Typically, conventional cell-based research focusses on the average response from a population of cells, without taking into account individual cell phenotypes. Single cell analysis is an alternative approach that can be used to gain a better understanding of variations from cell to cell. It can show that the same cell lines or tissues can present different genomes, transcriptomes and epigenomes. Perhaps the most major application of studying the genome at single-cell level is to explore the genetic evolution of cancer – patterns of somatic mutations and copy number aberration can be observed using single-cell sequencing.
Before conducting single cell analysis, individual cells need to be identified and isolated. Isolation techniques can be divided into two groups – by physical properties and by biological characteristics. Physical properties include size, density and electric changes. Biological characteristics are mainly based on biological protein expression properties. An Every Cell Matters webinar is available to watch on-demand – leaders in the field discuss the new and advanced approaches for high-throughput single cell sequencing!
There are a number of companies that market technologies for the isolation of single cells. One of the leaders in this field in 10x Genomics. Their instrument, called the Chromium Controller, is capable of analysing a number of different single cell parameters – single cell copy number variation, single cell gene expression, single cell immune profiling and the single cell epigenomic landscape. Mission Bio’s Tapestri Platform can analyse both single cell DNA and proteins simultaneously. Furthermore, S2 Genomics have developed a machine called the Singulator 100, which can effectively transform tissue into single cells or nuclei suspensions rapidly. These can then be used for library preparation of NGS.
More detail about single cell isolation technologies can be found in our Sample Preparation report.
Overview of single cell isolation technologies – each method is explained below in chronological order. Image credit: P. Hu, 2016
Single cell isolation technologies
Fluorescence-Activated Cell Sorting
Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry with a sorting capacity. It is a sophisticated technique that defines different cell types based on size, granularity and fluorescence. It is a particularly useful method because it enables the simultaneous analysis of single cells using multiple parameters. Since the late 1960’s, huge advances have been made in FACS technology and its capability has improved from measuring 1-2 fluorescent species per cell, to up to 15 species.
Firstly, a cell suspension is made and the target cells are labelled with fluorescent probes – usually fluorophore-conjugates monoclonal antibodies (mAb). The cell suspension runs through the cytometry and is exposed to a laser, allowing the florescence detector to identify cells based on certain characteristics. Fluorescence detectors typically apply a charge to the droplet containing the cell of interest. Then an electrostatic deflection system facilitates the collection of the charged droplets for analysis.
FACS is widely used in many research types, including clinical studies. However, the approach does require a large number of starting cells in suspension – over 10,000 cells! Therefore, it cannot be used to isolate single cells from a low-quantity population. Also, the rapid flow in the machine and non-specific fluorescent molecules can reduce the success of sorting.
Magnetic-Activated Cell Sorting
Magnetic-activated cell sorting (MACS) is a technique that isolates different cell types based on antibodies, enzymes, lectins or streptavidins using magnetic beads. Essentially, the magnetic beads are coated with antibodies against a particular surface antigen, and cells that express this antigen attach to them. After a period of isolation, the solution is exposed to a strong magnetic field. This means that cells not expressing the antigen flow through, whilst cells attached to the magnetic beads remain.
MACS is capable of isolating specific cell populations with a purity of over 90%. It is also a relatively simple and cost-effective approach. However, there is an initial cost for the separation magnet and also the running costs, including of the magnetic beads. Furthermore, the final purity of the isolated cells depends on the specificity and affinity of the antibodies used to select the target cells.
Laser Capture Microdissection
Laser capture microdissection (LCM) is an advanced technology that enables single cell isolation from solid tissue samples on a microscope slide. There are two classes of LCM systems – depending on the use of infrared lasers or ultraviolet lasers. Each system consists of an inverted microscope, a solid state near infrared diode, a laser control unit, a joystick-controlled microscope stage with a vacuum, a camera and a colour monitor.
A tissue sample is visualised under a microscope and individual cells are identified, either manually or in a semi-automated fashion, and a thin transparent thermoplastic film is placed above the targeted cells. Next, a pulsed laser, which is fixed in place, is used to melt the film, fusing with the underlying cells of interest. The laser width is less than 1 micrometre, so the target cells are not affected by the beam, even if they are live. When the film is removed, the target cells remain bound to the film and are transferred to a buffer solution for analysis.
There are various LCM technologies. For example, the ION LMD system follows a gravity-assisted microdissection method, which uses gravity to collect samples in a capture device underneath the slide used. Each method requires a different collection process and types of tissue preparation before imaging and isolation, depending on the application of the experiment and the type of sample used.
The greatest advantage of LCM is its speed, whilst maintaining precision. It provides a rapid and reliable method to generate pure populations of target cells from a wide range of tissue preparations. No adjacent tissues are destroyed and the morphology of captured cells is well preserved. Therefore, all the remaining tissue is available for further capture, allowing the comparative molecular analysis of adjacent cells. Also, LCM reduces the amount of human contact with the samples, reducing the risk of contamination. However, a major drawback of the approach is that visual microscopic inspection is needed to identify single cells in a complex tissue. This requires a technologist trained in cell identification.
Manual Cell Picking
Manual cell picking is a simple and convenient approach for single cell isolation. The system consists of an inverted microscope and micro-pipettes, which are movable through motorised mechanical stages. Essentially, cells in a suspension are viewed under a microscope and then individually picked using a micro-pipette. Each isolated single cell can be observed and photographed under the microscope, enabling unbiased isolation. The technique is particularly useful for isolating live cultures or embryo cells.
Manual cell picking can be easily performed in most labs, but the throughput is limited and skilled professionals are required to perform the system.
Microfluidics refers to a group of systems that process small amount of fluidics using channels. Advantages of these approaches include precise fluid control, low sample consumption, low analysis cost and easy handling.
Cell-affinity chromatography-based microfluidics is probably the most common method for microfluidic chip analysis. The channel in the chip is modified with specific antibodies that bind to specific cell surface antigens. As the sample flows through the channels, the cell surface antigens bind to the specific antibodies, immobilizing the cells of interest on the chip. Cells that are not relevant flow off the chip with a buffer and the immobilized cells can be washed for analysis.
Other popular separation methods include physical characteristic-based, immunomagnetic bead-based and dielectric-property-based microfluidics.
Microfluidic technologies have been increasingly utilised to study variations in single cell genomes – from cancer biology to neurobiology. For example, Fluidigm developed a commercially available microfluidic PCR system called the Dynamic Array, which provides low volume and high throughput methods and is utilised in large-scale single cell studies.
Image credit: BioCompare