scholarly journals Transcriptomic and Morphological Analysis of Cells Derived from Porcine Buccal Mucosa—Studies on an In Vitro Model

Animals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Artur Bryja ◽  
Grzegorz Latosiński ◽  
Maurycy Jankowski ◽  
Ana Angelova Volponi ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Transgenic Animals ◽  
Automated Analysis ◽  
Transcriptomic Analysis ◽  
Transcriptional Analysis ◽  
Suitable Model ◽  
Human Tissues ◽  
Differential Interference Contrast Microscopy ◽  
Vitro Model

Transcriptional analysis and live-cell imaging are a powerful tool to investigate the dynamics of complex biological systems. In vitro expanded porcine oral mucosal cells, consisting of populations of epithelial and connective lineages, are interesting and complex systems for study via microarray transcriptomic assays to analyze gene expression profile. The transcriptomic analysis included 56 ontological groups with particular focus on 7 gene ontology groups that are related to the processes of differentiation and development. Most analyzed genes were upregulated after 7 days and downregulated after 15 and 30 days of in vitro culture. The performed transcriptomic analysis was then extended to include automated analysis of differential interference contrast microscopy (DIC) images obtained during in vitro culture. The analysis of DIC imaging allowed to identify the different populations of keratinocytes and fibroblasts during seven days of in vitro culture, and it was possible to evaluate the proportion of these two populations of cells. Porcine mucosa may be a suitable model for reference research on human tissues. In addition, it can provide a reference point for research on the use of cells, scaffolds, or tissues derived from transgenic animals for applications in human tissues reconstruction.

240 QUALITY AND VIABILITY OF IVF BOVINE EMBRYOS AFTER INTRACYTOPLASMIC INJECTION OF DNA–LIPOSOME COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 232
Author(s):  
L. N. Moro ◽  
G. Vichera ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Dna Fragmentation ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Egfp Expression ◽  
Bovine Embryos ◽  
Exogenous Dna ◽  
Intracytoplasmic Injection

Transgenic animals have important applications in agriculture and human medicine; nevertheless the available techniques still remain inefficient and technically difficult. We have recently developed a novel method to transfect bovine embryos that consists of intracytoplasmic injection of exogenous DNA–liposome complexes (eDNA-LC) in IVF zygotes. This study was designed to evaluate the quality and viability of IVF bovine embryos, after intracytoplasmic injection of pCX-EGFP–liposome complexes (EGFP-LC) or pBCKIP2.8-liposome complexes (plasmid that codifies the human insulin gene, HI-LC). First, we evaluated embryo development and enhanced green fluorescent protein (EGFP) expression of IVF embryos injected with both plasmids separately. This treatment was analysed by Fisher's Exact test (P ≤ 0.05). Cleavage rates for EGFP-LC, HI-LC and IVF embryos injected with liposomes alone (IVF-L) and IVF control (IVF-C) were 62% (63/102), 67% (67/100), 66% (67/101) and 79% (98/124); blastocysts rates were 17% (17/102), 21% (21/100), 21% (21/101) and 23% (28/124), respectively. No statistical differences were seen among groups. The percentage of EGFP-positive embryos (EGFP+) after EGFP-LC injection was 42.9% after 3 days of culture and 41.8% at the blastocyst stage. In the second experiment, the blastocysts obtained, EGFP+ or EGFP-negative (EGFP–), were analysed by TUNEL assay at Day 6 (Bd6), 7 (Bd7) and 8 (Bd8) of in vitro culture, in order to evaluate the effect of the transgene and culture length, on DNA fragmentation. This treatment was analysed by the difference of proportions test (P ≤ 0.05) using statistical INFOSTAT software. All EGFP+ blastocysts showed TUNEL positive cells (T+). The percentage of T+ in Bd6, Bd7 and Bd8 were 91, 73.7 and 99.5%, respectively (P ≤ 0.05). EGFP– blastocysts showed lower fragmented nuclei (0, 44.6 and 85%, respectively; P ≤ 0.05). Groups IVF-L and IVF-C were also evaluated. In both groups, there was no evidence of DNA fragmentation in Bd6 and Bd7, but T+ were detected in Bd8 (66.4 and 85.8%, respectively; P ≤ 0.05). In the third experiment, bovine blastocysts obtained from the HI-LC group were individually transferred to recipient cows after 6 (n = 11), 7 (n = 5) and 8 (n = 5) days of culture post-IVF and HI-LC injection. The pregnancies obtained were from Bd6 [18.2% (2/11)] and Bd7 [40% (2/5)], although none of the recipients receiving Bd8 were diagnosed pregnant. Two pregnancies developed to term, one derived from Bd6 and the other from Bd7. Analysis by PCR determined that none of the born cows were transgenic. In summary, IVF bovine embryos could be easily transfected after the injection of eDNA-LC and the technique did not affect offspring viability. The results indicate that extended time in in vitro culture increases the percentage of fragmented nuclei in blastocysts. Moreover, this parameter increases in blastocysts with transgene expression compared with those without expression. Finally, more transfers are required in order to obtain the real efficiency of this new technique and to overcome the drawbacks generated by in vitro culture length and transgene expression.

An in Vitro Model for the Molecular Investigation of Varicose Veins: Use of Cultured Explants of Human Long Saphenous Vein

2001 ◽  
Vol 16 (2) ◽  
pp. 53-59 ◽  
Author(s):  
S. J. Hollingsworth ◽  
C. Bong Tang ◽  
S. G. E. Barker
Keyword(s):  
Venous Insufficiency ◽  
In Vitro Model ◽  
Fetal Calf Serum ◽  
Varicose Veins ◽  
Calf Serum ◽  
Suitable Model ◽  
Long Saphenous Vein ◽  
The Media ◽  
Vitro Model

Objective: To develop an in vitro model from explants of human varicose veins (VVs). Procedures: Segments of VVs were cultured for up to 14 days with 30% fetal calf serum. At 7 and 14 days, segments were analysed for changes in intima:media thickness and by immunohistochemistry. Comparisons were made with VVs at isolation and cultured explants of normal vein. Results: At isolation, VVs had a significantly thicker intima than normal veins ( p<0.001). By day 7 in culture, normal veins developed a significant (smooth muscle cell-derived) ‘neo-intima’ ( p<0.006). In contrast, in VVs there was little change in the intima but a significant increase in the thickness of the media ( p<0.001). Following 14 days in culture, both the neo-intima in normal veins and thickened media in VVs had regressed. Overall, there was a reduction in the intima:media ratio in VVs by day 14 ( p<0.03). Conclusions: Segments of VVs can be maintained in culture for up to 14 days without developing a neo-intima and may provide a suitable model to investigate mechanisms underlying the chronic venous insufficiency of varicosity.

scholarly journals The human SOX18 gene: Expression analysis and characterization of its 5’ flanking region

2007 ◽  
Vol 59 (4) ◽  
pp. 267-272 ◽  
Author(s):  
Isidora Petrovic ◽  
Milena Stevanovic
Keyword(s):  
Transcriptional Regulation ◽  
Regulatory Elements ◽  
Model System ◽  
Suitable Model ◽  
Flanking Region ◽  
Vitro Model ◽  
The Given ◽  
First Time

The aim of this study was to establish an adequate in vitro model system for studying transcriptional regulation of the human SOX18 gene. The paper presents an analysis of expression of this gene in cultured cell lines and characterization of its 5' flanking region. Using RT-PCR, Northern and Western blot analysis, we demonstrated SOX18 expression in HeLa cells, indicating that this cell line provides a suitable model system for studying transcriptional regulation of the given gene. We also cloned, sequenced and for the first time characterized the human SOX18 5? flanking region. It is shown that the region 892 bp in size immediately upstream from the start codone harbors regulatory elements sufficient for transcription and represents an SOX18 promoter region.

235 PROGESTERONE AFFECTS THE MESSENGER RNA EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 265
Author(s):  
K. Knauer ◽  
H. Stinshoff ◽  
S. Wilkening ◽  
C. Wrenzycki
Keyword(s):  
In Vitro Culture ◽  
Messenger Rna ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Morphological Development ◽  
Bovine Embryo ◽  
Development Rates ◽  
Vitro Model

It is known that the progesterone (P4) provided by the corpus luteum is essential for the maintenance of pregnancy. It has been suggested that supplying external P4 in vivo is beneficial to the establishment and upkeep of pregnancy. The aim of the present study was to assess the effects of supplementation with different concentrations of P4 on either of 2 days of in vitro culture (IVC) on early bovine embryo development in an in vitro model. A total of 5073 cumulus–oocyte complexes were matured and fertilized in vitro. Before culture, they were collected in groups of 30 and allocated to 1 of 9 groups. The groups were supplemented with 10, 20, or 100 ng of P4 on Days 4 or 5 of IVC (IVF = Day 0). Alcohol (ETOH) was used as the solvent, so 8 µL of ETOH was used per supplementation. Therefore, two additional groups were supplemented with only ETOH on Day 4 or 5 of IVC. The presumptive zygotes allocated to group 9 were not supplemented. A culture system without oil overlay was used to prevent the lipophilic P4 from moving into the oil. Embryo cleavage and development rates were determined solely on Day 8 of IVC. Single expanded blastocysts were stored at –80°C for RT-qPCR. Subsequently, the relative amounts of six developmentally important gene transcripts (IGF1R, SLC2A1, HSD3B1, IFNT, PGRMC1, and PGRMC2) were analysed in single embryos of all groups. Statistical analysis was performed using one-way and two-way ANOVA, and the level of significance was set at P ≤ 0.05. Cleavage and development rates did not differ among groups (see Table 1). The relative abundance of IGF1R, SLC2A1, PGRMC1, and PGRMC2 was not affected by either the concentration or the timing of P4 supplementation. Nevertheless, there was a statistically significant interaction between the day of treatment and the concentration used for the expression of HSD3B1 mRNA. When 20 ng of P4 was added on Day 5 of IVC, significantly more HSD3B1 transcripts were detected than if 10 ng, 100 ng, or ETOH alone was added. The expression of IFNT was not affected by the day of supplementation, only by the concentration used. Thus, supplementation with 20 ng of P4 resulted in a significantly higher level of transcripts than when 10 ng or ETOH was supplemented. The results indicate that the amount of P4 present during early embryonic development and the timing of its presence had an impact on molecular developmental competence. However, no effects concerning morphological development up to the blastocyst stage could be detected. Table 1.Cleavage and development rates (± SEM) of embryos supplemented with 10, 20, or 100 ng on Day 4 or 5 of in vitro culture (P ≥ 0.05) The financial support of the FBF e.V. is acknowledged.

scholarly journals Comparison of methods to experimentally induce opacification and elasticity change in ex vivo porcine lenses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Manuel Ruiss ◽  
Martin Kronschläger ◽  
Andreas Schlatter ◽  
Thomas Dechat ◽  
Oliver Findl
Keyword(s):  
Sodium Chloride ◽  
Microwave Heating ◽  
Culture Medium ◽  
Ex Vivo ◽  
Treatment Strategies ◽  
Suitable Model ◽  
Lens Opacification ◽  
Vitro Model ◽  
The Moment

AbstractAt the moment, cataract, which is the opacification of the eye’s lens, can only be treated by surgery. In order to develop and test new pharmacological treatment strategies for the disease, there is a need for an appropriate in vitro model using ex vivo animal lenses. In this study, porcine lenses were incubated in either culture medium, glucose, triamcinolone acetonide, sodium chloride, hydrogen peroxide, sodium selenite, neutral buffered formalin, or were exposed to microwave heating to experimentally induce lens opacification. Changes in the lens morphology, weight, size, and elasticity were monitored 7 days after treatment. The fastest induction of dense opacification was seen in lenses exposed to sodium chloride, neutral buffered formalin, and microwave heating. No change in the size and weight of the lenses were detected, whereas loss in elasticity could be detected in lenses treated with formalin solution or microwave heating. Thus, neutral buffered formalin- and microwave-treated ex vivo porcine lenses seem to be a suitable model for mature cataracts, whereas hypertonic sodium chloride may be useful for studies on osmolarity-induced lens opacification.

scholarly journals Extracellular Vesicles Function as Bioactive Molecular Transmitters in the Mammalian Oviduct: An Inspiration for Optimizing in Vitro Culture Systems and Improving Delivery of Exogenous Nucleic Acids during Preimplantation Embryonic Development

2020 ◽  
Vol 21 (6) ◽  
pp. 2189 ◽  
Author(s):  
Bo Fu ◽  
Hong Ma ◽  
Di Liu
Keyword(s):  
Nucleic Acids ◽  
In Vitro Culture ◽  
Early Development ◽  
Extracellular Vesicles ◽  
Genetic Material ◽  
Transgenic Animals ◽  
Preimplantation Embryos ◽  
New Horizons ◽  
Production Techniques

Two technologies, in vitro culture and exogenous gene introduction, constitute cornerstones of producing transgenic animals. Although in vitro embryo production techniques can bypass the oviduct during early development, such embryos are inferior to their naturally produced counterparts. In addition, preimplantation embryos are resistant to the uptake of exogenous genetic material. These factors restrict the production of transgenic animals. The discovery of extracellular vesicles (EVs) was a milestone in the study of intercellular signal communication. EVs in the oviduct, known as oviductosomes (OVS), are versatile delivery tools during maternal–embryo communication. In this review, we discuss the important roles of OVS in these interactions and the feasibility of using them as tools for transferring exogenous nucleic acids during early development. We hypothesize that further accurate characterization of OVS cargoes and functions will open new horizons for research on maternal–embryo interactions and enhance the production of transgenic animals.

An in vitro model for investigation of vitamin A effects on wound healing

2021 ◽  
pp. 1-6
Author(s):  
Hoda Keshmiri Neghab ◽  
Mohammad Hasan Soheilifar ◽  
Gholamreza Esmaeeli Djavid
Keyword(s):  
Wound Healing ◽  
Endothelial Cell ◽  
Vitamin A ◽  
In Vitro Model ◽  
Cellular Proliferation ◽  
Endothelial Cell Migration ◽  
Normal Fibroblast ◽  
Anti Inflammatory ◽  
Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inflammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inflammation responses.

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