Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).     

Cellular biosensors for drug discovery.

Recent advances in cell biology, fluorescent probe chemistry, miniaturization and automation have allowed the use of mammalian cells in a variety of medical and industrial applications. Here we describe the generation of cell-based biosensors, engineered to optically report specific biological activity. Cellular biosensors are comprised of living cells and can be used in various applications, including screening chemical libraries for drug discovery and environmental sensing. Panels of biosensors may also be useful for elucidating the function of novel genes. Here we describe two examples of the construction and use of engineered cell lines as biosensors for drug discovery.     

Advances in Cell‐Free Biosensors: Principle,Mechanism, and Applications

Synthetic biology has promoted the development of biosensors as tools for detecting trace substances. In the past, biosensors based on synthetic biology have been designed on living cells, but the development of cell biosensors has been greatly limited by defects such as genetically modified organism problem and the obstruction of cell membrane. However, the advent of cell‐free synthetic biology addresses these limitations. Biosensors based on the cell‐free protein synthesis system have the advantages of higher safety, higher sensitivity, and faster response time over cell biosensors, which make cell‐free biosensors have a broader application prospect. This review summarizes the workflow of various cell‐free biosensors, including the identification of analytes and signal output. The detection range of cell‐free biosensors is greatly enlarged by different recognition mechanisms and output methods. In addition, the review also discusses the applications of cell‐free biosensors in environmental monitoring and health diagnosis, as well as existing deficiencies and aspects that should be improved. In the future, through continuous improvement and optimization, the potential of cell‐free biosensors will be stimulated, and their application fields will be expanded.     

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).     

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.     

Embryonic stem cells as a novel cell source of cell-based biosensors

To investigate the use of embryonic stem cells as biosensor elements, mouse embryoid bodies were cultured on the surface of the light-addressable potentiometric sensor and induce to in vitro differentiate into cardiomyocytes and neurons. Extracellular potentials of the cells were recorded by sensor, to detect stem cells potential applications in drugs screening. The experimental results show that known cardiac stimulants (isoproterenol) and relaxants (carbamylcholine) have characteristic effects on the cardiomyocytes in terms of the changes of beat frequency, amplitude and duration. Thus, the embryonic stem cells potentially represent a renewable cell source for the cell-based biosensors.     

DNA水凝胶的制备及应用

脱氧核糖核酸(Deoxyribonucleic acid,DNA)不仅可作为生物遗传的物质基础,又以其可编程性、功能多样性、生物相容性和生物可降解性等优点,在生物材料的构建方面表现出巨大的潜力。DNA水凝胶是一种主要由DNA参与形成的三维网状聚合物材料,同时因其保留的DNA生物性能与自身骨架的机械性能的完美融合使得它成为近年来最受关注的新兴功能高分子材料之一。目前,基于各种功能核酸序列或通过结合不同的功能材料制备的单组分或多组分DNA水凝胶,已广泛用于生物医学、分子检测及环境保护的研究或应用领域中。文中主要总结了近十几年来DNA水凝胶制备方法上的研究进展,探讨了DNA水凝胶的分类策略,并进一步综述了DNA水凝胶在药物运输、生物传感、细胞培养等方面的应用研究。最后对DNA水凝胶未来的发展方向以及可能面临的挑战进行了展望。     

Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.     

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.     

Alginate Hydrogels as Scaffolds and Delivery Systems to Repair the Damaged Spinal Cord

Alginate (ALG) is a lineal hydrophilic polysaccharide present in brown algae cell walls, which turns into a gel state when hydrated. Gelation readily produces a series of three dimensional (3D) architectures like fibers, capillaries, and microspheres, used as biosensors and bio‐actuators in a plethora of biomedical applications like drug delivery and wound healing. Hydrogels have made a great impact on regenerative medicine and tissue engineering because they are able to mimic the mechanical properties of natural tissues due to their high water content. Recent advances in neurosciences have led to promising strategies for repairing and/or regenerating the damaged nervous system. Spinal cord injury (SCI) is particularly challenging, owing to its devastating medical, human, and social consequences. Although effective therapies to repair the damaged spinal cord (SC) are still lacking, multiple pharmacological, genetic, and cell‐based therapies are currently under study. In this framework, ALG hydrogels constitute a source of potential tools for the development of implants capable of promoting axonal growth and/or delivering cells or drugs at specific damaged sites, which may result in therapeutic strategies for SCI. In this mini‐review, the current state of the art of ALG applications in neural tissues for repairing the damaged spinal cord is discussed.     

High-throughput screening of cell-free riboswitches by fluorescence-activated droplet sorting

Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets.     

A genetically engineered cell-based biosensor for functional classification of agents.

Cell-based biosensors (CBBs) utilize whole cells to detect biologically active agents. Although CBBs have shown success in detecting the presence of biological agents, efforts to classify the type of agent based on functional activity have proven difficult because multiple biochemical pathways can lead to the same cellular response. However, a new approach using a genetically-engineered cell-based biosensor (GECBB) described in this paper translates this cross-talk noise into common-mode noise that can be rejected. The GECBB operates by assaying for an agent's ability to differentially activate two populations of cells, wild-type (WT) cells and cells genetically engineered to lack a specific receptor, knockout (KO) cells. Any biological agent that targets the knocked out receptor will evoke a response in the WT but not in the KO. Thus, the GECBB is exquisitely sensitive to agents that effect the engineered pathway. This approach provides the benefits of an assay for specific functional activity while simplifying signal analysis. The GECBB implemented was designed to be sensitive to agents that activate the beta 1-adrenergic receptor (beta 1-AR). This was achieved by using mouse cardiomyocytes in which the beta 1-AR had been knocked out. The cellular signal used in the GECBB was the spontaneous beat rate of the two cardiomyocyte syncitia as measured with microelectrode arrays. The GECBB was able to detect the beta-AR agonist isoproterenol (ISO) at a concentration of 10 microM (P<0.005).     

Micro 3D cell culture systems for cellular behavior studies: Culture matrices,devices, substrates,and in‐situ sensing methods

Microfabricated systems equipped with 3D cell culture devices and in‐situ cellular biosensing tools can be a powerful bionanotechnology platform to investigate a variety of biomedical applications. Various construction substrates such as plastics, glass, and paper are used for microstructures. When selecting a construction substrate, a key consideration is a porous microenvironment that allows for spheroid growth and mimics the extracellular matrix (ECM) of cell aggregates. Various bio‐functionalized hydrogels are ideal candidates that mimic the natural ECM for 3D cell culture. When selecting an optimal and appropriate microfabrication method, both the intended use of the system and the characteristics and restrictions of the target cells should be carefully considered. For highly sensitive and near‐cell surface detection of excreted cellular compounds, SERS‐based microsystems capable of dual modal imaging have the potential to be powerful tools; however, the development of optical reporters and nanoprobes remains a key challenge. We expect that the microsystems capable of both 3D cell culture and cellular response monitoring would serve as excellent tools to provide fundamental cellular behavior information for various biomedical applications such as metastasis, wound healing, high throughput screening, tissue engineering, regenerative medicine, and drug discovery and development.     

Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture

Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.     

An Automatable Hydrogel Culture Platform for Evaluating Efficacy of Antibody‐Based Therapeutics in Overcoming Chemoresistance

The microenvironment plays a major role in conferring chemoresistance to cancer cells. In order to better inform clinical response to chemoresistance, preclinical models that recapitulate its hallmark features are needed to enable screening for resistance‐specific therapeutic targets. A novel platform for seeding cancer cells in 3D hydrogels is presented utilizing derivatives of chitosan and alginate that, critically, is amenable to high throughput screening: cell seeding in hydrogels, media changes, dosing of anticancer compounds, and cell viability assays are all automated using a standard and commercially available liquid handling robot. Culture in these hydrogels elicits resistance in ovarian, lung, and prostate cancer cells to treatment by doxorubicin and paclitaxel. In correlation, proteomics analysis of SKOV3 cells cultured in 3D reveals enrichment of proteins associated with extreme drug resistance including HMOX1 and ALDH2. Subsequently, therapeutic antibodies targeted to tumor‐associated antigens upregulated in 3D cultures are shown to have higher efficacy compared to 2D cultures. Collectively, this automated 3D cell culture platform provides a powerful tool with utility in identification of drugs that may overcome chemoresistance.     

Cardiomyocyte-transistor-hybrids for sensor application.

An extracellular recording system has been designed for the detection of electrical cell signals using p-channel or n-channel field-effect transistor (FET) arrays with non-metallized gates. Signals from rat heart muscle cell were recorded by these devices and the results described on the basis of an equivalent circuit. This technique is sensitive enough to detect minute changes of the extracellular membrane voltage and has potential applications in drug screening. We show that known cardiac stimulants (isoproterenol, norepinephrine) and relaxants (verapamil, carbamylcholine) have characteristic effects on the heart cells in terms of the changes of beat frequencies in the absence or presence of corresponding agents.     

In vivo Wnt signaling tracing through a transgenic biosensor fish reveals novel activity domains

The creation of molecular tools able to unravel in vivo spatiotemporal activation of specific cell signaling events during cell migration, differentiation and morphogenesis is of great relevance to developmental cell biology. Here, we describe the generation, validation and applications of two transgenic reporter lines for Wnt/β-catenin signaling, named TCFsiam, and show that they are reliable and sensitive Wnt biosensors for in vivo studies. We demonstrate that these lines sensitively detect Wnt/β-catenin pathway activity in several cellular contexts, from sensory organs to cardiac valve patterning. We provide evidence that Wnt/β-catenin activity is involved in the formation and maintenance of the zebrafish CNS blood vessel network, on which sox10 neural crest-derived cells migrate and proliferate. We finally show that these transgenic lines allow for screening of Wnt signaling modifying compounds, tissue regeneration assessment as well as evaluation of potential Wnt/β-catenin genetic modulators.     

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.     

A new helicase assay based on graphene oxide for anti-viral drug development

Recently, graphene oxide (GO), one of the carbon nanomaterials, has received much attention due to its unique physical and chemical properties and high potential in many research areas, including applications as a biosensor and drug delivery vehicle. Various GO-based biosensors have been developed, largely based on its surface adsorption properties for detecting biomolecules, such as nucleotides and peptides, and real-time monitoring of enzymatic reactions. In this review, we discuss recent advances in GO-based biosensors focusing on a novel assay platform for helicase activity, which was also employed in high-throughput screening to discover selective helicase inhibitors.