69 EFFECTS OF CELL TYPE, PRE-ACTIVATION PROTOCOL, AND CULTURE CONDITIONS ON DEVELOPMENT OF PORCINE HANDMADE CLONED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 193
Author(s):  
J. C. Mezzalira ◽  
L. U. Ohlweiler ◽  
A. Massie ◽  
E. Monaco ◽  
E. P. Silva ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cytochalasin B ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Cell Type ◽  
Embryo Production ◽  
Distinct Cell

Despite the rather successful and widespread use of cloning in various species, distinct cell types from the same species and even the same genotype display differences in blastocyst yield. Moreover, variations in the protocol for embryo production can influence development to the blastocyst stage and subsequent fetal development. The aim of this study was to evaluate the effect of 2 cell types and 2 embryo pre-activation protocols with or without the presence of FCS in the in vitro culture medium on development of handmade pig cloned embryos to the blastocyst stage. Cumulus-oocyte complexes recovered from sow ovaries were in vitro-matured for 38 to 40 h. Denuded matured oocytes selected by the presence of a polar body had the zona pellucida removed in a 0.2% protease HEPES-buffered solution +25% FCS, followed by manual bisection and UV screening of enucleated halves using Hoechst stain. Clone embryo reconstruction was performed using a phytohemoagglutinin solution to adhere 2 cytoplasts and a somatic cell. Adipocyte-derived mesenchymal stem cells (ADMSC) from a Yorkshire pig or granulosa cells (GC) from an Ossabaw pig were used as nuclear donors. Following electrical fusion, couplets were pretreated with a brief exposure to cytochalasin B (CB) or cytochalasin B + cycloheximide (CB+CX) in the presence of serum before the electrical activation (Naruse et al. 2007 Theriogenology 68, 709-716; Du et al. 2009 Reprod. Fertil. Dev. 21, 114). Activated embryos were in vitro-cultured in the well of the well (WOW) system, with 2 embryos per microwell, for 7 days in PZM-3 medium +0.3% BSA in the presence (FBS+) or absence (FBS-) of 10% FCS. Cleavage (Day 2, chi-square test) and blastocyst (Day 7, Fisher test) rates, on a per WOW basis, were compared for a level of significance of 5%. Our preliminary data indicate that the presence of serum in the IVC affected cleavage and blastocyst yield in a cell-type-dependent manner. The presence of serum enhanced the blastocyst yield for ADMSC, whereas for GC, only the absence of serum allowed any blastocyst development. The cell type and the pre-activation protocol did not appear to affect cleavage and embryo development to the blastocyst stage. Despite the low number of replications, our results reinforce the importance of optimizing the embryo production system taking into consideration the individual requirements for distinct cell types, procedures, and culture conditions. Table 1.Effects of cell type, pre-activation process and in vitro culture (IVC) medium on development of handmade pig cloned embryos

scholarly journals 265EFFECT OF SPECIFIC GRAVITY OF CULTURE MEDIUM ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO

2004 ◽  
Vol 16 (2) ◽  
pp. 253
Author(s):  
D.S. Arathy ◽  
S. Ashis ◽  
G.T. Sharma ◽  
A.C. Majumdar ◽  
M.S. Chauhan
Keyword(s):  
Specific Gravity ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Cell Stage

In buffalo the success rate of transferable quality embryo production through in vitro procedure is very low as compared to cattle. Sub optimal culture conditions and physical conditions such as specific gravity of the culture medium may lead to a reduced rate of transferable buffalo embryo production from the oocytes matured and fertilized in vitro (Palta & Chauhan,1998 Reprod. Fertil. Dev. 10, 379–391). This experiment was therefore conducted to find out the role of specific gravity of the IVC medium on the development rate of the buffalo embryos in vitro. Follicles of slaughter house ovaries were aspirated and the collected oocytes with cumulus-oocytes complexes (COCs) were cultured in TCM-199 medium supplemented with 10% fetal calf serum, 10% buffalo follicular fluid and 0.5μgmL−1 FSH in 5% CO2 incubator at 38.5°C. The matured oocytes were then inseminated with frozen-thawed buffalo semen suspended in BO medium. After 42h of post-inseminations the cleavage rates were evaluated. The 2–4 cell-cleaved eggs (Day 2 of post-insemination) were randomly divided and cultured for eight days in vitro in 1) modified synthetic fluid (mSOF)+0.8 %BSA (control), 2) mSOF+0.8 % BSA+gelatin (1mgmL−1) 3) mSOF+0.8% BSA+1mgmL−1 gelatin+10ngmL−1 epidermal growth factor (EGF). Supplementation of gelatin increased the specific gravity of the mSOF medium from 0.9658±0.009 to 1.0331±0.013 without any change in pH (7.4). The development of embryos to the 8–16 cell-stage on day 4 of in vitro culture were significantly higher (P<0.05) in mSOF+0.8% BSA+1mgmL−1 gelatin (81.8%; 27/33) than that in mSOF + 0.8% BSA (75.7%; 28/37) and mSOF+0.8% BSA+10ng/mL EGF (68.7%; 22/32). When these embryos were further cultured for another four days (Day 8), the development of transferable quality embryos (morula/blastocyst) was 42.4% (14/33), 48.7% (18/37) and 46.9% (15/32), respectively. Supplementation of gelatin increased the cleavage of eggs up to the 8–16 cell-stage embryo, but did not significantly enhance the rate of development to the morula/blastocyst stage in comparison to control and EGF-supplemented group. However, the percentage of transferable quality embryos was slightly lower in the gelatin-added group but not statistically significant than other groups. The study concluded that increase in specific gravity of the in vitro culture medium enhanced initial cleavage rate but did not have any role in transferable embryo production in buffalo.

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

scholarly journals Human axial progenitors generate trunk neural crest cells in vitro

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Thomas JR Frith ◽  
Ilaria Granata ◽  
Matthew Wind ◽  
Erin Stout ◽  
Oliver Thompson ◽  
...  
Keyword(s):  
Neural Crest ◽  
Cell Types ◽  
Embryonic Cell ◽  
Axial Position ◽  
Dependent Manner ◽  
In Vitro Differentiation ◽  
Distinct Cell ◽  
Axis Elongation ◽  
Trunk Neural Crest

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

Selection of kidney cell types in primary glomerular explant outgrowths by in vitro culture conditions

1986 ◽  
Vol 84 (1) ◽  
pp. 69-92
Author(s):  
T.D. Oberley ◽  
A.H. Yang ◽  
J. Gould-Kostka
Keyword(s):  
Epithelial Cell ◽  
Cell Types ◽  
Defined Medium ◽  
Culture Conditions ◽  
Chemically Defined Medium ◽  
Cell Type ◽  
Epithelial Cell Type ◽  
Tubular Cells ◽  
Selection Of

Adult guinea pig glomeruli were grown in vitro either in serum or in a chemically defined medium. Glomeruli were plated either directly into plastic flasks or into plastic flasks that had been coated with the extracellular matrix produced by the PF-HR-9 mouse teratocarcinoma endodermal cell line. Both the composition of the medium and the nature of the culture substrate affected whole glomerular attachment and the type of cells produced in culture. Quantitative studies demonstrated selection of cell types by different culture conditions. Three colony types, each composed of distinctive cell types, could be identified by morphological features. The cells constituting two of these colony types were epithelial in nature, but they were identified as different epithelial types by both histochemical and ultrastructural criteria. Previous studies suggested that one epithelial cell type was derived from the glomerular visceral epithelial cell. This study demonstrates that this cell type could be selectively grown in defined medium on plastic. A second cell type showed several features of renal tubular epithelial cells, including histochemical staining for catalase, cell surface microvilli and cilia, and formation of hemicysts and structures that resembled tubules after prolonged periods in culture. To demonstrate that the ‘glomerulus-derived’ tubular cells were indeed tubular epithelium, we isolated purified renal cortical tubules (greater than 99% pure) and cultured them on the HR-9 matrix in a serum-free chemically defined medium. The resultant outgrowths had morphological properties identical to those of the glomerulus-derived tubular cells. It seems likely that small tubular fragments attached to a minority of the glomeruli are the source of these glomerulus-derived tubular cells. Neither epithelial cell type could be subcultured on plastic, but both could be passaged on the HR-9 matrix. A third cell type, the spindle-shaped cell, was easily propagated on both plastic and the HR-9 matrix. The origin of this cell type is not clear. Our results demonstrate the important effect of culture conditions on the selection, growth and differentiation of kidney cell types in vitro.

55 PRELIMINARY STUDIES ON THE DEVELOPMENT OF RECONSTRUCTED EMBRYOS AFTER NUCLEAR TRANSFER IN DROMEDARY CAMEL (CAMELUS DROMEDARIUS)

2009 ◽  
Vol 21 (1) ◽  
pp. 128 ◽  
Author(s):  
N. A. Wani ◽  
J. A. Skidmore ◽  
U. Wernery
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Light Exposure ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Reconstructed Embryos

Experiments were conducted to study the in vitro development of reconstructed dromedary camel embryos after nuclear transfer by a modified zona-free method. Cumulus oocyte complexes, collected from slaughterhouse ovaries were cultured in TCM199 at 38.5°C in an atmosphere of 5% CO2 in air for 32 to 36 h. Matured oocytes were denuded of cumulus cells by repeated pipetting and the zona pellucida was removed by brief incubation in 5 mg mL–1 pronase dissolved in Ca- and Mg-free PBS. Zona-free oocytes were stained with 5 mg mL–1 Hoechst 33342 in H199 supplemented with 7.5 μg mL–1 cytochalasin B and 10% FCS. They were enucleated under constant UV-light exposure in H199 supplemented with cytochalasin B and 10% FCS. The granulosa cells at passage numbers 4 to 15 were used as nuclear donors. The zona-free cytoplasts were individually washed for a few seconds in 300 μg mL–1 of Phytohemagglutinin in H199, then quickly dropped on a single donor cell settled to the bottom of a drop of H199 with 0.5% FCS and pushed together with the mouth pipette. Couplets were electrically fused, at room temperature, with two DC pulses of 100 V cm–1 for 15 μs. Reconstructs were activated 2 h post-fusion, with 5 μm ionomycin for 3 min followed by culture in 6-diethylaminopurine for 4 h. The reconstructs were then cultured individually in either 5 μL drops under oil, in agar wells or in wells of wells (WOW) in a well of 4-well culture plate. Embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 μg mL–1 gentamycine, 1% insulin-transferrin-selenium (ITS), and 15% estrous dromedary serum. The number of oocytes that had cleaved was recorded on day 2, whilst those developing to morulae and blastocysts were recorded on day 7 of culture. For cell count, the blastocysts were stained with Hoechst and cells counted under a fluorescent microscope at ×400. Data obtained was analysed by chi-square test. About 92% (349/380) of the oocytes were successfully enucleated and 76% (259/340) fused with the attached cells. The cleavage rate was significantly lower (P < 0.05) in reconstructed embryos cultured in droplets (10/72, 14%) as compared with those cultured in agar wells (37/87, 42%) or WOW system (42/96, 44%). The proportions of cleaved embryos reaching morula stage were 0, 83, and 89% in droplets, agar wells, and WOW, respectively. However, only 8% and 5% of the cleaved embryos developed to the blastocyst stage in the agar well and WOW culture systems, respectively. No difference was observed in the cell number of blastocysts produced in agar wells (77.3 ± 8.02) or WOW (78.0 ± 4.2) culture system. To the best of our knowledge, this is the first report of embryo production up to the blastocyst stage after NT in camelids and it shows that NT can be successfully applied for embryo production in camelids. Further studies are needed to optimize the parameters and to improve the efficiency for production of transferable blastocysts in this species. This study was kindly sponsored by H.H. General Sheikh Mohammed bin Rashid Al Maktoum, Ruler of Dubai.

scholarly journals Equol: A Microbiota Metabolite Able to Alleviate the Negative Effects of Zearalenone during In Vitro Culture of Ovine Preantral Follicles

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 652
Author(s):  
Talyne Emilia Santos Silva ◽  
Danielle Cristina Calado de Brito ◽  
Naiza Arcângelo Ribeiro de Sá ◽  
Renato Felix da Silva ◽  
Anna Clara Accioly Ferreira ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cell Types ◽  
Culture Conditions ◽  
Dna Double Strand Breaks ◽  
Female Reproduction ◽  
Negative Effects ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
The Impact

The impact of zearalenone (ZEN) on female reproduction remains an issue, since its effects may differ among exposed cell types. Besides the use of decontaminants in animal diet, other approaches should be considered to minimise ZEN effects after exposure. Since the first organ in contact with ZEN is the gastrointestinal tract, we hypothesise that products of microbiota metabolism may play a role in ZEN detoxification. We aimed to evaluate the effect of 1 µmol/L ZEN and 1 µmol/L equol (a microbial metabolite), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 12 sheep were collected at a local abattoir and fragmented, and the ovarian pieces were submitted to in vitro culture for three days in the presence or absence of the test compounds. The follicular morphology was impaired by ZEN, but equol could alleviate the observed degeneration rates. While ZEN decreased cell proliferation in primary and secondary follicles, as well as induced DNA double-strand breaks in primordial follicles, all these observations disappeared when equol was added to a culture medium containing ZEN. In the present culture conditions, equol was able to counteract the negative effects of ZEN on ovarian preantral follicles.

scholarly journals Human axial progenitors generate trunk neural crest cells

2018 ◽  
Author(s):  
Thomas J. R. Frith ◽  
Ilaria Granata ◽  
Erin Stout ◽  
Matthew Wind ◽  
Oliver Thompson ◽  
...  
Keyword(s):  
Neural Crest ◽  
Cell Types ◽  
Embryonic Cell ◽  
Axial Position ◽  
Dependent Manner ◽  
In Vitro Differentiation ◽  
Distinct Cell ◽  
Axis Elongation ◽  
Trunk Neural Crest

AbstractThe neural crest (NC) is a multipotent embryonic cell population generating distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and therefore current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

scholarly journals Role of leukemia inhibitor factor (LIF) in decidualisation of murine uterine stromal cells in vitro

2004 ◽  
Vol 181 (3) ◽  
pp. 477-492 ◽  
Author(s):  
AA Fouladi Nashta ◽  
CV Andreu ◽  
N Nijjar ◽  
JK Heath ◽  
SJ Kimber
Keyword(s):  
In Vitro Culture ◽  
Stromal Cells ◽  
Control Level ◽  
Calf Serum ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Alp Activity ◽  
Dose Dependent ◽  
In Cells

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

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