A Rapid Method to Immortalize Schwann Cells

Abstract

The study of cells aids in comprehending the pathophysiology of diseases. However, obtaining a large number of primary cells in a short period is challenging, and they senesce and die after repeated passages. Therefore, establishing immortalized cell lines is necessary for conducting cellular experiments. Researchers commonly use antibiotics to screen immortalized cell models upon construction. However, due to the low transfection rate of the immortalized genes, a significant number of nonimmortalized cells are killed. The connections between the cells act as a web that floats when many cells die. As a result, successfully transfected immortalized cells are lifted and carried away, leading to only a small number of immortalized cells surviving. The surviving cells survive in the absence of other cell-secreted factors. However, their proliferative ability is limited, which makes obtaining immortalized cell lines a time-consuming process. This study aimed to shorten the time required to obtain immortalized cell lines by constructing immortalized Schwann cells and improving the traditional screening method. The immortalized gene transfectants were first cultured, passaged, and then screened. A comparison with the traditional screening method demonstrated the feasibility and advantages of the improved method.

Keywords

immortalization / immortalized cells / improved method / SV40T antigen

References

[1] S. Aoki, T. Takezawa, H. Sugihara, and S. Toda, “Progress in Cell Culture Systems for Pathological Research,” Pathology International66, no. 10 (2016): 554-562, https://doi.org/10.1111/pin.12443.

[2] M. Abbas, F. Moradi, W. Hu, et al., “Vertebrate Cell Culture as an Experimental Approach–Limitations and Solutions,” Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology254 (2021): 110570, https://doi.org/10.1016/j.cbpb.2021.110570.

[3] K. Bacon, A. Lavoie, B. M. Rao, M. Daniele, and S. Menegatti, “Past, Present, and Future of Affinity-Based Cell Separation Technologies,” Acta Biomaterialia112 (2020): 29-51, https://doi.org/10.1016/j.actbio.2020.05.004.

[4] S. M. Cascio, “Novel Strategies for Immortalization of Human Hepatocytes,” Artificial Organs25, no. 7 (2001): 529-538, https://doi.org/10.1046/j.1525-1594.2001.025007529.x.

[5] M. Irfan Maqsood, M. M. Matin, A. R. Bahrami, and M. M. Ghasroldasht, “Immortality of Cell Lines: Challenges and Advantages of Establishment,” Cell Biology International37, no. 10 (2013): 1038-1045, https://doi.org/10.1002/cbin.10137.

[6] C. Lipps, T. May, H. Hauser, and D. Wirth, “Eternity and Functionality–Rational Access to Physiologically Relevant Cell Lines,” Biochemistry394, no. 12 (2013): 1637-1648, https://doi.org/10.1515/hsz-2013-0158.

[7] E. B. Uglow, S. Slater, G. B. Sala-Newby, et al., “Dismantling of Cadherin-Mediated Cell-Cell Contacts Modulates Smooth Muscle Cell Proliferation,” Circulation Research92, no. 12 (2003): 1314-1321, https://doi.org/10.1161/01.res.0000079027.44309.53.

[8] C. A. Baptista, M. E. Hatten, R. Blazeski, and C. A. Mason, “Cell-Cell Interactions Influence Survival and Differentiation of Purified Purkinje Cells In Vitro,” Neuron12, no. 2 (1994): 243-260, https://doi.org/10.1016/0896-6273(94)90268-2.

[9] K. Ramli, I. Aminath Gasim, A. A. Ahmad, et al., “Human Bone Marrow-Derived MSCS Spontaneously Express Specific Schwann Cell Markers,” Cell Biology International43, no. 3 (2019): 233-252, https://doi.org/10.1002/cbin.11067.

[10] U. Namgung, “The Role of Schwann Cell-Axon Interaction in Peripheral Nerve Regeneration,” Cells Tissues Organs200, no. 1 (2015): 6-12, https://doi.org/10.1159/000370324.

[11] P. A. Eccleston, R. Mirsky, and K. R. Jessen, “Spontaneous Immortalisation of Schwann Cells in Culture: Short-Term Cultured Schwann Cells Secrete Growth Inhibitory Activity,” Development112, no. 1 (1991): 33-42, https://doi.org/10.1242/dev.112.1.33.

[12] M. Bierlein De la Rosa, A. D. Sharma, S. K. Mallapragada, and D. S. Sakaguchi, “Transdifferentiation of Brain-Derived Neurotrophic Factor (BDNF)-Secreting Mesenchymal Stem Cells Significantly Enhance BDNF Secretion and Schwann Cell Marker Proteins,” Journal of Bioscience and Bioengineering124, no. 5 (2017): 572-582, https://doi.org/10.1016/j.jbiosc.2017.05.014.

[13] M. A. Dickson, W. C. Hahn, Y. Ino, et al., “Human Keratinocytes That Express hTERT and Also Bypass a P16INK4a-Enforced Mechanism That Limits Life Span Become Immortal Yet Retain Normal Growth and Differentiation Characteristics,” Molecular and Cellular Biology20, no. 4 (2000): 1436-1447, https://doi.org/10.1128/mcb.20.4.1436-1447.2000.

[14] Y. Huang, Y. Yang, M. Jiang, M. Lin, S. Li, and Y. Lin, “Immortalization and Characterization of Human Dental Mesenchymal Cells,” Journal of Dentistry43, no. 5 (2015): 576-582, https://doi.org/10.1016/j.jdent.2015.02.008.

[15] M. Kitagawa, H. Ueda, S. Iizuka, et al., “Immortalization and Characterization of Human Dental Pulp Cells With Odontoblastic Differentiation,” Archives of Oral Biology52, no. 8 (2007): 727-731, https://doi.org/10.1016/j.archoralbio.2007.02.006.

[16] B. M. Gumbiner, “Cell Adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis,” Cell84, no. 3 (1996): 345-357, https://doi.org/10.1016/S0092-8674(00)81279-9.

[17] B. Parekkadan, Y. Berdichevsky, D. Irimia, et al., “Cell–Cell Interaction Modulates Neuroectodermal Specification of Embryonic Stem Cells,” Neuroscience Letters438, no. 2 (2008): 190-195, https://doi.org/10.1016/j.neulet.2008.03.094.

[18] T. Gordon, “The Role of Neurotrophic Factors in Nerve Regeneration,” Neurosurgical Focus26, no. 2 (2009): E3, https://doi.org/10.3171/foc.2009.26.2.e3.

RIGHTS & PERMISSIONS

2025 The Author(s). Pediatric Discovery published by John Wiley & Sons Australia, Ltd on behalf of Children's Hospital of Chongqing Medical University.