scholarly journals Preservation and transplantation of porcine testis tissue

2009 ◽  
Vol 21 (3) ◽  
pp. 489 ◽  
Author(s):  
W. Zeng ◽  
A. K. Snedaker ◽  
S. Megee ◽  
R. Rathi ◽  
F. Chen ◽  
...  
Keyword(s):  
Cell Viability ◽  
Germ Cell ◽  
Slow Freezing ◽  
Freezing And Thawing ◽  
Developmental Potential ◽  
Long Term Storage ◽  
Germ Cell Differentiation ◽  
Testis Tissue

Grafting of immature mammalian testis tissue to mouse hosts can preserve the male germline. To make this approach applicable to a clinical or field situation, it is imperative that the testis tissue and/or spermatozoa harvested from grafted tissue are preserved successfully. The aim of the present study was to evaluate protocols for the preservation of testis tissue in a porcine model. Testis tissue was stored at 4°C for short-term preservation or cryopreserved by slow-freezing, automated slow-freezing or vitrification for long-term storage. Preserved tissue was transplanted ectopically to mouse hosts and recovered xenografts were analysed histologically. In addition, spermatozoa were harvested from xenografts and cryopreserved. Total cell viability and germ cell viability remained high after tissue preservation. Complete spermatogenesis occurred in xenografts preserved by cooling up to 48 h, whereas spermatogenesis progressed to round spermatids in the xenografts that were frozen–thawed before grafting. Approximately 50% of spermatozoa harvested from xenografts remained viable after freezing and thawing. The in vivo developmental potential of cryopreserved tissue was reduced despite high post-thaw viability. Therefore, it is important to evaluate germ cell differentiation in vivo in addition to cell viability in vitro when optimising freezing protocols for testis tissue.

193 IMPROVED POST-THAW SURVIVAL OF BOVINE EMBRYOS PRODUCED IN SERUM-FREE IN VITRO PRODUCTION SYSTEM

2016 ◽  
Vol 28 (2) ◽  
pp. 227
Author(s):  
M. Nõmm ◽  
E. Mark ◽  
O. Sarv ◽  
S. Kõks ◽  
Ü. Jaakma
Keyword(s):  
Survival Rates ◽  
Slow Freezing ◽  
In Vitro Fertilisation ◽  
Ministry Of Education ◽  
In Vitro Production ◽  
Freezing And Thawing ◽  
Embryo Survival ◽  
Serum Free

Over a few decades the bovine in vitro embryo production (IVP) systems have been improving rapidly. Still, the goal to produce the same quality embryos in vitro as in vivo has not yet been reached. The FCS is usually added to media during IVP to provide growth factors and energy sources. Currently, serum-free culture systems are often preferred due to the lower risk of contamination and prevention of the development of large offspring syndrome. The aim of this study was to establish whether complete elimination of FCS from the bovine IVP system has an effect on blastocyst rates, embryo quality, and embryo survival rates after slow freezing. We replaced our conventional in vitro maturation (IVM) medium [tissue culture medium-199, 10% (v/v) FCS, 10 µg mL–1 epidermal growth factor (EGF), 1500 U mL–1 serum gonadotropin and chorionic gonadotropin (PG600), Na-pyruvate 0.5 mM, gentamycin sulfate 50 µg mL–1 and l-glutamine 1 mM] with SOF (SOFaaci) supplemented with 0.4% fatty acid-free BSA fraction V, 10 µg mL–1 EGF, and 1500 U mL–1 PG600. Matured cumulus-oocyte complexes (COC) from both experimental groups (total of 1145 from serum-free IVP and 687 from our conventional IVP system) were used for in vitro fertilisation and culture. Blastocyst rates were similar in the serum-free and our usual IVP protocol, 18 and 22%, respectively. Seventy-seven Grade 1 (according to IETS) Day 7 blastocysts from the serum-free IVP system and 80 Grade 1 Day 7 blastocysts from our conventional IVP system were frozen in 1.5 M ethylene glycol and 0.1 M sucrose containing cryopreservation medium. The post-thaw survival rates after 24 h of culture and evaluated as percentages of re-expanded embryos were 63.6% for the serum-free IVP and 46.3% for the conventional IVP system (P < 0.05, Z Test for 2 population proportions). These results indicate that it is possible to have a completely serum-free bovine IVP system and based on the slow freezing and thawing results the quality of serum-free IVP embryos might be better than of the embryos matured in our conventional maturation media. However, more experiments and increased sample sizes are needed to confirm the results. This study was supported by Project 3.2.0701.12–0036 of Archimedes Foundation, AP 2.4 of CCRMB, and institutional research funding (IUT 08–01) of the Estonian Ministry of Education and Research.

Size heterogeneity of immunoreactive prolactin in patients with prolactinoma

1987 ◽  
Vol 114 (4) ◽  
pp. 475-482 ◽  
Author(s):  
B. Allolio ◽  
A. Hoeppener ◽  
U. Leonhardt ◽  
U. Deuβ ◽  
W. Winkelmann
Keyword(s):  
Tumour Size ◽  
Serum Prolactin ◽  
Gel Chromatography ◽  
Freezing And Thawing ◽  
Serum Samples ◽  
Elution Pattern ◽  
Long Term Storage ◽  
Repeated Freezing

Abstract. We investigated the chromatographic pattern of serum prolactin in 41 patients with prolactinoma and correlated the distribution of immunoreactive prolactin with the clinical variables sex, tumour size, age, and response to bromocriptine therapy. In addition, the effect of long-term storage and repeated freezing and thawing on the different molecular weight forms of prolactin was evaluated. Gel chromatography (column 100 cm × 1.5 cm) was performed in 0.1 mol/l phosphate buffer, pH 7.5, using Ultrogel ACA 54 (LKB). No correlation of age or the response to drug therapy to the elution pattern of prolactin was found. Females showed a higher percentage of big prolactin than males (10.4 ± 1.2% vs 6.8 ± 0.7%, x̄ ± sem, P <0.05) and patients with microprolactinomas too had a higher percentage of big prolactin than those with macroprolactinomas (11.3 ± 1.8% vs 7.7 ± 0.7%, P <0.05). Serum samples kept frozen for more than 2 years showed a higher percentage of bigbig prolactin (P < 0.01) than samples stored for less than 12 months suggesting formation in vitro. However, examination of fresh samples prior to freezing also demonstrated bigbig prolactin, indicating that bigbig prolactin circulates in vivo. Repeated freezing and thawing of bigbig prolactin led to almost complete interconversion to little prolactin without any increase in immunoreactivity. This finding supports the concept that bigbig prolactin represents little prolactin loosely associated to a carrier molecule.

scholarly journals In vivo and in vitro differentiation of male germ cells in the mouse

Reproduction ◽  
2004 ◽  
Vol 128 (2) ◽  
pp. 147-152 ◽  
Author(s):  
Orly Lacham-Kaplan
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Cellular Interactions ◽  
Germ Cell Differentiation

Primordial germ cells appear in the embryo at about day 7 after coitum. They proliferate and migrate towards the genital ridge. Once there, they undergo differentiation into germ stem cells, known as ‘A spermatogonia’. These cells are the foundation of spermatogenesis. A spermatogonia commit to spermatogenesis, stay undifferentiated or degenerate. The differentiation of primordial germ cells to migratory, postmigratory and germ stem cells is dependent on gene expression and cellular interactions. Some of the genes that play a crucial role in germ cell differentiation are Steel, c-Kit, VASA, DAZL, fragilis, miwi, mili, mil1 and mil2. Their expression is stage specific, therefore allowing solid identification of germ cells at different developmental phases. In addition to the expression of these genes, other markers associated with germ cell development are nonspecific alkaline phosphatase activity, the stage specific embryonic antigen, the transcription factor Oct3/4 and β1- and α6-integrins. Commitment of cells to primordial germ cells and to A spermatogonia is also dependent on induction by the bone morphogenetic protein (BMP)-4. With this knowledge, researchers were able to isolate germ stem cells from embryonic stem cell-derived embryoid bodies, and drive these into gametes either in vivo or in vitro. Although no viable embryos were obtained from these gametes, the prospects are that this goal is not too far from being accomplished.

scholarly journals Genetic and structural analysis of the in vivo functional redundancy between murine NANOS2 and NANOS3

Development ◽  
2020 ◽  
pp. dev.191916
Author(s):  
Danelle Wright ◽  
Makoto Kiso ◽  
Yumiko Saga
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Double Knockout ◽  
Germ Cell Differentiation ◽  
Evolutionarily Conserved ◽  
Male Specific

NANOS2 and NANOS3 are evolutionarily conserved RNA-binding proteins involved in murine germ cell development. NANOS3 is required for protection from apoptosis during migration and gonadal colonization in both sexes, whereas NANOS2 is male-specific and required for the male-type differentiation of germ cells. Ectopic NANOS2 rescues the functions of NANOS3, but NANOS3 cannot rescue NANOS2 function even though its expression is up-regulated in Nanos2-null conditions. It is unknown why NANOS3 cannot rescue NANOS2 function and it is unclear whether NANOS3 plays any role in male germ cell differentiation. To address these questions, we made conditional Nanos3/Nanos2 knockout mice and chimeric mice expressing chimeric NANOS proteins. Conditional double knockout of Nanos2 and 3 led to the rapid loss of germ cells, and in vivo and in vitro experiments revealed that DND1 and NANOS2 binding is dependent on the specific NANOS2 zinc finger structure. Moreover, murine NANOS3 failed to bind CNOT1, an interactor of NANOS2 at its N-terminal. Collectively, our study suggests that the inability of NANOS3 to rescue NANOS2 function is due to poor DND1 recruitment and CNOT1 binding.

Antitumor effects of AR-42, a novel histone deacetylase inhibitor, in embryonal carcinoma.

2011 ◽  
Vol 29 (7_suppl) ◽  
pp. 232-232
Author(s):  
A. S. Bhinder ◽  
V. Varma ◽  
B. Abbaoui ◽  
J. M. Thomas-Ahner ◽  
S. K. Kulp ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Germ Cell ◽  
Histone Deacetylase ◽  
Embryonal Carcinoma ◽  
Rodent Model ◽  
Antitumor Effects ◽  
Dose Dependent

232 Background: Histone deacetylase inhibitors (HDACIs) modulate gene expression and induce cellular differentiation, growth inhibition and apoptotic cell death by chromatin hyperacetylation. Developmental arrest of germ cell differentiation earlier in the life is responsible for the pathogenesis of germ cell tumors (GCT). With current treatment nearly 95% of patients with GCT can be cured. Yet, effective agents with less toxicity are desired. In addition, those with relapsed/refractory disease have a dismal prognosis, indicating a clear need for new, more effective agents. Here we assess the antitumor effects of AR-42, a novel HDACI in in vitro and in vivo models of embryonal carcinoma. Methods: In vitro effects of AR-42 and suberoylanilide hydroxamic acid (SAHA) were evaluated in NTERA-2, an embryonal carcinoma (EC) cell line derived from a human testicular cancer. Cell viability (MTS assay), apoptosis (caspase 3/7 activity and PARP cleavage), cell cycle analysis (flow cytometry) and HDAC inhibition (immunoblotting) were assessed. The in vivo efficacy of AR-42 was assessed in a NTERA-2 xenograft tumor model in male athymic nude mice. Mice were fed control diet and diet containing AR-42 at an average dose of 25 mg/kg/day. Tumor volumes and weights were used as in vivo endpoints. Results: Treatment of NTERA-2 cells with both agents at 0.1-10 μM concentrations showed a time- and dose-dependent reduction in cell viability. Both agents significantly induced apoptosis, cell cycle inhibition and hyperacetylation of histones H-3 and H-4 in a dose-dependent manner. In vitro studies showed that AR-42 was more potent than SAHA. In our rodent model, AR-42-containing diet resulted in a significant reduction in tumor volumes and weights (50% and 56%, respectively). The results for intratumoral changes of proliferation and apoptosis are pending. There were no significant toxicities associated with AR-42, except for testicular atrophy, known to be reversible. Conclusions: AR-42 appears to be a potent inhibitor of EC through different mechanisms, orally bioavailable and well tolerated in our rodent model. Our data indicates that AR- 42 may have clinical value in the treatment of GCT and requires further investigation in clinical trials. No significant financial relationships to disclose.

scholarly journals EXTH-46. ARTIFICIAL INTELLIGENCE-BASED IDENTIFICATION OF COMBINED VANDETANIB AND EVEROLIMUS IN THE TREATMENT OF ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii97-ii97
Author(s):  
Diana Carvalho ◽  
Peter Richardson ◽  
Nagore Gene Olaciregui ◽  
Reda Stankunaite ◽  
Cinzia Emilia Lavarino ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Cell Viability ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Serine Threonine Kinase ◽  
Orthotopic Xenografts ◽  
Extended Survival

Abstract Somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2 receptor, are found in a quarter of children with the currently incurable brain tumour diffuse intrinsic pontine glioma (DIPG). Treatment of ACVR1-mutant DIPG patient-derived models with multiple inhibitor chemotypes leads to a reduction in cell viability in vitro and extended survival in orthotopic xenografts in vivo, though there are currently no specific ACVR1 inhibitors licensed for DIPG. Using an Artificial Intelligence-based platform to search for approved compounds which could be used to treat ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an approved inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd=150nM) and reduce DIPG cell viability in vitro, but has been trialed in DIPG patients with limited success, in part due to an inability to cross the blood-brain-barrier. In addition to mTOR, everolimus inhibits both ABCG2 (BCRP) and ABCB1 (P-gp) transporter, and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination is well-tolerated in vivo, and significantly extended survival and reduced tumour burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Based on these preclinical data, three patients with ACVR1-mutant DIPG were treated with vandetanib and everolimus. These cases may inform on the dosing and the toxicity profile of this combination for future clinical studies. This bench-to-bedside approach represents a rapidly translatable therapeutic strategy in children with ACVR1 mutant DIPG.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Guoying Zhang ◽  
Cheng Xue ◽  
Yiming Zeng
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Cell Model ◽  
Protective Efficacy ◽  
Rabbit Model ◽  
Akt Pathway ◽  
Loss Of Function ◽  
Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

scholarly journals Rapid Fabrication of Microfluidic Devices for Biological Mimicking: A Survey of Materials and Biocompatibility

Micromachines ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 346
Author(s):  
Hui Ling Ma ◽  
Ana Carolina Urbaczek ◽  
Fayene Zeferino Ribeiro de Souza ◽  
Paulo Augusto Gomes Garrido Carneiro Leão ◽  
Janice Rodrigues Perussi ◽  
...  
Keyword(s):  
Shear Stress ◽  
Cell Viability ◽  
Cellular Response ◽  
Fluid Shear Stress ◽  
Umbilical Vein ◽  
Microfluidic Devices ◽  
In Vitro Assays ◽  
Stress Effect

Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept.

scholarly journals Effect of Different Titanium Dental Implant Surfaces on Human Adipose Mesenchymal Stem Cell Behavior. An In Vitro Comparative Study

2021 ◽  
Vol 11 (14) ◽  
pp. 6353
Author(s):  
Vittoria D’Esposito ◽  
Josè Camilla Sammartino ◽  
Pietro Formisano ◽  
Alessia Parascandolo ◽  
Domenico Liguoro ◽  
...  
Keyword(s):  
Cell Viability ◽  
Dental Implant ◽  
In Vivo Studies ◽  
Implant Surface ◽  
Cytokines And Chemokines ◽  
Titanium Dental Implant ◽  
Adhesive Ability ◽  
Stimulating Factor

Background: The aim of this research was to evaluate the effects of three different titanium (Ti) implant surfaces on the viability and secretory functions of mesenchymal stem cells isolated from a Bichat fat pad (BFP-MSCs). Methods: Four different Ti disks were used as substrate: (I) D1: smooth Ti, as control; (II) D2: chemically etched, resembling the Kontact S surface; (III) D3: sandblasted, resembling the Kontact surface; (IV) D4: blasted/etched, resembling the Kontact N surface. BFP-MSCs were plated on Ti disks for 72 h. Cell viability, adhesion on disks and release of a panel of cytokines, chemokines and growth factor were evaluated. Results: BFP-MSCs plated in wells with Ti surface showed a viability rate (~90%) and proliferative rate comparable to cells plated without disks and to cells plated on D1 disks. D2 and D4 showed the highest adhesive ability. All the Ti surfaces did not interfere with the release of cytokines, chemokines and growth factors by BFP-MSCs. However, BFP-MSCs cultured on D4 surface released a significantly higher amount of Granulocyte Colony-Stimulating Factor (G-CSF) compared either to cells plated without disks and to cells plated on D1 and D2. Conclusions: The implant surfaces examined do not impair the BFP-MSCs cell viability and preserve their secretion of cytokines and chemokines. Further in vitro and in vivo studies are necessary to define the implant surface parameters able to assure the chemokines’ optimal release for a real improvement of dental implant osseointegration.

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