scholarly journals A Microfluidic Single-Cell Cloning (SCC) Device for the Generation of Monoclonal Cells

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1482 ◽  
Author(s):  
Chuan-Feng Yeh ◽  
Ching-Hui Lin ◽  
Hao-Chen Chang ◽  
Chia-Yu Tang ◽  
Pei-Tzu Lai ◽  
...  
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Single Cells ◽  
A549 Cells ◽  
Cost Effective ◽  
In Vitro Models ◽  
Cell Cloning ◽  
Single Cell Cloning ◽  
Specialized Equipment

Single-cell cloning (SCC) is a critical step in generating monoclonal cell lines, which are widely used as in vitro models and for producing proteins with high reproducibility for research and the production of therapeutic drugs. In monoclonal cell line generation, the development time can be shortened by validating the monoclonality of the cloned cells. However, the validation process currently requires specialized equipment that is not readily available in general biology laboratories. Here, we report a disposable SCC device, in which single cells can be isolated, validated, and expanded to form monoclonal cell colonies using conventional micropipettes and microscopes. The monoclonal cells can be selectively transferred from the SCC chip to conventional culture plates, using a tissue puncher. Using the device, we demonstrated that monoclonal colonies of actin-GFP (green fluorescent protein) plasmid-transfected A549 cells could be formed in the device within nine days and subsequently transferred to wells in plates for further expansion. This approach offers a cost-effective alternative to the use of specialized equipment for monoclonal cell generation.

scholarly journals ETMR-17. SINGLE-CELL TRANSCRIPTOME ANALYSIS OF ETMR PATIENT SAMPLES

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii326-iii326
Author(s):  
Volker Hovestadt ◽  
McKenzie L Shaw ◽  
Alexander Beck ◽  
Sander Lambo ◽  
Olivia A Hack ◽  
...  
Keyword(s):  
Single Cell ◽  
Phenotypic Diversity ◽  
Single Cells ◽  
Pediatric Brain Tumor ◽  
Cell Types ◽  
Malignant Cell ◽  
Genetic Alterations ◽  
In Vitro Models ◽  
Normal Brain

Abstract Brain tumors are comprised of cells with heterogeneous genetic and transcriptional states, resulting in substantial phenotypic diversity. This diversity is particularly evident in embryonal tumor with multilayered rosettes (ETMR), which shows a striking bi-phasic pattern for which it is named. A better understanding of its underlying molecular makeup is urgently needed to develop more effective therapeutic strategies that eliminate all malignant cell types underlying ETMR initiation, maintenance, progression, and relapse. Furthermore, the cellular origin of ETMR is currently poorly understood. We used plate-based single-cell RNA sequencing to assess the intratumoral heterogeneity in 6 fresh and 4 snap-frozen surgical biopsies, following a workflow that we have previously established to study pediatric high grade gliomas, medulloblastomas, and ependymomas. Computational analyses conducted on >4,000 single cells identified cellular hierarchies ranging from a proliferative, undifferentiated cell population to more differentiated, predominantly neural-like progeny in all samples. Patient-derived cell line and xenograft models partially recapitulated this hierarchy. We further integrated transcriptional programs identified in single cells with available datasets of the developing normal brain, as well as with programs identified in other pediatric brain tumor entities, to inform both putative cellular origins and ETMR-specific oncogenic pathways. These timely results provide unparalleled insights into the molecular underpinnings of the phenotypic heterogeneity observed in ETMR. Analyses aimed at further integrating malignant cell type abundances with genetic alterations and clinical annotations, and therapeutical targeting of malignant cell populations using in-vitro models are currently ongoing.

scholarly journals Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karl Olofsson ◽  
Valentina Carannante ◽  
Madoka Takai ◽  
Björn Önfelt ◽  
Martin Wiklund
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Drug Distribution ◽  
In Vitro Models ◽  
Biological Information ◽  
Dimensional Structure ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Nuclear Segmentation

AbstractMulticellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

scholarly journals An epigenetic GPI anchor defect impairs TLR4 signaling in the B cell transdifferentiation model for primary human monocytes BLaER1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julia Wegner ◽  
Thomas Zillinger ◽  
Thais Schlee-Guimaraes ◽  
Eva Bartok ◽  
Martin Schlee
Keyword(s):  
Single Cell ◽  
Human Monocyte ◽  
Surface Expression ◽  
Human Monocytes ◽  
Innate Immune Responses ◽  
Loss Of Function ◽  
Cell Cloning ◽  
Gpi Anchor ◽  
Single Cell Cloning ◽  
Antigen Presenting

AbstractAntigen-presenting myeloid cells like monocytes detect invading pathogens via pattern recognition receptors (PRRs) and initiate adaptive and innate immune responses. As analysis of PRR signaling in primary human monocytes is hampered by their restricted expandability, human monocyte models like THP-1 cells are commonly used for loss-of-function studies, such as with CRISPR-Cas9 editing. A recently developed transdifferentiation cell culture system, BLaER1, enables lineage conversion from malignant B cells to monocytes and was found superior to THP-1 in mimicking PRR signaling, thus being the first model allowing TLR4 and inflammasome pathway analysis. Here, we identified an important caveat when investigating TLR4-driven signaling in BLaER1 cells. We show that this model contains glycosylphosphatidylinositol (GPI) anchor-deficient cells, which lack CD14 surface expression when differentiated to monocytes, resulting in diminished LPS/TLR4 but not TLR7/TLR8 responsiveness. This GPI anchor defect is caused by epigenetic silencing of PIGH, leading to a random distribution of intact and PIGH-deficient clones after single-cell cloning. Overexpressing PIGH restored GPI-anchored protein (including CD14) expression and LPS responsiveness. When studying CD14- or other GPI-anchored protein-dependent pathways, researchers should consider this anomaly and ensure equal GPI-anchored protein expression when comparing cells that have undergone single-cell cloning, e. g. after CRISPR-Cas9 editing.

scholarly journals Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs

2015 ◽  
Vol 75 (10) ◽  
pp. 1866-1875 ◽  
Author(s):  
Elisa Corsiero ◽  
Michele Bombardieri ◽  
Emanuela Carlotti ◽  
Federico Pratesi ◽  
William Robinson ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
B Cells ◽  
Single Cell ◽  
Cell Cloning ◽  
Single Cell Cloning

scholarly journals ZipSeq : Barcoding for Real-time Mapping of Single Cell Transcriptomes

2020 ◽  
Author(s):  
Kenneth H. Hu ◽  
John P. Eichorst ◽  
Chris S. McGinnis ◽  
David M. Patterson ◽  
Eric D. Chow ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Real Time ◽  
Dimensional Space ◽  
Single Cells ◽  
Expression Patterns ◽  
Live Cells ◽  
Glass Substrates ◽  
Complete Mapping

ABSTRACTSpatial transcriptomics seeks to integrate single-cell transcriptomic data within the 3-dimensional space of multicellular biology. Current methods use glass substrates pre-seeded with matrices of barcodes or fluorescence hybridization of a limited number of probes. We developed an alternative approach, called ‘ZipSeq’, that uses patterned illumination and photocaged oligonucleotides to serially print barcodes (Zipcodes) onto live cells within intact tissues, in real-time and with on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in-vitro wound healing, live lymph node sections and in a live tumor microenvironment (TME). In all cases, we discovered new gene expression patterns associated with histological structures. In the TME, this demonstrated a trajectory of myeloid and T cell differentiation, from periphery inward. A variation of ZipSeq efficiently scales to the level of single cells, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

scholarly journals Molecular characterization of human anti-hinge antibodies derived from single-cell cloning of normal human B cells

2017 ◽  
Vol 293 (3) ◽  
pp. 906-919 ◽  
Author(s):  
Tao Huang ◽  
Mary Mathieu ◽  
Sophia Lee ◽  
Xinhua Wang ◽  
Yee Seir Kee ◽  
...  
Keyword(s):  
B Cells ◽  
Single Cell ◽  
Molecular Characterization ◽  
Cell Cloning ◽  
Human B Cells ◽  
Single Cell Cloning ◽  
Normal Human

scholarly journals SCELLECTOR: ranking amplification bias in single cells using shallow sequencing

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Vivekananda Sarangi ◽  
Alexandre Jourdon ◽  
Taejeong Bae ◽  
Arijit Panda ◽  
Flora Vaccarino ◽  
...  
Keyword(s):  
Single Cell ◽  
Multiple Displacement Amplification ◽  
Single Cells ◽  
Sequencing Data ◽  
High Coverage ◽  
Amplification Bias ◽  
Single Cell Profiling ◽  
High Concordance ◽  
Human Neuronal Cells

Abstract Background The study of mosaic mutation is important since it has been linked to cancer and various disorders. Single cell sequencing has become a powerful tool to study the genome of individual cells for the detection of mosaic mutations. The amount of DNA in a single cell needs to be amplified before sequencing and multiple displacement amplification (MDA) is widely used owing to its low error rate and long fragment length of amplified DNA. However, the phi29 polymerase used in MDA is sensitive to template fragmentation and presence of sites with DNA damage that can lead to biases such as allelic imbalance, uneven coverage and over representation of C to T mutations. It is therefore important to select cells with uniform amplification to decrease false positives and increase sensitivity for mosaic mutation detection. Results We propose a method, Scellector (single cell selector), which uses haplotype information to detect amplification quality in shallow coverage sequencing data. We tested Scellector on single human neuronal cells, obtained in vitro and amplified by MDA. Qualities were estimated from shallow sequencing with coverage as low as 0.3× per cell and then confirmed using 30× deep coverage sequencing. The high concordance between shallow and high coverage data validated the method. Conclusion Scellector can potentially be used to rank amplifications obtained from single cell platforms relying on a MDA-like amplification step, such as Chromium Single Cell profiling solution.

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