scholarly journals A Cytotoxic Three-Dimensional-Spheroid, High-Throughput Assay Using Patient-Derived Glioma Stem Cells

2018 ◽  
Vol 23 (8) ◽  
pp. 842-849 ◽  
Author(s):  
Victor Quereda ◽  
Shurong Hou ◽  
Franck Madoux ◽  
Louis Scampavia ◽  
Timothy P. Spicer ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Three Dimensional ◽  
Treatment Resistance ◽  
Small Population ◽  
Molecular Probes ◽  
Glioma Stem Cells

Glioblastoma (GBM) is the most aggressive primary brain cancer with an average survival time after diagnosis of only 12–14 months, with few (<5%) long-term survivors. A growing body of work suggests that GBMs contain a small population of glioma stem cells (GSCs) that are thought to be major contributors to treatment resistance and disease relapse. Identifying compounds that modulate GSC proliferation would provide highly valuable molecular probes of GSC-directed signaling. However, targeting GSCs pharmacologically has been challenging. Patient-derived GSCs can be cultured as neurospheres, and in vivo these cells functionally recapitulate the heterogeneity of the original tumor. Using patient-derived GSC-enriched cultures, we have developed a 1536-well spheroid-based proliferation assay and completed a pilot screen, testing ~3300 compounds comprising approved drugs. This cytotoxic and automation-friendly assay yielded a signal-to-background (S/B) ratio of 161.3 ± 7.5 and Z′ of 0.77 ± 0.02, demonstrating its robustness. Importantly, compounds were identified with anti-GSC activity, demonstrating the applicability of this assay for large-scale high-throughput screening (HTS).

scholarly journals Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Hilary A. Kenny ◽  
Madhu Lal-Nag ◽  
Erin A. White ◽  
Min Shen ◽  
Chun-Yi Chiang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Organotypic Culture ◽  
Three Dimensional ◽  
In Vivo Efficacy

scholarly journals 3D Visualization and Volume-Based Quantification of Rice Chalkiness In Vivo by Using High Resolution Micro-CT

Rice ◽  
2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Yi Su ◽  
Lang-Tao Xiao
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
3D Visualization ◽  
Three Dimensional ◽  
Basic Research ◽  
Micro Ct ◽  
Milled Rice ◽  
Phenotype Analysis ◽  
Rice Seeds

Abstract Background Rice quality research attracts attention worldwide. Rice chalkiness is one of the key indexes determining rice kernel quality. The traditional rice chalkiness measurement methods only use milled rice as materials and are mainly based on naked-eye observation or area-based two-dimensional (2D) image analysis and the results could not represent the three-dimensional (3D) characteristics of chalkiness in the rice kernel. These methods are neither in vivo thus are unable to analyze living rice seeds for high throughput screening of rice chalkiness phenotype. Results Here, we introduced a novel method for 3D visualization and accurate volume-based quantification of rice chalkiness in vivo by using X-ray microcomputed tomography (micro-CT). This approach not only develops a novel volume-based method to measure the 3D rice chalkiness index, but also provides a high throughput solution for rice chalkiness phenotype analysis by using living rice seeds. Conclusions Our method could be a new powerful tool for rice chalkiness measurement, especially for high throughput chalkiness phenotype screening using living rice seeds. This method could be used in chalkiness phenotype identification and screening, and would greatly promote the basic research in rice chalkiness regulation as well as the quality evaluation in rice production practice.

scholarly journals 3D visualization and volume based quantification of rice chalkiness in vivo by using high resolution micro-CT

2020 ◽  
Author(s):  
Yi Su ◽  
Langtao Xiao
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
3D Visualization ◽  
Three Dimensional ◽  
Micro Ct ◽  
Kernel Quality ◽  
Production Practice ◽  
Phenotype Analysis ◽  
Novel Method

Abstract Background: Rice quality research attracts attention worldwide. Rice chalkiness is one of the key indexes determining rice kernel quality. The traditional rice chalkiness measurement methods are mainly based on naked-eye observation or two-dimensional (2D) image analysis and the results could not represent the three-dimensional (3D) characteristics of chalkiness in the rice kernel. These methods are neither in vivo thus are unable to provide technical support for high throughput screening of rice chalkiness phenotype. Results: Here, we introduced a novel method for 3D visualization and accurate volume-based quantification of rice chalkiness in vivo by using X-ray microcomputed tomography (micro-CT). This approach not only develops a novel method to measure the rice chalkiness index, but also provides a high throughput solution for rice chalkiness phenotype analysis. Conclusions: Our method could be a new powerful tool for rice chalkiness measurement, which would greatly help the research of rice chalkiness traits as well as the quality evaluation in rice production practice.

scholarly journals Identification of Small-Molecule Modulators of Mouse SVZ Progenitor Cell Proliferation and Differentiation Through High-Throughput Screening

2009 ◽  
Vol 14 (4) ◽  
pp. 319-329 ◽  
Author(s):  
Yaping Liu ◽  
Raul Lacson ◽  
Jason Cassaday ◽  
David A. Ross ◽  
Anthony Kreamer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Progenitor Cell ◽  
Large Scale ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Progenitor Cell Proliferation

Adult mouse subventricular zone (SVZ) neural stem/progenitor cells are multipotent self-renewing cells that retain the capacity to generate the major cell types of the central nervous system in vitro and in vivo. The relative ease of expanding SVZ cells in culture as neurospheres makes them an ideal model for carrying out large-scale screening to identify compounds that regulate neural progenitor cell proliferation and differentiation. The authors have developed an adenosine triphosphate—based cell proliferation assay using adult SVZ cells to identify small molecules that activate or inhibit progenitor cell proliferation. This assay was miniaturized to a 1536-well format for high-throughput screening (HTS) of >1 million small-molecule compounds, and 325 and 581 compounds were confirmed as potential inducers of SVZ cell proliferation and differentiation, respectively. A number of these compounds were identified as having a selective proliferative and differentiation effect on SVZ cells versus mouse Neuro2a neuroblastoma cells. These compounds can potentially be useful pharmacological tools to modulate resident stem cells and neurogenesis in the adult brain. This study represents a novel application of primary somatic stem cells in the HTS of a large-scale compound library. ( Journal of Biomolecular Screening 2009:319-329)

Abstract 19641: Novel Microprinted Elisa Platform for High Throughput Screening of Protein Secretion Reveals a Comprehensive Strategy to Prevent Ischemia Reperfusion Induced Apoptosis

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Kshitiz Kz ◽  
David E Ellison ◽  
Junaid Afzal ◽  
Maimon E Hubbi ◽  
Segun Bernard ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Stem Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Ischemia Reperfusion ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Small Population ◽  
Cardiac Differentiation

Introduction: A major challenge in using stem cells to treat myocardial infarction (MI) is the massive cell death post transplant. Many progenitor cell types limit disrepair post MI without significant cardiac differentiation, possibly by anti-apoptotic signals in their secretions. The signature of these secretions is however not well known. Methods: We created a novel high throughput microELISA platform by combining microprinting and microfluidics to precisely estimate secretions of a small population of cells over time. Screening the secretions in many stem cell types used previously to treat MI, i.e. BMSCs (Bone marrow derived stem cells), CDCs (Cardiosphere derived cells), and iPSC-CMs (induced pluripotent stem cell derived cardiomyocytes), we found a common preserved secretory signature of growth factors. Results: Using a high throughput screen of pro-apoptotic factors that prevent CDCs from peroxide induced cell death, we surprisingly found that these factors were identical to the preserved secretory signature. Using the constituents of the anti-apoptotic secretory signature in combination with ischemic and mechanical preconditioning in myocardium mimicking rigidity, we created a comprehensive cytoprotective cocktail to prevent CDCs from ischemia induced cell death. We tested the cocktail in a rat model of ischemia reperfusion, and found a stark reduction in CDC retention post injection. Figures: (A) MicroELISA schematic. Secretions from cells in left are detected by rows of microprinted capture antibodies; photograph in (C-D). Bioluminescence imaging of CDCs treated with comprehensive cocktail show significantly high cell retention vs untreated CDCs.

Targeting Pre-Leukemic Stem Cells in T-Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 527-527
Author(s):  
Bastien Gerby ◽  
Diogo F.T Veiga ◽  
Jana Krosl ◽  
Julianne Ouellette ◽  
André Haman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Lymphoblastic Leukemia ◽  
High Throughput ◽  
High Throughput Screening ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Primary Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Current chemotherapy of pediatric T cell acute lymphoblastic leukemia (T-ALL) efficiently reduces the tumor mass with, however, undesirable long term consequences and remains ineffective in adolescent and adult T-ALL. Furthermore, relapse can be caused by pre-leukemic stem cells (pre-LSCs) that were spared by current protocols and evolved to malignancy. A distinctive characteristic of pre-LSCs is their critical dependence on interactions with the microenvironment for survival, which guided our strategy to target pre-LSCs using niche-based screening assays. Using transgenic mouse models that closely reproduce the human disease, we showed that the SCL/TAL1 and LMO1 oncogenic transcription factors establish a pre-leukemic state by reprogramming normal pro-T cells into aberrantly self-renewing pre-LSCs (Gerby et al. PloS Genetics, 2014). We now provide direct evidence that pre-LSCs are much less chemosensitive than leukemic blasts to current drugs, due to a distinctive lower proliferative state as assessed by real-time imaging in a competitive assay. We therefore designed a robust protocol for high-throughput screening (HTS) of compounds targeting primary pre-LSCs that are maintained on stromal cells engineered for optimal NOTCH1 activation to mimick the thymic microenvironement. The multiparametric readout takes into account the intrinsic complexity of primary cells to specifically monitor pre-LSCs. We screened a targeted library of 1904 compounds and identified UM0119979 that disrupts both cell autonomous and non-cell autonomous pathways: UM0119979 abrogates pre-LSC viability and self-renewal activity in vivo by specifically inhibiting the translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remain functional. Moreover, in vivo administration of UM0119979 efficiently reduced the leukemia propagating activity of primary human T-ALL samples in xenografted mice. Finally, in addition to SCL-LMO-induced T-ALL, our results reveal a novel possibility of therapeutic intervention in MYC-dependent hematologic malignancies. In summary, our screening assay, built on the genetic dependencies of pre-LSCs, revealed their vulnerabilities to compounds that inhibit both the primary oncogenes and non-cell autonomous pathways triggered by the microenvironment. The results illustrate how recapitulating tissue-like properties of primary cells in high throughput screening is a promising avenue for innovation in cancer chemotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Complementary, Semi-automated Methods for Creating Multi-dimensional, PEG-based Biomaterials

2017 ◽  
Author(s):  
Elizabeth A. Brooks ◽  
Lauren E. Jansen ◽  
Maria F. Gencoglu ◽  
Annali M. Yurkevicz ◽  
Shelly R. Peyton
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Three Dimensional ◽  
Cancer Cell Line ◽  
Protein Composition ◽  
Cell Matrix ◽  
Hydrogel Matrix ◽  
Sense And Respond ◽  
Tunable Stiffness

ABSTRACTTunable biomaterials that mimic selected features of the extracellular matrix (ECM), such as its stiffness, protein composition, and dimensionality, are increasingly popular for studying how cells sense and respond to ECM cues. In the field, there exists a significant trade-off for how complex and how well these biomaterials represent the in vivo microenvironment, versus how easy they are to make and how adaptable they are to automated fabrication techniques. To address this need to integrate more complex biomaterials design with high-throughput screening approaches, we present several methods to fabricate synthetic biomaterials in 96-well plates and demonstrate that they can be adapted to semi-automated liquid handling robotics. These platforms include 1) glass bottom plates with covalently attached ECM proteins, and 2) hydrogels with tunable stiffness and protein composition with either cells seeded on the surface, or 3) laden within the three-dimensional hydrogel matrix. This study includes proof-of-concept results demonstrating control over breast cancer cell line phenotypes via these ECM cues in a semi-automated fashion. We foresee the use of these methods as a mechanism to bridge the gap between high-throughput cell-matrix screening and engineered ECM-mimicking biomaterials.

scholarly journals Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening

2018 ◽  
Vol 23 (6) ◽  
pp. 574-584 ◽  
Author(s):  
Shurong Hou ◽  
Hervé Tiriac ◽  
Banu Priya Sridharan ◽  
Louis Scampavia ◽  
Franck Madoux ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Anticancer Agents ◽  
High Throughput Screening ◽  
Large Scale ◽  
Ex Vivo ◽  
Cell Models ◽  
Tumor Models ◽  
Flat Bottom

Traditional high-throughput drug screening in oncology routinely relies on two-dimensional (2D) cell models, which inadequately recapitulate the physiologic context of cancer. Three-dimensional (3D) cell models are thought to better mimic the complexity of in vivo tumors. Numerous methods to culture 3D organoids have been described, but most are nonhomogeneous and expensive, and hence impractical for high-throughput screening (HTS) purposes. Here we describe an HTS-compatible method that enables the consistent production of organoids in standard flat-bottom 384- and 1536-well plates by combining the use of a cell-repellent surface with a bioprinting technology incorporating magnetic force. We validated this homogeneous process by evaluating the effects of well-characterized anticancer agents against four patient-derived pancreatic cancer KRAS mutant-associated primary cells, including cancer-associated fibroblasts. This technology was tested for its compatibility with HTS automation by completing a cytotoxicity pilot screen of ~3300 approved drugs. To highlight the benefits of the 3D format, we performed this pilot screen in parallel in both the 2D and 3D assays. These data indicate that this technique can be readily applied to support large-scale drug screening relying on clinically relevant, ex vivo 3D tumor models directly harvested from patients, an important milestone toward personalized medicine.

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