Single-cell analysis has become one of the most important applications of microfluidic technology in recent years. It refers to the process of isolation, manipulation, and analysis of the cells at single-cell resolution in microfluidic chips. Depending on the application, single-cell analysis chips can include any or all the above-mentioned steps.
The small size of the cells makes it difficult to conduct single-cell analysis using conventional cell culture methods. Also, conventional methods normally rely upon the bulk analysis of the culture while the cells are primarily heterogenous. Oftentimes, the concentration of the target cells can be very low making it even more challenging to isolate and work with individual cells. Therefore, these conventional techniques come short of capturing the cell-to-cell differences and the heterogeneity of the culture.
Microfluidics is associated with handling and manipulating very tiny amounts of fluids and chemical and biological analytes which makes it ideal for single-cell analysis. Microfluidics for single-cell analysis has gained significant attention lately. Various microfluidic devices have been developed for isolating, manipulating, and analyzing single cells.
Isolation is the first step for working with individual cells. Isolation is the process at which the target cells are separated and isolated to be manipulated or analyzed. Conventionally, it was done using FACS machines that require expensive and bulky equipments as well as trained personnel. Various microfluidic chip designs have been developed for isolating single cells. It should be noted that it might be needed to first separate two different cell types from each other followed by isolating a specific type. For this, microfluidic cell separation methods should be first employed to separate the desired cell type. Microfluidic single-cell isolation methods include but are not limited to the following:
Here, an array of microwells are required to isolate the cells. The parameters that matter here are the size of the pores and the modification of the surface. The pores allow the single cells to be trapped and released by inverting the flow upon the experiment. The density of the microwells can be quite high in each microfluidic chip. Microchips with ~10000 wells have been made.
Microfluidics fabrication allows creating active and passive trapping approaches. Passive methods do not require any external power source while active techniques rely on an external source. Among passive approaches, hydrodynamic traps are popular. Two common hydrodynamic traps are U-shaped traps where single cells are trapped inside a U-shaped barrier and bypass microfluidic channel traps where the cells are sucked into side channels.
Active microfluidic single-cell traps often use electrical or optical sources to capture the cells. Optical tweezers employ a highly focused laser beam to grab a single-cell and transfer it to a desired location.
Droplet-based microfluidics is indeed the most popular method for single-cell analysis. In the droplet microfluidic technique, a microfluidic droplet generator chip is employed to generate picoliter to nanoliter droplets at very high frequencies. The droplet generation frequency can reach up to thousands of droplets per second. These droplets can then be used in a variety of ways for high and ultrahigh throughput analysis. Among all applications of droplet microfluidics, single-cell analysis has revolutionized biology research.
In droplet microfluidics, the flow conditions can be adjusted to encapsulate single cells in each droplet. Then each droplet serves as a microchamber in which cells can undergo lysis, DNA/RNA extraction, etc. They can encapsulate microorganisms such as bacteria to perform antibiotic susceptibility tests or analyze the heterogeneity of the subpopulations. Or the droplets could be made of hydrogels and then crosslinked to house the cells for longer-term culturing and analysis. The concentration of the cells is often adjusted using Poisson distribution to ensure the droplets do not contain more than one cell. A variety of microfluidic methods have been developed for the post-modification of the droplets and analysis of these cell encapsulating droplets. For example, the droplets can contain a lysis reagent and barcodes to lyse the cells and sequence the RNA.
After isolation and encapsulation in droplets, the single cells can be incubated and analyzed. The range of the studies, however, is quite wide and includes genomics, epigenomics, transcriptomics, proteomics, and metabolomics. It should be noted that depending on the type of analysis, different reagents might be used in the droplets and different protocols will be employed for analysis.
Studying the genome is a powerful tool for understanding the functions of organisms as well as the mechanisms involved in their growth. Single-cell sequencing the genome can give a better understanding of the genotypic basis of the diverse phenotypes. One major challenge in single-cell sequencing is that heterogeneous systems contain a massive number of cells which in turn makes the isolation and analysis even more challenging. Droplet microfluidic single-cell genomics aims at high-throughput isolation of single-cells within picoliter to nanoliter droplets and amplification of their DNA contents. Depending on whether the target is the whole genome or parts of the genome, different approaches can be taken for nucleic acid amplification. Briefly, in the targeted genomics study, a primer targets a specific region while in whole-genome analysis, primers bind to various places to enable replication of the whole genome.
For targeted genome analysis, two methods can be employed. In the first method, single-cells along with dye-labeled forward primers and beads coated with reverse primers get encapsulated within droplets using a microfluidic droplet generator. The cell gets lysed within the droplet where the DNA content has the opportunity to interact with the beads and the primers. Next, the droplets undergo thermal cycling to amplify the nucleic acid content and generate beads with fluorescent amplicons. Finally, the droplets are broken down and the beads will be analyzed for their DNA contents. In the other method, the cells, primers, and the fluorescent probes get encapsulated within hydrogel droplets. The droplets then undergo PCR cycles followed by gelation and washing. The fluorescent intensity of the crosslinked gels will be used for analysis here. (Learn more about microfluidic PCR methods here)
Droplet-based microfluidic whole genome analysis, however, is more challenging since enzymatic DNA isolation/amplification within droplets and data analysis are more difficult. Therefore, whole genome analysis is more time-consuming and requires more steps compared to the targeted-genome analysis. But all the steps can be done using microfluidic chips to reduce the contamination. A droplet microfluidic technology called single-cell genome sequencing (SiC-seq) can process single cells at ultra-high throughput (>50000 cells in a few hours). This method is based on encapsulating cells in hydrogel microspheres which allow reagents below a certain size (enzymes, detergents, etc) to pass through while maintaining the larger molecules such as DNA fragments.
In this technique, first, the cells in the cell suspension get encapsulated in a mixture of agarose and upon solidification undergo lysis and DNA fragmentation. Next, they get merged with droplets containing PCR reagents and barcode droplets each containing ~10 million similar barcodes to tag the DNA fragments for further analysis. Here, the DNA fragments from the same originating cell get tagged with the same sequence identifier (barcode). This is followed by thermal-cycling and sequencing and computational analysis.
Single-cell transcriptomic aims at studying the messenger RNA (mRNA) content of single cells. Two major droplet-based microfluidic techniques that have revolutionized the single-cell transcriptomic research are called Drop-seq and inDrop. They both use droplet microfluidics to encapsulate the single-cells to analyze their mRNA contents.
Drop-seq first introduced in 2015 by McCarroll’s lab at Harward is one of the most famous droplet-based techniques for whole-transcriptome analysis. The McCarol’s lab has provided a comprehensive manual on their website including a step by step protocol, troubleshooting, and FAQs on their website. The dropseq microfluidic chips include three inlets for the oil, cells, and beads mixture and an outlet to collect the droplets as shown below.
Active microfluidic single-cell traps often use electrical or optical sources to capture the cells. Optical tweezers employ a highly focused laser beam to grab a single-cell and transfer it to the desired location.
The cells are co-encapsulated with a barcoded microparticle (bead) in a nanoliter droplet. Next, the cells are lysed and their mRNA content is captured on the bead’s surface to form STAMPS (single-cell transcriptomase attached to a microparticle). Next, the mRNA is reverse transcribed and amplified for sequencing.
A challenge in using droplets for transcriptomic research is determining the cell of the origin from which an mRNA was captured. Dropseq solves this issue by developing a barcoding strategy to keep track of the original cell. Therefore, the STAMP barcodes can determine each transcript’s cell of origin.
The barcoded microparticle includes a common PCR handle to initiate the nucleic acid amplification down the road. This is followed by a cell barcode that is similar on all 108 primers on each bead but different from bead to bead. Therefore, this cell barcode can be used to identify the cell of origin of each mRNA transcript. To digitally count the mRNA transcript, the cell barcode is followed by a unique molecular identifier (UMI). The UMI is a random sequence of 4-12 bp which is unique for each primer on the microparticle. In the end, a 30-bp oligo dT handle is placed to capture the mRNA within the droplets.