1. Microfluidic manipulation of artificial cells
1) Capture, movement, shape control
The capture and localization of artificial cells on a chip is the basis for experiments such as static observation and structural manipulation. The passive capture approach utilizes cell characterization, fluidic control, and microstructural design.
For example, the sedimentation method works by increasing the density of artificial cells and pulling them to the observation area with the help of gravity. This method is simple in design, but the high density of material may affect the structure and function of the lipid membrane and complete immobilization cannot be achieved.
Another way is chemical or biological coupling, such as the common biotin-affinity coupling, but this method requires pre-modification of the cells, which is a complicated step and may affect cell function.
Currently, more efficient means of immobilization have become available by assembling adhesion proteins (e.g., platelet integrins) on membranes. Such methods do not require external equipment, but there may be a risk of cellular escape and the flow process may have a shearing effect on the membrane structure.
Physical means also play an important role in artificial cell manipulation. Optical tweezers technology, with its high spatio-temporal control, can precisely localize single or multiple artificial cells and can also be combined with microfluidic cells to drive and measure the mechanical properties of lipid nanotubes.
However, optical tweezers have low flux and high equipment costs. Dielectric electrophoresis, on the other hand, has become mainstream by virtue of its high precision and flexibility, but the process of integrating microelectrodes is more complex and the electric field may introduce adverse effects such as electrolysis.
2) sorting
The preparation of artificial cells is often difficult to achieve the desired monodispersity, so it is necessary to screen cells with specific characteristics for subsequent studies. In addition, residues and by-products generated during vesicle preparation and manipulation may affect the application results.
Therefore, the sorting operation is crucial. Microfluidic sorting technology can select target cells from a polydisperse initial cell population, reducing the stringent requirements for preparation techniques.
These methods include active sorting based on optical features and specific markers, and passive sorting based on dielectrophoresis. Dielectrophoretic sorting is widely used due to its high throughput and ability to be combined with fluorescence detection.
In practice, size-based passive sorting methods are relatively simple. Cells are captured and sorted by gap size differences using microcapture columns, microtraps, etc., or by adjusting the height of microchannels to screen cells based on their behavior in different flow layers.
3) Local injection
In artificial cell construction and related research, it is often necessary to introduce specific materials into the cell membrane or interior to fulfill design requirements. Localized injection techniques are therefore a key means to precisely inject the desired components into the target object, supporting either sequential activation reactions or bottom-up stepwise assembly.
Microinjection techniques allow the introduction of specific components into artificial cells, enabling the manipulation of compartments or the stepwise addition of multiple components. Although this method offers high precision and good quantitative control, it is less efficient.
In order to improve the efficiency of substance delivery, arrayed electroporation technology has been developed, which allows for the simultaneous injection of a large number of artificial cells with precise control of the contents of each vesicle. This technique is simple, reliable and efficient, and has the prospect of wide application.
2. Application of artificial cells
1) Membrane Biology Research
The cell membrane serves as a barrier to prevent the random entry of external substances and maintains the stability of the intracellular environment, thus ensuring the orderly conduct of biochemical reactions. However, cells need to exchange information, substances and energy with the outside world, so they must have a well-developed substance transportation system.
The study of artificial cells provides a platform for in-depth exploration of core issues of cell membranes, such as membrane asymmetry, lipid raft structure, transmembrane transport, membrane proteins and their interactions with the environment.
Microfluidics can precisely manipulate membrane generation, prepare asymmetric membrane structures that are difficult to achieve by conventional methods, and study lateral heterogeneity inside and outside the membrane. However, asymmetric lipid vesicles of artificial cells are difficult to maintain asymmetry for a long time due to the lack of support from other components in natural cell membranes, and lipid diffusion destroys this structure in the short term.
In contrast, block polymers have the advantage that their structure, shape, size and charge can be tuned by synthetic means and that the spontaneous distribution of different polymers inside and outside the membrane contributes to long-term asymmetry. In addition to lateral heterogeneity, there is also lateral heterogeneity within the monolayer, forming so-called “lipid raft” structures.
2) Reaction vessels and microcarriers
The multicompartmental structure of artificial cells provides a highly practical platform for the study of complex reactions. Inside the liposome, the stability of the enzyme is much higher than in the free state, probably due to the ability to maintain the concentration of the reactants internally and the additional immobilization space provided for the enzyme at the membrane surface.
Through channel proteins, the membrane is able to realize cascading biochemical reactions controlled by multiple enzymes when stimulated by external signals. The multicompartmental structure also enables specific cascade reactions and efficient communication between compartments through selective release of chemicals.
To enhance the controllability of the release of intra-zone components, ternary lipid vesicles (composed of dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and cholesterol) were generated using emulsion phase-transfer technology to release the internal fluorescent dyes and peptides upon heating to a certain temperature, and the release temperature was adjusted by adjusting the composition of the vesicles.
The possibility of individually addressing and triggering the release using lasers and other technologies to realize on-demand regulated reaction processes has great potential in the development of smart synthetic biological systems. In addition, giant vesicles can be used not only as microreaction vessels but also as microcarriers to selectively protect payloads and release them under specific stimuli or precise spatio-temporal control, making them attractive delivery vehicles.
3) Information exchange and sensing analysis
Although artificial cells have made significant progress in signaling and communication, they still have many shortcomings compared to the complex communication mechanisms between natural cells.
In addition to enabling the exchange of information between artificial cells, artificial cells can also communicate with biological cells and thus influence their behavior.
The study of mutual regulation among artificial cells can help construct artificial cell populations with complex communication mechanisms and promote the development of novel sensing and detection functions through the perception, transmission and recognition of signals.
4) Medical Applications
Advances in artificial cells in the areas of gene and protein expression, intracellular cascade reactions and intercellular communication have laid a solid foundation for their application in medicine.
With artificial cell membrane camouflage, the interference of blood cells and proteins in the isolation of circulating tumor cells can be effectively reduced and the collection efficiency can be enhanced. In addition, artificial cells show great potential in drug development.
Using histones and cell membranes derived from cancer cells, they can mimic the gene protection and transfection functions of cancer cells. At the same time, the artificial cells can also be used as drug delivery carriers to induce intracellular cascade reactions.
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