Serge Ostrovidov

Gelatin methacrylate as a promising hydrogel for 3D microscale organization and proliferation of dielectrophoretically patterned cells

Establishing the 3D microscale organization of cells has numerous practical applications, such as in determining cell fate (e.g., proliferation, migration, differentiation, and apoptosis) and in making functional tissue constructs. One approach to spatially pattern cells is by dielectrophoresis (DEP). DEP has characteristics that are important for cell manipulation, such as high accuracy, speed, scalability, and the ability to handle both adherent and non-adherent cells. However, widespread application of this method is largely restricted because there is a limited number of suitable hydrogels for cell encapsulation. To date, polyethylene glycol-diacrylate (PEG-DA) and agarose have been used extensively for dielectric patterning of cells. In this study, we propose gelatin methacrylate (GelMA) as a promising hydrogel for use in cell dielectropatterning because of its biocompatibility and low viscosity. Compared to PEG hydrogels, GelMA hydrogels showed superior performance when making cell patterns for myoblast (C2C12) and endothelial (HUVEC) cells as well as in maintaining cell viability and growth. We also developed a simple and robust protocol for co-culture of these cells. Combined application of the GelMA hydrogels and the DEP technique is suitable for creating highly complex microscale tissues with important applications in fundamental cell biology and regenerative medicine in a rapid, accurate, and scalable manner.

Dielectrophoretically aligned carbon nanotubes to control electrical and mechanical properties of hydrogels to fabricate contractile muscle myofibers.

Dielectrophoresis is used to align carbon nanotubes (CNTs) within gelatin methacrylate (GelMA) hydrogels in a facile and rapid manner. Aligned GelMA-CNT hydrogels show higher electrical properties compared with pristine and randomly distributed CNTs in GelMA hydrogels. The muscle cells cultured on these materials demonstrate higher maturation compared with cells cultured on pristine and randomly distributed CNTs in GelMA hydrogels.

Hybrid hydrogels containing vertically aligned carbon nanotubes with anisotropic electrical conductivity for muscle myofiber fabrication

Biological scaffolds with tunable electrical and mechanical properties are of great interest in many different fields, such as regenerative medicine, biorobotics, and biosensing. In this study, dielectrophoresis (DEP) was used to vertically align carbon nanotubes (CNTs) within methacrylated gelatin (GelMA) hydrogels in a robust, simple, and rapid manner. GelMA-aligned CNT hydrogels showed anisotropic electrical conductivity and superior mechanical properties compared with pristine GelMA hydrogels and GelMA hydrogels containing randomly distributed CNTs. Skeletal muscle cells grown on vertically aligned CNTs in GelMA hydrogels yielded a higher number of functional myofibers than cells that were cultured on hydrogels with randomly distributed CNTs and horizontally aligned CNTs, as confirmed by the expression of myogenic genes and proteins. In addition, the myogenic gene and protein expression increased more profoundly after applying electrical stimulation along the direction of the aligned CNTs due to the anisotropic conductivity of the hybrid GelMA-vertically aligned CNT hydrogels. We believe that platform could attract great attention in other biomedical applications, such as biosensing, bioelectronics, and creating functional biomedical devices.

Myotube formation on gelatin nanofibers – Multi-walled carbon nanotubes hybrid scaffolds

Engineering functional muscle tissue requires the formation of densely packed, aligned, and mature myotubes. To enhance the formation of aligned myotubes with improved contractibility, we fabricated aligned electrospun gelatin multi-walled carbon nanotubes (MWNTs) hybrid fibers that were used as scaffolds for the growth of myoblasts (C2C12). The MWNTs significantly enhanced myotube formation by improving the mechanical properties of the resulting fibers and upregulated the activation of mechanotransduction related genes. In addition, the fibers enhanced the maturation of the myotubes and the amplitude of the myotube contractions under electrical stimulation (ES). Such hybrid material scaffolds may be useful to direct skeletal muscle cellular organization, improve cellular functionality and tissue formation.

Interdigitated array of Pt electrodes for electrical stimulation and engineering of aligned muscle tissue

Engineered skeletal muscle tissues could be useful for applications in tissue engineering, drug screening, and bio-robotics. It is well-known that skeletal muscle cells are able to differentiate under electrical stimulation (ES), with an increase in myosin production, along with the formation of myofibers and contractile proteins. In this study, we describe the use of an interdigitated array of electrodes as a novel platform to electrically stimulate engineered muscle tissues. The resulting muscle myofibers were analyzed and quantified in terms of their myotube characteristics and gene expression. The engineered muscle tissues stimulated through the interdigitated array of electrodes demonstrated superior performance and maturation compared to the corresponding tissues stimulated through a conventional setup (i.e., through Pt wires in close proximity to the muscle tissue). In particular, the ES of muscle tissue (voltage 6 V, frequency 1 Hz and duration 10 ms for 1 day) through the interdigitated array of electrodes resulted in a higher degree of C2C12 myotube alignment (∼80%) as compared to ES using Pt wires (∼65%). In addition, higher amounts of C2C12 myotube coverage area, myotube length, muscle transcription factors and protein biomarkers were found for myotubes stimulated through the interdigitated array of electrodes compared to those stimulated using the Pt wires. Due to the wide array of potential applications of ES for two- and three-dimensional (2D and 3D) engineered tissues, the suggested platform could be employed for a variety of cell and tissue structures to more efficiently investigate their response to electrical fields.

Bioconjugated Hydrogels for Tissue Engineering and Regenerative Medicine

Hydrogels are hydrophilic polymer networks with high water content, which have played an important role as scaffolds for cells, as carriers for various biomolecules (e.g., drugs, genes, and soluble factors), and as injectable biomaterials in tissue engineering (TE) and regenerative medicine. Bioconjugation is an approach for improving the performance of hydrogels using cell-responsive components, such as proteins and peptides, which have high affinity to regulate cellular behaviors and tissue morphogenesis. However, the current knowledge on the role of those bioconjugated moieties in controlling cellular functions and tissue morphogenesis and bioconjugation methods are limited in the context of TE and organogenesis. Moreover, micro- and nanofabrication techniques have been used to manipulate bioconjugated hydrogels for regulating cell behaviors and function. This Review therefore describes synthesis, characteristics, and manipulation of various bioconjugated hydrogels and their potential in TE applications with special emphasis on preclinical/clinical translation.

Electrical stimulation as a biomimicry tool for regulating muscle cell behavior.

There is a growing need to understand muscle cell behaviors and to engineer muscle tissues to replace defective tissues in the body. Despite a long history of the clinical use of electric fields for muscle tissues in vivo, electrical stimulation (ES) has recently gained significant attention as a powerful tool for regulating muscle cell behaviors in vitro. ES aims to mimic the electrical environment of electroactive muscle cells (e.g., cardiac or skeletal muscle cells) by helping to regulate cell-cell and cell-extracellular matrix (ECM) interactions. As a result, it can be used to enhance the alignment and differentiation of skeletal or cardiac muscle cells and to aid in engineering of functional muscle tissues. Additionally, ES can be used to control and monitor force generation and electrophysiological activity of muscle tissues for bio-actuation and drug-screening applications in a simple, high-throughput, and reproducible manner. In this review paper, we briefly describe the importance of ES in regulating muscle cell behaviors in vitro, as well as the major challenges and prospective potential associated with ES in the context of muscle tissue engineering.

Controlled release of drugs from gradient hydrogels for high-throughput analysis of cell-drug interactions.

In this paper, we report a method to fabricate microengineered hydrogels that contain a concentration gradient of a drug for high-throughput analysis of cell-drug interactions. A microfluidic gradient generator was used to create a concentration gradient of okadaic acid (OA) as a model drug within poly(ethylene glycol) diacrylate hydrogels. These hydrogels were then incubated with MC3T3-E1 cell seeded glass slides to investigate the cell viability through the spatially controlled release of OA. The drug was released from the hydrogel in a gradient manner and induced a gradient of the cell viability. The drug concentration gradient containing hydrogels developed in this study have the potential to be used for drug discovery and diagnostics applications due to their ability to simultaneously test the effects of different concentrations of various chemicals.

Stretchable and micropatterned membrane for osteogenic differentation of stem cells.

Stem cells have emerged as potentially useful cells for regenerative medicine applications. To fully harness this potential, it is important to develop in vitro cell culture platforms with spatially regulated mechanical, chemical, and biological cues to induce the differentiation of stem cells. In this study, a cell culture platform was constructed that used polydopamine (PDA)-coated parafilm. The modified parafilm supports cell attachment and proliferation. In addition, because of the superb plasticity and ductility of the parafilm, it can be easily micropatterned to regulate the spatial arrangements of cells, and can exert different mechanical tensions. Specifically, we constructed a PDA-coated parafilm with grooved micropatterns to induce the osteogenic differentiation of stem cells. Adipose-derived mesenchymal stem cells that were cultured on the PDA-coated parafilm exhibited significantly higher osteogenic commitment in response to mechanical and spatial cues compared to the ones without stretch. Our findings may open new opportunities for inducing osteogenesis of stem cells in vitro using the platform that combines mechanical and spatial cues.

A microfluidic-based neurotoxin concentration gradient for the generation of an in vitro model of Parkinson’s disease

In this study, we developed a miniaturized microfluidic-based high-throughput cell toxicity assay to create an in vitro model of Parkinsons disease (PD). In particular, we generated concentration gradients of 6-hydroxydopamine (6-OHDA) to trigger a process of neuronal apoptosis in pheochromocytoma PC12 neuronal cell line. PC12 cells were cultured in a microfluidic channel, and a concentration gradient of 6-OHDA was generated in the channel by using a back and forth movement of the fluid flow. Cellular apoptosis was then analyzed along the channel. The results indicate that at low concentrations of 6-OHDA along the gradient (i.e., approximately less than 260 μM), the neuronal death in the channel was mainly induced by apoptosis, while at higher concentrations, 6-OHDA induced neuronal death mainly through necrosis. Thus, this concentration appears to be useful for creating an in vitro model of PD by inducing the highest level of apoptosis in PC12 cells. As microfluidic systems are advantageous in a range of properties such as throughput and lower use of reagents, they may provide a useful approach for generating in vitro models of disease for drug discovery applications.