Cryopreservation of transfected primary dorsal root ganglia neurons

Seggio, A M; Ellison, Karen; Hynd, Matthew R.; Shain, William; Thompson, Deanna M.

doi:10.1016/j.jneumeth.2008.05.017

Cited by 20 publications

(27 citation statements)

References 14 publications

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“…Previous studies have shown that neurite length is reduced by 25–60% at 24- and 72 h time points post-transfection in postnatal DRG neurons and adult retinal ganglion cells, respectively (Leclere et al, 2005; Seggio et al, 2008). Under our protocol, electroporation indeed diminished neurite extension by approximately 44% at 48 h (Student's t -test p < 0.05), compared to untreated naïve cultures, but not the percentage of neurons extending neurites (data not shown).…”

Section: Resultsmentioning

confidence: 93%

Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth

McCall¹,

Nicholson²,

Weidner³

et al. 2012

Front. Mol. Neurosci.

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The development of eukaryotic transfection technologies has been rapid in recent years, providing the opportunity to better analyze cell-autonomous mechanisms influencing various cellular processes, including cell-intrinsic regulators of regenerative neurite growth and survival. Electroporation is one of the more effective methodologies for transfection of post-mitotic neurons demonstrating sufficient neuronal survival and transfection efficiency. To further maximize the number of transfected neurons especially with large plasmids, to limit the cellular exposure to serum, and to minimize the number of animals required for cell isolation per experiment, we compared two state-of-the-art electroporation devices for in vitro transfection of adult rat dorsal root ganglion (DRG) neuron cultures. By refining different parameters, transfection efficiencies of 39–42% could be achieved using the Lonza 4D-Nucleofector X-unit system, 1.5–2-fold higher rates than those that have been previously published for adult DRG neurons using smaller plasmid sizes. Our protocol further limits the number of cells required to 3 × 105 cells per 20 μl reaction using only 2 μg DNA/reaction and allows for the complete omission of serum post-transfection. Application of this optimized protocol will contribute to furthering the study of neuron-intrinsic mechanisms responsible for growth and survival under physiological and pathophysiological conditions.

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Section: Resultsmentioning

confidence: 93%

Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth

McCall¹,

Nicholson²,

Weidner³

et al. 2012

Front. Mol. Neurosci.

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“…Dorsal root ganglia (DRG) were isolated from Sprague Dawley P2 neonatal rats as previously described (Taconic Farms, Inc.) (Seggio, Ellison et al 2008; Seggio, Narayanaswamy et al 2010; Koppes, Seggio et al 2011). Whole DRG were first digested in a trypsin-collagenase A solution (0.1% trypsin (MediaTech, Inc.) and 1 mg/mL collagenase (Sigma Aldrich) diluted in 1x Hanks Balanced Salt Solution (HBSS; Fisher Scientific, Pittsburgh, PA), followed by a second 0.1% trypsin digestion and subsequent mechanical trituration in Dulbecco’s Modified Eagle Medium (DMEM; MediaTech, Inc.).…”

Section: 2 Materials and Methodsmentioning

confidence: 99%

Neurite outgrowth on electrospun PLLA fibers is enhanced by exogenous electrical stimulation

Koppes¹,

Zaccor²,

Rivet³

et al. 2014

J. Neural Eng.

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Objective Both electrical stimuli (endogenous and exogenous) and topographical cues are instructive to axonal extension. This report, for the first time, investigated the relative dominance of directional topographical guidance cues and directional electrical cues to enhance and/or direct primary neurite extension. We hypothesized the combination of electrical stimulation with electrospun fiber topography would induce longer neurite extension from DRG neurons than the presence of electrical stimulation or aligned topography alone. Approach To test the hypothesis, neurite outgrowth was examined on laminin-coated poly-L-lactide (PLLA) films or electrospun fibers (2 μm in diameter) in the presence or absence of electrical stimulation. Immunostained neurons were semi-automatically traced using Neurolucida software and morphology was evaluated. Results Neurite extension increased 74% on the aligned fibers compared to film controls. Stimulation alone increased outgrowth by 32% on films or fibers relative to unstimulated film controls. The co-presentation of topographical (fibers) with biophysical (electrical stimulation) cues resulted in a synergistic 126% increase in outgrowth relative to unstimulated film controls. Field polarity had no influence on the directionality of neurite, indicating topographical cues are responsible to guide neurite extension. Significance Both cues (electrical stimulation and fiber geometry) are modular in nature and can be synergistically applied in conjunction with other common methods in regenerative medicine such as controlled release of growth factors to further influence axonal growth in vivo. The combined application of electrical and aligned fiber topographical guidance cues described herein, if translated in vivo, could provide a more supportive environment for directed and robust axonal regeneration following peripheral nerve injury.

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“…38,41,43 The collected DRG were digested in trypsin and collagenase solution (0.1% trypsin [Mediatech, Inc.] and 1 mg/mL collagenase A [Sigma Aldrich] solution diluted in 1 · HBSS [Fisher Scientific]). The tissue was further digested in 0.1% trypsin and mechanically triturated in base medium.…”

Section: Neuron Isolationmentioning

confidence: 99%

“…The dissociated cells were cryopreserved at a concentration of 1 · 10 6 cells/mL in freezing medium. 43 …”

Section: Neuron Isolationmentioning

confidence: 99%

“…Neuronal morphology was quantified using Neurolucida software (MBF Bioscience) as previously described in detail. 43 This software allows quantification of neuronal morphometrics, including total neurite outgrowth, number of primary neurites, number of branch points, length of longest neurite, and directionality of neurite outgrowth.…”

Section: Microscopy and Image Analysismentioning

confidence: 99%

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Electrical Stimulation of Schwann Cells Promotes Sustained Increases in Neurite Outgrowth

Koppes

Nordberg²,

Paolillo³

et al. 2013

Tissue Engineering Part A

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Endogenous electric fields are instructive during embryogenesis by acting to direct cell migration, and postnatally, they can promote axonal growth after injury (McCaig 1991, Al-Majed 2000. However, the mechanisms for these changes are not well understood. Application of an appropriate electrical stimulus may increase the rate and success of nerve repair by directly promoting axonal growth. Previously, DC electrical stimulation at 50 mV/mm (1 mA, 8 h duration) was shown to promote neurite outgrowth and a more pronounced effect was observed if both peripheral glia (Schwann cells) and neurons were co-stimulated. If electrical stimulation is delivered to an injury site, both the neurons and all resident non-neuronal cells [e.g., Schwann cells, endothelial cells, fibroblasts] will be treated and this biophysical stimuli can influence axonal growth directly or indirectly via changes to the resident, non-neuronal cells. In this work, non-neuronal cells were electrically stimulated, and changes in morphology and neuro-supportive cells were evaluated. Schwann cell response (morphology and orientation) was examined after an 8 h stimulation over a range of DC fields (0-200 mV/mm, DC 1 mA), and changes in orientation were observed. Electrically prestimulating Schwann cells (50 mV/mm) promoted 30% more neurite outgrowth relative to co-stimulating both Schwann cells with neurons, suggesting that electrical stimulation modifies Schwann cell phenotype. Conditioned medium from the electrically prestimulated Schwann cells promoted a 20% increase in total neurite outgrowth and was sustained for 72 h poststimulation. An 11-fold increase in nerve growth factor but not brain-derived neurotrophic factor or glial-derived growth factor was found in the electrically prestimulated Schwann cell-conditioned medium. No significant changes in fibroblast or endothelial morphology and neuro-supportive behavior were observed poststimulation. Electrical stimulation is widely used in clinical settings; however, the rational application of this cue may directly impact and enhance neuro-supportive behavior, improving nerve repair.

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Cryopreservation of transfected primary dorsal root ganglia neurons

Cited by 20 publications

References 14 publications

Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth

Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth

Neurite outgrowth on electrospun PLLA fibers is enhanced by exogenous electrical stimulation

Electrical Stimulation of Schwann Cells Promotes Sustained Increases in Neurite Outgrowth

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