1. Microfluidic cell culture
1) Three-dimensional training
Traditional cell culture mostly uses a two-dimensional approach, where cells form a monolayer on a substrate. This method is easy to operate and low cost, but it cannot accurately simulate the three-dimensional growth environment of cells in vivo, and it is difficult to maintain physiologically relevant phenotypic characteristics. Therefore, the transition from two-dimensional to three-dimensional culture has become a critical step in simulating the in vivo microenvironment.
The Microfluidic Chip provides advanced technology support for three-dimensional cell culture, with a core strategy of encapsulating cells in a hydrogel for culture.
Commonly used hydrogel materials include agarose, sodium alginate, collagen, and Matrigel gels, which possess good biocompatibility and permeability to support the diffusion and exchange of gases, nutrients, and metabolites, as well as to effectively mimic the interactions of cells with the extracellular matrix.
In specific operations, structures such as ridges, columns or piles are usually set up in the microfluidic chip to ensure stable filling of the hydrogel in the channels or chambers. After the hydrogel is added to the chip and solidified and finalized, a cell suspension is introduced, and cells can attach to the surface of the hydrogel and grow.
2) Cell co-culture
Microfluidic chip-based cell co-culture can be categorized into contact and non-contact modes.
Contact co-culture is suitable for studying various cellular interactions by controlling different cells in the same tiny space and realizing direct contact between cells through microdroplet technology or trap trapping technology.
Non-contact co-culture utilizes structures such as micro-valves, hydrogels, semi-permeable membranes or narrow channels to segregate different cells in different areas of the chip, avoiding the interference of direct contact and making it more suitable for paracrine and endocrine signaling studies.
Semi-permeable membranes are widely used in co-culture systems and common materials include polycarbonate (PC) and polyethylene (PE) membranes. These membranes carry micropores with a pore size of 0.1-12.0 μm, allowing small molecules and proteins to pass through, but preventing cells from crossing.
Through microfluidic technology, cells can be inoculated on both sides of the semipermeable membrane separately, one cell on the bottom of the chip and the other on the semipermeable membrane. The layered structure of the microfluidic chip supports long-term stable co-culture of cells.
3) Tumor Microenvironment
Tumor microenvironment refers to the sum of internal and external environments in which tumors are located in the process of occurrence and development, and it is a complex and variable system that distinguishes itself from the survival environment of normal cells and tissues.
Its main features include tissue hypoxia, peripheral mesenchymal hypertension, and the aggregation of a large number of cell growth factors, chemokines, protein hydrolases, and immunoinflammatory factors. The tumor microenvironment plays a key role in tumor growth, proliferation, invasion, metastasis and angiogenesis.
Microfluidic chips are able to mimic chemical and physical factors in physiology due to their micro-nanostructures that match the cell size, sealed perfusion cultures that mimic physiological environments, as well as highly efficient mass and heat transfer properties and flexible and controllable microfluidic networks.
Through structural design and functional integration, microfluidic chips enable co-culture of multiple cell types and introduction of soluble cytokines in a concentration-gradient manner, thus constructing complex tumor microenvironments in vitro. Due to its ability to highly simulate the near-physiological environment, microfluidic chip has become an important platform for tumor microenvironment research.
2. Tissue/organ chips
With the popularization of microfluidic chip technology and the development of three-dimensional cell culture and co-culture technologies based on it, bionic systems capable of mimicking the main functions of human tissues or organs, i.e., “Tissue/Organ Chips,” have emerged.
Tissue/organ microarrays enable precisely controlled fluidic shear stress and mechanical forces through microfluidics. In these chips, cells are placed within specific microstructures, either individually or in co-culture, and are stimulated by a variety of biological factors and mechanical actions to mimic the multi-cellular structure of a tissue or organ and the in vivo microenvironment.
Compared with animal models, tissue/organ microarrays are more relevant to humans, solving the ethical controversy of animal experiments and overcoming the problems of long cycle time, high cost, and difficulty in accurately predicting human drug responses.
This technology offers the possibility of reducing and replacing animal testing in preclinical trials and accelerating the process of drug discovery while reducing R&D costs.
Currently, many types of tissue/organ microarrays have been established, including blood vessels, intestines, kidneys, lungs, hearts and so on. Tissue/organ microarrays combine multidisciplinary technologies such as cell biology, engineering and biomaterials to simulate the main structural and functional characteristics of a variety of tissues and organs, and are widely used in biomedical research, environmental toxicology testing, drug screening and personalized therapy.
3. Microfluidic single-cell separation
1) Separation of micro-pits and micro-dams
Mechanical capture is an efficient physical method for the capture and separation of single cells by designing special microstructures such as micro-pits and micro-dams.
In micro-pit capture, fluid enters the micro-pit and exits through the bottom pore. The hydrodynamic force directs individual cells to a small hole in the center of the micro-pit.
Since the cell size is larger than the pore size, when a cell enters a micro-pit, it blocks the bottom pore, preventing the liquid from continuing through that micro-pit and forcing the remaining cell suspension to flow to other micro-pits, resulting in rapid and uniform distribution of single cells.
Another commonly used method is microdam capture. Microdams utilize a U-shaped cup structure to capture individual cells within a microfluidic chip and incorporate “Quake Valve” technology, which closes the channel by gas pressure, sealing individual cells in separate chambers and allowing for cell isolation.
Within the chip, these single cells can be further lysed and support repetitive processing and washing steps for quantitative analysis of target proteins.
2) Droplet Separation
In recent years, droplet separation of single cells has become a popular method. A large number of studies have used T-crossing structures or focused flow structures to generate droplets to suspend cells in solution. When the cell suspension is wrapped by hydrophobic buffer, the connection between cells is severed, completing the encapsulation and separation of single cells.
Due to the stochastic nature of cell distribution in space, the generated droplets contain an uneven number of cells with a Poisson distribution, thus requiring further sorting of the droplets.
4. Microfluidic single-cell gene sequencing
Microfluidic technology has brought new breakthroughs in single-cell research. The microfluidic chip is small in size, with an operating volume in the range of microliters to nanoliters, which not only maintains a high sample concentration, but also reduces the consumption of reagents and lowers the cost of experiments. At the same time, the closed operating space of microfluidic device effectively avoids cross contamination between reagents.
Single-cell sequencing typically includes the following steps: single-cell isolation, genomic DNA/RNA extraction, whole-genome/whole-transcriptome amplification, sequencing, and subsequent analysis.
Since the gene content of a single cell is only in the pg range, which is difficult to detect directly by conventional gene sequencers, the genome needs to be amplified first. Single-cell whole genome amplification (WGA) is required to generate enough copies of DNA to accommodate library preparation for current sequencing protocols.
Commonly used WGA techniques include three categories: PCR-based amplification (e.g., primer extension pre-amplification PCR, PEP-PCR, and parsimonious oligonucleotide-primed PCR), multiple-displacement amplification (MDA), and multiple annealing loop cycle amplification (MALBAC).
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