Studying Cells and Microtissues Using Micro- and Nanofabricated Biomaterials

Biomaterials science encompasses designing and fabricating intelligent materials for studying, directing, or mimicking biology. When it comes to successfully integrating biomaterials into biological research, a meaningful understanding of biological systems is necessary. 

Furthermore, at the interface of materials science and biology, advancements in the latter are often sparked by breakthroughs in the former. The progress in micro- and nanotechnology has thus significantly improved the understanding of cells and microtissues.

This article will delve into interesting advances in biomaterials in the context of cellular and multicellular systems.

At the Cellular Level: Fluorescent and Inorganic Probes

Fluorescent probes are widely utilized at the cellular level to observe and measure biological events and signals. Unlike simple organic dyes, fluorescent protein-based indicators can pinpoint subcellular compartments and can be used across a broader range of tissues and intact organisms.

In addition, fluorescent probes maintain a superior temporal and spatial resolution while causing less photodynamic toxicity compared to organic dyes.

Numerous fluorescent probes are derived from the jellyfish Aequorea victoria (AFPs), like green fluorescent protein (GFP) (Product No. 11814524001), red fluorescent protein (RFP), and cyan fluorescent protein (CFP). These probes have become invaluable tools for investigating various intricate processes within live cells.

However, rapid photobleaching and high sensitivity to chloride fluctuations and pH have driven the development of more stable AFP variants.¹

Coral green fluorescent protein (CGFP), despite its lower brightness and broader emission and excitation peaks, boasts high pH resistance, making it an excellent choice for labeling acidic cellular organelles.

AFPs have proven especially useful as Fluorescence Resonance Energy Transfer (FRET)-based indicators.

FRET manifests when a pair of fluorophores are situated in proximity (approximately 80 Å), resulting in an overlap between the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore.

The ratio of acceptor to donor fluorescence functions as a key indicator for quantifying biochemical events in cells. Continuous enhancements in fluorescent probe technology are increasingly enabling deeper insights into the biology of living cells.

For instance, a new generation of highly sensitive near-infrared fluorescent probes has been developed with the capability to detect subtle conformational changes in proteins, improving the detection limits and applicability of AFP-based reporters.²

Additionally, single-cell and single-molecule imaging can elucidate cell-to-cell variability and enable the visualization of individual molecular interactions, both of which can be rather challenging for existing techniques.

As an alternative to fluorescent-based probes, inorganic nanoparticles, like gold nanoparticles, iron oxide nanoparticles, and quantum dots (QDs), can be synthesized in various sizes, shapes, and yields by adjusting reaction times and solvents.

Various techniques exist for creating inorganic nanoparticles, such as microemulsions, thermal decomposition, hydrothermal, and solvothermal synthesis.³ Collectively, these particles have found application in biomedical fields like fluorescence imaging, magnetic resonance imaging, cell targeting, and delivering drugs.³

In one study, Simovic et al. investigated the properties and in vivo drug delivery abilities of nanoparticles. These particles had a hydrophobic phospholipid core and a PEG hydrophilic corona.

These PEGylated phospholipid nanoparticles can be used to dissolve and maintain water-insoluble anticancer drugs while staying close in size to drug-free particles.⁴

QDs, or Quantum Dots, have recently gained prominence in cellular and molecular biology methodologies, marking a significant advancement in the field. They consist of a semiconductor core (Cd or Se) with a ZnS shell, offering better optical features than organic fluorescent dyes.

QDs with adjustable core sizes produce various narrow emission peaks and wide excitation spectra. These qualities make them suitable for single-molecule tracking (SPT), fluorescence multiplexing, and FRET. The use of quantum dots in SPT has also revealed new insights into cell surface receptor dynamics.

For instance, water-stabilized QDs help observe receptor-mediated endocytic trafficking in live cells using fluorescence microscopy.⁵ The use of QDs as FRET donors, however, is still in its infancy.

Eventually, advancements in the generation of brighter and smaller QDs will allow for the development of combined SPT and FRET techniques, revolutionizing the field of membrane receptor dynamics, activation, and transport.⁵

At the Cellular Level: Cellular Interactions

At the cellular level, the progress in smart materials and sensors has enhanced the exploration of physical forces between cells and their surroundings. Measuring the forces involved in cell–cell and cell–matrix interactions has deepened our understanding of how external-to-internal physical cues influence cell behavior.

Originally, Harris et al. pioneered a system involving cell culture on soft silicone substrates to investigate cell locomotion and the associated forces on the surrounding matrix.⁶ The soft substrate allows visualization of forces exerted by cells through wrinkle formation on the substrate surface.

By using microneedles and counterweights, researchers can recreate these wrinkles and calculate the shear forces exerted on these surfaces.⁶

Current technology for studying cellular forces has become more sophisticated, as various forms of force microscopy now enable quantitative measurement of forces exerted by cells, either on one another or on the matrix around them. Traction Force Microscopy (TFM) represents an advancement of Harris et al.'s system.

Progress in TFM, such as the ability to analyze more than two dimensions, has expanded the study beyond traditional shear traction to include inward forces exerted by cells.⁷ 

Legant et al. introduced a system to measure forces within a three-dimensional (3D) matrix using TFM.⁷

Briefly, GFP-expressing cells were encapsulated in a PEG hydrogel (with known mechanical properties) containing fluorescent beads in the vicinity of each cell. Tracking the displacement of the beads within the surrounding matrix enabled linear elastic theory and finite element methods to be used to calculate the cellular traction forces.⁷

This powerful tool quantifies cell–matrix interactions in a spatio-temporal manner, offering numerous possibilities for future studies on various cell types, cell–ligand interactions, and even multicellular interactions.

Cell Adhesion Force Microscopy (CAFM) is another method that can be used to examine cell–matrix interactions. Initially devised for measuring the forces needed to displace inert objects from surfaces, it was adapted to assess cell–substrate adhesion forces.

Developed by Sagvolden et al., this system employs an inclined Atomic Force Microscopy (AFM) cantilever and laser deflection to gauge the force needed to displace a cell adhered to a substrate.⁸ Combined with an inverted microscope, CAFM can produce force curves related to dislodging adhered cells.⁸

This system eliminates the necessity of a gel-bead construct while seamlessly integrating with a typical two-dimensional (2D) cell culture setup. However, CAFM lacks the superior spatio-temporal resolution and 3D measurements provided by TFM.

Unsurprisingly, many technologies for studying cellular mechanics have been borrowed from other fields. Optical tweezers, initially developed in physics to detect forces on micron-sized particles, have found application in studying cellular mechanics.

Specifically, the effects of the mechanical properties of the extracellular matrix on cell surface proteins can be studied using optical tweezers and microbeads. Choquet et al. first used optical tweezers to manipulate ligand-coated latex microbeads, observing their interaction with murine fibroblasts.⁹

This setup allowed the tracking of individual microbead movement in response to cell force and precise measurement of the forces required to detach beads from adhered cells.⁹

Wang et al. also employed magnetic twisting cytometry with microbeads to explore cell–ligand interactions. Ferromagnetic microbeads coated with ligands were utilized to investigate the impact of varying mechanical loads on specific integrins.¹⁰

In this system, a robust magnetic field was applied to the ferromagnetic microbeads, and a second weaker magnetic field was introduced orthogonally to induce twisting.¹⁰

This approach allowed for higher-throughput mechanical loading on a cell population compared to optical tweezers, which opens up the opportunity to study multicellular interactions.

Beyond microbead manipulation, micropads also offer insights into the interaction between a cell and its environment.

Galbraith et al. devised a force-measuring device comprising laminin-coated micron-scale pads attached to cantilevers. The displacement of these pads in response to fibroblast-generated force was measured, enabling precise quantification of cell-generated force.¹¹

The use of microstructures for understanding cellular interactions extends beyond the study of physical forces.

Pinney et al. demonstrated that microrods and microcubes influenced the fibrotic response of cardiac cells.¹² By modifying the microenvironment with which cells interact, a therapeutic response could be achieved without the need for drug administration.¹²

Similarly, a nanotubular coating was found to enhance endothelial healing while reducing smooth muscle cell proliferation.¹³ These investigations into cellular interactions with micro- and nanostructures provided insights into the effects of the physical environment on cellular functions.

The resourceful use of advanced materials and technologies has advanced our comprehension of cellular interactions. As the methodologies are refined at the nanoscale and beyond, the opportunities for addressing complex biological questions will persistently expand.

At the Microtissue Level: Characterization

Accurate characterization of cellular interactions within microtissues is crucial for advancing tissue engineering.

While biosensors have significantly advanced the understanding of biology at the cellular level, their sensitivity and resolution have been ingeniously applied to explore the complexities of multicellular constructs.

Fluorescent probes offer a specific means to characterize cell phenotype or biochemical signaling events in multicellular constructs with minimal disruption to the tissue or 3D structure.

For instance, Wartenberg et al. utilized confocal laser scanning microscopy to assess cell viability in the core of multicellular glioma spheroids, an important in vitro model for studying multicellular microtissues.¹⁴

Although limitations like diffusion, light scattering, and dye absorption exist, fluorescent probes are still the best way to characterize the geometry and viability of multicellular constructs without disturbing cells or the microenvironment's structure.

Inorganic micro- and nanoparticles have also exhibited versatility and usefulness in sensing and imaging at the tissue level. Silica nanoparticles coated with a lipid bilayer have significantly enhanced the functionality of fluorescent dyes in various biosensing applications.¹⁵

Typically, lipid bilayers rupture in the presence of silica microspheres, enveloping the microsphere and forming a lipid bilayer coating. Porous silica microspheres allow fluorophores to be integrated into the internal pore space.

Research indicates that microspheres supported by a lipid bilayer can safeguard a pH-sensitive dye, preserving its fluorescence intensity even in higher pH environments surrounding them. This quality showcases the stability and protective nature of these microspheres.¹⁵

Contrarily, gold nanoparticles demonstrate heightened absorption and scattering intensity compared to most organic molecular dyes.¹⁶ These characteristics, coupled with their size-dependent optical and photothermal properties, position gold nanoparticles as ideal contrast agents for in vivo imaging.

Variants like gold nanoshells, nanocages, and nanorods can absorb near-infrared (NIR) light, capable of penetrating human tissue to depths of a few centimeters.

Upon NIR light absorption, gold nanoparticles disperse heat to the adjacent tissue, paving the way for applications in imaging, cancer treatment, and tumor ablation.¹⁶

The application of inorganic particles in tissue engineering is also worth mentioning. For instance, Saldanha et al. utilized iron oxide particles to label and track mesenchymal stem cells (MSCs) through magnetic resonance imaging (MRI), monitoring articular cartilage regeneration.¹⁷

Pre-transplantation labeling of MSCs with super-paramagnetic iron oxides facilitated longitudinal non-invasive in vivo assessment of transplanted cell populations' distribution.

This study underscores the necessity of developing techniques to monitor cell-based tissue engineering strategies across various applications.

At the Microtissue Level: Biosensors

As well as for the characterization of microtissues, micro- and nanotechnologies are actively used in the advancement of biosensors.

Specifically designed organic particles and molecules can function as biosensors, either as recognition elements for detecting enzymatic or antibody activity or as delivery platforms for other recognition elements.

Peptides or proteins linked to fluorophores, QDs, or nanoparticles have been effective in detecting HIV antibodies, bacterial components, and disease-related molecules like EGFR in cancer or amyloid plaques in Alzheimer's patients.¹⁸

Polymer-based particulates, commonly shaped into spheres and capsules, are also utilized extensively in biosensor applications. For instance, poly(lactic-co-glycolic acid) (PLGA), a prevalent polymer, forms nanoparticles via a double emulsion method, allows for the incorporation of hydrophilic or hydrophobic cargo.

Nehilla et al. developed QD and drug-loaded PLGA nanoparticles for improved imaging and drug release purposes.¹⁹

Aside from their applications as nanoparticulate-based carriers, polymers can also be engineered to serve as biosensors. Common stimulus-responsive polymers include pNIPAM,²⁰ pHEMA,^21, and pVA-pAa.²²

These polymers can be integrated into hydrogels, polymer brushes, and molecularly imprinted polymer coatings to detect changes in analytes at cellular (DNA, FRET, antibody fragments) or multicellular (pH, glucose, oxygen) levels.²³

Oligonucleotides also function as platforms for biosensors. Single-stranded DNA (ssDNA) is typically immobilized onto surfaces such as glass, polymers, or gold nanoparticles through various means like physisorption, chemical crosslinking, or covalent attachment.

The phosphate groups of DNA can be used to detect positively charged analytes or dye molecules.²⁴ Furthermore, DNA strands can form secondary structures that act as molecular beacons to detect DNA, small molecules, and proteins for antibody detection and signal amplification.²⁴ 

Additionally, ssDNA and their complementary counterparts contribute to DNA-directed assembly, enabling the creation of novel antibodies and multicellular tissues.²⁶ These assemblies find application in biosensing and tissue characterization investigations.²⁷

Conclusion

Micro- and nanotechnologies have become invaluable tools for biologists and engineers engaged in in vitro studies of cells and tissues.

However, it is increasingly evident that the physicochemical properties of biomaterials employed in studying biological systems can influence the system's structure and functional performance. Understanding the limitations and considerations when using such materials is thus crucial.

For instance, at the multicellular level, the platform utilized to accommodate and monitor tissues in vitro might trigger unexpected phenomena like tissue inversion, potentially compromising biological performance.²⁸

The exact mechanisms underlying these occurrences are still not fully understood. However, one thing is certain: the continued symbiotic progression of materials science and biology is bound to yield numerous enlightening discoveries and provoke further intriguing questions.

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