Raquel Obregón

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.

A Pt layer/Pt disk electrode configuration to evaluate respiration and alkaline phosphatase activities of mouse embryoid bodies.

A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E(1/2)) for [Fe(CN)(6)](4-) oxidation and O(2) reduction measured versus the internal Pt pseudo reference was shifted by about -0.2V, compared with the E(1/2) measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at -0.7 V versus the Pt pseudo reference and compared with those monitored at -0.5 V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O(2))) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R(2)=0.90). The slope of the least squares method was 1. In a 1.0mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E(1/2) for PAP oxidation measured versus the Pt layer was not shifted, compared with the E(1/2) versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3 V versus the Pt layer and compared with those monitored at +0.3 V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier.

Facile and rapid generation of 3D chemical gradients within hydrogels for high-throughput drug screening applications

We propose a novel application of dielectrophoresis (DEP) to make three-dimensional (3D) methacrylated gelatin (GelMA) hydrogels with gradients of micro- and nanoparticles. DEP forces were able to manipulate micro- and nanoparticles of different sizes and materials (i.e., C2C12 myoblasts, polystyrene beads, gold microparticles, and carbon nanotubes) within GelMA hydrogels in a rapid and facile way and create 3D gradients of these particles in a microchamber. Immobilization of drugs, such as fluorescein isothiocyanate-dextran (FITC-dextran) and 6-hydroxydopamine (6-OHDA), on gold microparticles allowed us to investigate the high-throughput release of these drugs from GelMA-gold microparticle gradient systems. The latter gradient constructs were incubated with C2C12 myoblasts for 24h to examine the cell viability through the release of 6-OHDA. The drug was released from the microparticles in a gradient manner, inducing a cell viability gradient. This novel approach to create 3D chemical gradients within hydrogels is scalable to any arbitrary length scale. It is useful for making anisotropic biomimetic materials and high-throughput platforms to investigate cell-microenvironment interactions in a rapid, simple, cost-effective, and reproducible manner.

Non-invasive measurement of glucose uptake of skeletal muscle tissue models using a glucose nanobiosensor

Skeletal muscle tissues play a significant role to maintain the glucose level of whole body and any dysfunction of this tissue leads to the diabetes disease. A culture medium was created in which the muscle cells could survive for a long time and meanwhile it did not interfere with the glucose sensing. We fabricated a model of skeletal muscle tissues in vitro to monitor its glucose uptake. A nanoporous gold as a high sensitive nanobiosensor was then successfully developed and employed to detect the glucose uptake of the tissue models in this medium upon applying the electrical stimulation in a rapid, and non-invasive approach. The response of the glucose sensor was linear in a wide concentration range of 1-50 mM, with a detection limit of 3 μM at a signal-to-noise ratio of 3.0. The skeletal muscle tissue was electrically stimulated during 24 h and glucose uptake was monitored during this period. During the first 3 h of stimulation, electrically stimulated muscle tissue consumed almost twice the amount of glucose than counterpart non-stimulated sample. In total, the glucose consumption of muscle tissues was higher for the electrically stimulated tissues compared to those without applying the electrical field.

Clinical/preclinical aspects of nanofiber composites

Nanofiber composite materials play an important role in tissue engineering (TE) and regenerative medicine, mimicking the structure and properties of the extracellular matrix at nanoscale. They serve as scaffolds for cells, as carriers for various biomolecules (e.g., drugs, genes, and soluble factors), and as injectable biomaterials in TE and regenerative medicine. Clinical and preclinical studies of nanofiber composite materials have recently emerged to further develop therapeutic applications of these materials. Topics of these studies range from tissue regeneration and wound healing to biosensing and monitoring, demonstrating the great promise of nanofiber composites for a wide variety of clinical applications. This book chapter describes the characteristics and applications of nanofiber composite materials in clinical and preclinical studies. Results of relevant research are highlighted and potential research directions are discussed.

Nanofiber composites in blood vessel tissue engineering

Abstract Tissue engineering (TE) aims to restore function or replace damaged tissue through biological principles and engineering. Nanofibers are attractive substrates for tissue regeneration applications because they structurally mimic the native extracellular matrix. Composite nanofibers, which are hybrid nanofibers blended from natural and synthetic polymers, represent a major advancement in TE and regenerative medicine, since they take advantage of the physical properties of the synthetic polymer and the bioactivity of the natural polymer while minimizing the disadvantages of both. Although various nanofibrous matrices have been applied to almost all the areas of TE, in this chapter we will focus on nanofiber composites scaffolds for vascular TE.

Gradient Biomaterials as Tissue Scaffolds

Abstract Gradient biomaterials have been developed and employed as an important tool in tissue engineering and biology research since the discovery that tissues and organs are non-homogeneous, exhibiting natural functional gradients in their structure or composition. Gradient biomaterials consist of relatively gradual continuous transitions in either compositional or mechanical properties. They have been used to study cellular responses such as cell adhesion, migration, proliferation, and differentiation, and may also be useful tools in drug discovery and development. Gradients made of hydrogels and nanofibers are widely used scaffolds in tissue engineering, which have aroused great interest owing to their tunable properties and analogy to the microenvironment of native tissues. In this chapter, we classify gradient biomaterials into two main cohorts, physical and chemical/biological gradients, and describe their features and applications, particularly as tooth or bone tissue scaffolds.

3.2.2 Toward functional engineered tissues as biosensors using hydrogels and dielectrophoretic technique

Microscale technologies have been emerged as powerful tools for tissue engineering. Such technologies render precise positioning for the cells in order to define the cell-cell and cell-extracellular matrix (ECM) interactions mimicking the structure of native tissue constructs. Dielectrophoresis (DEP) method is a suitable microscale technology to do that having notable characteristics in cell manipulation such as being high accurate, rapid, scalable, and capable of handling both adherent and non-adherent cells. DEP could be used in combination with new biomaterials to embed the cells within a given pattern, allowing precise positioning of cells in order to define interactions between neighboring cells. In this investigation, we propose the gelatin methacrylate (GelMA) as a promising hydrogel for the cell dielectropatterning due to low viscosity and ionic concentration. Combined application of the GelMA hydrogel and DEP technique could be useful for precisely creating complex and cell-responsive microtissues in a rapid, accurate, and scalable manner. In this study, we proposed also the interdigitated array of electrodes as a novel platform to electrically stimulate the 3D engineered muscle tissue. The attained muscle myofibers were analyzed and quantified in terms of myotube characteristics and gene expression. These engineered tissues have the potential to serve as biosensors to examine biologically active reagents and could be applied to pharmacological screening and environmental monitoring.