Electrophysiological characterization of embryonic hippocampal neurons cultured in a 3D collagen hydrogel.

Rat embryonic hippocampal neurons were cultured in (1) 3D collagen hydrogels as 'entrapped' evenly distributed cells, (2) at the interface of two collagen layers (sandwich model), and (3) on the surface of collagen coated coverslips (2D model). In the 'entrapment' model the neuronal processes grew out of the plane of the cell body and extended into the collagen matrix, in contrast to the sandwich model where the cells and their processes rarely left the plane in which they were seeded. Hippocampal neurons 'entrapped' in the 3D collagen gel grew the same number, but shorter, processes and exhibited improved survival compared to neurons cultured in the 2D model. There was no difference in the electrophysiological properties of the neurons cultured in the 3D compared to the 2D model except in the resting membrane potential and in the duration of the after-hyperpolarization. Spontaneous postsynaptic currents were recorded in 14- and 21-day-old 3D cultures evidencing functional synapse formation. Our results indicate that the physiological characteristics of 3D neuronal cultures are similar to traditional 2D cultures. However, functional 3D networks of hippocampal neurons will be necessary for multi-level circuit formation, which could be essential for understanding the basis of physiological learning and memory.

Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation.

This work describes the step-by-step development of a novel, serum-free, in vitro cell culture system resulting in the formation of robust, contracting, multinucleate myotubes from dissociated skeletal muscle cells obtained from the hind limbs of fetal rats. This defined system consisted of a serum-free medium formulation developed by the systematic addition of different growth factors as well as a nonbiological cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine. Each growth factor in the medium was experimentally evaluated for its effect on myotube formation. The resulting myotubes were evaluated immunocytochemically using embryonic skeletal muscle, specifically the myosin heavy chain antibody. Based upon this analysis, we propose a new skeletal muscle differentiation protocol that reflects the roles of the various growth factors which promote robust myotube formation. Further observation noted that the proposed skeletal muscle differentiation technique also supported muscle-nerve coculture. Immunocytochemical evidence of nerve-muscle coculture has also been documented. Applications for this novel culture system include biocompatibility and skeletal muscle differentiation studies, understanding myopathies, neuromuscular disorders, and skeletal muscle tissue engineering.

Electrophysiological and immunocytochemical characterization of DRG neurons on an organosilane surface in serum-free medium.

We are attempting to recreate a stretch reflex circuit on a patterned Bio-MEMS (bio-microelectromechanical systems) chip with deflecting micro-cantilevers. The first steps to recreate this system is to be able to grow individual components of the circuit (sensory neuron, motoneuron, skeletal muscle, and muscle spindle) on a patternable, synthetic substrate coating the MEMS device. Sensory neurons represent the afferent portion of the stretch reflex arc and also play a significant role in transmitting the signal from the muscle spindle to the spinal cord motoneurons. We have utilized a synthetic silane substrate N-1[3-(trimethoxysilyl) propyl) diethylenetriamine (DETA) on which to grow and pattern the cells. DETA forms a self-assembled monolayer on a variety of silicon substrates, including glass, and can be patterned using photolithography. In this paper, we have evaluated the growth of sensory neurons on this synthetic silane substrate. We have investigated the immunocytochemical and electrophysiological properties of the sensory neurons on DETA and compared the resultant properties with a biological control substrate (ornithine/laminin). Immunocytochemical studies revealed the survival and growth of all three subtypes of sensory neurons: trkA, trkB, and trkC on both surfaces. Furthermore, whole-cell patch clamp recordings were used to study the electrophysiological properties of the sensory neurons on the two surfaces. There were no significant differences in the electrical properties of the neurons grown on either surface. This is the first study analyzing the immunocytochemical and electrophysiological properties of sensory neurons grown long-term in a completely defined environment and on a nonbiological substrate.

A defined system to allow skeletal muscle differentiation and subsequent integration with silicon microstructures.

This work documents the development of an in vitro cell culture model consisting of a novel serum-free medium and a non-biological growth substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA), to enable functional myotube integration with cantilevers fabricated using MEMS technology. This newly developed, defined in vitro model was used to study the differentiation of fetal rat skeletal muscle and it promoted the formation of myotubes from the dissociated rat fetal muscle cells. The myotubes were characterized by morphological analysis, immunocytochemistry and electrophysiology. Further, it was demonstrated that when the dissociated muscle cells were plated on fabricated microcantilevers, the muscle cells aligned along the major axis of the cantilever and formed robust myotubes. This novel system could not only find applications in skeletal muscle differentiation and biocompatibility studies but also in bioartificial muscle engineering, hybrid actuation system development, biorobotics and for a better understanding of myopathies and neuromuscular disorders.

Viability and electrophysiology of neural cell structures generated by the inkjet printing method.

Complex cellular patterns and structures were created by automated and direct inkjet printing of primary embryonic hippocampal and cortical neurons. Immunostaining analysis and whole-cell patch-clamp recordings showed that embryonic hippocampal and cortical neurons maintained basic cellular properties and functions, including normal, healthy neuronal phenotypes and electrophysiological characteristics, after being printed through thermal inkjet nozzles. In addition, in this study a new method was developed to create 3D cellular structures: sheets of neural cells were layered on each other (layer-by-layer process) by alternate inkjet printing of NT2 cells and fibrin gels. These results and findings, taken together, show that inkjet printing is rapidly evolving into a digital fabrication method to build functional neural structures that may eventually find applications in neural tissue engineering.

Inkjet printing of viable mammalian cells.

The purpose of this study was to explore the use of a commercial thermal printer to deposit Chinese Hamster Ovary (CHO) and embryonic motoneuron cells into pre-defined patterns. These experiments were undertaken to verify the biocompatibility of thermal inkjet printing of mammalian cells and the ability to assemble them into viable constructs. Using a modified Hewlett Packard (HP) 550C computer printer and an HP 51626a ink cartridge, CHO cells and rat embryonic motoneurons were suspended separately in a concentrated phosphate buffered saline solution (3 x). The cells were subsequently printed as a kind of "ink" onto several "bio-papers" made from soy agar and collagen gel. The appearance of the CHO cells and motoneurons on the bio-papers indicated an healthy cell morphology. Furthermore, the analyses of the CHO cell viability showed that less than 8% of the cells were lysed during printing. These data indicate that mammalian cells can be effectively delivered by a modified thermal inkjet printer onto biological substrates and that they retain their ability to function. The computer-aided inkjet printing of viable mammalian cells holds potential for creating living tissue analogs, and may eventually lead to the construction of engineered human organs.

Adult rat spinal cord culture on an organosilane surface in a novel serum-free medium.

In this study, we have documented by morphological analysis, immunocytochemistry, and electrophysiology, the development of a culture system that promotes the growth and long-term survival of dissociated adult rat spinal cord neurons. This system comprises a patternable, nonbiological, cell growth-promoting organosilane substrate coated on a glass surface and an empirically derived novel serum-free medium, supplemented with specific growth factors (acidic fibroblast growth factor, heparin sulfate, neurotrophin-3, brain-derived neurotrophic factor, glial-derived neurotrophic factor, cardiotrophin-1, and vitronectin). Neurons were characterized by immunoreactivity for neurofilament 150, neuron-specific enolase, Islet-1 antibodies, electrophysiology, and the cultures were maintained for 4-6 wk. This culture system could be a useful tool for the study of adult mammalian spinal neurons in a functional in vitro system.

Long-term culture of embryonic rat cardiomyocytes on an organosilane surface in a serum-free medium.

Potential applications of engineered, functional, cardiac muscle extends from basic research through drug discovery to engineering heart tissue for transplantation. One of the central questions in cardiac tissue engineering is to understand and control the complex interactions between the cardiac muscle cells and their environment. Recent progress in chemistry, material science, and cell biology have made possible the control of the extracellular environment (soluble factors and also cell-substrate signaling) in in vitro systems. In this study we report on the development of a defined system (artificial surface, serum-free medium combination, consistent cell preparation), which promotes the differentiation and long-term survival of rat embryonic cardiomyocytes. Cardiac muscle cells plated on a N-1 (3-(trimethoxysilyl) propyl) diethylenetriamine (DETA) surface in serum-free medium survived for more than 8 weeks in vitro and maintained their contractile and electrophysiological properties. Our methods are also compatible with advanced cell patterning techniques such as microcontact printing and photolithography which now could enable systematic spacial modifications to create growth substrates for the study of the role of contact signaling in cardiac myocyte development and physiology. It also provides a test-bed for the long-term evaluation of soluble compounds such as toxins and drug candidates in a defined system.