Magnetic Nanoparticle‐Mediated Gene Delivery to Two‐ and Three‐Dimensional Neural Stem Cell Cultures: Magnet‐Assisted Transfection and Multifection Approaches to Enhance Outcomes

Mark R. Pickard1, Christopher F. Adams2, Divya M. Chari2

1 Institute of Medicine, University of Chester, Chester, 2 Institute for Science and Technology in Medicine, Keele University, Keele
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.19
DOI:  10.1002/cpsc.23
Online Posting Date:  February, 2017
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Abstract

Neural stem cells (NSCs) have high translational potential in transplantation therapies for neural repair. Enhancement of their therapeutic capacity by genetic engineering is an important goal for regenerative neurology. Magnetic nanoparticles (MNPs) are major non‐viral vectors for safe bioengineering of NSCs, offering critical translational benefits over viral vectors, including safety, scalability, and ease of use. This unit describes protocols for the production of suspension (neurosphere) and adherent (monolayer) murine NSC cultures. Genetic engineering of NSCs with MNPs and the application of ‘magnetofection’ (magnetic fields) or ‘multifection’ (repeat transfection) approaches to enhance gene delivery are described. Magnetofection of monolayer cultures achieves optimal transfection, but neurospheres offer key advantages for neural graft survival post‐transplantation. A protocol is presented which allows the advantageous features of each approach to be combined into a single procedure for transplantation. The adaptation of these protocols for other MNP preparations is considered, with emphasis on the evaluation of procedural safety. © 2017 by John Wiley & Sons, Inc.

Keywords: gene delivery; magnetofection; magnetic nanoparticles; neural stem cells; transplantation

     
 
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Table of Contents

  • Significance Statement
  • Introduction
  • Basic Protocol 1: Preparation of Neural Stem Cell Suspension and Monolayer Cultures
  • Support Protocol 1: Coating of Culture Surfaces and Coverslips with Polyornithine‐Laminin
  • Basic Protocol 2: Magnetofection of NSC Monolayer Cultures
  • Alternate Protocol 1: Magnetofection and Multifection of NSC Suspension Cultures
  • Support Protocol 2: Formation of Neurospheres from Transfected Monolayer Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Neural Stem Cell Suspension and Monolayer Cultures

  Materials
  • Mouse pups (e.g., CD1 strain), 1‐ to 3‐days old
  • Phosphate‐buffered saline (PBS; see recipe)
  • NS‐M medium (see recipe), for neurosphere cultures
  • 10× DNase I (see recipe)
  • Accutase/DNase I (see recipe)
  • 0.4% (w/v) trypan blue
  • ML‐M medium (see recipe), for monolayer cultures
  • Stereomicroscope or dissecting microscope
  • Inverted microscope
  • Dissection equipment: watchmaker forceps (110‐mm); scalpel blades (no. 10)
  • Universal tubes (Greiner Bio‐One, cat. no. 201120)
  • Petri dishes, 10‐cm and 3.5‐cm diameter
  • Kimberley Clark paper hand towels
  • Transfer pipets
  • 1.5‐ml microcentrifuge tubes
  • Cell sieves, 40‐µm mesh
  • 50‐ml conical tubes
  • Neubauer counting chamber and coverslip
  • Compound microscope
  • Non‐treated Nunc T‐25 flasks for suspension cultures (Thermo Fisher Scientific, cat. no. TKT‐300‐010 J)
  • Non‐treated Nunc 24‐well plates for suspension cultures (Thermo Fisher Scientific, cat. no.TKT‐220‐024P)
  • Cell culture‐treated Nunc 24‐well plates for monolayer cultures (Thermo Fisher Scientific, cat. no. 142475)
IMPORTANT NOTE: Be sure all animal protocols are approved by your institutional animal care and use committee.

Support Protocol 1: Coating of Culture Surfaces and Coverslips with Polyornithine‐Laminin

  Materials
  • 0.01% poly‐L‐ornithine solution (Sigma‐Aldrich, cat. no. P4957)
  • PBS (see recipe)
  • 1 mg/ml laminin (Sigma‐Aldrich, cat. no. L‐2020), stored as 25 µl aliquots at −20°C
  • Circular coverslips, 13‐mm, acid‐washed and sterilized
  • Cell culture‐treated Nunc 24‐well plates (Thermo Fisher Scientific, cat. no. 142475)

Basic Protocol 2: Magnetofection of NSC Monolayer Cultures

  Materials (also see protocol 1)
  • NSC monolayers plated in 24‐well plates ( protocol 1, step 21b)
  • ML‐M medium (as per recipe, but minus antibiotics)
  • 0.5 µg/µl plasmid DNA in water; store at −20°C in 5‐ to 10‐µl aliquots and vortex before use
  • DMEM:Ham's F12 medium (1:1)
  • NeuroMag transfection reagent (Oz Biosciences); store at −20°C in 5‐ to 10‐µl aliquots and vortex for 30 sec before use
  • Magnefect‐Nano oscillating magnetic array system (nanoTherics Ltd), with a 24‐magnet array (NdFeB, grade N42, field strength 421 ± 20 mT)

Alternate Protocol 1: Magnetofection and Multifection of NSC Suspension Cultures

  Materials (also see protocol 3)
  • NSC neurospheres plated in 24‐well plates ( protocol 1, step 21a)
  • NS‐M medium (as per recipe, but minus antibiotics)
  • DMEM: Ham's F12 medium (3:1)

Support Protocol 2: Formation of Neurospheres from Transfected Monolayer Cultures

  Materials
  • Magnetofected NSC monolayers ( protocol 3)
  • PBS (see recipe)
  • Accutase/DNase I (see recipe)
  • NS‐M medium (see recipe)
  • 0.4% (w/v) trypan blue
  • Inverted microscope
  • Neubauer counting chamber and coverslip
  • Compound microscope
  • Untreated Nunc T‐25 flasks (Thermo Fisher, cat. no. TKT‐300‐010 J) or Nunc 24‐well plates (Thermo Fisher, cat. no. TKT‐220‐024P) for suspension cultures
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Figures

Videos

Literature Cited

Literature Cited
   Adams, C. F. , Pickard, M. R. , & Chari, D. M. (2013). Magnetic nanoparticle mediated transfection of neural stem cell suspension cultures is enhanced by applied oscillating magnetic fields. Nanomedicine, 9, 737–741. doi: 10.1016/j.nano.2013.05.014
   Berman, S. M. C. , Walczak, P. , & Bulte, J. W. M. (2011). Tracking stem cells using magnetic nanoparticles. Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology, 3, 343–355. doi: 10.1002/wnan.140
   Carenza, E. , Barceló, V. , Morancho, A. , Levander, L. , Boada, C. , Laromaine, A. ,... Rosell, A. (2014). In vitro angiogenic performance and in vivo brain targeting of magnetized endothelial progenitor cells for neurorepair therapies. Nanomedicine, 10, 225–234. doi: 10.1016/j.nano.2013.06.005
   Conti, L. , & Cattaneo, E. (2010). Neural stem cell systems: Physiological players or in vitro entities? Nature Reviews Neuroscience, 11, 176–187. doi: 10.1038/nrn2938
   de Filippis, L. (2011). Neural stem cell‐mediated therapy for rare brain diseases: Perspectives in the near future for LSDs and MNDs. Histology and Histopathology, 26, 1093–1109. doi: 10.14670/HH‐26.1093
   Elsabahy, M. , Nazarali, A. , & Foldvari, M. (2011). Non‐viral nucleic acid delivery: Key challenges and future directions. Current Drug Delivery, 8, 235–244. doi: 10.2174/156720111795256174
   Fernandes, A. R. , & Chari, D. M. (2016a). Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy. Journal of Controlled Release, 238, 289–299. doi: 10.1016/j.jconrel.2016.06.024
   Fernandes, A. R. , & Chari, D. M. (2016b). Part II: Functional delivery of a neurotherapeutic gene to neural stem cells using minicircle DNA and nanoparticles: Translational advantages for regenerative neurology. Journal of Controlled Release, 238, 300–310. doi: 10.1016/j.jconrel.2016.06.039
   Jensen, J. , & Parmar, M. (2006). Strengths and limitations of the neurosphere culture system. Molecular Neurobiology, 34, 153–161. doi: 10.1385/MN:34:3:153
   Kim, S. U. , & de Vellis, J. (2009). Stem cell‐based cell therapy in neurological diseases: A review. Journal of Neuroscience Research, 87, 2183–2200. doi: 10.1002/jnr.22054
   Meng, X.‐L. , Shen, J.‐S. , Ohashi, T. , Maeda, H. , Kim, S. U. , & Eto, Y. (2003). Brain transplantation of genetically engineered human neural stem cells globally corrects brain lesions in the mucopolysaccharidosis type VII mouse. Journal of Neuroscience Research, 74, 266–277. doi: 10.1002/jnr.10764
   Mintzer, M. A. , & Simanek, E. E. (2009). Nonviral vectors for gene delivery. Chemical Reviews, 109, 259–302. doi: 10.1021/cr800409e
   Mothe, A. J. , Kulbatski, I. , Parr, A. , Mohareb, M. , & Tator, C. H. (2008). Adult spinal cord stem/progenitor cells transplanted as neurospheres preferentially differentiate into oligodendrocytes in the adult rat spinal cord. Cell Transplantation, 17, 735–751. doi: 10.3727/096368908786516756
   Phillips, M. I. , & Tang, Y. (2012). Genetic modification of stem cells for cardiac, diabetic, and hemophilia transplantation therapies. Progress in Molecular Biology and Translational Science, 111, 285–304. doi: 10.1016/B978‐0‐12‐398459‐3.00013‐7
   Pickard, M. R. , & Chari, D. M. (2010). Enhancement of magnetic nanoparticle‐mediated gene transfer to astrocytes by “magnetofection”: Effects of static and oscillating fields. Nanomedicine, 5, 217–232. doi: 10.2217/nnm.09.109
   Pickard, M. R. , Barraud, P. , & Chari, D. M. (2011). The transfection of multipotent neural precursor/stem cell transplant populations with magnetic nanoparticles. Biomaterials, 32, 2274–2284. doi: 10.1016/j.biomaterials.2010.12.007
   Pickard, M. R. , Adams, C. F. , Barraud, P. , & Chari, D. M. (2015). Using magnetic nanoparticles for gene transfer to neural stem cells: Stem cell propagation method influences outcomes. Journal of Functional Biomaterials, 6, 259–276. doi: 10.3390/jfb6020259
   Plank, C. , Zelphati, O. , & Mykhaylyk, O. (2011). Magnetically enhanced nucleic acid delivery. Ten years of magnetofection—Progress and prospects. Advanced Drug Delivery Reviews, 63, 1300–1331. doi: 10.1016/j.addr.2011.08.002
   Polyak, B. , Fishbein, I. , Chorny, M. , Alferiev, I. , Williams, D. , Yellen, B. ,... Levy, R. J. (2008). High field gradient targeting of magnetic nanoparticle‐loaded endothelial cells to the surfaces of steel stents. Proceedings of the National Academy of Sciences of the United States of America, 105, 698–703. doi: 10.1073/pnas.0708338105
   Yiu, H. H. P. (2011). Engineering the multifunctional surface on magnetic nanoparticles for targeted biomedical applications: A chemical approach. Nanomedicine, 6, 1429–1446. doi: 10.2217/nnm.11.132
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