.

scholarly journals 118VITRIFICATION OF PRONUCLEAR-STAGE MOUSE EMBRYOS ON ALUMINUM FOIL FLOATING ON LIQUID NITROGEN

2004 ◽  
Vol 16 (2) ◽  
pp. 181 ◽  
Author(s):  
H. Sagirkaya ◽  
F. Ergin ◽  
H. Bagis ◽  
S. Arat
Keyword(s):  
Liquid Nitrogen ◽  
Culture Media ◽  
Aluminum Foil ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Mouse Embryos ◽  
Morphological Appearance ◽  
Development Rates ◽  
Developmental Rates ◽  
And Control

The cryopreservation of pronuclear-stage embryos has a special importance in transgenic technology, cloning, and human-assisted reproductive technology. The objective of this study was to investigate the efficiency of a vitrification method modified in our lab for pronuclear-stage embryos. In experiment I, groups of 10 pronuclear-stage mouse embryos were cultured in 20μL drops of three different culture media (G1.3/G2.3, CZB and M16) covered with mineral oil (Sigma M-8410, St. Louis, MO, USA). Twenty-four hours later, embryos cultured in G1.3 were transferred into G2.3 medium. In experiment II, 25–30 pronuclear-stage embryos were transferred into a 50-μL drop of equilibration medium containing 4% ethylene glycol (EG, Sigma E-9129) in TCM-199 (Sigma M-2520) supplemented with 10% FCS at 37°C for 12–15min; then they were rinsed three times in 30-μL drops of vitrification medium containing 35% EG, 5% polyvinylpyrrolidone (PVP, Sigma P-0930) and 0.4M trehalose (Sigma T-0167) in TCM-199 supplemented with 10% FCS at 37°C for 20–30s. Embryos rinsed in vitrification solution were aspirated into a micropipette as a 1–2-μL drop containing 25–30 embryos and dropped onto aluminum foil floating on liquid nitrogen (LN2). Vitrified droplets were stored in cryovials in LN2. Warming was performed by moving the vitrified droplets into 0.3M trehalose in TCM-199 supplemented with 10% FCS at 37°C. Embryos having normal morphological appearance under stereomicroscope examination were cultured in G1.3/G2.3 medium. Differences in the two experiments were analyzed by one-way ANOVA. In experiment I, development rates to the blastocyst stage were 26%, 10% and 4% for G1.3/G2.3, CZB and M16 media, respectively. The highest development rate in experiment I was obtained in G1.3/G2.3 culture media (P<0.05). Therefore, G1.3/G2.3 media were used for culturing of vitrified-warmed and control embryos. In experiment II, the rate of embryos having normal morphology was 98.5%. There were no significant differences between the development rates of vitrified (13.1%) and control (18.7%) embryos to the blastocyst stage (P>0.05). Although the vitrification method resulted in a high survival rate based on the morphological appearance, developmental rates of vitrified and control embryos were found to be lower than expected and reported previously by other researchers. We believe that the low developmental rates in this study were due to our culture conditions but not our vitrification method. Therefore, it could be concluded that this vitrification method is an efficient one for pronuclear-stage embryo cryopreservation and better development rates could be obtained by improving the culture conditions. This study was supported by a grant from TUBITAK, Turkey (VHAG-1908-102V048). F. Ergin is a young volunteer researcher.

scholarly journals Physiological Microenvironmental Conditions in Different Scalable Culture Systems for Pluripotent Stem Cell Expansion and Differentiation

2019 ◽  
Vol 13 (1) ◽  
pp. 41-54
Author(s):  
Fuad G. Torizal ◽  
Ikki Horiguchi ◽  
Yasuyuki Sakai
Keyword(s):  
Culture System ◽  
Large Scale ◽  
Culture Conditions ◽  
High Yield ◽  
Culture Systems ◽  
Physiological Conditions ◽  
Advantages And Disadvantages ◽  
Wide Range ◽  
Recent Developments ◽  
Cell Propagation

Human Pluripotent Stem Cells (PSCs) are a valuable cell type that has a wide range of biomedical applications because they can differentiate into many types of adult somatic cell. Numerous studies have examined the clinical applications of PSCs. However, several factors such as bioreactor design, mechanical stress, and the physiological environment have not been optimized. These factors can significantly alter the pluripotency and proliferation properties of the cells, which are important for the mass production of PSCs. Nutritional mass transfer and oxygen transfer must be effectively maintained to obtain a high yield. Various culture systems are currently available for optimum cell propagation by maintaining the physiological conditions necessary for cell cultivation. Each type of culture system using a different configuration with various advantages and disadvantages affecting the mechanical conditions in the bioreactor, such as shear stress. These factors make it difficult to preserve the cellular viability and pluripotency of PSCs. Additional limitations of the culture system for PSCs must also be identified and overcome to maintain the culture conditions and enable large-scale expansion and differentiation of PSCs. This review describes the different physiological conditions in the various culture systems and recent developments in culture technology for PSC expansion and differentiation.

scholarly journals Toxin Production in Soybean (Glycine max L.) Plants with Charcoal Rot Disease and by Macrophomina phaseolina, the Fungus that Causes the Disease

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 645 ◽  
Author(s):  
Hamed K. Abbas ◽  
Nacer Bellaloui ◽  
Cesare Accinelli ◽  
James R. Smith ◽  
W. Thomas Shier
Keyword(s):  
Culture Media ◽  
Leaf Disc ◽  
Toxin Production ◽  
Macrophomina Phaseolina ◽  
Economic Losses ◽  
Limit Of Quantification ◽  
Charcoal Rot ◽  
Wide Range ◽  
Soybean Plants ◽  
Rot Disease

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, results in major economic losses in soybean production in southern USA. M. phaseolina has been proposed to use the toxin (-)-botryodiplodin in its root infection mechanism to create a necrotic zone in root tissue through which fungal hyphae can readily enter the plant. The majority (51.4%) of M. phaseolina isolates from plants with charcoal rot disease produced a wide range of (-)-botryodiplodin concentrations in a culture medium (0.14–6.11 µg/mL), 37.8% produced traces below the limit of quantification (0.01 µg/mL), and 10.8% produced no detectable (-)-botryodiplodin. Some culture media with traces or no (-)-botryodiplodin were nevertheless strongly phytotoxic in soybean leaf disc cultures, consistent with the production of another unidentified toxin(s). Widely ranging (-)-botryodiplodin levels (traces to 3.14 µg/g) were also observed in the roots, but not in the aerial parts, of soybean plants naturally infected with charcoal rot disease. This is the first report of (-)-botryodiplodin in plant tissues naturally infected with charcoal rot disease. No phaseolinone was detected in M. phaseolina culture media or naturally infected soybean tissues. These results are consistent with (-)-botryodiplodin playing a role in the pathology of some, but not all, M. phaseolina isolates from soybeans with charcoal rot disease in southern USA.

scholarly journals Feasibility and safety study of 22-gauge endoscopic ultrasound (EUS) needles for portal vein sampling in a swine model

2020 ◽  
Vol 08 (11) ◽  
pp. E1717-E1724
Author(s):  
Kenneth Park ◽  
Daniel Lew ◽  
Christopher Chapman ◽  
Ashley Wachsman ◽  
Matthew Bloom ◽  
...  
Keyword(s):  
Portal Vein ◽  
Endoscopic Ultrasound ◽  
Large Volume ◽  
Ethylenediaminetetraacetic Acid ◽  
Standard Procedure ◽  
Bleeding Risk ◽  
Needle Aspiration ◽  
Swine Model ◽  
Volume Blood ◽  
Collection Methods

Abstract Background and study aims Endoscopic ultrasound (EUS) has been used for portal vein sampling in patients with pancreaticobiliary cancers for enumerating circulating tumor cells but is not yet a standard procedure. Further evaluation is needed to refine the methodology. Therefore, we evaluated the feasibility and safety of 19-gauge (19G) versus a 22-gauge (22 G) EUS fine-needle aspiration needles for portal vein sampling in a swine model. Methods Celiotomy was performed on two farm pigs. Portal vein sampling occurred transhepatically. We compared 19 G and 22 G needles coated interiorly with saline, heparin or ethylenediaminetetraacetic acid (EDTA). Small- (10 mL) and large- (25 mL) volume blood collections were evaluated. Two different collection methods were tested: direct-to-vial and suction syringe. A bleeding risk trial for saline-coated 19 G and 22 G needles was performed by puncturing the portal vein 20 times. Persistent bleeding after 3 minutes was considered significant. Results All small-volume collection trials were successful except for 22 G saline-coated needles with direct-to-vial method. All large-volume collection trials were successful when using suction syringe; direct-to-vial method for both 19 G and 22 G needles were unsuccessful. Collection times were shorter for 19 G vs. 22 G needles for both small and large-volume collections (P < 0.05). Collection times for saline-coated 22 G needles were longer compared to heparin/EDTA-coated (P < 0.05). Bleeding occurred in 10 % punctures with 19 G needles compared to 0 % with 22 G needles. Conclusion The results of this animal study demonstrate the feasibility and the safety of using 22 G needles for portal vein sampling and can form the basis for a pilot study in patients.

scholarly journals Adipose and Muscle Cell Co-Culture System: A Novel In Vitro Tool to Mimic the In Vivo Cellular Environment

Biology ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 6
Author(s):  
Palaniselvam Kuppusamy ◽  
Dahye Kim ◽  
Ilavenil Soundharrajan ◽  
Inho Hwang ◽  
Ki Choon Choi
Keyword(s):  
Drug Development ◽  
Culture System ◽  
Cell Types ◽  
Culture Cell ◽  
In Vitro Techniques ◽  
Culture Systems ◽  
Complex Interactions ◽  
Cellular Environment

A co-culture system allows researchers to investigate the complex interactions between two cell types under various environments, such as those that promote differentiation and growth as well as those that mimic healthy and diseased states, in vitro. In this paper, we review the most common co-culture systems for myocytes and adipocytes. The in vitro techniques mimic the in vivo environment and are used to investigate the causal relationships between different cell lines. Here, we briefly discuss mono-culture and co-culture cell systems and their applicability to the study of communication between two or more cell types, including adipocytes and myocytes. Also, we provide details about the different types of co-culture systems and their applicability to the study of metabolic disease, drug development, and the role of secretory factors in cell signaling cascades. Therefore, this review provides details about the co-culture systems used to study the complex interactions between adipose and muscle cells in various environments, such as those that promote cell differentiation and growth and those used for drug development.

P–227 Fatty acid regulation of Nrf2/Keap1 pathway during mouse preimplantation embryo development

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Dionne ◽  
A J Watson ◽  
D H Betts ◽  
B A Rafea
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Embryo Development ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Control Group ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development

Abstract Study question Our objective is determining whether supplementing embryo culture media with palmitic acid and/or oleic acid impacts Nrf2/Keap1 antioxidant response pathways during preimplantation mouse embryo development. Summary answer Supplementation of embryo culture media with palmitic acid increases cellular Nrf2 levels per embryo after 48-hour culture, while oleic acid reverses this effect. What is known already Obese women experience higher incidence of infertility than women with healthy BMIs. The obese reproductive tract environment supporting preimplantation embryo development is likely to include enhanced free fatty acid (FFA) levels and increased accumulation of reactive oxygen species. Exposure to palmitic acid (PA) in vitro significantly impairs mouse embryo development while increasing ER stress mRNAs. Oleic acid (OA) reverses these effects. To further define effects of FFA exposure, we are characterizing the influence of FFAs on the Nrf2–Keap1 pathway and its downstream antioxidant defense systems. We hypothesize that PA treatment induces Nrf2-Keap1 activity, while OA treatment alleviates pathway activity. Study design, size, duration Female CD–1 mice (4–6 weeks) were super-ovulated via intraperitoneal injections of PMSG, followed 48 hours later by hCG. Female mice were mated with male CD–1 mice (6–8 months) overnight. Females were euthanized using CO2 and two-cell embryos were collected by flushing oviducts. Two-cell embryos were placed into KSOMaa-based treatment groups: 1) BSA (control); 2) 100µM PA; 3) 100µM OA; 4) 100µM PA+OA, and cultured for 48 hours (37 °C; 5% O2, 5% CO2, 90% N2). Participants/materials, setting, methods After 48-hour embryo culture, developmental stages of all mouse embryos were recorded. Immunofluorescence analysis of Nrf2 and Keap1 localization was performed for embryo treatments (BSA, 100µM PA, 100µM OA & 100µM PA+OA) using rabbit polyclonal anti-Nrf2 antibody, with Rhodamine-Phalloidin and DAPI staining. Embryos were imaged using confocal microscopy and Nrf2-positive cells were counted using ImageJ. Nrf2 and Keap1 mRNA abundances were assessed after culture in each treatment condition using RT-qPCR and the delta-delta Ct method. Main results and the role of chance Inclusion of 100µM PA in embryo culture significantly decreased blastocyst development frequency from 70.06±16.38% in the BSA (control) group to 11.61±8.19% in the PA-treated group (p &lt; 0.0001). Embryo culture with 100µM OA and 100µM PA+OA co-treatment did not significantly impair blastocyst development (OA: 61.59±8.07%, p = 0.4053; PA+OA: 63.53±7.63%, p = 0.6204). Embryo culture with PA treatment significantly increased the mean percentage of Nrf2-positive cells to 56.83±30.49% compared with 21.22±15.63% in the control group (p &lt; 0.0001). Conversely, 100µM OA and 100µM PA+OA treatments did not significantly affect Nrf2-positive cell frequencies compared with the control group (OA: 33.28±21.83%, p = 0.1825; PA+OA: 34.84±12.66%, p = 0.0691). Immunofluorescence results show that treating embryos with 100µM PA for 48 hours results in increased levels of cellular Nrf2, while combining 100µM PA with 100µM OA reversed these effects. Preliminary qPCR analysis showed no significant differences in Nrf2 or Keap1 relative transcript abundance between any embryo treatment groups. Nrf2 and Keap1 mRNA levels were both higher after embryo culture with 100µM OA than all other culture groups (p = 0.6268; p = 0.3201). Notably, Keap1 relative transcript levels dropped to undetectable levels after culture with 100µM PA, which suggests an increase in Nrf2 activation.Limitations, reasons for caution: While immunofluorescence localization of Nrf2/Keap1 provides insight into how the proteins behave during preimplantation embryo development, confocal images cannot determine protein-protein interactions or activity levels. Similarly, transcript information from RT-qPCR analysis only provides information about Nrf2 and Keap1 at the transcript level. Nrf2 activity will be assessed via downstream targets. Wider implications of the findings: The Nrf2–Keap1 pathway coordinates numerous cellular defence mechanisms, and is implicated in various diseases, including cancer. Establishing an impact of free fatty acid exposure on Nrf2–Keap1 during preimplantation embryo development will provide valuable information regarding the effects of maternal obesity on outcomes for embryos produced from these patients. Trial registration number Not applicable

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

scholarly journals Iron nanoparticles on growth and acclimatization of Chrysanthemum morifolium Ramat. cv. "Jimba" in different culture systems

2020 ◽  
Vol 18 (2) ◽  
pp. 307-319
Author(s):  
Hoang Thanh Tung ◽  
Truong Hoai Phong ◽  
Phan Le Ha Nguyen ◽  
Luong Thien Nghia ◽  
Ha Thi My Ngan ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Antioxidant Enzyme ◽  
Chlorophyll A ◽  
Culture System ◽  
Iron Nanoparticles ◽  
Cultivated Plants ◽  
Ms Medium ◽  
Culture Systems ◽  
Optimal Activity

In plant tissue culture, iron nanoparticles (FeNPs) was one of the first types of nano to be used in plants. Previous reports have identified the effect of FeNPs on many different plant species. In this study, FeNPs was used to replace Fe-EDTA in MS (Murashige, Skoog, 1962) medium to assess their effects on growth, chlorophyll (a, b and a+b) accumulation, antioxidant activity of ascorbate peroxidase (APX) and superoxide dismutase (SOD) enzymes, and acclimatization in greenhouse conditions in different culture systems (in vitro solid, in vitro hydroponic and microponic culture). The obtained results show that FeNPs added to MS medium was higher growth, chlorophyll (a, b and a+b) content, antioxidant activity of SOD and APX enzymes than Fe-EDTA in MS medium as control treatment. The effect of FeNPs are differences between culture systems. In vitro solid and microponic culture systems, the optimal concentration is 75 mM FeNPs and in vitro hydroponic culture system is 100 mM FeNPs. The optimal activity of the antioxidant enzyme SOD (35.04 U.mg−1 prot) obtained in the roots of cultured plants in microponic culture system; meanwhile, the optimal activity of the antioxidant enzyme APX (2.11 μmol.min−1.mg−1 prot) obtained in leaves cultivated in solid culture system. The plantlets derived from MS medium added FeNPs were transfered into greenhouse conditions, the microponic cultivated plants supplemented with FeNPs at a concentration of 100 mM gave the highest survival rate (94.67%). The results of this study showed that FeNPs can replace Fe-EDTA salt in MS medium, and iron deficiency in culture media will reduce chlorophyll content.

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