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In JoVE (2)
- The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds
- Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters
Other Publications (6)
Articles by Michelle K. Leach in JoVE
JoVE Neuroscience
The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds
1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatric Research, Education and Clinical Center, Veterans Affairs Ann Arbor Health System
Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.
JoVE Bioengineering
Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters
1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatrics Research, Education and Clinical Center, Veterans Affairs Ann Arbor Healthcare Center
Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.
Other articles by Michelle K. Leach on PubMed
Rat Hepatocyte Aggregate Formation on Discrete Aligned Nanofibers of Type-I Collagen-coated Poly(L-lactic Acid)
Primary hepatocytes cultured in three dimensional tissue constructs composed of multicellular aggregates maintain normal differentiated cellular function in vitro while cultured monolayers do not. Here, we report a technique to induce hepatocyte aggregate formation using type-I collagen-coated poly(L-lactic acid) (PLLA) discrete aligned nanofibers (disAFs) by providing limited cell-substrate adhesion strength and restricting cell migration to uniaxial movement. Kinetics of aggregate formation, morphology and biochemical activities of rat hepatocyte aggregates were tested over a 15 day culture period. Evidence was provided that physical cues from disAFs quickly induced the formation of aggregates. After 3 days in culture, 88.3% of free hepatocytes on disAFs were incorporated into aggregates with an average diameter of 61 +/- 18 microm. Hepatocyte aggregates formed on disAFs displayed excellent cell retention, cell activity and stable functional expression in terms of albumin secretion, urea synthesis and phase I and II (CYP1A and UGT) metabolic enzyme activity compared to monolayer culture of hepatocytes on tissue culture plastic (TCP) with type-I collagen as well as on meshes of type-I collagen-coated PLLA random nanofibers (meshRFs). These results suggest that disAFs may be a suitable method to maintain large-scale hepatic cultures with high activity for tissue engineering research and potential therapeutic applications, such as bioartificial liver devices.
Accelerated Neuritogenesis and Maturation of Primary Spinal Motor Neurons in Response to Nanofibers
Neuritogenesis, neuronal polarity formation, and maturation of axons and dendrites are strongly influenced by both biochemical and topographical extracellular components. The aim of this study was to elucidate the effects of polylactic acid electrospun fiber topography on primary motor neuron development, because regeneration of motor axons is extremely limited in the central nervous system and could potentially benefit from the implementation of a synthetic scaffold to encourage regrowth. In this analysis, we found that both aligned and randomly oriented submicron fibers significantly accelerated the processes of neuritogenesis and polarity formation of individual cultured motor neurons compared to flat polymer films and glass controls, likely due to restricted lamellipodia formation observed on fibers. In contrast, dendritic maturation and soma spreading were inhibited on fiber substrates after 2 days in vitro. This study is the first to examine the effects of electrospun fiber topography on motor neuron neuritogenesis and polarity formation. Aligned nanofibers were shown to affect the directionality and timing of motor neuron development, providing further evidence for the effective use of electrospun scaffolds in neural regeneration applications.
The Influence of Type-I Collagen-coated PLLA Aligned Nanofibers on Growth of Blood Outgrowth Endothelial Cells
Nanofibrous scaffolds have been applied widely in tissue engineering to simulate the nanostructure of natural extracellular matrix (ECM) and promote cell bioactivity. The aim of this study was to design a biocompatible nanofibrous scaffold for blood outgrowth endothelial cells (BOECs) and investigate the interaction between the topography of the nanofibrous scaffold and cell growth. Poly(L-lactic acid) (PLLA) random and aligned nanofibers with a uniform diameter distribution were fabricated by electrospinning. NH(3) plasma etching was used to create a hydrophilic surface on the nanofibers to improve type-I collagen adsorption; the conditions of the NH(3) plasma etching were optimized by XPS and water contact angle analysis. Cell attachment, proliferation, viability, phenotype and morphology of BOECs cultured on type-I collagen-coated PLLA film (col-Film), random fibers (col-RFs) and aligned fibers (col-AFs) were detected over a 7 day culture period. The results showed that collagen-coated PLLA nanofibers improved cell attachment and proliferation; col-AFs induced the directional growth of cells along the aligned nanofibers and enhanced endothelialization. We suggest that col-AFs may be a potential implantable scaffold for vascular tissue engineering.
Electrospun Chitosan Nanofibers for Hepatocyte Culture
In this study, we developed a method to obtain high surface area nanofiber meshes composed of chitosan of a number of molecular weights. This method required decreasing the viscosity and surface tension of the chitosan solution as well as optimization of the electrospinning parameters such as applied voltage and environmental humidity. These chitosan nanofiber meshes were developed as a culture substrate for hepatocytes. The fibers exhibited a uniform diameter distribution (average diameter: 112 nm) and FTIR results indicate that the chemical structure of chitosan is stable during the electrospinning process. The attachment, morphology and activity of hepatocytes cultured on the chitosan nanofiber meshes were tested. The results showed that the chitosan nanofibers are biocompatible with hepatocytes and that these chitosan nanofiber meshes could be useful tissue culture substrates for various applications, including bioartificial liver-assist devices and tissue engineering for liver regeneration.
Stages of Neuronal Morphological Development in Vitro--an Automated Assay
Following plating in vitro, neurons pass through a series of morphological stages as they adhere and mature. These morphological stage transitions can be monitored as a function of time to evaluate the relative health and development of neuronal cultures under different conditions. While morphological development is usually quite obvious to the experienced eye, it can often be difficult to quantify in a meaningful way. Morphology quantification typically relies on manual image measurement and can therefore be tedious, time consuming and prone to human error. Here we report the successful development of an automated process using the commercially available image analysis program MetaMorph(®) to analyze the morphology and quantify the growth of embryonic spinal motor neurons in vitro. Our process relied on the Neurite Outgrowth and Cell Scoring modules included in MetaMorph(®) and on analyzing the exported data with an algorithm written in MATLAB(®). We first adopted a series of stages of motor neuron development in vitro. Neurons were classified into these stages directly from the available output of MetaMorph(®) using the algorithm written in MATLAB(®). We validated the results of the automated analysis against a manual analysis of the same images and found no statistically significant difference between the two methods. When properly configured, automated image analysis with MetaMorph(®) is a rapid and reliable alternative to manual measurement and has the potential to accelerate the research process.
Erythropoietin-loaded Oligochitosan Nanoparticles for Treatment of Periventricular Leukomalacia
In this study, a single intraperitoneal injection of erythropoietin (EPO) loaded oligochitosan nanoparticles (epo-NPs) (average diameter 266 nm) was investigated as a treatment for periventricular leukomalacia (PVL). Nanoparticles were fabricated using a gelation technology process. PVL rats models were prepared to examine the therapeutic efficacy of epo-NPs and analyze the mechanism by which epo-NPs protect white matter. The metabolization of epo-NPs in the liver was also investigated. The pathology and behavioral data show that this single injection of a low quantity of epo-NPs had an excellent therapeutic effect on the rat model of PVL. The EPO release curve in phosphate buffered saline solution was a good fit with the zero-order kinetics distribution and was maintained at around 25% in 48 h. In vivo experiments demonstrated that 50 IU/kg epo-NPs had the same effect as a 5000 IU/kg direct injection of free EPO. Nanoparticles prolonged the time course of EPO metabolization in the liver and the stable release of EPO from the nanoparticles kept the plasma concentration of EPO at around 100 IU/ml during the 8-12h post-injection. Therefore, we suggest that oligochitosan based nanoparticles are an effective vehicle for drug delivery.
