Give Your Frozen Cells A Vitality Boost
Asking cells to come back to life after cryopreservation is like asking your teenage son or daughter to be out of bed before 10 AM on a Saturday morning. Chances are you will have to coax, bribe, charm, or physically wriggle them back to life…and even then, you may be conversing with someone operating at 50% capacity for the first 45 minutes. This may render your teen useless at performing basic tasks like putting their dishes away or cleaning up their room. Your favorite cells can unfortunately also be rendered mostly useless by the process of long-term “sleep.” In fact, many will not survive the procedure and cannot be used for downstream analysis. With the LeviCell™ platform, samples with an abundance of dead or dying cells are gently processed to remove these unwanted cells, enriching only the viable cells that have survived the freeze/thaw journey. In our research snapshot “Enrichment of Viable PBMCs with Levitation Technology,” we demonstrate the successful revival of cryopreserved peripheral blood mononuclear cells (PBMCs) through cellular enrichment. After initial thawing, the starting viability of these cells was as low as 20%. After a short enrichment with the LeviCell system, we were able to boost the viability to ~90%, a recovery that was independent of the number of cells loaded. It’s the equivalent of a glass of fresh juice, a cup of coffee, and a hearty breakfast. Enough to bring even the laziest sample back to life! Download Research Snapshot | Request More Info
Clean-Up of Dissociated Tumor Cells Prior to Flow Cytometry Analysis
Dissociated cells from solid tumor samples (DTCs) are a critical source of valuable, actionable information about the fundamentals of cancer biology. Understanding the makeup of the tumor and its resident phenotypes leads researchers to new discoveries, and can lead to meaningful, targeted therapeutics in the future for specific cancer types. However, solid tissues in general, and solid tumors in particular, present many processing hurdles that render many precious samples unusable. Common challenges with tissue dissociation Tissue dissociation often exacerbates the percentage of dead cells and debris in an already stressed cellular environment, as does cryo-preservation of the dissociated single cell suspension. However, these are necessary evils of the sample collection process, particularly when many of the most valuable samples are coming from clinical specimens and may need to be cryo-preserved prior to performing analysis at a centralized facility. When the goal is to transfer these samples to a downstream analytical workflow, such as single cell library preparation or high-parameter cytometric analysis, what can we do to improve the sample quality? The quality of DTC samples can be improved through proper debris removal In our latest Research Snapshot, we illustrate how the LeviCell™ system can help to clear away the residual dead and dying cells, along with the cellular debris left behind in the sample, improving the quality of the DTC suspension for downstream analysis. In the example provided, flow cytometry analysis of the sample was performed before and after enrichment on the LeviCell, illustrating a clear reduction in the percentage of dead cells and overall debris. This label-free, gentle method of viable cell enrichment prepares DTC samples for their ultimate destination: high-quality data generation that will lead cancer biologists to new and relevant insights! >> Download Research Snapshot
Optimization of a Tissue Dissociation Workflow for Single Cell Analysis
New Tissue Dissociation Workflow for Single Cell Analysis The complexity of dissociating complex tissue into single cells Isolating cells for cancer diagnostics is critical if personalized medicine is to flourish. However, dissociating complex tissues into single cells while maintaining cellular integrity and stable genetic expression is poorly understood and most present approaches in the lab do not have the efficiency that is needed to be clinically relevant for processing of samples of heterogeneous tissue. A new approach: 15 minute chemical-mechanical dissociation protocol Writing in a recent issue of Cellular and Molecular Bioengineering, (Vol. 14, No. 3, June 2021 pp. 241–258; https://doi.org/10.1007/s12195-021-00667-y) researchers from the Center for Biomedical Engineering at Brown University reveal a 15 minute chemical-mechanical dissociation protocol for clinically relevant preparation of single-cell suspensions from frozen biopsy cores of complex tissues. Dissociation and analysis of frozen bovine liver biopsy cores Frozen bovine liver biopsy cores were normalized by weight, dimension, and calculated cellular composition. Various chemical reagents were tested for their capability to dissociate the tissue via confocal microscopy, hemocytometry and quantitative flow cytometry. Images were processed using ImageJ. Quantitative flow cytometry with gating analysis was also used for the analysis of dissociation. Physical modeling simulations were conducted in COMSOL Multiphysics. The researchers established that a combination of 1% type-1 collagenase and pronase or hyaluronidase in 100 U/lL HBSS solution is the most effective at dissociating 2.5 mm thawed bovine liver biopsy cores in 15 min, with dissociation efficiency of 37-42% and viability >90% as verified using live MDA-MB-231 cancer cells. Cellular dissociation is significantly improved by adding a controlled mechanical force during the chemical process, to dissociate 93 + 8% of the entire tissue into single cells. The protocol demonstrates that controlled mechanical force in combination with chemical treatment produces high quality tissue dissociation. Applicability to different tissue and cell types According to the researchers, the applicability of this workflow is likely to hold for most soft tissues, which are the tissue type that is most frequently dissociated in practice. However, the success of this protocol across different tissue and cell types, especially fibrous, crosslinked, and necrotic or diseased tissues, should be investigated further.
Tumor Dissociation of Highly Viable Cell Suspensions
Tumor Dissociation of Highly Viable Cell Suspensions in Breast Cancer Research Overview – Incidence of Breast Cancer National Breast Cancer Awareness Month started October 1st. This annual spotlight serves as a reminder that breast cancer is the second leading cause of cancer death in women. Only lung cancer kills more women each year. The chance that a woman will die from breast cancer is about 1 in 39 (about 2.6%). According to American Cancer Society estimates (U.S. only) in 2021 about 282,000 new cases of invasive breast cancer will be diagnosed in women and about 44,000 women will die from breast cancer. The tremendous human cost of this disease drives a multi-billion dollar annual research effort for cures, including basic R&D and lab breakthroughs such as the just published single cell protocol that is highlighted below. Optimizing Single-cell Multiomic Workflows for Breast Cancer Research Writing in STAR Protocols, an open access journal from Cell Press, Laura Rodriguez de la Fuente, Andrew M.K. Law, David Gallego-Ortega, and Fatima Valdes-Mora disclose a new protocol that yields cell suspensions with 90% viability of live cells from mouse mammary tumors. Cell preparation with a high rate of viable cells is required to obtain reliable single-cell transcriptomic and epigenomic data. This protocol describes a technique for digestion and single-cell isolation from mouse mammary tumors to achieve 90% of viable cells, which can be subsequently processed in a diverse array of high-throughput single-cell ‘‘omic platforms,’’ both in an unbiased manner or after selection of a specific cell population. Broadly, the first step in the protocol involves tumor harvesting and enzymatic digestion, where tumor samples are collected and dissociated in single-cell suspension; the second step involves quality control and sample selection of the tumor areas with high-cellular content to represent heterogeneity as much as possible; the third step can either involve unbiased enrichment of all viable cells from tumors or antibody-based selection of specific cell subpopulations, including rare cells. The fourth step consists of the evaluation of cell viability and preparation of cells for single-cell capture for downstream sequencing and analysis. The researchers have tested the new protocol in primary tumors of several mammary mouse models, including transgenic models such as MMTV-PyMT (see Valdes-Mora et al., 2021) and syngeneic models of orthotopically injected cancer cell lines, such as the 4T1.2 and 67NR cell lines. This method has been also used in tumor metastasis from lungs, and in normal mouse lungs, liver and intestines. See further: STAR Protocols 2, 100841, December 17, 2021 (Tumor dissociation of highly viable cell suspensions for single-cell omic analyses in mouse models of breast cancer).