Genome engineering using CRISPR/Cas9 has transformed the way scientific biological research has progressed over the years. However, the protocols for isolating edited-pluripotent stem cells subject those cells to rough conditions in which gene expression, genomic stability and overall cell-health can become compromised. These issues are compounded additionally with intrinsic challenges in culturing single cells for monoclonal outgrowth.
Microfluidic cell sorting with the WOLF®, along with single cell deposition using the N1 single cell dispenser, eliminates the challenges that pluripotent stem cells face by sorting at gentle pressures without decompression shock and shear stress, and allow for efficient single cell deposition for high monoclonal outgrowth. This improved workflow will allow for higher throughput pluripotent stem cell research, which is valuable for both basic and clinical research.
CRISPR and disease modelling
Induced pluripotent stem cells provide considerable advantages when it comes to in vitro modelling of human diseases. The ability for iPSCs to be reprogrammed and differentiate into virtually any cell type will allow for patient-specific disease models. This holds a powerful promise to perform high throughput drug screening, and other testing of therapeutics. The WOLF has been used to isolate healthy populations of Autism and Alzheimer’s iPSC CRISPR/Cas9 edited cell lines for disease modelling. The viability and proliferation of these fragile cell lines fared significantly better than those that were sorted on a conventional electrostatic sorter. The challenge of low proliferation and low viability can be overcome using a microfluidic sorter like the WOLF in sorting fluorescent CRISPR/Cas9 edited iPSC cell lines.
Protection of Karyotype
In the pursuit of creating banks of iPSCs or other stem cells for various disease models, specific screening assays must be performed. Karyotype analyses are one such screen to determine genome stability. Pluripotent stem cells are often unstable in long-term culture, so it is advisable to authenticate genomic integrity prior to any sort of master cell banking, differentiation into cell lines for disease modelling, therapeutics, etc. As an example, Autism spectrum and Alzheimer’s iPSCs were comparatively sorted using the gentle microfluidic WOLF cell sorter versus a traditional electrostatic sorter. G-banding was performed to complete chromosome counts, and it was found that all traditionally sorted iPSCs had an abnormal karyotype, while the WOLF had significantly less, implying that conventional sorting selects for unstable cells in the harsh sorting process. Chromosomal aberrations are an extremely important screening tool for researchers attempting to create long standing stem cell lines, and the WOLF cell sorter facilitates that difficult selection process.
Gentle Sorting Accelerates Generation of Genome-Edited iPSCs
Fluorescent activated cell sorting, or FACS, is a commonly used method to separate and purify populations of cells from a heterogenous mixture but has traditionally been avoided due to the fragile and delicate nature of iPSCs. Electrostatic pressure, shear stress and decompression shock have all been factors that have made scientists working with iPSCs and other stem cell types typically shy away from traditional cell sorting. The WOLF allows users who are not experts in flow cytometry, to isolate their population of interest simply, efficiently, and gently to preserve the endogenous nature of CRISPR edited iPSCs. In one such example, WOLF sorted iPSCs showed significant one week and 1+ week survival compared to conventionally sorted iPSCs. WOLFViewer software allows users to sort based on 3 fluorescence parameters, like GFP expression, as well as sorting away dead cells and debris, making it ideal for reliable cloning of iPSCs.
Recombinant protein production in Cho Cells
To produce therapeutic and recombinant proteins, the most widely used mammalian expression system in industrial production is gene amplification using dihydrofolate reductase-deficient (DHFR−) CHO cells (Chinese hamster ovary mammalian cells) with DHFR-mediated gene amplification. Following standard molecular biology plasmid construction methods and stable transfection of cells (such as the host cell line CHO-DG44), the expression of a recombinant protein becomes stable. The next step requires sorting single-cells to establish recombinant protein producing clones. With traditional cytometers, the number of CHO cell colonies is low due to cell death with high-pressure cell sorting systems. With low-pressure cell sorting, the WOLF results in up to 95% single cell deposition efficiency and more clonal outgrowth.
The WOLF is a technology that enables small companies to do things they could not do before.” “The microfluidics capability is something that I have been excited about. The nature of how the WOLF works is being extremely attractive to us. Some of the experiments we’d like to do in the future could not be done on any other device.Justin Chartron, Ph.D. Protabit LLC
Neurons are extremely delicate so the selection step should be the least disturbing of all if we want to have reliable results. When we tried the WOLF, we were extremely surprised that our cells were very healthy and viability was above 90%, sometimes above 95% which is very impressive.Dorota Skowronska-Krawczyk, M.Sc.,Ph.D. University of California Irvine
A great machine that has helped us take our cell-engineering pipeline to the next level!Ronan O'Connell Rice University
The NanoCellect team and technical specialist have been awesome. After two test runs with very involved training/assistance, we have been very pleased with the results. The equipment is intuitive and the sorts have been very clean.B. Davenport, Ph.D. University of Colorado
NanoCellect Customer Support team has been fantastic in helping us get up and running. The instrument is a helpful tool in our cell line development workflow.Douglas Austen Takeda Pharmaceuticals
The NanoCellect customer support team is really helpful, knowledgeable and efficient; the WOLF is a great tool in our stem cell research workflowHelen Miranda, Ph.D. Case Western Reserve University
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