scholarly journals Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies

2021 ◽  
Author(s):  
David Choy Buentello ◽  
Lina Sophie Koch ◽  
Grissel Trujillo-de Santiago ◽  
Mario Moisés Alvarez ◽  
Kerensa Broersen
Keyword(s):  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Cost Effective ◽  
Embryoid Bodies ◽  
High Yield ◽  
Final Size ◽  
Surface Attachment ◽  
Cellular Aggregation ◽  
Centrifugation Step ◽  
Lower Variability

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×10 3 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×10 3 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×10 3 and 11×10 3 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages

scholarly journals Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in

2017 ◽  
Author(s):  
Yi-Jiun Chen ◽  
Weikang Wang ◽  
Xiao-Jiun Tian ◽  
Daniel E. Lefever ◽  
David A. Taft ◽  
...  
Keyword(s):  
Disease Modeling ◽  
Directed Assembly ◽  
Experimental Tests ◽  
Cost Effective ◽  
High Yield ◽  
Gibson Assembly ◽  
Knock Out ◽  
Quantitative Studies ◽  
Repair Template ◽  
Assembly Procedure

ABSTRACTCRISPR-based gene knock-in at endogenous sites is desirable in multiple fields such as quantitative studies of signal transduction pathways and gene regulation, synthetic biology, and disease modeling. Contrasting the knock-out procedure, a key step of CRISPR knock-in procedure relies on the homology-directed repairing (HDR) process that requires a donor construct as repair template. Therefore, it is desirable to generate a series of donor DNA constructs efficiently and cost-effectively. In this study, we developed a general Gibson assembly procedure that combines strengths of a Modular Overlap-Directed Assembly with Linkers (MODAL) strategy and a restriction enzyme based hierarchical framework. This procedure also allows fusing sgRNAs to the constructs for enhanced homology-directed repairing efficiency. Experimental tests on multiple constructs achieved from 3-8 folds of increase in assembly efficiency to high yield of constructs that failed to make with conventional Gibson assembly. The modularized procedure is simple, fast and cost-effective while making multiple constructs, and a computer package is provided for customized design.

scholarly journals Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ratchapong Netsrithong ◽  
Siriwal Suwanpitak ◽  
Bootsakorn Boonkaew ◽  
Kongtana Trakarnsanga ◽  
Lung-Ji Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Drug Screening ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Induced Pluripotent

Abstract Background Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. Methods Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. Results Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. Conclusion This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

scholarly journals Cost Effectiveness of Transbronchial Needle Aspiration

1999 ◽  
Vol 6 (4) ◽  
pp. 332-335 ◽  
Author(s):  
Jennifer A Crocket ◽  
Eric YL Wong ◽  
Dale C Lien ◽  
Khanh Gia Nguyen ◽  
Michelle R Chaput ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Cost Effective ◽  
Diagnostic Procedures ◽  
Needle Aspiration ◽  
Benign Disease ◽  
University Hospital ◽  
High Yield ◽  
Transbronchial Needle Aspiration ◽  
Computed Tomographic ◽  
The Cost

OBJECTIVE: To evaluate the yield and cost effectiveness of transbronchial needle aspiration (TBNA) in the assessment of mediastinal and/or hilar lymphadenopathy.DESIGN: Retrospective study.SETTING: A university hospital.POPULATION STUDIED: Ninety-six patients referred for bronchoscopy with computed tomographic evidence of significant mediastinal or hilar adenopathy.RESULTS: Ninety-nine patient records were reviewed. Three patients had two separate bronchoscopy procedures. TBNA was positive in 42 patients (44%) and negative in 54 patients. Of the 42 patients with a positive aspirate, 40 had malignant cytology and two had cells consistent with benign disease. The positive TBNA result altered management in 22 of 40 patients with malignant disease and one of two patients with benign disease, thereby avoiding further diagnostic procedures. The cost of these subsequent procedures was estimated at $27,335. No complications related to TBNA were documented.CONCLUSIONS: TBNA is a high-yield, safe and cost effective procedure for the diagnosis and staging of bronchogenic cancer.

scholarly journals Development of Quantum Dot (QD) Based Color Converters for Multicolor Display

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1089
Author(s):  
Muhammad T. Sajjad ◽  
Ashu K. Bansal ◽  
Francesco Antolini ◽  
Eduard Preis ◽  
Lenuta Stroea ◽  
...  
Keyword(s):  
Polymer Matrix ◽  
Cost Effective ◽  
Final Size ◽  
Force Microscopy ◽  
Morphological Studies ◽  
Color Displays ◽  
Transmission Electron ◽  
Different Temperatures ◽  
Color Conversion ◽  
Temperature Time

Many displays involve the use of color conversion layers. QDs are attractive candidates as color converters because of their easy processability, tuneable optical properties, high photoluminescence quantum yield, and good stability. Here, we show that emissive QDs with narrow emission range can be made in-situ in a polymer matrix, with properties useful for color conversion. This was achieved by blending the blue-emitting pyridine based polymer with a cadmium selenide precursor and baking their films at different temperatures. To achieve efficient color conversion, blend ratio and baking temperature/time were varied. We found that thermal decomposition of the precursor leads to highly emissive QDs whose final size and emission can be controlled using baking temperature/time. The formation of the QDs inside the polymer matrix was confirmed through morphological studies using atomic force microscopy (AFM) and transmission electron microscopy (TEM). Hence, our approach provides a cost-effective route to making highly emissive color converters for multi-color displays.

scholarly journals Recent Advances in Disease Modeling and Drug Discovery for Diabetes Mellitus Using Induced Pluripotent Stem Cells

2016 ◽  
Vol 17 (2) ◽  
pp. 256 ◽  
Author(s):  
Mohammed Kawser Hossain ◽  
Ahmed Abdal Dayem ◽  
Jihae Han ◽  
Subbroto Kumar Saha ◽  
Gwang-Mo Yang ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Stem Cells ◽  
Drug Discovery ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Recent Advances ◽  
Induced Pluripotent

scholarly journals Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bei Liu ◽  
Shi Chen ◽  
Yaxing Xu ◽  
Yulin Lyu ◽  
Jinlin Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Human Fibroblast ◽  
Culture System ◽  
Disease Modeling ◽  
Directed Differentiation

AbstractExtended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

scholarly journals The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin.

1991 ◽  
Vol 11 (8) ◽  
pp. 4196-4206 ◽  
Author(s):  
A Roy ◽  
C F Lu ◽  
D L Marykwas ◽  
P N Lipke ◽  
J Kurjan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Signal Sequence ◽  
Complementation Group ◽  
Alpha Cells ◽  
Specific Expression ◽  
Surface Attachment ◽  
Cellular Aggregation ◽  
Specific Regulation ◽  
Binding Subunit

Saccharomyces cerevisiae a and alpha cells express the complementary cell surface glycoproteins a-agglutinin and alpha-agglutinin, respectively, which interact with one another to promote cellular aggregation during mating. Treatment of S. cerevisiae a cells with reducing agents releases the binding subunit of a-agglutinin, which has been purified and characterized; little biochemical information on the overall structure of a-agglutinin is available. To characterise a-agglutinin structure and function, we have used a genetic approach to clone an a-agglutinin structural gene (AGAI). Mutants with a-specific agglutination defects were isolated, the majority of which fell into a single complementation group, called aga1. The aga1 mutants showed wild-type pheromone production and response, efficient mating on solid medium, and a mating defect in liquid medium; these phenotypes are characteristic of agglutinin mutants. The AGA1 gene was cloned by complementation; the gene sequence indicated that it could encode a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors. Active a-agglutinin binding subunit is secreted by aga1 mutants, indicating that AGA1 is involved in cells surface attachment of a-agglutinin. This result suggests that AGA1 encodes a protein with functional similarity to the core subunits of a-agglutinin analogs from other budding yeasts. Unexpectedly, the AGA1 transcript was expressed and induced by pheromone in both a and alpha cells, suggesting that the a-specific expression of active a-agglutinin results only from a-specific regulation of the a-agglutinin binding subunit.

scholarly journals The Promise of Human Induced Pluripotent Stem Cells in Dental Research

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Thekkeparambil Chandrabose Srijaya ◽  
Padmaja Jayaprasad Pradeep ◽  
Rosnah Binti Zain ◽  
Sabri Musa ◽  
Noor Hayaty Abu Kasim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Patient Specific ◽  
Dental Research ◽  
Cell Based Therapy ◽  
Induced Pluripotent ◽  
Disease Specific

Induced pluripotent stem cell-based therapy for treating genetic disorders has become an interesting field of research in recent years. However, there is a paucity of information regarding the applicability of induced pluripotent stem cells in dental research. Recent advances in the use of induced pluripotent stem cells have the potential for developing disease-specific iPSC linesin vitrofrom patients. Indeed, this has provided a perfect cell source for disease modeling and a better understanding of genetic aberrations, pathogenicity, and drug screening. In this paper, we will summarize the recent progress of the disease-specific iPSC development for various human diseases and try to evaluate the possibility of application of iPS technology in dentistry, including its capacity for reprogramming some genetic orodental diseases. In addition to the easy availability and suitability of dental stem cells, the approach of generating patient-specific pluripotent stem cells will undoubtedly benefit patients suffering from orodental disorders.

Application of Epoxy Resin for Improvement of the Banana Stem-Acoustic Panel

2021 ◽  
Vol 15 ◽  
Author(s):  
Mohd Azril Riduan ◽  
Mohd Jumain Jalil ◽  
Intan Suhada Azmi ◽  
Afifudin Habulat ◽  
Danial Nuruddin Azlan Raofuddin ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Kinetic Modelling ◽  
Low Cost ◽  
Cost Effective ◽  
Performic Acid ◽  
High Yield ◽  
Renewable Materials ◽  
Runge Kutta Method ◽  
To Come

Background: Greener epoxidation by using vegetable oil to create an eco-friendly epoxide is being studied because it is a more cost-effective and environmentally friendly commodity that is safer than non-renewable materials. The aim of this research is to come up with low-cost solutions for banana trunk acoustic panels with kinetic modelling of epoxy-based palm oil. Method: In this study, the epoxidation of palm oleic acid was carried out by in situ performic acid to produce epoxidized palm oleic acid. Results: Banana trunk acoustic panel was successfully innovated based on the performance when the epoxy was applied. Lastly, a mathematical model was developed by using the numerical integration of the 4th order Runge-Kutta method, and the results showed that there is a good agreement between the simulation and experimental data, which validates the kinetic model. Conclusion: Overall, the peracid mechanism was effective in producing a high yield of epoxy from palm oleic acid that is useful for the improvement of acoustic panels based on the banana trunk.

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