Selecting Monoclonal Cell Lineages from Somatic Reprogramming Using Robotic-Based Spatial-Restricting Structured Flow.

Somatic cell reprogramming generates induced pluripotent stem cells (iPSCs), which serve as a crucial source of seed cells for personalized disease modeling and treatment in regenerative medicine. However, the process of reprogramming often causes substantial lineage manipulations, thereby increasing cellular heterogeneity. As a consequence, the process of harvesting monoclonal iPSCs is labor-intensive and leads to decreased reproducibility. Here, we report the first in-house developed robotic platform that uses a pin-tip-based micro-structure to manipulate radial shear flow for automated monoclonal iPSC colony selection (~1 s) in a non-invasive and label-free manner, which includes tasks for somatic cell reprogramming culturing, medium changes; time-lapse-based high-content imaging; and iPSCs monoclonal colony detection, selection, and expansion. Throughput-wise, this automated robotic system can perform approximately 24 somatic cell reprogramming tasks within 50 days in parallel via a scheduling program. Moreover, thanks to a dual flow-based iPSC selection process, the purity of iPSCs was enhanced, while simultaneously eliminating the need for single-cell subcloning. These iPSCs generated via the dual processing robotic approach demonstrated a purity 3.7 times greater than that of the conventional manual methods. In addition, the automatically produced human iPSCs exhibited typical pluripotent transcriptional profiles, differentiation potential, and karyotypes. In conclusion, this robotic method could offer a promising solution for the automated isolation or purification of lineage-specific cells derived from iPSCs, thereby accelerating the development of personalized medicines.

Accurate non-invasive image-based cytotoxicity assays for cultured cells.

BACKGROUND: The CloneSelect Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay. RESULTS: Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. However, the image-based technique was found non-invasive, considerably quicker and more accurate than the MTT assay. CONCLUSIONS: This new image-based technique has the potential to replace the cumbersome MTT assay when fast, unbiased and high-throughput cytotoxicity assays are requested.

Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic breast cancer patients.

INTRODUCTION: Circulating tumor cells (CTCs) are detectable in most cancer patients and they can meet an existing medical need to monitor cancer patients during a course of treatment and to help determine recurrent disease. CTCs are rarely found in the blood of cancer patients and enrichment is necessary for sensitive CTC detection. Most CTC enrichment technologies are anti-EpCAM antibody based even though CTC identification criteria are cytokeratin positive (CK+), CD45 negative (CD45-) and 4'6-diamidino-2-phenylindole (nuclear stain) positive (DAPI+). However, some tumor cells express low or no EpCAM. Here we present a highly sensitive and reproducible enrichment method that is based on binding to anti-CK alone or a combination of anti-CK and anti-EpCAM antibodies. METHODS: Blood samples from 49 patients with metastatic breast cancer were processed using the CellSearchtrade mark system (Veridex, LLC, Raritan, NJ, USA), in parallel with our CTC assay method. We used anti-CK alone or in combination with anti-EpCAM antibodies for CTC enrichment. Brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images were used for CTC identification. The Ariol(R) system (Genetix USA Inc, San Jose, CA, USA) was used for automated cell image capture and analysis of CTCs on glass slides. RESULTS: Our method has the capability to enrich three types of CTCs including CK+&EpCAM+, CK+&EpCAM-/low, and CK-/low&EpCAM+ cells. In the blind method comparison, our anti-CK antibody enrichment method showed a significantly higher CTC positive rate (49% vs. 29%) and a larger dynamic CTC detected range (1 to 571 vs. 1 to 270) than that of the CellSearchtrade mark system in the total of 49 breast cancer patients. Our method detected 15 to 111% more CTCs than the CellSearchtrade mark method in patients with higher CTC counts (>20 CTCs per 7.5 ml of blood). The three fluorescent and brightfield images from the Ariol(R) system reduced the number of false-positive CTC events according to the established CTC criteria. CONCLUSION: Our data indicate that the tumor-specific intracellular CK marker could be used for efficient CTC enrichment. Enrichment with anti-CK alone or combined with anti-EpCAM antibodies significantly enhances assay sensitivity. The three fluorescent and brightfield superior images with the Ariol(R) system reduced false-positive CTC events.

Whole-genome analysis of 60 G protein-coupled receptors in Caenorhabditis elegans by gene knockout with RNAi.

G protein-coupled receptors (GPCRs) are the largest family of genes in animal genomes and represent more than 2% of genes in humans and C. elegans. These evolutionarily conserved seven-transmembrane proteins transduce a diverse range of signals. In view of their pivotal role in cell signaling, it is perhaps surprising that decades of genetic analysis in C. elegans, and recent genome-wide RNAi screens, have identified very few GPCR mutants. Therefore, we screened all GPCRs predicted to bind either small-molecule neurotransmitters or neuropeptides by using RNAi and quantitative behavioral assays. This shows that C16D6.2, C25G6.5, C26F1.6, F35G8.1, F41E7.3, and F59C12.2 are likely to be involved in reproduction, whereas C15B12.5, C10C6.2, C24A8.4, F15A8.5, F59D12.1, T02E9.1, and T05A1.1 have a role in locomotion. Gene deletions for F35G8.1 and T05A1.1 resulted in the same phenotype as that seen with RNAi. As some GPCRs may be resistant to RNAi, or may result in abnormalities not screened for here, the actual proportion of nonredundant receptors with an assayable function is probably greater. Strikingly, most phenotypes were observed for NPY-like receptors that may bind neuropeptides. This is consistent with the known actions of neuropeptides on the body wall muscle and reproductive tract in nematodes.