Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: stemness gene expression and structural features

Zygote ◽  
2021 ◽  
pp. 1-7
Author(s):  
Masoud Hemadi ◽  
Vahideh Assadollahi ◽  
Ghasem Saki ◽  
Afshin Pirnia ◽  
Masoud Alasvand ◽  
...  
Keyword(s):  
Gene Expression ◽  
3D Culture ◽  
Enzymatic Digestion ◽  
Structural Features ◽  
Alginate Hydrogel ◽  
Scaffold Structure ◽  
Polymerase Chain ◽  
Magnetic Activated Cell Sorting

Summary The quality and quantity of a spermatogonial stem-cell (SSC) culture can be measured in less time using a 3D culture in a scaffold. The present study investigated stemness gene expression and the morphological and structural characterization of SSCs encapsulated in alginate. SSCs were harvested from BALB/c neonatal mice testes through two-step mechanical and enzymatic digestion. The spermatogonial populations were separated using magnetic-activated cell sorting (MACS) using an anti-Thy1 antibody and c-Kit. The SSCs then were encapsulated in alginate hydrogel. After 2 months of SSC culturing, the alginate microbeads were extracted and stained to evaluate their histological properties. Real-time polymerase chain reaction (PCR) was performed to determine the stemness gene expression. Scanning electron microscopy (SEM) was performed to evaluate the SSC morphology, density and scaffold structure. The results showed that encapsulated SSCs had decreased expression of Oct4, Sox2 and Nanos2 genes, but the expression of Nanog, Bcl6b and Plzf genes was not significantly altered. Histological examination showed that SSCs with pale nuclei and numerous nucleolus formed colonies. SEM evaluation revealed that the alginate scaffold structure preserved the SSC morphology and density for more than 60 days. Cultivation of SSCs on alginate hydrogel can affect Oct4, Sox2 and Nanos2 expression.

scholarly journals Preparation, Modification, and Characterization of Alginate Hydrogel with Nano-/Microfibers: A New Perspective for Tissue Engineering

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Bianca Palma Santana ◽  
Fernanda Nedel ◽  
Evandro Piva ◽  
Rodrigo Varella de Carvalho ◽  
Flávio Fernando Demarco ◽  
...  
Keyword(s):  
Cell Viability ◽  
Chemical Properties ◽  
Chemical Characteristics ◽  
X Ray Diffraction ◽  
Alginate Hydrogel ◽  
X Ray ◽  
New Perspective ◽  
Nih 3T3

We aimed to develop an alginate hydrogel (AH) modified with nano-/microfibers of titanium dioxide (nfTD) and hydroxyapatite (nfHY) and evaluated its biological and chemical properties. Nano-/microfibers of nfTD and nfHY were combined with AH, and its chemical properties were evaluated by FTIR spectroscopy, X-ray diffraction, energy dispersive X-Ray analysis, and the cytocompatibility by the WST-1 assay. The results demonstrate that the association of nfTD and nfHY nano-/microfibers to AH did not modified the chemical characteristics of the scaffold and that the association was not cytotoxic. In the first 3 h of culture with NIH/3T3 cells nfHY AH scaffolds showed a slight increase in cell viability when compared to AH alone or associated with nfTD. However, an increase in cell viability was observed in 24 h when nfTD was associated with AH scaffold. In conclusion our study demonstrates that the combination of nfHY and nfTD nano-/microfibers in AH scaffold maintains the chemical characteristics of alginate and that this association is cytocompatible. Additionally the combination of nfHY with AH favored cell viability in a short term, and the addition of nfTD increased cell viability in a long term.

scholarly journals Recovery of Salmonella from biofilms in a headwater spring ecosystem

2011 ◽  
Vol 9 (3) ◽  
pp. 458-466 ◽  
Author(s):  
James P. Gaertner ◽  
Joseph A. Mendoza ◽  
Michael R. J. Forstner ◽  
Dittmar Hahn
Keyword(s):  
Pathogenic Bacteria ◽  
Chain Reaction ◽  
Selective Enrichment ◽  
Animal Wastes ◽  
San Marcos ◽  
Polymerase Chain ◽  
Spring Lake ◽  
Precipitation Events

Salmonellae are pathogenic bacteria often detected in waters impacted by human or animal wastes. In order to assess the fate of salmonellae in supposedly pristine environments, water and natural biofilm samples along with snails (Tarebia granifera) and crayfish (Procambarus clarkia) were collected before and up to 7 days following four precipitation events from sites within the headwater springs of Spring Lake, San Marcos, TX. The samples were analyzed for the presence of salmonellae by polymerase chain reaction (PCR) after semi-selective enrichment. Salmonellae were detected in one water sample directly after precipitation only, while detection in ten biofilm and two crayfish samples was not related to precipitation. Salmonellae were not detected in snails. Characterization of isolates by rep-PCR revealed shared profiles in water and biofilm samples, biofilm and crayfish samples, and biofilm samples collected 23 days apart. These results suggest that salmonellae are infrequently washed into this aquatic ecosystem during precipitation runoff and can potentially take up residency in biofilms which can help facilitate subsequent long-term persistence and eventual transfer through the food chain.

scholarly journals Characterization of Blueberry shock virus, an Emerging Ilarvirus in Cranberry

Plant Disease ◽  
2018 ◽  
Vol 102 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Sara Thomas-Sharma ◽  
Lindsay Wells-Hansen ◽  
Rae Page ◽  
Victoria Kartanos ◽  
Erika Saalau-Rojas ◽  
...  
Keyword(s):  
Virus Transmission ◽  
Systemic Infection ◽  
Seed Transmission ◽  
Economic Consequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymerase Chain ◽  
Marketable Fruit ◽  
Mixed Virus Infection

Blueberry shock virus (BlShV), an Ilarvirus sp. reported only on blueberry, was associated with scarring, disfigurement, and premature reddening of cranberry fruit. BlShV was detected by triple-antibody sandwich enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction, and isometric virions of 25 to 28 nm were observed in cranberry sap. The virus was systemic, although unevenly distributed in plants. The coat protein of BlShV from cranberry shared 90% identity compared with BlShV accessions from blueberry on GenBank. Phylogenetic analysis of isolates of BlShV from cranberry collected from Wisconsin and Massachusetts did not indicate grouping by state. BlShV was detected in cranberry pollen, and seed transmission of up to 91% was observed. Artificial inoculation of cranberry flowers by pollination did not cause virus transmission. In some Nicotiana spp., rub inoculation of leaves with homogenized BlShV-positive cranberry flowers resulted in systemic infection. Cranberry plants recovered from symptoms the year after berry scarring occurred but continued to test positive for BlShV. The virus caused significant reduction in the average number of marketable fruit and average berry weight in symptomatic cranberry plants but recovered plants yielded comparably with healthy plants. Although recovery may limit the immediate economic consequences of BlShV, long-term implications of single- or mixed-virus infection in cranberry is unknown.

Deformation Mechanism of Basic Rock During Long-Term Compression: Area of Hlw Repository Design, Chelyabinsk District, Russia

1995 ◽  
Vol 412 ◽  
Author(s):  
V. A. Petrov ◽  
L. I. Zviagintsev ◽  
V. V. Poluektov
Keyword(s):  
Compressive Strength ◽  
Deformation Mechanism ◽  
Near Field ◽  
Structural Features ◽  
Behavior Changes ◽  
Underground Disposal ◽  
High Level ◽  
Compression Area

AbstractA combination of ultrasound, mechanical and petrographic results for long-term experimental compression of greenschist facies porphyritic andesite tuffs indicate a deformation mechanism that depends upon the mineral composition, textural-structural features of the rocks and the orientation of compression relative to the rock textures. Three dry samples of rock were investigated. Coaxial compression of a massive sample for 816 hours and a foliated sample for 1176 hours (pressure orthogonal to foliation) is characterized by solidification when the rocks are temporarily metastable. Compressive strength of the first sample is 850 kg/cm2 and of the second one, 800 kg/cm2. Experimentally, the rock behavior changes from a plastic to a brittle regime of deformation. In contrast, compression of the foliated sample parallel to foliation causes disintegration along the foliation within 480 hours without solidification. The rock is liable to brittle deformation and its compressive strength is 500 kg/cm2. These results may have implications for characterization of near-field processes in connection with numerous subhorizontal zones of schistosity within the strata that are targeted for underground disposal of high-level wastes (HLW) in the Mayak radiochemical complex area.

scholarly journals Memory immune response: a major challenge in vaccination

2012 ◽  
Vol 3 (5) ◽  
pp. 479-486 ◽  
Author(s):  
Antonella Prisco ◽  
Piergiuseppe De Berardinis
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Vaccine Development ◽  
Immune Memory ◽  
Gene Expression Signatures ◽  
Antibody Titers ◽  
Memory Immune Response ◽  
Protective Threshold

AbstractA crucial challenge for vaccine development is to design vaccines that induce a long-lasting protective immune response, i.e., immune memory. The persistence of antigen-specific antibody titers over a protective threshold, and the ability to exibit a ‘recall response’ to a subsequent encounter with an antigen have long been the only measurable correlates of vaccine take and immune memory development, suffering from the disadvantage of relying on long-term monitoring of the immune response. In the last few years, advances in the technologies for the identification and characterization of the cell subsets and molecular pathways involved in the immune response to vaccination have allowed innovative approaches to the identification of early correlates of immune memory. In this review, we discuss recent data and hypotheses on early correlates of the development of immune memory, with special emphasis on the gene expression signatures that underlie the self-renewal ability of some lymphocyte subsets, and their similarities with gene expression signatures in stem cells.

scholarly journals Ing4-deficiency enhances HSC quiescence and confers resistance to inflammatory stress

2021 ◽  
Author(s):  
Zanshé Thompson ◽  
Georgina A. Anderson ◽  
Melanie Rodriguez ◽  
Seth Gabriel ◽  
Vera Binder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Malignant Cells ◽  
Short Term ◽  
Regenerative Capacity ◽  
Hematopoietic Stem ◽  
Inflammatory Stress ◽  
Identification And Characterization

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the development and maintenance of hematopoietic stem cells (HSCs). We recently identified the tumor suppressor, Ing4, as a critical regulator of HSC homeostasis. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to Ing4 mediated regulation of several major signaling pathways, including NF-kB and c-Myc. In murine hematopoiesis, Ing4 deficiency induces G0 arrest in HSCs, while simultaneously promoting gene expression signatures associated with differentiation. This results in a poised state for Ing4-deficient HSCs. Long term HSCs are unable to overcome this block, but short-term HSCs convert the poised state into regenerative capacity during hematopoietic challenges, including irradiation and transplantation. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis. Our model provides key tools for further identification and characterization of pathways that control quiescence and differentiation in HSCs.

scholarly journals 3D Bioprinting Allows the Establishment of Long-Term 3D Culture Model for Chronic Lymphocytic Leukemia Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Francesca Vittoria Sbrana ◽  
Riccardo Pinos ◽  
Federica Barbaglio ◽  
Davide Ribezzi ◽  
Fiorella Scagnoli ◽  
...  
Keyword(s):  
Gene Expression ◽  
3D Culture ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
3D Bioprinting ◽  
Culture Model ◽  
3D Culture Model

Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia in the western world and remains incurable. Leukemic cells organize and interact in the lymphoid tissues, however what actually occurs in these sites has not been fully elucidated yet. Studying primary CLL cells in vitro is very challenging due to their short survival in culture and also to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited for the first time three-dimensional (3D) bioprinting to advance in vitro models for CLL. This technology allowed us to print CLL cells (both primary cells and cell lines) mixed with the appropriate, deeply characterized, hydrogel to generate a scaffold containing the cells, thus avoiding the direct cell seeding onto a precast 3D scaffold and paving the way to more complex models. Using this system, we were able to efficiently 3D bioprint leukemic cells and improve their viability in vitro that could be maintained up to 28 days. We monitored over time CLL cells viability, phenotype and gene expression, thus establishing a reproducible long-term 3D culture model for leukemia. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings. In particular, we identified pathways upregulated in 3D, at both day 7 and 14, associated with immunoglobulins production, pro-inflammatory molecules expression, activation of cytokines/chemokines and cell-cell adhesion pathways, paralleled by a decreased production of proteins involved in DNA replication and cell division, suggesting a strong adaptation of the cells in the 3D culture. Thanks to this innovative approach, we developed a new tool that may help to better mimic the physiological 3D in vivo settings of leukemic cells as well as of immune cells in broader terms. This will allow for a more reliable study of the molecular and cellular interactions occurring in normal and neoplastic conditions in vivo, and could also be exploited for clinical purposes to test individual responses to different drugs.

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