Single-Cell Analysis – 3 Pitfalls All Researchers Face
Single-cell analysis has become a key driver of scientific discovery. The move from studying populations of cells to studying single-cells has powered a more detailed and accurate understanding of cellular processes – and fueled numerous advances in immunology, oncology, and neuroscience. However, there are 3 pitfalls to single-cell analysis that can lead to incorrect biological conclusions, stalled research, and wasted resources – a true research nightmare. 1. Samples with low viability can lead to wasted sequencing reads and fewer cells sequenced2. Samples with low cell numbers may result in unusable data3. Samples with excess dead cells and contaminants can produce data that is not statistically [or bioinformatically] sound, adversely affecting your findings
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
The Power of Visualization
In science, as in many other disciplines, seeing is believing. Fluorescent cell staining is a common and reliable technique to visualize many different aspects of a cell’s state. There are many stains which can report if a cell is viable, dead or dying, or expressing a particular surface protein or reporter gene. The LeviCellTM System provides three modes of light detection: brightfield, as well as fluorescence in both green and red excitation and emission spectra. This makes commonly used viability stains such as Acridine Orange (AO) and Propidium Iodide (PI) detectable during levitation. Researchers can use fluorescence to easily see where the live and dead cell fractions are respectively positioned inside the LeviCell cartridge. Staining can also assist in setting the appropriate cutoff when collecting the live cell fraction. In this Technical Note, we review some of the commonly used cellular stains and provide guidelines for coupling these with the LeviCell system. When getting started with the LeviCell, this can be a great way to visualize how your desired cell population responds to levitation. It can also help to show you, very clearly, the power of viable cell enrichment with Levitation Technology! Download Technical Note | Request More Info
Giving Your Cells a Smoother Ride
Pursuing cell analysis often means trying to give your target cells a ride with the least turbulence: a smooth and steady experience that leaves your cell population calm, relaxed, and as close to their native state as possible. For some tissues, this is a challenging thing to achieve. Some cells must take many forms of transportation before they arrive at their destination, which makes for a stressful experience. I am sure you can relate if you have ever taken a multi-leg flight journey, followed by a train, followed by a car or taxi. It is enough to switch anyone’s immune system into fight mode. Microglia cells are an attractive target of study as the main immunomodulators of the brain, and because of their role in many neurodegenerative disorders, such as Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease. Standard isolation methods however involve many steps and can often cause unwanted transcriptional activation due to mechanical stressors. In this Research Snapshot, we illustrate how the LeviPrepTM Tissue Dissociation Kit, together with the LeviCellTM system, can help to enrich microglia cells while minimizing stress and maintaining homeostasis. Now doesn’t that sound like a more pleasant journey? >> Download Research Snapshot
Reduce the Garbage in Your scRNA-seq Data
Reduce the Garbage (aka Noise) in Your scRNA-seq Data to Ensure Meaningful Data Single-cell transcriptional data sets can illuminate the path to that rare, elusive cell type lurking in the shadows, undiscovered by bulk sequencing, waiting to stand in the spotlight. To optimize the return of unique phenotypic signatures, it is important to start with a healthy cell population free of dead cells and residual debris. Unfortunately, some of the most interesting problems require tissues that contain notoriously high amounts of these contaminates, such as solid tumors, brain tissue, or any research samples that must be cryopreserved before processing. Given that library preparation costs tend to be fixed, getting the best return on your investment means maximizing the number of usable cells and reducing the noise. In other words, steering clear of the old adage, “garbage in, garbage out.” Since apoptotic cells, dead cells, and residual ambient RNA can all make their way into single-cell sequencing data sets, the raw data is screened via a set of standard QC metrics to help filter these contaminants out before proceeding to downstream analyses. In this latest Research Snapshot, we have evaluated some of these quality control (QC) metrics in a single-cell data set from whole mouse brain that has undergone both viable cell enrichment on the LeviCellTM system as well as no enrichment. We demonstrate that enrichment using Levitation Technology elevates the single-cell suspension by increasing the ratio of viable cells to dead cells and debris, improving the quality of the data set across a total of eight recommended QC parameters. Higher quality data means more usable information, which ultimately can lead to more accurate and reproducible discoveries. >> Download Research Snapshot
Basics of Cell Separation Methods: A Deep Dive on Density
Cellular density, defined here as the mass of a cell per unit volume and typically expressed in units of grams per mL or grams per cubic centimeter, is an intrinsic property of a cell. It is generally thought to be a highly regulated property of a given cell type, as cells maintain tight control over the concentration of cellular components (Relevance & Regulation of Cell Density). Cellular composition can change, leading to overall changes in cellular density by way of physiological changes such as stages of differentiation, malignant transformation, and entry into senescence. Thus, cell density can be used both as a tool to measure a population of cells as well as to separate distinct populations of cells. This blog focuses on some frequently asked questions around the importance of cell density and the role density plays in cell separation. How is cellular density related to cell health & viability? Cell health is frequently measured with a variety of stains that interact with cells in different physiological states. For example, trypan blue is frequently used to distinguish live cells from dead. Trypan blue interacts with intracellular proteins in the cytoplasm of dead cells, giving them a dark blue appearance. However, trypan blue is excluded from live cells, which are observed to have a clear cytoplasm readily distinguishable from dead cells. Cells that are viable can be separated from those that have been damaged and are undergoing apoptosis, though not yet dead, through use of markers. Scientists can assess the state of individual cells using a protein (Annexin V) that binds cell-surface exposed phosphatidylserine for apoptosis or dyes for DNA replication (ethidium monoazide or propidium iodide (PI)) which are indicative of cell division/proliferation. What is the difference between cell mass and cell volume, whose ratio makes up cell density? Cell mass is the amount of matter that makes up a cell. Water constitutes the majority of a cell’s mass at about 70%. Cells are usually classified based on their organic macromolecule content (lipid, carbohydrate, protein, nucleic acids), which constitute the majority of a cell’s weight. The organic macromolecule composition of a cell determines its basic chemistry, ultimately defining its structure and function and how it interacts with the external world. While inorganic ions of a cell are the least abundant at 1% or less of the cell mass, they play a significant role in cell metabolism and overall cell function. Cell volume is a cellular characteristic defined as the amount of space a cell occupies. A balance between intracellular osmolarity and extracellular tonicity determines a cell’s volume which is controlled by water influx/efflux for homeostatic function. Cell volume can define not only cell shape, but also modulate other cellular functions such as cell proliferation, migration, and death. Apoptosis is related to volume shrinkage and changes in cell deformability. How does cellular osmotic stress impact cell density and thus cell function? Regulation of cell volume is a critical function of cells. When cells are exposed to osmotically active environments, normal cellular function is to maintain equilibrium through regulation of osmotic stress. Movement of water via osmosis (influx or efflux) alters intracellular macromolecule concentrations. These changes in extracellular osmolarity alter cell volume, and therefore, cell density. The inability to respond to an osmotic challenge can result in impaired function of a cell. What are some common cell separation methods? What are the benefits of cell separation? Cells can be separated by physical properties like cell size, density, and cell surface markers. Cellular density is a specific physical property that allows cell populations to be isolated from each other. Use of cell surface markers enable identification and specific isolation of subpopulations. Cell separation methods, also referred to as cell isolation, provide avenues of cellular enrichment for scientists. Cell isolations can be performed using density gradient centrifugation, separating cells based on their buoyant density in solutions such as sucrose. Specific cell types, such as blood cells that are already in single-cell suspensions, can be easily separated in density gradient mediums. Altering the concentration of the density gradient media or layering several different gradients on top of each other can influence where the cells eventually settle after centrifugation. Cells that are separated based on properties such as protein expression are often isolated with analytical techniques such as flow cytometry or Fluorescence-activated cell sorting (FACS). This is typically done by flowing the cells under pressure in a stream, hydrodynamically focusing cells to partition single cells into individual droplets. Single cells in droplets are then electrostatically deflected into tubes to sort their respective populations based on user defined gating strategies. Magnetic cell isolation is used via magnetic beads attached to target antibodies that bind to cellular proteins to identify the cells of interest. Magnetic forces attract magnetically labeled cells to either a column bead matrix or to the side of the tube within the magnetic field. Both labeled and unlabeled fractions can be collected. Using this technique, cells of interest can be separated from undesired cells. Levitation technology is a unique enrichment method that is label-free. Cells are mixed with an inert, paramagnetic compound then exposed to an externally applied magnetic field via a specifically designed cartridge. The cells levitate in solution to specific heights determined by the cells’ intrinsic properties, including density and magnetic susceptibility. Viable cells levitate higher than dead cells or debris, due to permeability of the membranes of dead cells, which allows for reproducible separation. Single-cell suspensions from both blood and tissue samples are often used to determine cell function, cell state (I.e. disease), or even how cells respond to treatments and drugs. Cellular enrichment and purification improve downstream single-cell molecular analysis on desired cell types such as single-cell RNA sequencing (scRNA-seq), immunocytochemistry (ICC), or single-cell protein analysis.
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).
Elevating Organoid Research with Levitation Technology
In 2009, Sato et al. study reported the establishment of the first 3D intestinal organoid culture derived from adult stem cells. Since then, many subsequent organoid protocols for other organs have appeared and are being widely used. These 3D in vitro cultures provide a powerful tool for a broad swath of research applications as they recapitulate organ functionality and recreate disease physiology in a simplified model system. In developing drugs or treatment plans, organoids can also be used in high-throughput screening assays to see an array of effects induce on varying tissue types. Working with Organoids The maintenance of the organoids is a critical step that still has its challenges. Passaging organoid lines for their continued survival relies on the enrichment of highly viable cells, and specific cell types from dissociated organoids for re-culture. If organoid lines are not properly passaged, and become non-viable, research efforts can be set back by months as new lines need to be derived or thawed. Organoids can be frozen as a whole and dissociated in single cells. When researchers thaw a frozen organoid line, it takes time to get it to grow in a robust way. Factors like cell viability, the amount of cells frozen down and years of storage can impact the recovery of the line. Levitation technology using the LeviCell System can select for the existent viable cells of the thawed organoids thus increasing the chances of a fast and a more robust recovery of the organoid line. Levitation of all viable cells (fully differentiated, progenitor, and stem cells) away from dead/dying cells and cellular debris is a new approach that offers exciting opportunities to both improve organoid passaging efficiency as well as reestablishing a frozen organoid line. Apart from these routine processes where gentle levitation can help organoid work, there are other applications where the LeviCell System can be a powerful tool in organoid research. Organoids cells can be genetically modified to target cellular pathways or gene expression. Example of such transfection techniques commonly used with organoids is via CRISPR/Cas9 gene editing. Transfection relies on the growth of post-transfected viable cells to form the desired modified organoids. After this process, the LeviCell System can help to enrich transfected viable cells for further culturing and formation of organoids, while removing the non-viable cells that would delay growth. Organoids in Translational Research Organoids can be a valuable tool for disease modelling and can generate data regarding the correct treatment in screening assays. Drugs can be added into organoid cultures to study its effects on the different cell types or different patient-derived organoids. This is a key application for the field of personalized medicine as some treatments can fail depending on the patient. The LeviCell System can isolate the drug resistant cells from the organoids for further study to understand the action of a specific compound. Understanding why certain microbes live in certain parts of the body and why others make people sick is key to developing new treatments while providing a solution to the antibiotic resistant crisis. To understand the microbiome world better, organoids can be infected and the progression of certain diseases studied on them. The analysis of the infected organoid cells can be difficult since it requires isolation away from the uninfected cells. Using levitation technology, infected organoid cells can be separated from the uninfected cells for further analysis. Moreover, this technology provides the key safety benefit where the infectious agent has no contact with the machine – only with a single-use cartridge. This enables work with certain agents that require BSL 2+ containment and cannot be run on shared equipment because of their biosafety level. Levitating organoid cells can be used in for routine maintenance, establishment, and formation of modified organoid lines. Also, it can be useful to investigate deeper into cellular response after drug treatment or infectious agents. Stay tuned for more details on how the LeviCell System can separate whole organoids or organoid derived cells before other downstream analysis methods like sequencing or flow cytometric analysis.
Hepatocyte Isolation and Purification
Hepatocyte Isolation and Purification – A Worldwide Research Priority The largest solid organ in the body is the liver. Made up primarily (i.e., at least 80%) of hepatocytes of various types, the liver is the site of hepatocellular carcinoma (HCC), the fourth ranked cause of cancer deaths worldwide. A large and growing HCC caseload has led to a vast World-wide research effort to better understand hepatocyte structure, function and pathology. Hepatocyte research is also driven by another major factor, namely the need to better manage preclinical testing of pharmaceutical compounds for hepatotoxicity, a major reason for retractions of marketed drugs not to mention the outright failure of drugs during clinical trials. Both oncology and drug testing would benefit if the culturing of human hepatocytes could be perfected. Finally, cultured human hepatocytes hold the promise of development of bioengineered livers and use in cell transplantation in diseased livers for correction of metabolic disorders or as a bridge to organ transplantation. Experiments in the late 1960’s resulted in the first successful hepatocyte isolation protocols and this laboratory work culminated around 1976 when Seglen published the two-step perfusion protocol that is the basis for the hepatocyte isolation protocols used today. However, even with improved protocols, and newer techniques to culture isolated primary hepatocytes, the result is often loss of phenotype and altered gene expression, problems unresolved to this day. Better isolation/purification methods must be invented. Despite decades of research, it is still not possible to maintain the maturity of hepatocytes in long-term culture. During culture, primary hepatocytes undergo a process called dedifferentiation where they rapidly lose expression of a multitude of genes, including those that are involved in key functions such as xenobiotic metabolism, glucose metabolism, urea synthesis, and amino acid metabolism. Current research suggests that the loss of phenotype for cultured hepatocytes occurs via two distinct processes: 1) an inflammatory response centered around NF-κβ (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. This is induced by ischemia/reperfusion damage, hypoxia, and tissue disruption during the isolation process, resulting in the formation of reactive compounds such as reactive oxygen species (ROS), which can directly lead to injury in hepatocytes or induce the secretions of pro-inflammatory cytokines from the liver cells, especially Kupffer cells. Notably, this inflammatory response is often worsened by the contamination of the collagenase used during perfusion by bacterial lipopolysaccharide activating Kupfer cells and inducing the release of pro-inflammatory cytokines; and 2) Loss of phenotype occurs during a proliferative phase that is based on activation of mitogen-activated protein kinase (MAPK) signaling among other pathways. A result is massive changes involving mRNA, proteins, and noncoding RNAs driving the hepatocytes away from their normally highly differentiated state toward a proliferative state concurrently downregulating genes responsible for metabolic processes associated with mature liver functions such as cytochrome P450s, UGTs, SULTs, and enzymes involved in glucose metabolism. A sub-optimal extracellular matrix can also contribute to the dedifferentiation of mature hepatocytes. For example, a stiff extracellular matrix can lead to the formation of stress fibers and tension in the cells. Many methods have been investigated to extend the mature hepatic phenotype in culture, including media additives and novel culturing techniques. Media additives are classified as physiological versus non-physiological. The physiological additives include insulin, growth factors (e.g., EGF and HGF), glucocorticoids (especially dexamethasone), glutamine, selenium and transferrin, and fetal bovine serum. The nonphysiological additives include dimethyl sulfoxide (DMSO) and enzyme inducers such as rifampicin and phenobarbital. Liver ECM is also a Principal Research Focus Concurrent with research to culture hepatocytes, perfecting a liver extracellular matrix (ECM) is also a goal. A biomimetic ECM is necessary to simulate healthy versus damaged liver for liver disease research, drug toxicity studies and cancer metastasis modelling. Currently used liver ECM hydrogels are based on time-consuming thermal gelation and that limits the control of mechanical properties. A new technique though (see Ravichandran A, Murekatete B, Moedder D, Meinert C, Bray LJ. Photocrosslinkable liver extracellular matrix hydrogels for the generation of 3D liver microenvironment models. Sci Rep. 2021 Jul 30;11(1):15566. doi: 10.1038/s41598-021-94990-z. PMID: 34330947; PMCID: PMC8324893) demonstrates use of detergent-based protocols to produce a decellularized porcine liver ECM, which in turn can be solubilized and functionalized with methacrylic anhydride to generate photocrosslinkable methacrylated liver ECM (LivMA) hydrogels. The research demonstrated the efficacy of two protocols to decellularize porcine liver tissue using varying combinations of commonly used chemical agents such as Triton X-100, Sodium Dodecyl Sulphate (SDS) and Ammonium hydroxide. A successful formation of stable, reproducible LivMA hydrogels was demonstrated via both protocols by photocrosslinking. The LivMA hydrogels obtained from the two decellularization protocols showed distinct mechanical properties. The compressive modulus of the hydrogels was directly dependent on the hydrogel concentration, thereby demonstrating the tunability of mechanical properties of these hydrogels. Immortalized Human Hepatocytes were encapsulated in the LivMA hydrogels and cytocompatibility of the hydrogels was demonstrated after one week of culture. In summary, the LivMA hydrogel system demonstrated in this research provides a simple, photocrosslinkable platform, which can potentially be used to simulate healthy versus damaged liver for liver disease research, drug studies and cancer metastasis modelling.
Technical Challenges of Conventional Cell-Sorting Methods
Cell sorting and separation technologies are ubiquitous in molecular biology labs. But their popularity occurred despite serious limitations. Here, we look at some of the major challenges in conventional cell analysis tools. Built-In Restrictions Flow cytometry is one of the simplest and least expensive techniques for single-cell analysis and isolation, but the approach inherently limits the universe of possible answers. Users can’t look holistically at a population; they must select an antibody as part of the process. This pre-defines the cell population and naturally limits the questions that can be answered. For instance, selecting circulating tumor cells based on EpCAM+ status ignores many potential tumor-derived cells that may be useful for analysis but don’t express the same markers. Cell Modification Most cell sorting technologies require the use of a label or stain to detect the cells of interest. Unfortunately, this fundamentally changes the cells and limits the real-world applicability of the information generated in downstream analyses. Cell assays are already known for their broad range of variability, and labeling or staining only adds to the difficulties in predicting outcomes. Damage or Death Another hallmark of conventional cell sorting tools is the harm they inflict on the cells passing through. Damage in the sorting process is widely accepted by scientists, but it risks skewing results if some particularly rare but useful cells get lost in the process. Even something as basic as separating live from dead cells can kill so many of the live cells that the experiment is hardly worthwhile. Magnetic levitation technology from LevitasBio addresses these challenges. The approach gently separates cells based on their response to a magnetic field, no stains, labels, or antibodies required. The unique magnetic and density profiles of different cell types allow for accurate, harmless sorting and collection. This technique is especially useful with precious or difficult samples where losing cells would be a major setback. To learn more, see how magnetic levitation works.