Influence of nutrient medium composition on the morphological characteristics of culture of dorsal root ganglion cells of neonatal piglets
Abstract
Dorsal root ganglion (DRG) is a potential source of neural stem cells because it contains neural crest derived cells that are capable to differentiate into neurons and glial cells. Cell cultures obtained from animals that are close to humans by physiological characteristics can be regarded as an adequate modern model for in vitro studies. In this respect, DRG cell culture obtained from the domestic pig (Sus scrofa domesticus) is a convenient model. The aim of the work was to obtain a primary cell culture of DRG of neonatal piglets and to study its morphological and proliferative properties depending on culture medium composition. The composition of the media prepared on the basis of α-MEM varied depending on the presence of fetal calf serum (FCS) or its modern supplements B-27 and NeuroMax. It is established that morphological differences of primary DRG cell cultures of neonatal pigs depend on the composition of the nutrient medium. When cultured in the presence of 10% FCS, the formation of monolayer which includes satellite glial cells (SGC) and fibroblast-like cells was observed. Small colonies of neurons producing long processes were on the monolayer. When cultured in the presence of NeuroMax and B-27 supplements, the bulk of the cells is not attached, but organized into floating multicellular spheroids (MS). With the passage of culture obtained in the presence of 10% FCS, rapid attachment and proliferation of cells was observed. When MS obtained in the presence of NeuroMax and B-27 were transferred to the medium with 10% FCS, the attachment of MS to the substrate and cell migration were observed. The cells retain the ability to actively proliferate, because the monolayer achieves confluence by 5–7 days of subculture. Regardless of the composition of the primary culture medium, there were 3 morphologically different types of cells in the subcultures: SGC, neuron-like and fibroblast-like cells. The type of cells prevailing in the subculture depends on the composition of the nutrient medium. When MS is transferred from a B-27-containing medium, a significant growth of fibroblast-like cells is observed, whereas when MS is transferred from NeuroMax-containing medium MG and neuron-like cells were abundant.
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References
Backström E., Chambers B.J., Kristensson K., Ljunggren H.G. Direct NK cell-mediated lysis of syngenic dorsal root ganglia neurons in vitro // J. Immunol. – 2000. – Vol.165, no. 9. – P. 4895–4900.
Bassols A., Costa C., Eckersall P.D. et al. The pig as an animal model for human pathologies: A proteomics perspective // Proteomics Clin. Appl. – 2014. – Vol.8, no. 9. – P. 715–731.
Belzer V., Shraer N., Hanani M. Phenotypic changes in satellite glial cells in cultured trigeminal ganglia // Neuron Glia Biology. – 2010. – Vol.6, no. 4. – P. 1–7.
Capuano A., De Corato A., Lisi L. et al. Proinflammatory-activated trigeminal satellite cells promote neuronal sensitization: relevance for migraine pathology // Mol. Pain. – 2009. – Vol.5. – P.43.
Ciaroni S., Cecchini T., Cuppini R. et al. Are there proliferating neuronal precursors in adult rat dorsal root ganglia? // Neurosci. Lett. – 2000. – Vol.281, no. 1. – P. 69–71.
de Luca A.C., Faroni A., Reid A.J. Dorsal root ganglia neurons and differentiated adipose-derived stem cells: An in vitro co-culture model to study peripheral nerve regeneration // J. Vis. Exp. – 2015. – Vol.96. – e52543.
Farel P.B. Late differentiation contributes to the apparent increase in sensory neuron number in juvenile rat // Brain Res. Dev. Brain. Res. – 2003. – Vol.144, no. 1. – P. 91–98.
Gu Y., Chen Y., Zhang X. et al. Neuronal soma-satellite glial cell interactions in sensory ganglia and the participation of purinergic receptors // Neuron Glia Biol. – 2010. – Vol.6. – P. 53–62.
Hanani M. Intercellular communication in sensory ganglia by purinergic receptors and gap junctions: implications for chronic pain // Brain Res. – 2012. – Vol.1487. – P. 183–191.
Hanani M. Satellite glial cells in sensory ganglia: from form to function // Brain Res. Dev. Brain. Res. – 2005. – Vol.48, no. 3. – P. 457–476.
Hanani M. Satellite glial cells in sympathetic and parasympathetic ganglia: in search of function // Brain Res. Rev. – 2010. – Vol.64, no. 2. – P. 304–327.
Hayden S.M., Seeds N.W. Modulated expression of plasminogen activator system components in cultured cells from dissociated mouse dorsal root ganglia // J. Neurosci. – 1996. – Vol.16, no. 7. – P. 2307–2317.
Lagares A., Li H.Y., Zhou X.F., Avendano C. Primary sensory neuron addition in the adult rat trigeminal ganglion: Evidence for neural crest glio-neuronal precursor maturation // J. Neurosci. – 2007. – Vol.27, no. 30. – P. 7939–7953.
Lawson S.N. Morphological and biochemical cell types of sensory neurons / S.A.Scott (Ed.) Sensory neurons, diversity, development and plasticity. – Oxford University Press, New York, 1992. – P. 27–59.
Le Douarin N., Dulac C., Dupin E., Cameron-Curry P. Glial cell lineages in the neural crest // Glia. – 1991. – Vol.4, no. 2. – P. 175–184.
Li H.Y., Say E.H., Zhou X.F. Isolation and characterization of neural crest progenitors from adult dorsal root ganglia // Stem Cells. – 2007. – Vol.25, no. 8. – P. 2053–2206.
Mudge A.W. Effect of non-neuronal cells on peptide content of cultured sensory neurones // J. Exp. Biol. – 1981. – Vol.95. – P. 195–203.
Ogawa R., Fujita K., Ito K. Mouse embryonic dorsal root ganglia contain pluripotent stem cells that show features similar to embryonic stem cells and induced pluripotent stem cells // Biol. Open. – 2017. – Vol.6, no. 5. – P. 602–618.
Poulsen J.N., Larsen F., Duroux M., Gazerani P. Primary culture of trigeminal satellite glial cells: a cell-based platform to study morphology and function of peripheral glia // Int. J. Physiol. Pathophysiol. Pharmacol. – 2014. – Vol.6, no. 1. – P. 1–12.
Singh R.P., Cheng Y.-H., Nelson P., Zhou F.C. Retentive multipotency of adult dorsal root ganglia stem cells // Cell Transplant. – 2009. – Vol.18, no. 1. – P. 55–68.
Svennigsen A.F., Colman D.R., Pedraza L. Satellite cells of dorsal root ganglia are multipotential glial precursors // Neuron Glia Biol. – 2004. – Vol.1, no. 1. – P. 85–93.
Tongtako W., Lehmbecker A., Wang Y. et al. Canine dorsal root ganglia satellite glial cells represent an exceptional cell population with astrocytic and oligodendrocytic properties // Sci. Rep. – 2017. – Vol.7, no. 1. – P.13915.
Warwick R.A., Hanani M. The contribution of satellite glial cells to chemotherapy-induced neuropathic pain // Eur. J. Pain. – 2013. – Vol.17, no. 4. – P. 571–580.
Citations
SOME PHENOTYPIC CHARACTERISTICS OF THE DORSAL ROOT GALGLIA CELL CULTURE OF NEONATAL PIGLETS
Ali S. G., Kovalenko I. F. & Bozhok G. A. (2019) Bulletin of Problems Biology and Medicine
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