Magselectofection [Elektronische Ressource] : a novel integrated technology of magnetic separation and genetic modification of target cells / vorgelegt von Yolanda Sánchez Antequera

Yolanda Sánchez Antequera

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Dissertation
zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie der
Ludwig-Maximilians-Universität München





Magselectofection: A novel integrated technology of magnetic separation
and genetic modification of target cells

Vorgelegt von
Yolanda Sánchez Antequera
aus Valencia
2010

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von PD Dr. Christian Plank, TUM, betreut und von Prof. Dr.
Ernst Wagner an der Fakultät für Chemie und Pharmazie der LMU vertreten.


Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, am 13.09.2010




Yolanda Sánchez Antequera





Dissertation eingereicht am 13.09.2010
1. Gutacher: Prof. Dr. Ernst Wagner
2. Gutacher: PD Dr. Christian Plank
Mündliche Prüfung am 23.11.2010
TABLE OF CONTENT I

TABLE OF CONTENT

1. INTRODUCTION ............................................................................................................................... 1
1.1. A brief history of gene therapy 1
1.2. Gene and cell therapies, ex vivo and in vivo ........................................................................... 3
1.2.1. Ex vivo and in vivo nucleic acid (gene) therapy ....... 3
1.2.2. Cell therapies ........................................................................................................................... 5
1.3. Vectors and physical methods for nucleic acid delivery ......................... 6
1.3.1. Viral vectors ............................................................................................................................. 6
1.3.2. Non-viral vectors ..................... 7
1.3.3. Physical methods, Magnetofection ....................................................................................... 10
1.4. Magnetic cell separation ....................................................................................................... 12
1.5. Objectives of this thesis ......... 14
2. MATERIALS AND METHODS .......................................................................................................... 15
2.1. Material ................................. 15
2.1.1. Chemicals and reagents ......................................................................................................... 15
2.1.2. Cell culture reagents .............. 15
2.1.3. Plasmids ................................................................................................................................. 16
2.1.4. Chemical reagents and buffers .............................. 16
2.2. Cell isolation and culture ....................................................................................................... 17
+2.2.1. Isolation of the PBMC and CD34 by Ficoll gradient ............................. 17
2.2.2. Isolation of Wharton’s Jelly stem cells (hUC-MSC) ............................................................... 18
2.2.3. Cell culture ............................................................................................. 19
2.3. Magnetic nanoparticles ......................................................................... 20
2.3.1. Synthesis ................................................................ 20
2.3.2. Characterization of magnetic nanoparticles ......................................................................... 21
2.3.3. Determination of magnetic nanoparticle concentration in terms of dry weight and iron
content ..................................................................... 21
2.4. Lentivirus production and titer estimation ........................................................................... 22
2.4.1. Packaging of lentivirus vectors .............................................................. 22
TABLE OF CONTENT II

2.4.2. Biological titer of the lentivirus ............................................................................................. 23
2.4.3. Physical titer of the lentivirus ................................ 23
2.5. Preparation and characterisation of transfection complexes ............................................... 24
2.5.1. Assembling of magnetic non-viral complexes for magnetofection ....... 24
2.5.2. Assembling of magnetic non-viral complexes for magselectofection .................................. 24
2.5.3. Assembling of magnetic virus complexes for magnetofection and standard infection ........ 25
2.5.4. Assembling of magnetic virus complexes for magselectofection ......................................... 26
2.6. Characterization of magnetic vectors ................................................... 27
2.6.1. Size and zeta potential measurements ................................................. 27
2.6.2. Testing vector association and magnetic sedimentation in complexes with magnetic
nanoparticles ......................................................................................... 27
2.6.2.1. Non-viral vectors .................................................... 27
2.6.2.2. Viral vectors ........................................................................................... 28
2.7. Evaluation of magnetophoretic mobility............................................... 28
2.8. Non-heme iron determination .............................................................................................. 30
2.8.1. Non-heme iron determination in cells .................. 30
2.8.2. Non-heme iron determination in tissue samples .................................................................. 31
2.9. Transfection/transduction experiments of Jurkat T cells and hUC-MSCs using magnetofection
............................................................................................................................................... 31
2.9.1. Standard magnetofection of suspension cells ...... 31
2.9.2. Magnetofection combined with glycerol shock .................................................................... 31
2.9.3. Magnetofection using lentivirus and adenovirus vectors ..................................................... 32
2.9.4. Standard infection protocols for lenti- and adenovirus ........................ 33
2.10. Modification of the Miltenyi LS separation column with magnetic complexes .................... 33
2.10.1. Standard protocol .................................................................................................................. 33
2.10.2. Freeze-drying protocol .......... 34
2.11. Genetic modification of magnetically labelled cells by viral and non-viral magselectofection
............................................................................................................................................... 34
2.11.1. Magnetic labeling of the cells before magselectofection ..................... 34
2.11.2. Magselectofection general protocol ................................................................ 34
2.11.3. Analysis of the magnetic cell separation and gene transfer efficiency ................................. 35
TABLE OF CONTENT III

2.11.4. Magselectofection experiments using XS columns ............................................................... 37
2.11.5. Lentiviral magselectofection versus standard infection of hCB-HSCs ................................... 37
®2.11.6. Testing of immobilization of the magnetic complexes at the Miltenyi column and
association with cells upon magnetic field application ......................................................... 39
2.11.6.1. Using non-viral complexes ..................................................................... 39
2.11.6.2. Using viral complexes ............................................ 39
2.11.7. Analysis of cell association and internalization efficiency of the non-viral transfection
complexes by flow cytometry ............................................................................................... 40
2.11.8. Analysis of pDNA internalization using radioactively labeled pDNA ..................................... 40
2.12. Immunocharacterization of cells ........................................................................................... 41
2.13. Reporter gene expression analysis ........................ 41
2.13.1. Luciferin & luciferase assay ................................................................................................... 41
2.13.2. eGFP expression analysis ....... 41
2.13.2.1. Fluorescence microscopy ....................................................................................................... 41
2.13.2.2. Analysis of transfection/transduction efficiency by fluorescence-activated cell sorting (FACS)
............................................... 42
2.14. Cell viability ........................................................................................................................... 42
2.14.1. Trypan blue dye exclusion test .............................. 42
2.14.2. MTT assay ............................................................................................................................. 43
2.15. Different