Cryopreservation of Cell Culture Derived from Dorsal Root Ganglia of Neonatal Pigs
Keywords:cryopreservation, cell culture, spinal ganglia, satellite glial cells, dimethyl sulfoxide, glutamine synthetase
The effect of cryopreservation with various concentrations of dimethyl sulfoxide (DMSO) on morphofunctional properties of the dorsal root ganglia cell culture (DRGCC) was investigated in this research. Cells were obtained from the dorsal root ganglia of neonatal piglets and cultured for 7 days in -MEM with 10% fetal calf serum (FCS). These conditions promote a predominant growth of satellite glial cells (SGCs). The resulting culture was cryopreserved at a rate of 0.5 deg / min to –20°C at stage 1, and at 1 deg / min to -80°C at stage 2, afterwards the samples were immersed into liquid nitrogen. Cryoprotective solutions based on α-MEM, 25% FBS, and DMSO at final concentrations of 5, 7.5, and 10% were used. After warming on day 10 of subculturing, the cell viability, relative monolayer area, and glutamine synthetase expression as a marker of SGCs were evaluated. It has been established that cryopreservation of DRGCC using 7.5% DMSO provided 87.7% of viable cells after warming and 85% relative monolayer area in respect of an intact control. The amount of SGCs was about 95%. The obtained results allow us to recommend the chosen regimen for low temperature storage of cell cultures enriched with MG.
Probl Cryobiol Cryomed 2020; 30(2): 158–168
Ali SG, Sidorenko OS, Bozhok GA. Influence of nutrient medium composition on the morphological characteristics of culture of dorsal root ganglion cells of neonatal piglets. The Journal of V. N. Karazin Kharkiv National University. Series "Biology". 2018; 30: 49-59. CrossRef
Arora DK, Cosgrave AS, Howard MR et al. Evidence of postnatal neurogenesis in dorsal root ganglion: role of nitric oxide and neuronal restrictive silencer transcription factor. Journal of Molecular Neuroscience. 2007; 32 (2): 97-107. CrossRef
Belzer V, Shraer N, Hanani M. Phenotypic changes in satellite glial cells in cultured trigeminal ganglia. Neuron Glia Biol. 2010; 6 (4): 237-43. CrossRef
Capuano A, De Corato A, Lisi L, et al. Proinflammatory-activated trigeminal satellite cells promote neuronal sensitization: relevance for migraine pathology. Molecular Pain. 2009; 5:43. CrossRef
de Luca AC, Faroni A, Reid AJ. 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; 96: e52543. [Cited 02.05.2019]. Available from: https://www.jove.com/video/52543/dorsal-root-ganglia-neurons-differentiated-adipose-derived-stem-cells CrossRef
Feldman-Goriachnik R, Wu B, Hanani M. Cholinergic responses of satellite glial cells in the superior cervical ganglia. Neurosci Lett. 2018; 671: 19-24. CrossRef
Fernandes MR, Pedroso AR. Animal experimentation: A look into ethics, welfare and alternative methods. Rev Assoc Med Bras [Internet]. 2017 Nov [cited 2019 June 20]; 63(11): 923-8. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0104-42302017001100923&lng=en CrossRef
Hornberger K, Yu G, McKenna D, et al. Cryopreservation of hematopoietic stem cells: Emerging assays, cryoprotectant agents, and technology to improve outcomes. Transfus Med Hemother. 2019; 46 (3): 188-96. CrossRef
Humphray SJ, Scott CE, Clark R, et al. A high utility integrated map of the pig genome. Genome Biology. 2007; 8 (7): R139. CrossRef
Li HY, Say EH, Zhou XF. Isolation and characterization of neural crest progenitors from adult dorsal root ganglia. Stem Cells. 2007; 25 (8): 2053-65. CrossRef
Lin YT, Chen JC. Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release. J Vis Exp [Internet]. 2018 [cited 02.05.2019]; 140: e57569. Available from: https://www.jove.com/video/57569/dorsal-root-ganglia-isolation-primary-culture-to-study CrossRef
Malin SA, Davis BM, Molliver DC. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat Protoc. 2007; 2 (1): 152-60. CrossRef
Meller K. The reaggregation of neurons and their satellite cells in cultures of trypsin-dissociated spinal ganglia. Cell Tiss. Res. 1974; 152 (2): 175-83. CrossRef
Miller KE, Richards BA, Kriebel RM. Glutamine-, glutaminesynthetase-, glutamate dehydrogenase- and pyruvate carboxylaseimmunoreactivities in the rat dorsal root ganglion and peripheral nerve. Brain Res. 2002; 945 (2): 202-11. CrossRef
Nagoshi N, Shibata S, Kubota Y, et al. Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem Cell. 2008; 2 (4): 392-403. CrossRef
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; 6 (5): 602-18. CrossRef
Ohara PT, Vit JP, Bhargava A, et al. Gliopathic pain: when satellite glial cells go bad. Neuroscientist. 2009; 15 (5): 450-63. CrossRef
Plaksina EM, Sidorenko OS, Legach Ð•I, et al. [Expression of ÃŸ-III-tubulin in the neonatal adrenal cell culture: comparison of monolayer and 3D-culture]. The Journal of V. N. Karazin Kharkiv National University. Series "Biology". 2018; 28: 76-86. Russian. CrossRef
Poulsen JN, Larsen F, Duroux M, et al. 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; 6 (1): 1-12.
Schwarz S, Spitzbarth I, BaumgÃ¤rtner W, Lehmbecker A. Cryopreservation of canine primary dorsal root ganglion neurons and its impact upon susceptibility to paramyxovirus infection. Int J Mol Sci [Internet]. 2019 Feb 28 [cited 02.05.2019]; 20 (5): 1058. Available from: https://www.mdpi.com/1422-0067/20/5/1058 CrossRef
Seggio AM, Ellison KS, Hynd MR, et al. Cryopreservation of transfected primary dorsal root ganglia neurons. J Neurosci Methods. 2008; 173 (1): 67-73. CrossRef
Singh RP, Cheng YH, Nelson P, et al. Retentive multipotency of adult dorsal root ganglia stem cells. Cell Transplant. 2009; 18 (1): 55-68. CrossRef
Smith RA, McInnes IB. Phase contrast and electron microscopical observations of adult mouse dorsal root ganglion cells maintained in primary culture. J Anat. 1986; 145:1-12.
Svenningsen A, Colman DR, Pedraza L. Satellite cells of dorsal root ganglia are multipotential glial precursors. Fex Neuron Glia Biol. 2004; 1 (1): 85-93. CrossRef
Takeda M, Takahashi M, Matsumoto S. Contribution of the activation of satellite glia in sensory ganglia to pathological pain. Neurosci Biobehav Rev. 2009; 33 (6): 784-92. CrossRef
Wang XB, Ma W, Luo T, et al. A novel primary culture method for high-purity satellite glial cells derived from rat dorsal root ganglion. Neural Regen Res. 2019; 14 (2): 339-45. CrossRef
How to Cite
Copyright (c) 2020 Sabina G. Ali, Natalia M. Moiseieva, Galina A . Bozhok
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).