scholarly journals Physicochemical and Functional Characterization of Female Reproductive Fluids: A Report of the First Two Infants Born Following Addition of Their Mother's Fluids to the Embryo Culture Media

2021 ◽  
Vol 12 ◽  
Author(s):  
Analuce Canha-Gouveia ◽  
Maria Teresa Prieto-Sánchez ◽  
Maria Luisa Sánchez-Ferrer ◽  
Marta Mollá ◽  
Juan Carlos Martínez-Soto ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Abdominal Hysterectomy ◽  
Functional Assay ◽  
Uterine Fluid ◽  
Human Reproductive

Culture media supplemented with reproductive fluids (RF) have been used in livestock species, improving the efficiency and quality of in vitro produced embryos. However, usefulness in humans is still unknown. In this study, we collected human reproductive fluids (HRFs) ex vivo (from 25 patients undergoing abdominal hysterectomy plus bilateral salpingectomy) and in vivo (from 31 oocyte donors). Afterward, protocols to evaluate their osmolality, pH, total protein concentration, endotoxin level, and sterility were optimized, establishing security ranges for their use as natural additives. In addition, a functional assay was developed with bovine embryos grown in vitro in a medium supplemented with 1% of collected HRFs. Finally, a proof of concept was performed with six patients on post ovulation day 2 to evaluate the full-term viability of embryos grown in media supplemented with autologous uterine fluid, collected under in vivo conditions. Two of the embryos resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study establishes a complete quality control sheet of HRFs as additives for embryo culture media and shows first preliminary data on obtaining healthy offspring derived from embryos grown in media supplemented with HRFs.

The Impact of Two Embryo Culture Media, SOF and Commercial BO, on Pre- and Post-Implantation Development of Cloned Sannen Goat Embryos

2020 ◽  
Author(s):  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Sayed Morteza Aghamiri ◽  
Shiva Rouhollahi Varnosfaderani ◽  
Mohsen Rahimi ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Specific Gene ◽  
Limited Information ◽  
Major Drawback ◽  
Animal Cloning ◽  
The Impact ◽  
Post Implantation

Abstract Background: The ingredients of embryo culture media developed by different companies are disclosed. Thus, it is impossible to determine which ingredients might be responsible for differences in pre-and post-implantation embryo development. To address this gap, we performed an experiment to compare two embryo culture media, namely, SOF and commercial BO, on pre- and post-implantation development of cloned Sannen goat embryos. Cumulus oocyte complexes derived from slaughterhouse ovaries were used for in vitro embryo production . In vitro development of IVF, parthenogenetic and SCNT embryos were assessed in both BO and SOF media. The expression of 16 genes, including AKT , OCT4 , SOX2 , BMPR1 , FGFR4 , CDC25 , CDX2 , GCN5 , PCAF , FOXD3 , SMAD5 , FZD , LIFR1 , CTNNB , ERK1 , and IFNT , belonging to 7 important pathways, i.e. pluripotency, FGF, TGFβ, cell cycle and proliferation, histone transferase, trophectoderm, and WNT, were examined in the goat SCNT and IVF blastocysts from both BO and SOF media. Results: The blastocyst rate in BO medium was significantly higher than that of the SOF medium in SCNT embryos ( P < 0.05). All of the genes examined showed increased expression levels in SCNT embryos compared to IVF embryos. In the IVF group, OCT4 , BMPR1 , and GCN5 showed significantly higher expression in the SOF medium compared to the BO medium. In this group, AKT , FGFR4 , SOX2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, FGFR4 , GCN5 , FZD , CTNNB , BMPR1 , and FGFR4 showed significantly higher expression in SOF medium compared to BO medium. In vivo development did not differ significantly between the two groups. Conclusions: Based on these results, we concluded that the limited information available on the allocations of ICM and TE cells in SCNT embryos and embryo-specific gene expression may be the major drawback IVC medium and an impediment to successful animal cloning.

Natural fluids collected from the reproductive tract of women can be used as additives for human in vitro embryo culture media

2020 ◽  
Author(s):  
Analuce Canha-Gouveia ◽  
María Teresa Prieto-Sánchez ◽  
Maria Luisa Sánchez-Ferrer ◽  
Marta Molla Silva ◽  
Juan Carlos Martínez Soto ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Culture Media ◽  
Full Range ◽  
Preimplantation Embryos ◽  
Physico Chemical ◽  
Uterine Fluid ◽  
Pregnancy And Delivery ◽  
Human Reproductive ◽  
Physiological Values

Abstract Reproductive fluids have been recently proposed as the most adequate natural additive to complement the chemically defined in vitro culture media in different animal models, since they contain the full range of molecules necessary for the fertilization and development of preimplantation embryos. However, reproductive fluids potentially are problematic in terms of biosecurity and reproducibility of results, and their availability is scarce. Therefore, we have optimized the protocols to characterize the physico-chemical and functional properties of human reproductive fluids to establish the security ranges that could allow their use as natural additives and using the least possible volume of fluids collected. The results of this study offer, for the first time, a complete quality control guideline for human reproductive fluids, including the security levels (referred from physiological values) that should be taken into account for further use – i.e., osmolality, pH, total protein concentrations, presence of endotoxins and sterility conditions. In addition, a functional test has been developed evaluating the in vitro growth of bovine embryos in medium with human reproductive fluids. Finally, as a proof of concept, four embryos cultured with uterine fluid of their own mothers were transferred and two resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study paves the way towards the future use of more natural culture media in human ART.

scholarly journals BLI-Based Functional Assay in Phage Display Benefits the Development of a PD-L1-Targeting Therapeutic Antibody

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 684
Author(s):  
Jong Rip Choi ◽  
Min Jung Kim ◽  
Nara Tae ◽  
Tae Min Wi ◽  
Se-Ho Kim ◽  
...  
Keyword(s):  
Phage Display ◽  
High Efficiency ◽  
Functional Characterization ◽  
Scfv Antibody ◽  
Functional Assay ◽  
Inhibition Assay ◽  
Antibody Screening ◽  
Mammalian Expression

The therapeutic functionality of the antibodies from phage display is verified after an initial screening. Several immunological assays such as ELISA, flow cytometry, the western blot, and surface plasmon resonance (SPR) assay are commonly used; the IgG-format antibody is usually preferred to verify the functionality of antibodies, which need elaborative mammalian expression and purification work. Here, we describe a biolayer interferometry (BLI)-based assay that can evaluate the inhibitory functions of antibodies at an earlier stage of screening. To develop a PD-L1-targeting antibody from phage display, we applied the BLI assay to the initial scFv antibody screening, in addition to common ELISA and fluorescence-activated cell sorting (FACS) assays, which showed high advantages and relevance with the in vitro cell-based PD-1/PD-L1 inhibition assay. The same assays for IgG-format antibodies showed high efficiency of the BLI assay in the functional characterization of antibodies, and one candidate selected from the BLI assay resulted in highly efficacious antitumor activity in an in vivo syngeneic mouse study. The BLI assay was also beneficial when searching for antibodies with diverse epitopes. These results demonstrated that the BLI-based inhibition assay is an excellent technique for high-throughput scFv antibody screening in earlier stages and can make phage-display antibody screening more efficient to develop therapeutic candidates.

scholarly journals Adult human circulating CD34−Lin−CD45−CD133− cells can differentiate into hematopoietic and endothelial cells

Blood ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2105-2115 ◽  
Author(s):  
Elisa Ciraci ◽  
Silvia Della Bella ◽  
Ombretta Salvucci ◽  
Cristina Rofani ◽  
Marta Segarra ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Adult Life ◽  
Hematopoietic Tissue ◽  
Developmental Potential ◽  
Adult Human

Abstract A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34−Lin−CD45−CD133− cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34−Lin−CD45−CD133− cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34−Lin−CD45−CD133− cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34−Lin−CD45−CD133− cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34−Lin−CD45−CD133− cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

VRQ397 (CRAVKY): a novel noncompetitive V2 receptor antagonist

2009 ◽  
Vol 297 (4) ◽  
pp. R1009-R1018 ◽  
Author(s):  
L. Rihakova ◽  
C. Quiniou ◽  
F. F. Hamdan ◽  
R. Kaul ◽  
S. Brault ◽  
...  
Keyword(s):  
Arginine Vasopressin ◽  
Ex Vivo ◽  
Dissociation Rate ◽  
Functional Assay ◽  
Natural Ligand ◽  
Oxytocin Receptors ◽  
Negative Allosteric Modulator ◽  
V2 Receptor

Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC50 = 0.69 ± 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-l-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI2 generation but not that of cAMP or recruitment of β-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.

scholarly journals Development and Functional Characterization of Fetal Lung Organoids

2021 ◽  
Vol 8 ◽  
Author(s):  
Mandy Laube ◽  
Soeren Pietsch ◽  
Thomas Pannicke ◽  
Ulrich H. Thome ◽  
Claire Fabian
Keyword(s):  
Ex Vivo ◽  
Functional Characterization ◽  
Fetal Lung ◽  
Therapeutic Strategies ◽  
Lung Epithelial Cells ◽  
Alveolar Type ◽  
Cell Marker ◽  
Lung Maturation

Preterm infants frequently suffer from pulmonary complications due to a physiological and structural lung immaturity resulting in significant morbidity and mortality. Novel in vitro and in vivo models are required to study the underlying mechanisms of late lung maturation and to facilitate the development of new therapeutic strategies. Organoids recapitulate essential aspects of structural organization and possibly organ function, and can be used to model developmental and disease processes. We aimed at generating fetal lung organoids (LOs) and to functionally characterize this in vitro model in comparison to primary lung epithelial cells and lung explants ex vivo. LOs were generated with alveolar and endothelial cells from fetal rat lung tissue, using a Matrigel-gradient and air-liquid-interface culture conditions. Immunocytochemical analysis showed that the LOs consisted of polarized epithelial cell adhesion molecule (EpCAM)-positive cells with the apical membrane compartment facing the organoid lumen. Expression of the alveolar type 2 cell marker, RT2-70, and the Club cell marker, CC-10, were observed. Na+ transporter and surfactant protein mRNA expression were detected in the LOs. First time patch clamp analyses demonstrated the presence of several ion channels with specific electrophysiological properties, comparable to vital lung slices. Furthermore, the responsiveness of LOs to glucocorticoids was demonstrated. Finally, maturation of LOs induced by mesenchymal stem cells confirmed the convenience of the model to test and establish novel therapeutic strategies. The results showed that fetal LOs replicate key biological lung functions essential for lung maturation and therefore constitute a suitable in vitro model system to study lung development and related diseases.

scholarly journals Ex Vivo Murine Skin Model for B. burgdorferi Biofilm

Antibiotics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 528
Author(s):  
Jason P. Torres ◽  
Alireza G. Senejani ◽  
Gauri Gaur ◽  
Michael Oldakowski ◽  
Krithika Murali ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Culture Media ◽  
Ex Vivo ◽  
Microbial Interactions ◽  
Rabbit Serum ◽  
Skin Model ◽  
Ex Vivo Model ◽  
Skin Biopsies

Borrelia burgdorferi, the causative agent of Lyme disease, has been recently shown to form biofilm structures in vitro and in vivo. Biofilms are tightly clustered microbes characterized as resistant aggregations that allow bacteria to withstand harsh environmental conditions, including the administration of antibiotics. Novel antibiotic combinations have recently been identified for B. burgdorferi in vitro, however, due to prohibiting costs, those agents have not been tested in an environment that can mimic the host tissue. Therefore, researchers cannot evaluate their true effectiveness against B. burgdorferi, especially its biofilm form. A skin ex vivo model system could be ideal for these types of experiments due to its cost effectiveness, reproducibility, and ability to investigate host–microbial interactions. Therefore, the main goal of this study was the establishment of a novel ex vivo murine skin biopsy model for B. burgdorferi biofilm research. Murine skin biopsies were inoculated with B. burgdorferi at various concentrations and cultured in different culture media. Two weeks post-infection, murine skin biopsies were analyzed utilizing immunohistochemical (IHC), reverse transcription PCR (RT-PCR), and various microscopy methods to determine B. burgdorferi presence and forms adopted as well as whether it remained live in the skin tissue explants. Our results showed that murine skin biopsies inoculated with 1 × 107 cells of B. burgdorferi and cultured in BSK-H + 6% rabbit serum media for two weeks yielded not just significant amounts of live B. burgdorferi spirochetes but biofilm forms as well. IHC combined with confocal and atomic force microscopy techniques identified specific biofilm markers and spatial distribution of B. burgdorferi aggregates in the infected skin tissues, confirming that they are indeed biofilms. In the future, this ex vivo skin model can be used to study development and antibiotic susceptibility of B. burgdorferi biofilms in efforts to treat Lyme disease effectively.

scholarly journals Year-Long Phenotypical Study of Calves Derived From Different Assisted-Reproduction Technologies

2022 ◽  
Vol 8 ◽  
Author(s):  
Jordana S. Lopes ◽  
Cristina Soriano-Úbeda ◽  
Evelyne París-Oller ◽  
Sergio Navarro-Serna ◽  
Analuce Canha-Gouveia ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Growth And Development ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Embryo Production ◽  
Cattle Industry ◽  
Entire Experiment

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro-derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro-derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro-derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation.

scholarly journals Inactivation and Elimination of SARS-CoV-2 in Biosamples Using Simple Fixatives and Ultrafiltration

2021 ◽  
Vol 4 (1) ◽  
pp. 18
Author(s):  
Ranjeet Kumar ◽  
Afsal Kolloli ◽  
Selvakumar Subbian
Keyword(s):  
Biological Samples ◽  
Culture Media ◽  
Ex Vivo ◽  
Host Cells ◽  
Laboratory Research ◽  
Health Economy ◽  
Safety Level ◽  
In Vivo Models

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) causes Coronavirus disease-2019 (COVID-19), which is an ongoing pandemic that has significantly affected the health, economy, and socio-economic status of individuals worldwide. Laboratory research using in vitro, ex vivo and in vivo models has been accelerated to understand the pathogenesis of SARS-CoV-2 infection. However, such experimental research involving SARS-CoV-2 is restricted to biocontainment/safety level-3 (BSL-3) settings, due to the high pathogenicity of this virus. Since many of the downstream analyses of SARS-CoV-2-infected biological samples need to be conducted in a non-BSL3 setting, it is important to ensure that the samples are fully decontaminated and safe for subsequent analysis. Here, we report the effectiveness of standard procedures used to fix cells and tissues for pathological analysis, including 2% or 4% paraformaldehyde, 50%–70% ethanol, 10% neutral buffered formalin and ultrafiltration using membranes with a molecular weight cut-off (MWCO) ranging from 3 to 30 kDa, for inactivating or eliminating SARS-CoV-2. We validated these methods in experimental laboratory samples, such as viral inoculum in cell culture media, SARS-CoV-2 infected host cells and animal tissue lysates. We found that 15 minutes’ treatment of viral inoculum (105 plaque-forming units; PFU) or SARS-CoV-2 infected cells with paraformaldehyde or 70% ethanol resulted in complete inactivation of the virus. The treatment of infected hamster lung tissues with 10% neutral buffered formalin also fully inactivated the virus. However, only 3 kDa ultracentrifuge filter was effective in eliminating the virus to an undetectable limit in the filtrate. Our validated methods are useful for decontaminating biological samples to reduce infection risk and safe handling in BSL2 facilities.

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