Automated Cell Selection and Retrieval Opens a Universe of Possibilities

Single-cell isolation is critical to numerous research areas, including rare-cell analysis, biologics discovery, and stem cell studies. Yet, the process of identifying and isolating productive single cells continues to be a hurdle, due in part to resource-intensive, low-success techniques like limiting dilution. Limiting dilution is a basic technique for generating a monoclonal cell line from a mixed cell population. It works based on the principal of Poisson distribution; essentially cells are serially diluted until there is a high probability of plating a volume that contains only one cell. Sometimes, multiple rounds are needed to isolate a single clone successfully. It’s well known that single cell cloning by limiting dilution is time consuming and difficult, but scientists choose it because it does not involve complicated lab equipment. Still, the process takes weeks to months and lots of manual steps without any guarantees. Limiting dilution is also stressful to cells because it deprives cells of crosstalk via growthpromoting factors in the media. In industrial applications where speed and monoclonality matter, limiting dilution can be—limiting. Automated robotic instruments are revolutionizing scientific research and bringing more productivity and throughput to routine workflows, thus accelerating important discoveries. This eBook highlights how automated, image-based systems, like CellCelector, are simplifying progress in a variety of research applications. Unlike dilution techniques (left), CellCelector Nanowell Cell Culture Plates (right) allow growth-promoting cellular crosstalk in spite of the local separation. Read more about this technique on page 8.4 Isolation of Rare Circulating Tumor Cells Automated technologies for single-cell isolation can vastly simplify protocols and isolate individual CTCs or CTC clusters from enriched cell suspensions for molecular characterization at the single-cell level. Understanding the heterogeneity in a cell population can reveal a wealth of insight into cell fate and function. In cancer, tumor heterogeneity plays a crucial role in both disease progression and resistance to therapies. One of the main obstacles in the treatment of cancer is metastasis, which is how new tumors originating from the primary site get established at secondary sites. Advances in high-throughput genome sequencing, gene editing, advanced cell models and instrument technology are enabling scientists to dissect the underlying mechanisms that support metastasis, including circulating tumor cells (CTCs) and CTC clusters. Studying tumors at the single-cell level can help inform tailored therapeutic strategies that improve outcomes for patients. The Role of CTCs in Cancer CTCs are cells that break away from the primary tumor and enter the bloodstream. Once in the blood, CTCs can adapt to the microenvironment of additional sites, forming a new tumor. This process, called metastasis, is responsible for over 90% of cancer-related deaths and is an active area of research. In order to colonize a secondary site, CTCs must first survive circulation in the blood, exit the circulatory system, and colonize a new site. This requires a host of advantageous mutations that allow CTCs to escape from immune surveillance in the blood and hijack other processes in their favor. To understand the mechanisms behind metastasis, scientists isolate CTCs to study their functional, biochemical, and biophysical properties. This is the first step in developing new diagnostic tools and therapeutic strategies to block metastasis. Finding the Needle in a Haystack CTCs obtained through a simple blood draw can serve as a “liquid biopsy” to monitor tumor characteristics in real-time, including inter- and intra-tumor heterogeneity. Isolated cells are then used for DNA, RNA or proteome analysis. A liquid biopsy is a non-invasive approach that is complementary to a solid tumor biopsy in providing data to clinicians. However, CTC isolation and subsequent characterization are technically challenging due to the low CTC cell numbers among an abundance of white and red blood cells (RBCs). For example, a one milliliter sample may contain as little as one CTC in a background of 107 white blood cells (WBCs). Leukocyte contamination interferes with downstream analysis of CTC-specific transcripts, and other markers, making enrichment a necessary step in single-CTC studies.5 CTC Enrichment and Isolation A typical CTC isolation and analysis workflow involves the following steps: 1- Blood draw and sample processing 2- CTC enrichment and staining 3- Imaging and isolation of pure CTCs 4- Single-CTC characterization A wide range of analytical methods have been developed for CTC detection, enrichment, and isolation. These methods exploit CTC-specific properties such as surface marker expression or physical features (e.g. size, density, or deformability). Following enrichment, CTCs are stained for immunofluorescence (IF) detection by microscopy and single-cell isolation. Having viable pure CTCs is critical to getting high-quality data in subsequent analyses. Limitations in CTC Workflows Cell culture protocols can influence the health, viability, and function of cells. Cell loss is common during CTC enrichment protocols that include many filtration steps to remove contaminating blood cells. Further, the added processing time can alter the expression profiles of CTCs due to environmental factors. Another limitation of common CTC enrichment methods is that they all carry over some amount of contaminating background cells, which interfere with downstream studies. Automated technologies for single-cell isolation can vastly simplify protocols and isolate individual CTCs or CTC clusters from enriched cell suspensions for molecular characterization at the single-cell level. Automated Rare-Cell Isolation Automated systems for the identification and isolation of pure single cells offer many advantages over traditional methods for rare-cell isolation and retrieval. Platforms like the CellCelector can reliably deliver 100% pure single CTCs or CTC clusters from samples processed using any of the common enrichment techniques. The CellCelector utilizes liquid buffered single-use glass capillaries that provide gentle aspiration with extremely high precision down to the nanoliter range. Each cell retrieval event is fully documented and traceable from the source to the destination, complete with images before and after picking. Fast, Yet Gentle on Cells Unlike manual or semi-automatic picking setups that rely on user skill, automated systems speed up the process, limiting manipulation of delicate cells. Vacuum‐-based or microdissection recovery systems, for example, cause stress to cells from shear stress or excessive heat, respectively. The CellCelector system scans cells in brightfield, phase contrast or fluorescence channels to identify the cells of interest. Putative live CTCs are recovered into the destination vessel of choice for downstream analysis or recultivation. The cells spend no more than 10 seconds inside the capillary, allowing for a fast yet very gentle recovery process.