Hostname: page-component-586b7cd67f-t8hqh Total loading time: 0 Render date: 2024-11-23T07:03:54.708Z Has data issue: false hasContentIssue false
  • English
  • Français

Non-viral genetic transfection of rat Schwann cells with FuGENE HD© lipofection and AMAXA© nucleofection is feasible but impairs cell viability

Published online by Cambridge University Press:  07 June 2011

Armin Kraus ,
Joachim Täger ,
Konrad Kohler ,
Max Haerle ,
Frank Werdin ,
Hans-Eberhard Schaller  and
Nektarios Sinis
Armin Kraus*
Affiliation:
Department of Hand, Plastic, Reconstructive and Burn Surgery, Eberhard-Karls-University of Tübingen, BG-Trauma Center, Germany
Joachim Täger
Affiliation:
Center for Regenerative Biology and Regenerative Medicine, Eberhard-Karls-University of Tübingen, Germany
Konrad Kohler
Affiliation:
Center for Regenerative Biology and Regenerative Medicine, Eberhard-Karls-University of Tübingen, Germany
Max Haerle
Affiliation:
Department of Hand and Plastic Surgery, Orthopaedic Hospital Markgroeningen, Kurt-Lindemann-Weg 10, D-71706 Markgroeningen, Germany
Frank Werdin
Affiliation:
Department of Hand, Plastic, Reconstructive and Burn Surgery, Eberhard-Karls-University of Tübingen, BG-Trauma Center, Germany
Hans-Eberhard Schaller
Affiliation:
Department of Hand, Plastic, Reconstructive and Burn Surgery, Eberhard-Karls-University of Tübingen, BG-Trauma Center, Germany
Nektarios Sinis
Affiliation:
Department of Hand, Plastic, Reconstructive and Burn Surgery, Eberhard-Karls-University of Tübingen, BG-Trauma Center, Germany
*
Correspondence should be addressed to: Dr. Armin Kraus, Department of Hand, Plastic, Reconstructive and Burn Surgery, Eberhard-Karls-Universität Tübingen, BG-Trauma Center, Schnarrenbergstrasse 95, 72076 Tübingen, Germany phone: + 49 7071 606 1036 fax: + 49 7071 606 1037 email: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Purpose:

To determine transfection efficiency of FuGENE HD© lipofection and AMAXA© nucleofection on rat Schwann cells (SC).

Methods:

The ischiadic and median nerves of 6-8 week old Lewis rats were cultured in modified melanocyte-growth medium. SCs were genetically transfected with green fluorescent protein (GFP) as reporter gene using FuGENE HD© lipofection and AMAXA© nucleofection. Transfection rates were determined by visualization of GFP fluorescence under fluorescence microscopy and cell counting. Transfected cell to non-transfected cell relation was determined.

Results:

Purity of Schwann cell culture was 88% as determined by immunohistologic staining. Transfection rate of FuGENE HD© lipofection was 2%, transfection rate of AMAXA© nucleofection was 10%. With both methods, Schwann cells showed pronounced aggregation behavior which made them unfeasible for further cultivation. Settling of Schwann cells on laminin and poly-l-ornithine coated plates was compromised by either method.

Conclusion:

Non-viral transfection of rat SC with FuGENE HD© lipofection and AMAXA© nucleofection is basically possible with a higher transfection rate for nucleofection than for lipofection. As cell viability is compromised by either method however, viral transfection is to be considered if higher efficiency is required.

Keywords

Schwann cellperipheral nervenon-viral transfectionmethodology
Type
Neurotechniques
Information
Neuron Glia Biology , Volume 6 , Issue 4 , November 2010 , pp. 225 - 230
Copyright
Copyright © Cambridge University Press 2011

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Abdellatif, A.A., Pelt, J.L., Benton, R.L., Howard, R.M., Tsoulfas, P., Ping, P. et al. (2006) Gene delivery to the spinal cord: comparison between lentiviral, adenoviral, and retroviral vector delivery systems. Journal of Neuroscience Research 84, 553–567.CrossRefGoogle Scholar
Barbu, A. and Welsh, N. (2007) Lipofection of insulin-producing RINm5F cells: methodological improvements. Journal of Liposome Research 17, 49–62.CrossRefGoogle ScholarPubMed
Bergen, J.M., Park, I.K., Horner, P.J. and Pun, S.H. (2008) Nonviral approaches for neuronal delivery of nucleic acids. Pharmaceutical Research 25, 983–998.CrossRefGoogle ScholarPubMed
Chang, C.J. (2009) Effects of nerve growth factor from genipin-crosslinked gelatin in polycaprolactone conduit on peripheral nerve regeneration – in vitro and in vivo. Journal of Biomedical Materials Research A 91, 586–596.CrossRefGoogle ScholarPubMed
Dean, D.A. (2005) Nonviral gene transfer to skeletal, smooth, and cardiac muscle in living animals. American Journal of Physiology. Cell Physiology 289, C233–C245.CrossRefGoogle ScholarPubMed
Gravvanis, A.I., Lavdas, A.A., Papalois, A., Tsoutsos, D.A. and Matsas, R. (2007) The beneficial effect of genetically engineered Schwann cells with enhanced motility in peripheral nerve regeneration: review. Acta Neurochirurgica Supplement 100, 51–56.CrossRefGoogle ScholarPubMed
Haastert, K., Grosheva, M., Angelova, S.K., Guntinas-Lichius, O., Skouras, E., Michael, J. et al. (2009) Schwann cells overexpressing FGF-2 alone or combined with manual stimulation do not promote functional recovery after facial nerve injury. Journal of Biomedicine and Biotechnology 2009, 1–11.CrossRefGoogle Scholar
Haastert, K., Mauritz, C., Chaturvedi, S. and Grothe, C. (2007) Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol. Nature Protocols 2, 99–104.CrossRefGoogle ScholarPubMed
Haastert, K., Mauritz, C., Matthies, C. and Grothe, C. (2006) Autologous adult human Schwann cells genetically modified to provide alternative cellular transplants in peripheral nerve regeneration. Journal of Neurosurgery 104, 778–786.CrossRefGoogle ScholarPubMed
Jordan, E.T., Collins, M., Terefe, J., Ugozzoli, L. and Rubio, T. (2008) Optimizing electroporation conditions in primary and other difficult-to-transfect cells. Journal of Biomolecular Techniques 19, 328–334.Google ScholarPubMed
Joung, I., Kim, H.S., Hong, J.S., Kwon, H. and Kwon, Y.K. (2000) Effective gene transfer into regenerating sciatic nerves by adenoviral vectors: potentials for gene therapy of peripheral nerve injury. Molecules and Cells 10, 540–545.CrossRefGoogle ScholarPubMed
Kraus, A., Taeger, J., Kohler, K., Manoli, T., Haerle, M., Werdin, F. et al. (2010) Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann cell cultures. Journal of Neurotrauma 27, 197–203.CrossRefGoogle ScholarPubMed
Lakshmipathy, U., Pelacho, B., Sudo, K., Linehan, J.L., Coucouvanis, E., Kaufman, D.S. et al. (2004) Efficient transfection of embryonic and adult stem cells. Stem Cells 22, 531–543.CrossRefGoogle ScholarPubMed
Li, Q., Ping, P., Jiang, H. and Liu, K. (2006) Nerve conduit filled with GDNF gene-modified Schwann cells enhances regeneration of the peripheral nerve. Microsurgery 26, 116–121.CrossRefGoogle ScholarPubMed
Magg, T., Hartrampf, S. and Albert, M.H. (2009) Stable nonviral gene transfer into primary human T cells. Human Gene Therapy 20, 989–998.CrossRefGoogle ScholarPubMed
Marchenko, S. and Flanagan, L. (2007) Transfecting human neural stem cells with the AMAXA nucleofector. Journal of Visual Expression 6, 240.Google Scholar
Mauritz, C., Grothe, C. and Haastert, K. (2004) Comparative study of cell culture and purification methods to obtain highly enriched cultures of proliferating adult rat Schwann cells. Journal of Neuroscience Research 77, 453–461.CrossRefGoogle ScholarPubMed
Millesi, H. (1984) Nerve grafting. Clinics in Plastic Surgery 11, 105–113.CrossRefGoogle ScholarPubMed
Muller, O.J., Katus, H.A. and Bekeredjian, R. (2007) Targeting the heart with gene therapy-optimized gene delivery methods. Cardiovascular Research 73, 453–462.CrossRefGoogle ScholarPubMed
Nakayama, A., Sato, M., Shinohara, M., Matsubara, S., Yokomine, T., Akasaka, E. et al. (2007) Efficient transfection of primarily cultured porcine embryonic fibroblasts using the AMAXA Nucleofection system. Cloning Stem Cells 9, 523–534.CrossRefGoogle ScholarPubMed
Nie, X., Zhang, Y.J., Tian, W.D., Jiang, M., Dong, R., Chen, J.W. et al. (2007) Improvement of peripheral nerve regeneration by a tissue-engineered nerve filled with ectomesenchymal stem cells. International Journal of Oral & Maxillofacial Implants 36, 32–38.CrossRefGoogle ScholarPubMed
Noble, J., Munro, C.A., Prasad, V.S. and Midha, R. (1998) Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. Journal of Trauma 45, 116–122.CrossRefGoogle Scholar
Rutkowski, G.E., Miller, C.A., Jeftinija, S. and Mallapragada, S.K. (2004) Synergistic effects of micropatterned biodegradable conduits and Schwann cells on sciatic nerve regeneration. Journal of Neural Engineering 1, 151–157.CrossRefGoogle ScholarPubMed
Shy, M.E., Tani, M., Shi, Y.J., Whyatt, S.A., Chbihi, T., Scherer, S.S. et al. (1995) An adenoviral vector can transfer lacZ expression into Schwann cells in culture and in sciatic nerve. Annals of Neurology 38, 429–436.CrossRefGoogle ScholarPubMed
Sinis, N., Schaller, H.E., Schulte-Eversum, C., Schlosshauer, B., Doser, M., Dietz, K. et al. (2005) Nerve regeneration across a 2-cm gap in the rat median nerve using a resorbable nerve conduit filled with Schwann cells. Journal of Neurosurgery 103, 1067–1076.CrossRefGoogle ScholarPubMed
Streppel, M., Azzolin, N., Dohm, S., Guntinas-Lichius, O., Haas, C., Grothe, C. et al. (2002) Focal application of neutralizing antibodies to soluble neurotrophic factors reduces collateral axonal branching after peripheral nerve lesion. European Journal of Neuroscience 15, 1327–1342.CrossRefGoogle ScholarPubMed
Terris, D.J., Toft, K.M., Moir, M., Lum, J. and Wang, M. (2001) Brain-derived neurotrophic factor-enriched collagen tubule as a substitute for autologous nerve grafts. Archives of Otolaryngology – Head & Neck Surgery 127, 294–298.CrossRefGoogle ScholarPubMed