Using Microfluidic Lab-on-a-Chip Technology for Single Cell Analysis (SCA)

Benefits of Single Cell Analysis Using Microfluidics

Single cell analysis technologies allow the study of circulating tumor cells and stem cells and makes it possible to reveal variations in gene expression including the presence of specific biomarkers. It is critical to understanding cell subtypes which helps us better understand disease diagnosis and treatment.

Liquid biopsy has recently and rapidly emerged as a less invasive and safer alternative sampling method to standard and more traditional tissue-biopsies for molecular testing. In such technologies, within which microfluidics plays an important role, circulating tumor cell, abnormal cells, or cancer stem cells are purified from peripheral blood samples. Most downstream analysis/characterization methods for liquid biopsy technologies suffer from low purities or low signal to noise ratio. Where the former is total number of cancer cells captures, and the latter is number of normal cells captured.

Using single cell analysis techniques, as a downstream post processing step to  liquid biopsy techniques, address this issue by eliminating more background/noise which in turn provides higher quality samples with maximum improved signal to noise ratios.

While there are numerous techniques for conducting single cell analysis, including flow cytometry and Fluorescence Activated Cell Sorting (FACS), a key advantage of using microfluidic cartridges (lab-on-a-chip) for SCA is the precise control over cellular flow, using far less sample. This makes it possible to get results quickly, using methods that are highly accurate and reproducible.

Microfluidic cartridges and associated instrumentation for SCA offer:

• Precise fluid control – Handing fluids at picoliter scale
• Accurate representation – Better replication of in vivo environments
• High sorting accuracy – Very high-throughput analysis of cells
• Small physical size – Cartridges are compact and disposable
• Less risk of contamination – Less physical handling decreases chances of contaminants
• Minimal sample needs – Very little sample required to conduct analysis
• Low reagent consumption – Lowering the overall cost of assays
• Fast speed – Up to 1,000 times faster than manual clone picking procedures
• Integration – Cell sampling, capture, fluid control, lysis, mixing and detection
• Discovery - Potential identification of new cell subsets

Primary SCA Applications

Microfluidic single cell analysis has been embraced by researchers focused on cell-to-cell interaction, cell metastasis, genetic analysis, protein analysis, drug discovery, immunotherapy and other areas. Here’s how the technology is most often applied.

Biomechanics - Microfluidic devices can be used to apply mechanical forces to examine the effects of various physical factors, such as temperature, pH, and nutrient concentration, on cell behavior and function. It also allows for precise control over the size and shape of the microenvironment in which the cells are grown or studied.

Cell sorting - There are several ways that microfluidic devices can be used for cell sorting (example: circulating tumor cells, or white blood cells). One method is called "dielectrophoresis" which involves using an electric field to manipulate the movement of cells through the channels. Cells can be sorted based on their dielectric properties, which are related to the electrical conductivity of the cell. Another method is called "hydrodynamic focusing," which uses the flow of a fluid to align and focus cells within the channels. This can be used to sort cells based on their size or shape and can be combined with lab-on-a-chip formats for diagnosis and therapeutical applications.

Other methods that can be used in combination with microfluidic devices to sort cells, such as Fluorescence-Activated Cell Sorting (FACS), which uses lasers and fluorescent dyes to sort cells based on the expression of specific markers.

Cell analysis - Microfluidic lab-on-a-chip devices can be used for cell analysis by performing cell culture experiments. The small size of the devices allows for the isolation and cultivation of individual cells or small groups of cells, which can be useful for studying cell behavior and function. This can prove invaluable when evaluating complex tumors that may mutate. Microfluidic devices can also be used to measure the presence, expression or activity of specific molecules, cellular processes, specific proteins or genes in cells, or to detect the presence of specific pathogens.
 

Droplet Traps

Single cell analysis using droplet-based microfluidics involves using microstructures designed with specific geometries and flow rate manipulation of various fluids, to generate droplets that encapsulate individual cells. The resulting droplets are typically on the order of a few hundred micrometers in size. Cells within the droplets might be lysed and the resulting cellular contents used for DNA or RNA sequencing, or the cells might be incubated with different reagents in order to study their behavior or gene expression. One of the key benefits of droplet-based microfluidics for single cell analysis is that it allows researchers to perform high-throughput analysis on a large number of cells in a relatively short amount of time and precise control over the conditions each cell is exposed to.

Microwells

Microwells are small, shallow wells that are etched or molded into the surface of a microfluidic device. They are designed to hold and manipulate small volumes of fluids, typically in the range of picoliters to nanoliters. They can be used to isolate individual cells or small groups of cells based on their physical or biochemical properties.

Microwells are used to extend cell viability, reduce cell stress or culture individual cells. In this case, cells are placed in the microwells, which are filled with a culture medium to support the proliferation of the cells. The microwells can also be used to perform various assays on the cells, such as measuring cell viability or gene expression.
 

Mechanical traps

Traps are microstructure barriers or chambers and are a highly-efficient way of using microfluidic devices to isolate and analyze individual cells in a sample. Once the cells are trapped, they can be analyzed using various techniques, such as fluorescence microscopy, spectroscopy, etc. These techniques allow researchers to study the properties of individual cells, such as their size, shape, and molecular makeup.

There are several ways this technique can be applied to single cell analysis. One method is to use passive microtraps, which rely on physical barriers or obstacles in the microfluidic device to capture and hold cells in place. Hydrodynamic traps are most commonly used and can be set up in parallel for high-throughput cell manipulations. They work by using microscale structures to remove the cell from bulk cell suspension.

Another method is to use active microtraps, which use external forces, such as electrical or magnetic fields, to capture and hold cells in place.
 

Valves

To perform this technique, a microfluidic device is used to separate cells based on their physical properties, such as size or shape. The microfluidic device uses channels and valves to control the flow of cells and direct them through a constriction, or "throat," in the device, where they can be trapped and directed into a separate channel for analysis.