Cell Sorting Overview

Cell sorting is a vital process in biological research involving isolating a single cell or a homogeneous population of cells from a complex mixture. It is an analytical process that employs techniques such as fluorescence-activated cell sorting (FACS) to accurately separate cells based on their specific functional and immunophenotypic characteristics. Cells can also be sorted based on their physicochemical properties, such as size, pH, density, volume, electrical impedance and light scatter properties.

It's important to note that cell sorting is not a final or end-point assessment, but instead, it allows researchers to perform further experimentation and analysis on the separated cells. This process has enabled researchers to make significant breakthroughs in various fields, such as oncology, regenerative medicine, infectious diseases and immunology.

Cell Sorting Techniques

Cell sorting techniques can be divided into various categories based on the principle they follow to sort cells.

Positive/Negative Selection

In this process, cells are tagged with dyes, fluorescent molecules or biomarkers for isolation and characterization. It includes the following techniques:

FACS

This cell sorting technique operates based on flow cytometry principles. In the workflow, the cells are either fluorescently labeled with conjugated antibodies or labeled with dyes that can coat or penetrate. As part of the process, the cell sample passes through a laser beam that scatters light from cells. The emitted light is then detected, and through digital conversion, measurable data is displayed on a computer screen that can be used to isolate specific cells. FACS allows for both positive and negative selection.

Magnetic-activated cell sorting (MACS)

MACS is an affinity-based technique. In this process, target cells are sorted by using antigen-antibody interaction. The magnetic particles are attached to antibodies with an affinity for antigens on the cell surface. The magnetic beads and cell suspension are incubated to facilitate antibodies binding with the antigens of interest in the presence of the magnetic field. This magnetic-assisted cell sorting ensures that only cells with the antigen of interest are sorted and eluted after the process. MACS cell sorting can be used to sort a subpopulation of cells based on their protein expression level.

Selective Media

This technique utilizes an antibiotic resistance marker for selection across all plasmids maintained in a host organism’s genome. The successful integration of antibiotic-resistant markers simplifies sorting and purifying cell populations. However, this method often needs to be combined with other strategies for downstream analysis.

Label-free methods

This technique does not use dyes or fluorescent molecules but instead uses the cells’ physical or optical properties for their isolation. This method can be grouped into the following techniques:

Visual identification

This technique sorts cells based on their physical properties and operator selection. A few examples of the technique include laser capture microdissection (LCM) and manual cell picking (also known as micromanipulation). LCM technique uses an inverted microscope and focused laser pulse to sort cells from solid tissue samples using microscope slides. The micromanipulation technique uses a microscope and micropipettes to isolate cells from tissue or cell culture samples.

Sedimentation

This method separates cells based on their density and diameter. Two types of sedimentation techniques sort cells: isopycnic sedimentation and velocity sedimentation. These techniques are often used to separate cells from various mixtures.

Centrifugation

It’s a conventional cell sorting technique that separates cells based on their densities. For example, in blood samples, the separation process can sort erythrocytes from leukocytes, platelets and plasma.

Filtering

This technique is used to separate cells based on their size using a fibrous membrane filter with the desired pore size. However, clogging and heterogeneity of cell sizes remain the major limitations of this conventional cell sorting approach.

Microfluidics

It is a high-throughput label-free cell sorting technique that uses a microfluidic chip to sort cells based on different parameters, such as physical characteristics or dielectric properties. It has applications in various fields, including stem cell research, cancer research and drug discovery.

Applications of Cell Sorting

Cell sorting enables a broad range of research and clinical applications. Below are some applications that benefit from cell sorting.

Drug Discovery & Development

Cell sorting plays a pivotal role in drug development as it facilitates the identification and screening of drug targets using cell-based assays. These assays are indispensable for every phase of drug discovery, ranging from the identification of targets to lead optimization.

Sequencing

Cell sorting is crucial for sequencing as it produces pure samples. Sorted cells can be analyzed using omics data (e.g., genomics, epigenomics, transcriptomics, proteomics, metabolomics and lipidomics) to understand the importance of cell populations.

Immunophenotyping

Cell profiling is a technique used to determine the characteristics of specific cells through the interaction between antibodies and cell surface markers. Sorting techniques can facilitate the identification and isolation of rare cell populations, such as circulating tumor cells, for non-invasive diagnostic purposes. This technique is useful in diagnosing critical diseases like immunodeficiency disorders and leukemias.

Enzyme engineering

FACS can be used to facilitate screening enzyme variants with high specificity using fluorogenic substrates. Sorting engineered mammalian or bacterial cells for desired enzyme activity holds immense potential in the development of new research tools or therapies, such as enzyme replacement.

Biotechnology and Medical Research

The technique of cell sorting has revolutionized the ability to isolate and study specific T cell and leukocyte populations from both blood and tumor tissues. This powerful technique has enabled researchers to develop more targeted CAR T cell therapies, which are now being used to treat a range of cancers. Moreover, cell sorting has also facilitated the detailed characterization of tumor-infiltrating leukocytes (TILs), allowing for a better understanding of how these cells interact with cancer cells and the tumor microenvironment.

Cell Sorting Challenges

The success of downstream applications can be significantly impacted by the choice of cell sorting technique. Some techniques may introduce challenges that need to be addressed to ensure accurate sorting and reliable results. For instance, certain methods may not be suitable for fragile or rare cells, while others may alter cell properties and affect downstream assays. Thus, it is crucial to carefully evaluate the advantages and disadvantages of different cell sorting methods and select the most appropriate one for the specific application and sample type.

Quality control is of utmost importance, especially for FACS, where doublets or cell-cell complexes can occur, leading to contamination that can negatively affect the analysis of cells and downstream applications. Therefore, it is essential to apply strict quality control protocols to produce precise and dependable data.

Sample preparation can destroy cells and tissues, resulting in a decrease in cell counts and debris contamination, which can also affect gene expression in other cells.

While advanced cell sorting techniques are valuable for biological research, their high operating costs, substantial sample volume requirements and long operation times can restrict access to labs with limited funding.

Future Directions

With technological advancements, high-throughput cell sorting techniques have been developed to sort and purify many cells with high accuracy. Some of these techniques include the precise sorting method, Surface acoustic wave (SAW), which includes traveling SAW (TSAW), standing SAW (SSAW) and microfluidic approaches. Surface acoustic wave cell sorting is an active cell sorting method that uses an acoustic field to sort cells.

Microfluidic platforms are considered next-generation cell sorters because of their miniaturized form, lowered cost, and efficient sorting of individual cells. Microfluidic systems (or cell sorting on a chip) can overcome challenges associated with conventional cell sorting techniques. It requires a lower volume of samples and reagents that don’t impact sensitivity and combines the ease of automation and integration with approaches such as single-cell sequencing. Innovations in microfluidics continue to lead the way for cell sorting applications because of its advantages. However, further research is required to understand the parameters associated with the process and design effective protocols for various applications.

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