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The generation of NGF-secreting primary rat monocytes: a comparison of different transfer methods.

ABSTRACT: Nerve growth factor (NGF), a member of the neurotrophin family, is responsible for the maintenance and survival of cholinergic neurons in the basal forebrain. The degeneration of cholinergic neurons and reduced acetycholine levels are hallmarks of Alzheimer's disease (AD) as well as associated with learning and memory deficits. Thus far, NGF has proven the most potent neuroprotective molecule against cholinergic neurodegeneration. However, delivery of this factor into the brain remains difficult. Recent studies have begun to elucidate the potential use of monocytes as vehicles for therapeutic delivery into the brain. In this study, we employed different transfection and transduction methods to generate NGF-secreting primary rat monocytes. Specifically, we compared five methods for generating NGF-secreting monocytes: (1) cationic lipid-mediated transfection (Effectene and FuGene), (2) classical electroporation, (3) nucleofection, (4) protein delivery (Bioporter) and (5) lentiviral vectors. Here, we report that classical transfection methods (lipid-mediated transfection, electroporation, nucleofection) are inefficient tools for proper gene transfer into primary rat monocytes. We demonstrate that lentiviral infection and Bioporter can successfully transduce/load primary rat monocytes and produce effective NGF secretion. Furthermore, our results indicate that NGF is bioactive and that Bioporter-loaded monocytes do not appear to exhibit any functional disruptions (i.e. in their ability to differentiate and phagocytose beta-amyloid). Taken together, our results show that primary monocytes can be effectively loaded or transduced with NGF and provides information on the most effective method for generating NGF-secreting primary rat monocytes. This study also provides a basis for further development of primary monocytes as therapeutic delivery vehicles to the diseased AD brain.

SUBMITTER: Hohsfield LA

PROVIDER: S-EPMC3638233 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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The generation of NGF-secreting primary rat monocytes: a comparison of different transfer methods.

Hohsfield Lindsay A LA Geley Stephan S Reindl Markus M Humpel Christian C

Journal of immunological methods 20130305 1-2

Nerve growth factor (NGF), a member of the neurotrophin family, is responsible for the maintenance and survival of cholinergic neurons in the basal forebrain. The degeneration of cholinergic neurons and reduced acetycholine levels are hallmarks of Alzheimer's disease (AD) as well as associated with learning and memory deficits. Thus far, NGF has proven the most potent neuroprotective molecule against cholinergic neurodegeneration. However, delivery of this factor into the brain remains difficult ...[more]

PMID: 23474426

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Project description:For in vitro culture of plant and animal cells, one of the critical steps is to adjust the initial cell density. A typical example of this is isolated microspore culture, where specific cell densities have been determined for different species. Out of these ranges, microspore growth is not induced, or is severely reduced. A similar situation occurs in many other plant and animal cell culture systems. Traditionally, researchers have used counting chambers (hemacytometers) to calculate cell densities, but little is still known about their technical advantages. In addition, much less information is available about other, alternative methods. In this work, using isolated eggplant microspore cultures and fluorescent beads (fluorospheres) as experimental systems, we performed a comprehensive comparison of six methods to calculate cell densities: (1) a Neubauer improved hemacytometer, (2) an automated cell counter, (3) a manual-counting method, and three flow cytometry methods based on (4) autofluorescence, (5) propidium iodide staining, and (6) side scattered light (SSC).Our results show that from a technical perspective, hemacytometers are the most reasonable option for cell counting, which may explain their widely spread use. Automated cell counters represent a good compromise between precision and affordability, although with limited accuracy. Finally, the methods based on flow cytometry were, by far, the best in terms of reproducibility and agreement between them, but they showed deficient accuracy and precision.Together, our results show a thorough technical evaluation of each counting method, provide unambiguous arguments to decide which one is the most convenient for the particular case of each laboratory, and in general, shed light into the best way to determine cell densities for in vitro cell cultures. They may have an impact in such a practice not only in the context of microspore culture, but also in any other plant cell culture procedure, or in any process involving particle counting.

Dataset Information

The generation of NGF-secreting primary rat monocytes: a comparison of different transfer methods.

Publications

The generation of NGF-secreting primary rat monocytes: a comparison of different transfer methods.

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