Mild surfection of neural cells, especially motoneurons, in primary culture and cell lines

Rakotoarivelo, Clovis; Petite, Didier; Weille, Jan de; Lumbroso, Serge; Privat, Alain; Sultan, Charles; Mersel, Marcel

doi:10.1016/j.expneurol.2006.09.027

Cited by 5 publications

(5 citation statements)

References 29 publications

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“…As it has no known direct integrin-mediated affect on neuronal differentiation, it is considered a more neutral substrate coating [38]. Since motor neurons in particular are very sensitive to environmental conditions and therefore difficult to maintain in culture [39][40][41], these results are promising and suggest that most neuronal types may survive and grow on this substrate design. Further experimentation with other types of neurons will be an integral step in elucidating the principles of neuronal behavior on nanofiber scaffolds and should lead to optimal designs that best promote regeneration in the nervous system.…”

Section: Discussionmentioning

confidence: 99%

The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons

et al. 2008

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Aligned electrospun nanofibers direct neurite growth and may prove effective for repair throughout the nervous system. Applying nanofiber scaffolds to different nervous system regions will require prior in vitro testing of scaffold designs with specific neuronal and glial cell types. This would be best accomplished using primary neurons in serum-free media; however, such growth on nanofiber substrates has not yet been achieved. Here we report the development of poly(L-lactic acid) (PLLA) nanofiber substrates that support serum-free growth of primary motor and sensory neurons at low plating densities. In our study, we first compared materials used to anchor fibers to glass to keep cells submerged and maintain fiber alignment. We found that poly(lactic-co-glycolic acid) (PLGA) anchors fibers to glass and is less toxic to primary neurons than bandage and glue used in other studies. We then designed a substrate produced by electrospinning PLLA nanofibers directly on cover slips pre-coated with PLGA. This substrate retains fiber alignment even when the fiber bundle detaches from the cover slip and keeps cells in the same focal plane. To see if increasing wettability improves motor neuron survival, some fibers were plasma etched before cell plating. Survival on etched fibers was reduced at the lower plating density. Finally, the alignment of neurons grown on this substrate was equal to nanofiber alignment and surpassed the alignment of neurites from explants tested in a previous study. This substrate should facilitate investigating the behavior of many neuronal types on electrospun fibers in serum-free conditions.

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Section: Discussionmentioning

confidence: 99%

The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons

et al. 2008

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“…In vitro studies of gene transfection into glial cells provide important insights into how to accomplish the in vivo delivery of therapeutic genes to treat spinal cord injuries. Studies showed that nanocarriers promote the efficiency of gene transfection into cultured primary glial cells (Table 1) [37–40]. An early study showed that liposomes would enable transfection of mature astrocyte monolayers with efficiencies of 3.3% [37].…”

Section: Non-viral Vector Gene Transfection Of Glial Cellsmentioning

confidence: 99%

“…The rapid intracellular degradation of the lipid increased the content of free DNA and decreased the toxicity of the reagent [39]. A recent study reported a 65% transfection efficiency into a rat spinal cord-derived glial cell line on lipoplex [DOTAP:PC (10:1)-pGFP]-precoated coverslips [40]. …”

Section: Non-viral Vector Gene Transfection Of Glial Cellsmentioning

confidence: 99%

Non-viral gene therapy for spinal cord regeneration

Yao¹,

Yao²,

Daly³

et al. 2012

Drug Discovery Today

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Spinal cord injury normally results in life-long disabilities and a broad range of secondary complications. Advances in therapeutic delivery during the past few decades offer hope for such victims. However, the limited functional improvement shown in in vivo studies hinders effective therapeutic application in clinical practice. Recent studies showed that gene vectors can transfect cells present in the lesion of an injured spinal cord (endogenous cells) and thereby produce therapeutic molecules with long-lasting biological effects that promote neural tissue regeneration. In this article we review recent advances in non-viral gene delivery into neural cells and their use for gene therapy in spinal cord injury.

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“…Cultures of rodent motoneurons have been employed to investigate the action mechanisms of neurotrophic factors (Arakawa et asl., 1990; Arce et al, 1999; Garces et al, 2000; Cisterni et al, 2000) and steroid hormones (Brooks et al, 1998; Pozzi et al, 2003; Rakotoarivelo et al, 2004), mechanisms underlying apoptosis (Appert‐Collin et al, 2006), programmed motoneuron death (Raoul et al, 1999; Barthelemy et al, 2004), and motoneuron–muscle interactions (Braun et al, 1997; Guettier‐Sigrist et al, 2001; Miles et al, 2004). In man, certain neuropathologies result from selective degeneration of motoneurons (Hand and Rouleau, 2002) and primary cultures of motoneurons have been employed to study the mechanisms underlying amyotrophic lateral sclerosis (Raoul et al, 2006) and spinal bulbar muscular atrophy (Kennedy's disease; Brooks et al, 1997; Piccioni et al, 2001, 2002; Rakotoarivelo et al, 2007). A prerequisite for successful use of such experimental models is long‐term motoneuron survival and maturation.…”

mentioning

confidence: 99%

“…The classical method of obtaining motoneuron primary cultures from rodent embryos consists of enrichment by density centrifugation and purification by immunopanning (Camu and Henderson, 1992). Alternative approaches omit the immunopanning step (Bataille et al, 1998; Rakatoarivelo et al, 2007). Nevertheless, addition of muscle extracts (Henderson et al, 1983) and growth factors is required for even relatively short‐term motoneuron survival and development (Zurn et al, 1994; Sendtner, 1995).…”

mentioning

confidence: 99%

A human spinal cord cell promotes motoneuron survival and maturation in vitro

Rouleau

Mersel

Weille

et al. 2008

J of Neuroscience Research

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Primary cultures of motoneurons represent a good experimental model for studying mechanisms underlying certain spinal cord pathologies, such as amyotrophic lateral sclerosis and spinal bulbar muscular atrophy (Kennedy's disease). However, a major problem with such culture systems is the relatively short cell survival times, which limits the extent of motoneuronal maturation. In spite of supplementing culture media with various growth factors, it remains difficult to maintain motoneurons viable longer than 10 days in vitro. This study employs a new approach, in which rat motoneurons are plated on a layer of cultured cells derived from newborn human spinal cord. For all culture periods, more motoneurons remain viable in such cocultures compared with control monocultures. Moreover, although no motoneurons survive in control cultures after 22 days, viable motoneurons were observed in cocultures even after 7 weeks. Although no significant difference in neurite length was observed between 8-day mono- and cocultures, after 22 and 50 days in coculture motoneurons had a very mature morphology. They extended extremely robust, very long neurites, which formed impressive branched networks. Data obtained using a system in which the spinal cord cultures were separated from motoneurons by a porous polycarbonate filter suggest that soluble factors released from the supporting cells are in part responsible for the beneficial effects on motoneurons. Several approaches, including immunocytochemistry, immunoblotting, and electron microscopy, indicated that these supporting cells, capable of extending motoneuron survival and enhancing neurite growth, had an undifferentiated or poorly differentiated, possibly mesenchymal phenotype.

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Mild surfection of neural cells, especially motoneurons, in primary culture and cell lines

Cited by 5 publications

References 29 publications

The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons

The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons

Non-viral gene therapy for spinal cord regeneration

A human spinal cord cell promotes motoneuron survival and maturation in vitro

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