As the technique is named as single cell genomics (SCG), so the first step is to isolate single cells for analysis. There are different principles used for cell isolation and on the basis of these we can classify cell isolation techniques into two groups. The first group of techniques the principle of isolation is based on physical properties like size, density, charge and physical deformation. These isolation techniques include microchip-based platforms and density gradient centrifugation. The advantage for these techniques is that the cells can be isolated without any need of labelling. In second group of techniques, the principle is based on biological properties and characteristics of cells. These techniques include affinity-based cell sorting i.e., magnetic-activated and fluorescence activated cell sorting. All the methods have certain limitations. Some of the isolation techniques are as follows:
Membrane Filtration: In this cell isolation method, a membrane filter having pores is used. The cells are isolated on the base of size. The membranes consist of all shapes and sizes of pores, it depends upon the size and shape of cell we want to trap on porous membrane. This method is very laborious, time-consuming and error-prone. These limitations are overcome by using other cell isolation techniques.
Fluorescently activated Cell sorter: It is a modern and easy cell isolation technique. It is a special type of flow cytometry which has ability to sort different cells on the characteristics like size, granular properties and fluorescence. This technique allows the analysis of single cells both quantitative and qualitative simultaneously.
For this method, cell suspension is prepared with fluorophore conjugated monoclonal antibodies labelled target cell. When they are exposed to laser light, the detectors identify the target cells and their particular properties. Then, the device gave the charge on target cell, either a positive charge or a negative charge. This charged particle is then collected by electrostatic deflection system in tubes.
Magnetic-Activated Cell sorting (MACs): The cells are isolated by this technique on the basis of their surface antigen. It allows the separation of pure cell population at about 90%. It uses magnetic beads bounded enzymes, antibodies or lectins. These molecules will associate with specific type of protein present on the target cell. After their association, when these magnetically labelled cells are placed under the outside magnetic field, then, they are polarized and are collected by elution. While all the unpolarized cells are washed out. This technique is faster and more efficient as compared with fluorescently labelled cell sorting. But on the other hand, it is quite expensive.
Laser Capture Microdissection (LCM): It is considered to be a most efficient and accurate cell sorting method where microscopic slide tissue samples are used. LCM has two types i.e., ultraviolet or infrared. The system of LCM is composed of an ultraviolet or infrared laser diode, inverted microscope, a laser control unit, CCD camera, microscope stage which is controlled by joystick supplemented with vacuum chuck for immobilization of slide and color monitor.
In this method, the target cells containing tissue samples are present on slide which can be viewed under microscope. Afterwards, these tissue sample slides are exposed to laser pulse for short duration. This results in liquification of thin and transparent thermoplastic film from the cells of interest. The film, which is liquified, then fuse with target cells. Then, as the thermoplastic film is removed which now have the cells of interest. In addition to this, these isolated cells can be centrifuged with appropriate buffer solution for analysis. Overall, LCM is an accurate, fast and efficient method to isolate pure populations of cells.
Also, it is important to know that, while we isolate our desired cells by using LCM, the adjacent cells present in the tissue samples are not destroyed or affected. So, if the analysis required isolation of the adjacent cells, then, it can be easily done. But the limitation faced in overall LCM is the high technical skills are required for accurate and precise isolation of cells. As we need a close microscopic inspection of the cells for their required characteristics, therefore, without a proper trained cytologist or pathologist, proper identification of cells cannot take place.
Manual Cell Picking (MCP): It is also known as the micromanipulation. MCP is a very important technique which allow us to isolate pure cells from cultures or embryos. This method contains movable micropipettes with an inverted microscope. Microscope is use to observe cells and then they are photographed, allowing to be isolated accurately. This is a very simple, easy and precise technique for single cell isolation. But as it is a manual method, so the output is limited or low and high level of professional skills are required.
Microfluidics (MF): It is considered as one of the powerful techniques which enables us to understand the intrinsic complexity present in cellular system by means of a very small amount of sample. Microfluidic cell sorting can be divided into four types:
Cell affinity chromatography based microfluidic isolation
Immunomagnetic beads based microfluidic isolation
Physical features based microfluidic isolation
Dielectric properties of cells based microfluidic separation
In these above-mentioned methods, most frequently used is the cell affinity chromatography based microfluidic isolation. This method contains specific types of interactions as present between antibody and antigen or ligand and the receptor. In this first step, chip’s microchannel are altered or modified by using specific antibodies. These antibodies have affinity to bind with the cell surface antigen of the target cell. So, as the antigen antibody interaction takes place, the cell becomes immobilized on the chip. The other cells are eluted by using a buffer. In the end, immobilized cells are eluted for downstream analysis by using a different buffer. These affinity-based methods are rapid, efficient and accurate due to binding by specific recognition. Presently, MF is being used in conjunction with techniques like MACS, filtration etc.