Automated Embryoid Body Generation powered by LEAP Generation of EBs of Specific Size

Embryoid body (EB) formation is a common intermediate during the in vitro differentiation of human embryonic and induced pluripotent stem cells (ESCs and iPSCs) into specialized cell types. EBs are typically generated by removing stem cell colonies from feeder/matrix with collagenase treatment after 5-6 days of culture. Removed colonies are then grown in suspension culture on low attachment plates in differentiation medium. EBs are formed within a few hours to days of suspension culture after which various differentiation strategies are employed. Cultures maintained by enzymatic passage contain variable sized colonies and result in formation of a heterogeneous EB population, varying in size and morphology. Heterogeneous EBs exhibit limited differentiation potential and low production yields (Dang et al, 2004; Ng et al, 2005; Falconnet et al, 2006).
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Automated Stem Cell Passage powered by LEAP Physical Passage of ES and iPS Cell Colonies

Passaging of stem cells is typically performed using enzymatic and/or physical techniques. Enzymatic techniques, such as using collagenase or trypsin, are the most widely used and allow rapid passaging for large-scale expansion, but result in variable-sized colonies and significant cellular trauma which may be associated with increased rates of genetic instability (Mitalipova et al, 2005). In contrast, physical passage of stem cells without enzymes is thought to better maintain genetic stability in long-term culture. Benefits of physical passaging include generation of similar sized clumps, decreased cellular trauma, and selective transfer of specific colonies (Oh et al, 2005; Joannides et al, 2006). In addition, physical passaging is essential for production of new GMP-quality, clinical-grade stem cell lines (Thomson, 2007). However, manual passaging of stem cell colonies is labor-, cost-, and time-intensive, and therefore not suitable for large scale culture.
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CellXpress™ powered by LEAP Accelerated Development of Highly-Secreting Cell Lines

The generation of stable cell lines with high protein secretion is challenging and represents a major bottleneck in biopharmaceutical process development. The inefficiencies of current methods for cell line selection and generation have created an urgent need for innovative approaches that shorten timelines and improve productivity. This application note presents a novel approach to cell line generation and characterization, based on in situ measurement of specific protein secretion followed by in situ cloning, which meets this critical need.
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High Throughput Stable Cell Line Generation

Stably transfected cell lines are used extensively in drug discovery. Cell lines expressing a target of interest, such as a G-protein coupled receptor (GPCR) or a reporter gene, form the basis for most cell-based compound screening campaigns. In establishing new assays for high throughput screening, creation of the appropriate cell line is a bottleneck. Typically, a stable cell line is created by transfection with a plasmid encoding the target of interest or reporter gene construct, and an additional gene which allows for chemical selection of successfully transfected cells (usually an antibiotic resistance gene). Through a lengthy selection process and subsequent limiting dilution to obtain clones, the desired stable cell line is generated. This process is time consuming and takes approximately 2-3 months, usually yielding 5-10 usable clones and allowing little control over the end result throughout the process.
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In Situ purification of human embryonic stem cell colonies

There is a significant need for the purification of specific subpopulations of adherent cells in complex cultures. Often the unique property of the specific subpopulation is destroyed by conventional approaches to purification. To date, a robust system that can adequately address this problem has been lacking. Even when markers are available, most systems (e.g., flow cytometry, magnetic beads, etc.) require that the desired cells be physically manipulated, often leading to changes in cell morphology and/or decreased cell survival, particularly with sensitive cells like human embryonic stem cell lines. The LEAP™ (Laser-Enabled Analysis and Processing) system has been developed to address current limitations in cell purification. LEAP operates through laser-mediated in situ elimination of undesired cells without physically manipulating the cells that are preserved. LEAP has been used for high-throughput laser-mediated cell elimination for general cell purification (Koller et al, 2004), as well as purification of cells based on direct measurement of antibody secretion by individual cells (Hanania et al, 2005). Further, LEAP purification can be efficiently performed on small samples with very low cell numbers.
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Automated high-throughput wound healing assay

Study of the migration of multi-cellular sheets is significantly important in tissue development and repair after injury (DiPietro and Burns, 2003). The in vitro wound healing process involves a complex and orderly sequence of events involving cell migration and proliferation. Currently, it is difficult to use the classic scratch-wound assay for large-scale applications, such as screening libraries of small molecules or siRNAs. In this type of assay, a confluent monolayer is `injured' by forcibly removing a strip of cells, and the remaining monolayer `heals' through some combination of cell migration, spreading, and proliferation. The wound is typically created by scraping with a needle, pipette tip, or razor blade. This crude process is limiting in a number of ways, including being very time consuming for a large number of samples, difficulty in delivering reproducible wounds over large number of samples, potentially damaging the plate surface, and limited implementation in formats smaller than 96-well plates.
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Automated in situ purification of primary rat brain microvascular endothelial cells

Primary cells can potentially provide more physiologically relevant systems for study in discovery biology and drug discovery applications, as compared to immortalized cell lines. However, it is very difficult to obtain primary cell preparations with sufficient purity due to limited cell numbers, and particularly with adherent cell populations. Most current approaches to purifying cells, such as flow cytometry, require enzyme treatment to suspend cells for processing, generally leading to reducing or removing important cell surface markers. Substrate-attached differentiated cell types are particularly challenging as manipulations can directly alter their biology, and forcing them into suspension leads to altered gene expression and decreased cell survival.
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Optoinjection of siRNA and functional knockdown of erythropoietin receptor in primary human CD34+ cells

RNA interference (RNAi) is a pathway for specific and efficient post-transcriptional gene silencing which is now in widespread use for a variety of applications. Despite the extensive utilization of RNAi through small interfering (si)RNA delivery, several limitations remain including: (i) inefficient introduction of siRNA into many important cell types; (ii) potential for off-target & toxic-response gene effects; and (iii) low cell viability & yield with standard transfection techniques.
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