Cell Separation: Techniques and Applications

1 3 4 2 separation Techniques and Applications C e l l 1 2 3 “ “ 1 2 3 There are many reasons why scientists might want to isolate individual cells or cell populations. In this infographic, we’ll dive into those reasons, discuss various cell separation methods and explore key downstream applications. Dr. S. H. Seal is credited with developing the first cell separation method in using a sieve to separate large tumor cells from smaller blood cells. At the time, he expressed that the sieve “leaves much to be desired” but offers a simple method for the isolation of free-floating cancer cells. One of the most widely used centrifugation methods for cell separation is density gradient centrifugation (DGC). This method can be used to separate cells, organelles or macromolecules based on their density as they travel through a density gradient while under a centrifugal force. Here’s a simple overview of the technique: FACS is a sophisticated technique that uses flow cytometry to purify cell populations of a known phenotype based on size, granularity and fluorescence, in a heterogenous sample of cells. Another affinity-based method for cell isolation, MACS utilizes magnetic separation to isolate cells based on specific cell surface markers. Here’s a simple overview of the technique: Over recent years, approaches to sort cells using spatial and time-resolved data have become increasingly popular and novel workflows continue to emerge. Collectively, these methods are known as image-based cell sorting, IBCS, also referred to as image-enabled cell sorting or ICS. Combining ICS with other omics assays, such as protein‐ (e.g., CITE‐seq, scMS), transcript‐ (e.g., scRNA‐seq) and genome‐centric (e.g., scATAC‐seq, Strand‐seq) readouts, will further increase the resolution of single‐cell profiling experiments. – Schraivogel and Steinmetz write. Machine learning algorithms can be combined with real-time imaging to support automated and precise sorting of cells in IBCS. DGC workflow for separation of cells from a whole blood sample: • DGC is commonly used to isolate peripheral blood mononuclear cells (PBMCs) for the generation and expansion of CAR T cells for CAR T-cell therapy. • DGC is also used to separate superior motile spermatozoa from total sperm populations before assisted reproductive technology (ART) procedures. Labeling Cells of interest must first be labeled with fluorescent markers, which are typically conjugated to antibodies, which bind to unique surface markers on the cell, such as surface proteins. FSC measures the intensity of light scattered in the forward direction as cells pass through the laser beam, providing information on the cell’s size. Larger cells scatter more light, resulting in higher FSC signals. SSC measures the intensity of light scattered at a 90-degree angle to the laser. It provides information on the complexity of the cell. Cells with higher SSC signals contain more internal structures, such as organelles. Sample of heterogenous cells. Microfluidic flow Cells move within a fluid stream and are imaged before being sorted into collection containers. Microfluidic containment Cells are isolated and captured in droplets or stationary traps for standard imaging and medium throughput sorting via fluid flow. Microarrays Microarrays are loaded with cells, imaged and then are isolated using low-throughput mechanical or optical retrieval methods. Select cells Sort and collect of interest Extract features Individualize Image cells Sample population Direct labeling Cell sample is incubated with magnetic particles that are conjugated to antibodies targeting specific cell surface markers. Indirect labeling Primary antibodies that bind to a cell surface marker are added to the sample. Secondary antibodies, conjugated to magnetic nanoparticles, are added to the sample. They bind to the primary antibodies. Flow cytometry The labeled cells are passed through a flow cytometer. Lasers excite the fluorophores, causing them to emit light at specific wavelengths. Detectors in the flow cytometer then measure the color and intensity of the emitted light, providing information on the cell such as size, granularity and the presence of specific markers. Cell sorting – unique to FACS The instrument applies a charge to the cell of interest, and an electrostatic deflection system ensures the deflection of the charged cells into appropriate collection tubes, enabling cell sorting. FACS is also being used in combination with ultra-fast imaging techniques to enhance the sorting process. • FACS has been used to sort cells when generating knock-out and knock-in clonal populations of human induced pluripotent stem cells (hiPSCs) using CRISPR-Cas9 technology. • Schardt et al. used FACS to purify antigen-specific memory B cells from mice that had been immunized with SARS-CoV-2 antigens. This method could identify clones that produced potentially useful neutralizing antibodies. • MACS offers a quick and reliable method for collecting human mesenchymal stem cell exosomes at high purity for use in cell therapy. • Welzel et al. established that MACS can be used as a large-scale method to generate zebrafish neuronal cell cultures for neuroscience research. • IBCS has been used in combination with CRISPR-pooled screens to rapidly isolate cells that have complex phenotypes. • Label free. • Useful for rough bulk separation using samples that contain a lot of different cell types (such as blood), before applying other, more specific cell separation techniques. • Can be inexpensive compared to other separation techniques depending on the cost of the medium used, the size of the experiment and equipment required. • Single-cell precision and high purity. • Can separate more than one cell population at the same time. • Can isolate cells based on surface marker expression and intracellular marker expression. • High-throughput. • Can be used in combination with other methods for downstream applications. • Relatively low cost. • Simple to operate. • High selectivity. • Relatively high speed compared to other techniques. • Can be used in combination with other methods, such as FACS. A whole blood sample is diluted and gently layered above the centrifugation medium, avoiding mixing. There are a variety of different commercialized media types that can be used for different DGC applications. A step gradient can be used directly, or it can be allowed to diffuse to form a continuous gradient. The sample is passed through a magnetic field. The magnetically labeled cells are retained in the field, while the unlabeled cells flow through and can be discarded (positive selection). The sample is then centrifuged, and each cell type travels through the gradient until they reach a point where their density matches the gradient media. Cells can then be removed from the interphase for downstream applications. Plasma Centrifuge Mononuclear cells Centrifugation medium Erythrocytes and granulocytes • Resolution can be an issue as differences in density are not always large enough to separate individual cell types. • Potential for sample loss during centrifugation. • Preparing the appropriate gradient for the centrifugation medium can be challenging. • Can be time-consuming and laborious. • Requires specialized equipment and training. • Varied recovery rates. • Requires exogenous labeling. • The cell sorting process can be slow. • Lacks the sensitivity and cell-specific data that can be provided by FACS. • Medium throughput. • Requires exogenous labeling. • The process of using magnets can be harsh on fragile cells. To meet growing demands across various research areas, there has been a push for the development of efficient and high-throughput cell separation methods in recent decades. There are now many ways to separate cells from complex samples. The techniques used by researchers will depend on factors such as: Next, we’ll explore four of the commonly used and emerging methods for cell separation. The method of choice also depends on either: Sample size The physical properties of the cell Cell size, shape or density. Purity requirements Cell affinity The electric, magnetic or adhesive properties specific to each cell type. Downstream applications Cell separation, also known as cell isolation, refers to processes that are used to separate a single cell – or a population of cells – from a heterogenous cell mixture. Why separate cells? Cell separation methods Cell separation is important across all major fields of modern biology. Cell separation has come a long way since then. Density gradient centrifugation (DGC) Fluorescence-activated cell sorting (FACS) Magnetic-activated cell sorting (MACS) Image-based cell sorting (IBCS) How it works How it works How it works The three main strategies used in IBCS are: Here is an example of a typical workflow: Research examples of downstream applications: Research examples of downstream applications: Research examples of downstream applications: Research examples of downstream applications: Advantages: Advantages: Advantages: Drawbacks: Drawbacks: Drawbacks: A multitude of different cell types could be present in a biological sample, each expressing their own unique genes, transcripts, proteins and metabolites that affect their function. Single-cell analysis requires high-throughput methods for single-cell separation. To isolate cells from a tumor sample. To analyze the effects of a drug on a specific group of cells. To create cell-based therapies. Researchers may want to genetically modify and expand a specific cell type to generate disease models. Mixed cell population Isolated cell populations Sample loading Laser beam Laser Electrode Positive charged plate Negative charged plate Detectors FSC Size of cells SSC Fluorescence/ granularity 1964 1 2 3 4 Cell number Density (g/ml) 1.060 1.070 1.080 1.090 1.100 Monocytes Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes 1 2 3 Sponsored by ! ! ! Magnet High-resolution microscopy enables researchers to visualize cell morphology, fluorescence signals, or other markers in real-time, to identify and select target cells. Automated tools can be used to target and pick cells of interest based on specific criteria such as size, shape, spatial location of fluorescence intensity, increasing the throughput of cell selection. Sophisticated tools have been developed that enable gentle transfer of cells, preserving cell viability for downstream applications.