Implementing microwell slides for detection and isolation of single circulating tumor cells from complex cell suspensions.

Cell loss during detection and isolation of circulating tumor cells (CTCs) is a challenge especially when label-free pre-enrichment technologies are used without the aid of magnetic particles. Although microfluidic systems can remove the majority of "contaminating" white blood cells (WBCs), their remaining numbers are still impeding single CTC isolation, thus making additional separation steps needed. This study aimed to develop a workflow from blood-to-single CTC for complex cell suspensions by testing two microwell formats. In the first step, different cell lines were used to compare the performances of Sievewell™ 370 K (TOK, Japan) and CellCelector™ Nanowell U25 (ALS Automated Lab Solutions, Germany) slides for cell labelling and single-cell micromanipulation. Confounding levels of auto-fluorescence inherent to different plastic materials used to cast the microwells, staining recovery rates, and cell isolation rates were determined. In the second step, three different blood preservation tubes were tested for RNA analysis. Lastly, the established workflow was applied to isolate CTCs from peripheral blood samples obtained from metastasized breast cancer (mBC) patients for single-cell DNA and RNA analysis. The detection of CTCs in Sievewell slides profit from better signal-to-noise ratios in the fluorescence channels mainly used for CTC detection. In addition, due to its design, Sievewell supports direct in situ CTC labelling, which minimizes cell loss and leads to single-cell recovery rates after staining of approx. 94%. Detection of PIK3CA mutations in single CTCs verified the applicability of the workflow for the analysis of genomic DNA of CTCs. Furthermore, combined with blood preservation up to 48 h at room temperature in LBguard tubes, panel RT-PCR transcript analysis was successful for single cell line cells and CTCs, respectively. The combined use of Sievewell microwell slides and CellCelector™ automated micromanipulation system improves single CTC detection, labelling and isolation from complex cell suspensions. This approach is especially valuable when samples of high cellular content are processed.

Automated rare single cell picking with the ALS cellcelector™.

Molecular analysis of rare single cells like circulating tumor cells (CTCs) from whole blood patient samples bears multiple challenges. One of those challenges is the efficient and ideally loss-free isolation of CTCs over contaminating white and red blood cells. While there is a multitude of commercial and non-commercial systems available for the enrichment of CTCs their cell output does not deliver the purity most molecular analysis methods require. Here we describe the ALS CellCelector™ which can solve this challenge allowing the retrieval of 100% pure single CTCs from blood processed by different upstream enrichment techniques. It is a multifunctional, extremely flexible system for automated screening of cell culture plates, Petri dishes, and microscope slides. Fixed or live single cells or multicellular clusters detected during screening can be picked out of those plates automatically. The complete scan and picking process is fully documented hence allowing highest standardization and reproducibility of all processes. Use of CellCelector allowed the isolation of pure single tumor cells or clusters from liquid biopsies of breast, prostate, ovarian, colorectal, lung, and brain cancers for their subsequent molecular analysis. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

DanioCTC: Analysis of Circulating Tumor Cells from Metastatic Breast Cancer Patients in Zebrafish Xenografts.

Circulating tumor cells (CTCs) serve as crucial metastatic precursor cells, but their study in animal models has been hindered by their low numbers. To address this challenge, we present DanioCTC, an innovative xenograft workflow that overcomes the scarcity of patient-derived CTCs in animal models. By combining diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the CellCelector setup, DanioCTC effectively enriches and isolates CTCs from metastatic breast cancer (MBC) patients for injection into zebrafish embryos. Validation experiments confirmed that MDA-MB-231 cells, transplanted following the standard protocol, localized frequently in the head and blood-forming regions of the zebrafish host. Notably, when MDA-MB-231 cells spiked (i.e., supplemented) into DLA aliquots were processed using DanioCTC, the cell dissemination patterns remained consistent. Successful xenografting of CTCs from a MBC patient revealed their primary localization in the head and trunk regions of zebrafish embryos. DanioCTC represents a major step forward in the endeavors to study the dissemination of individual and rare patient-derived CTCs, thereby enhancing our understanding of metastatic breast cancer biology and facilitating the development of targeted interventions in MBC. Summary statement: DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies of the capacity of these rare tumor cells to induce metastases.

Automated selection and harvesting of pluripotent stem cell colonies.

The ability of pluripotent stem cells to differentiate into specialized cells of all three germ layers, their capability to self-renew, and their amenability to genetic modification provide fascinating prospects for the generation of cell lines for biomedical applications. Therefore, stem cells must increasingly suffice in terms of industrial standards, and automation of critical or time-consuming steps becomes a fundamental prerequisite for their routine application. Cumbersome manual picking of individual stem cell colonies still represents the most frequently used method for passaging or derivation of clonal stem cell lines. Here, we explore an automated harvesting system (CellCelector™) for detection, isolation, and propagation of human embryonic stem cells (hESCs) and murine induced pluripotent stem cells (iPSCs). Automatically transferred hESC colonies maintained their specific biological characteristics even after repeated passaging. We also selected and harvested primary iPSCs derived from mouse embryonic fibroblasts expressing the green fluorescent protein (GFP) under the control of the Oct4 promotor using either morphological criteria or GFP fluorescence. About 80% of the selected and harvested primary iPSC colonies gave rise to homogenously GFP-expressing iPSC lines. To validate the iPSC lines, we analyzed the expression of pluripotency-associated markers and multi-germ layer differentiation potential in vitro. Our data indicate that the CellCelector™ technology enables efficient identification and isolation of pluripotent stem cell colonies at the phase contrast or fluorescence level.

Cartilaginous spheroid-assembly design considerations for endochondral ossification: towards robotic-driven biomanufacturing.

Spheroids have become essential building blocks for biofabrication of functional tissues. Spheroid formats allow high cell-densities to be efficiently engineered into tissue structures closely resembling the native tissues. In this work, we explore the assembly capacity of cartilaginous spheroids (d∼ 150µm) in the context of endochondral bone formation. The fusion capacity of spheroids at various degrees of differentiation was investigated and showed decreased kinetics as well as remodeling capacity with increased spheroid maturity. Subsequently, design considerations regarding the dimensions of engineered spheroid-based cartilaginous mesotissues were explored for the corresponding time points, defining critical dimensions for these type of tissues as they progressively mature. Next, mesotissue assemblies were implanted subcutaneously in order to investigate the influence of spheroid fusion parameters on endochondral ossification. Moreover, as a step towards industrialization, we demonstrated a novel automated image-guided robotics process, based on targeting and registering single-spheroids, covering the range of spheroid and mesotissue dimensions investigated in this work. This work highlights a robust and automated high-precision biomanufacturing roadmap for producing spheroid-based implants for bone regeneration.

Development and optimization of a process for automated recovery of single cells identified by microengraving.

Microfabricated devices are useful tools for manipulating and interrogating large numbers of single cells in a rapid and cost-effective manner, but connecting these systems to the existing platforms used in routine high-throughput screening of libraries of cells remains challenging. Methods to sort individual cells of interest from custom microscale devices to standardized culture dishes in an efficient and automated manner without affecting the viability of the cells are critical. Combining a commercially available instrument for colony picking (CellCelector, AVISO GmbH) and a customized software module, we have established an optimized process for the automated retrieval of individual antibody-producing cells, secreting desirable antibodies, from dense arrays of subnanoliter containers. The selection of cells for retrieval is guided by data obtained from a high-throughput, single-cell screening method called microengraving. Using this system, 100 clones from a mixed population of two cell lines secreting different antibodies (12CA5 and HYB099-01) were sorted with 100% accuracy (50 clones of each) in approximately 2 h, and the cells retained viability.