Name: methods for separating specific cell types
Uploaded: 2023-04-30T11:57:12.000Z
Duration: 1 min 20 s
Channel: JoVE

Researchers can isolate cells from a tissue and separate them to obtain a population containing only a single type of cells.
Other than blood, all tissue samples must first be minced into pieces and incubated with proteolytic enzymes to release individual cells.
These cells can then be further isolated based on their physical and biochemical properties.
Density gradient centrifugation uses an inert medium, such as Ficoll, to create a density gradient and separate cells into layers based on their density upon centrifugation.
Other techniques, such as cell affinity chromatography and fluorescence-activated cell sorting or FACS, use affinity interactions of cell surface molecules to isolate cells.
Cell affinity chromatography uses ligands, such as antibodies, immobilized on a stationary matrix to separate target cells that express a specific receptor while the rest of the cells flow through.
Similarly, FACS uses fluorescence-tagged antibodies to sort cells from a mixture based on their cell-surface antigens.&#160;
The homogeneous cell populations obtained from cell separation methods can be grown in the lab to establish cell cultures or used directly for analysis.

overview of cell separation and isolation

Overview Of Cell Separation And Isolation

Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, Pohl and Hawk performed experiments on how cells respond differently to a nonuniform electric field based on the cell type. Such observations were the inception of cell separation methods, which allow isolating a single cell type from a heterogeneous sample.

Commonly used cell separation techniques include filtration, density gradient centrifugation, affinity-based separation using antibodies, and flow cytometric cell sorting. Additional specialized techniques include aptamer technology, buoyancy-activated cell sorting, and laser capture microdissection. The choice of separation method is based on the characteristics of the cell type to be isolated, such as surface charge, cell size, density, morphology, physiology, and surface markers. Usually, one or more of these characteristics are employed to isolate the particular cell type efficiently.

Applications of Cell Separation and Isolation
Isolated cells can be grown in vitro to produce cell lines, which have numerous applications in areas like pharmacology, immunology, and stem cell therapy. For example, the in vitro effects of drugs can be analyzed on specific cell populations. Cell separation is also essential in screening for the appropriate B-cells for monoclonal antibody production. Single-cell analysis, such as studying gene expression patterns and epigenetic effects, also relies on the isolation of specific cells to study. Many oncological studies that contribute to our understanding of cancer cells require isolating particular tumor cells from the tissue. Thus, cell separation and isolation methods are used virtually in all major fields of modern biology.

Challenges of Cell Separation Methods
Cell separation techniques face a high noise-to-signal ratio, with a small number of specific cells (target) against a large number of varied components that form the tissue. Various factors affect the purity of the final isolate; hence, the protocols require standardization for each target cell type and application. For example, the efficiency of cells dissociating from the tissue decides the yield of isolated cells. Excess digestion increases the presence of dead cells in the isolate, whereas incomplete digestion results in cell type contamination. Cell type contamination can also occur in affinity-based cell separation. While labeling a particular cell type with specific antibodies, non-specific binding may occur, introducing different cells into&#160; the preparation. Additionally, the population size of the target cell type in the tissue is also a limiting factor &#8212; low-abundance cells often need tedious protocols to yield sufficient numbers in the isolated cell populations.

Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, ...

overview-of-cell-separation-and-isolation

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JoVE Core

Cell Biology

Unit 1

Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

methods for separating specific cell types

Cell separation is used to isolate one cell type from a mixed sample. Early work in this field began in 1964, when S. H. Seal separated large tumor cells from smaller blood cells by filtration. Two years later, Pohl and Hawk showed that different cell types can respond differently to a nonuniform electric field. These findings helped launch modern cell separation methods.

Several common techniques are used to separate cells. Filtration removes cells based on size, while density gradient centrifugation separates them by density. Affinity-based separation uses antibodies to bind specific surface markers on cells. Flow cytometric cell sorting can then sort cells one by one. More specialized methods include aptamer technology, buoyancy-activated cell sorting, and laser capture microdissection.

The best method depends on the properties of the target cell. Scientists may use surface charge, cell size, density, morphology, physiology, or surface markers to choose a protocol. In many cases, more than one feature is used to improve isolation. The goal is to collect the desired cell type as efficiently as possible.

Isolated cells have many uses in biology and medicine. They can be grown in vitro, which means outside the body, to make cell lines. These cell lines are useful in pharmacology, immunology, and stem cell therapy. Researchers also use isolated cells to test drug effects on specific cell populations and to find the right B-cells for monoclonal antibody production. Single-cell analysis, such as studies of gene expression and epigenetic effects, also depends on cell isolation. Many cancer studies use these methods to separate tumor cells from tissue.

Cell separation can be difficult because the target cells are often rare and mixed with many other tissue components. Purity depends on how well the tissue is dissociated, so each protocol must be standardized for the target cell type and use. Too much digestion can add dead cells to the sample, but too little digestion can leave contaminating cell types behind. In antibody-based methods, non-specific binding can also introduce unwanted cells. Low-abundance cell populations are especially challenging because they may require long and careful protocols to obtain enough cells.

Methods for Separating Specific Cell Types

methods-for-separating-specific-cell-types

Cell separation is used to isolate one cell type from a mixed sample. Early work in this field began in 1964, when S. H. Seal separated large tumor cells from smaller blood cells by filtration. Two years later, Pohl and Hawk showed that different cell types can respond differently to a nonuniform electric field. These findings helped launch modern cell separation methods. Several common techniques are used to separate cells. Filtration removes cells based on size, while density gradient...

cell culture

Growing Cells in the Lab

Growing cells in the lab depends on the right culture type and the right growth conditions. Most vertebrate cells grow attached to a surface as adherent cultures. These cells are grown in flasks and plates that are chemically treated to help the cells attach. Some cells, such as hematopoietic cells, grow in suspension instead. Suspension cultures can be kept in non-treated cultureware with magnetic stirrers or spinner flasks to keep the medium moving. The growth medium is a key part of...

cell lines

How Cell Lines Change and Are Verified

Cell lines are populations of cells grown in vitro that can be subcultured over many generations. Normal cells eventually stop dividing after a set number of cell divisions. This stopping point is called replicative senescence. The division limit is known as the Hayflick limit. Leonard Hayflick described this limit in 1961 after he found that fetal cells in culture could divide only about 40 to 60 times. Telomeres help explain why this happens. Telomeres shorten with each round of cell...

hybridoma technology

Monoclonal Antibody Production with Hybridomas

Hybridoma technology makes monoclonal antibodies in large amounts. Monoclonal antibodies are antibodies that bind to just one antigenic determinant, or epitope. These antibodies are important in research, diagnostics, and disease therapy. The method was developed in 1975 by Georges Köhler and Cesar Milstein, and it received the Nobel Prize in Medicine in 1984 for changing research and treatment. The process starts by fusing cells with different strengths. Common fusion methods include...

tissue homogenization and cell lysis

Lysis Methods for Preparing Tissue Samples

Tissue sample preparation often starts with homogenization and cell lysis. This step breaks tissue structure and opens cells so their parts can be studied. The best method depends on the sample type, the amount of sample, the analyte to be collected, and how sensitive the method is. Tissue homogenization methods are usually grouped into mechanical and non-mechanical methods. Mechanical methods use external physical force to disrupt tissues and cells. These methods may use grinding, shearing,...

subcellular fractionation

Separating Cell Organelles by Centrifugation

Separating cell organelles by centrifugation lets scientists isolate membrane-bound parts of a cell after lysis. The starting material is a homogenate, which is the mixture left after cells are broken open. These isolated fractions help researchers study specific cell parts, measure protein activity in one location, and support some diagnostic tests. Fractionation usually uses centrifugation, which spins the sample to separate its contents. The two main methods are differential centrifugation...

flow cytometry

Flow Cytometry in Cell Sorting

Flow cytometry in cell sorting tracks cells as they move through a laser beam. The instrument measures light scatter and fluorescence, then uses those signals to separate cells based on their properties. It is also used to count cells and study cell populations in detail. The method began in 1934 with Andrew Moldavan, a bacteriologist who counted cells in a flowing capillary system. He pushed cells through a capillary tube and viewed them under a microscope. Later, photometry made it possible...

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Column Chromatography: Separation Factors

Column chromatography separates compounds in a sample mixture by passing a solvent through a packed column. The technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, who used organic solvents to separate plant pigments. In 1941, Archer John Porter Martin and R. L. M. Synge improved the method by packing silica gel into a column and separating amino acids with a chloroform-water mobile phase. That work became the first report on column chromatography. Today, column...

types of column chromatography

Column Chromatography for Protein Purification

Column chromatography is used to purify proteins by matching the column material to the sample. The stability and compatibility of the material matter because pH, temperature, and solvent can change how the packed column performs. The right column choice helps improve purification efficiency and can be adjusted for the protein being isolated. Gel filtration chromatography separates proteins by size when the protein’s chemical nature is unknown. It uses matrices such as agarose, polyacrylamide,...

immunoprecipitation

Protein Capture with Antibodies

Immunoprecipitation uses antibodies to capture a specific protein or protein complex from a sample. The protein stays in its native state, which helps scientists study protein-protein interactions, quaternary structure, and larger supramolecular complexes. Several related methods build on this idea. Chromatin immunoprecipitation, or ChIP, is used to study protein-DNA and protein-RNA interactions. Cross-linking immunoprecipitation, or CLIP, is used to find where proteins bind endogenous RNA...

tagging and fusion proteins

Protein Tags for Purification and Detection

Protein tags help scientists isolate and study a specific protein from the other proteins in a cell. The target gene is overexpressed in a suitable host so the cell makes large amounts of the protein of interest. A tag, or label, is added to the gene to create a fusion protein that combines the target protein with the tag. These tags make detection and purification easier. Common tag types include affinity tags, epitope tags, reporter tags, fluorescent tags, and self-splicing intein tags. In...

sds-page

Protein Size Separation by SDS-PAGE

SDS-PAGE separates proteins by size using a polyacrylamide gel and an electric field. It is a type of gel electrophoresis, which moves biological macromolecules through a gel matrix. In PAGE, proteins pass through polyacrylamide gel and can be separated by size, charge, or both, depending on the method used. In native PAGE, intact proteins keep their natural charges and are separated by both net charge and size. In SDS-PAGE, proteins are first denatured and coated with sodium dodecyl sulfate,...

western blotting

Western Blotting for Protein Detection

Western blotting is a protein detection method used in biology and medicine. It helps identify proteins in biological samples and can support disease detection, including bovine spongiform encephalopathy, human immunodeficiency virus, and feline immunodeficiency virus. The process starts with SDS-PAGE, which stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This step separates proteins in a sample before they are moved to a membrane. The proteins are then transferred to...

two-dimensional gel electrophoresis

Protein Separation by Charge and Mass

Two-dimensional gel electrophoresis separates proteins by charge first and then by mass. It was first introduced by O’Farrell and Klose in 1975. Because it uses two separating steps, it gives a higher-resolution result than one-dimensional gel electrophoresis. The first dimension uses isoelectric focusing, or IEF, on immobilized pH gradient, or IPG, strips. Before this step, biological samples such as cells and tissues are treated with a buffer that contains urea, dithiothreitol, detergents,...

enzyme-linked immunosorbent assay

ELISA Test Types and Signal Detection

ELISA is an antibody-based test used to detect and measure specific molecules in a sample. It was developed in 1971 by Peter Perlman and Eva Engvall. Unlike western blot, ELISA is done in microtiter plates or in vivo rather than on an absorbent membrane. In ELISA, an antibody has an enzyme attached to its constant region. The variable region stays free to bind a matching antigen. After binding, an enzyme-substrate reaction makes the target visible or measurable. In many tests, the substrate is...

maldi-tof mass spectrometry

MALDI-TOF for Rapid Sample Analysis

MALDI-TOF mass spectrometry analyzes compounds by their mass-to-charge ratio, or m/z. It is a powerful way to detect and separate many chemical and biological samples. A mass spectrometer has three main parts: an ion source, a mass analyzer, and a detector. These parts work together, and the design can change depending on the source and analyzer used. Matrix-assisted laser desorption ionization, or MALDI, is a common ion source in these instruments. It creates soft ionization, which helps...

peptide identification using tandem mass spectrometry

peptide-identification-using-tandem-mass-spectrometry

Reading Peptide Fragments with Tandem MS

Tandem mass spectrometry helps identify peptides by measuring their mass and breaking them into smaller pieces. It is also called MS/MS or MS2. The method uses two mass analyzers to isolate a biomolecule and study its chemical properties. This approach is important in proteomics, the study of proteins on a large scale. Peptides from a complex mixture are often first separated as much as possible after enzymatic digestion. Instruments such as gel electrophoresis or liquid chromatography can...

x-ray diffraction of biological samples

Reading Crystal Patterns with X-Ray Diffraction

X-ray diffraction, or XRD, is used to study ordered structures in crystalline organic and inorganic samples, polycrystalline materials, proteins, carbohydrates, and drugs. The method shows how atoms are arranged in a sample by measuring how X-rays scatter from it. When X-rays hit a sample on a stage, the beams interact with the electron clouds around the atoms. The scattering follows Bragg’s law, and it comes from constructive interference of X-ray waves reflected from internal crystal planes.

applications of nmr in biology

NMR Spectroscopy in Biology and MRI

Nuclear magnetic resonance (NMR) spectroscopy is a useful analytical tool in biology. Researchers have used it for more than 50 years. F. Bloch and E. Purcell developed NMR in 1946 and received the 1952 Nobel Prize in Physics for this work. NMR studies biological macromolecules and small molecules. These include proteins, nucleic acids, lipids, and organic compounds such as pharmaceutical drugs. The technique works by using the magnetic properties of certain nuclei. The basic idea behind NMR...

proteomics

Proteomics and Protein Expression

Proteomics studies the proteins made by a cell type and how those proteins function. A proteome is the full set of proteins produced by a cell type, and proteomics is the large-scale study of that protein set. The term was coined by Mark Wilkins, who described it as the “PROTein complement expressed by a genOME.” Proteomics is closely linked to genomics because genes lead to mRNAs, and mRNAs usually encode proteins. However, not every mRNA is translated into a protein. This means mRNA data can...

key terms and concepts

Learn how scientists isolate specific cell types using filtration, centrifugation, antibodies, and cell sorting, plus the key challenges of purity and yield.

Methods for Separating Specific Cell Types | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Methods for Separating Specific Cell Types

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