Biochemistry, physiology and genetics of mammalian cell culture
Die in vitro Zellkulturmethoden gehören nicht zu den exakten Wissenschaften, sondern beruhen auf sehr viel Empire. Wie aus dem Titel der Vorlesung ersichtlich, sind die Beiträge etwas unterschiedlich zu den klassischen Lehrbüchern der Zellkultur. Obgleich die methodischen Aspekte der Säugerzellkulturen im Detail besprochen werden, liegt der Blickwinkel schwerpunktmäßig auf der Biologie der Zelle um zu besser zu verstehen wie sich Zellen in einem artifiziellen "Plastikmilieu" verhalten und wie relevant dieses für eine in vivo Korrelation ist. Die Vorlesung ist geeignet für Studenten der Naturwissenschaften und Medizin im Hauptstudium, Masterstudenten und Doktoranden.
Fakultät für Biologie, Universität Würzburg, Wahlpflichtvorlesung, Masterprogramm, Vorlesungsnummer: 0611019
Veranstaltungsort: Universität Würzburg, Biozentrum Hubland, Hörsaal A103 (WS 2013/2014), Hörsaal A102 (SS 2014)
Vorlesungstermin Blockvorlesung WS 2013/2014: 2-tägige Blockvorlesung: 17. und 18. März 2014, 8:30 - 17:00 Uhr. Sprache: Englisch oder Deutsch. Schriftliche Prüfung: 2. April 2014, 18:30 Uhr, Biozentrum Hörsaal A103. 5 ECTS Punkte.
Vorlesungstermin Blockvorlesung SS 2014: 2-tägige Blockvorlesung: 16. und 17. Juli 2014, 8:30 - 17:00 Uhr. Sprache: Englisch oder Deutsch. Schriftliche Prüfung: Termin nach Vereinbarung. 5 ECTS Punkte.
Fakultät für Biologie, Inst. f. Humanbiologie und BioImaging, LMU München, Wahlpflichtvorlesung, Masterprogramm
Veranstaltungsort: LMU München, Großhaderner Str. 4, Seminarraum B 01.027.
Vorlesungstermine WS 2013/2014: 6.11./13.11./20.11./27.11./4.12./11.12. 2013, 8.1./15.1./22.1./29.1. 2014. Sprache: Englisch. Schriftliche Prüfung: Termin: 29. 1. 2014 (Hörsaal B 01.027). 3 ECTS Punkte.
Technische Fakultät, Inst. f. Bioverfahrenstechnik, Universität Erlangen, Wahlpflichtvorlesung, Masterprogramm
Veranstaltungsort: Universität Erlangen, Konrad-Zuse-Str. 3/5, Seminarraum SR 00.030
Vorlesungstermin Blockvorlesung WS 2013/2014: 3-tägige Blockvorlesung: 10. bis 13. Februar 2014, 8:30 - 16:30 Uhr. Sprache: Deutsch. Schriftliche Prüfung: Termin nach Vereinbarung. 5 ECTS Punkte.
1. Introduction: cell culture history
Amphibian-bird-mammalian cells
History of media
Readings/books in mammalian cell culture
2. Essentials of cellular biochemistry (special focus on in vitro)
DNA/RNA (e. g. uptake of exogenous nucleosides, nucleotide pool imbalances, nucleic acid uptake into cells)
RNA (e.g. RNA uptake into cells, siRNA)
Amino acids (e.g. essential amino acids, amino acid transporter, protein solubility)
Lipids (e.g. membrane-micelle-liposome, lipofection, osmolarity)
Sugars (e.g. energy sources, sugar transporter, protein modification)
3. Cell structures (special focus on in vitro)
Membranes, molecule and vesicle transport (e.g. structure, molecule permeability/uptake, transporter, endo-/exocytosis or receptors)
Adhesion (e.g. structure extracellular matrix, cadherin/integrin)
Cytoskeleton (e.g. actin/microtubuli, cell origin and intermediate filam.)
Mitochondria (e.g. quantity of mitochondrial energy supply and anaerobic metabolism in vitro), energy metabolism aerob and anaerob in vivo/in vitro
4. Cell proliferation (special focus on in vitro)
Mitosis (e.g. mitotic index, aberrant mitosis, genome ploidies)
Cell cycle (e.g. cycling/non-cycling cells, G0/G1/S/G2M, genetics of cell cycle, aberrant cell cycles, endoreduplication, growth curves)
Cell cycle synchronization techniques (e.g. inhibitors)
5. Commercial sources and preparation of in vitro cell models
Sources of cells (e.g. commercial sources, homemade)
Establishment of cell culture (e.g. cell isolation, separation/purification, passaging, cell line description, in vitro evolution. Some examples: blood, bone marrow, skin, umbilical cord, liver, embryonic and adult stem cells)
Primary vs permanent vs immortalized cells
6. Cells and environment
Chemical, physical and biochemical parameters (e.g .osmolarity, pO2, pCO2, temperature, pH and buffering)
Basal-/minimal media for serum cultivation, defined-/protein reduced media for serum free cultivation
Medium supplements (e.g. L-glut, Na-pyruvate, Hepes , phenol red, amino acids)
Sera (e.g. fetal vs adult, different animals)
Serum free media and supplements (e.g. adhesion factors, transport proteins, growth factors)
pH regulation of in vitro cell cultures (e.g. CO2/bicarbonate, Hepes)
Cell adhesion and coating technologies of flasks
Cryoconservation (e.g. principle, temperature gradient, storage of cells, toxicity)
Cell culture contaminants: bacteria (antibiotics), fungi (antimycotics), yeast, mycoplasma, viruses, chemicals (e.g. endotoxin)
Cell culture flask type (e.g. plastic material: T-flasks, microtiter plates)
Cell culture problems
7. Cell culture formats
Suspension and adherent cells
Subcultivation, medium change
Bulk cultures and clones (e.g. biochemical and mechanical cloning techniques, genetic/chromosomal drift of clones)
Cocultivation techniques (e.g. feeder layer, sandwich techniques, 3D cocultures)
Aggregates and spheroids (e.g. +/- matrigel)
Softagar colony formation assays (e.g. tumorigenicity assay, bone marrow differentiation)
In vitro tumor stem cell (?) assays; genetics of dedifferentiation during EMT process in sphere cultures
Stem cell assay (e.g. embryonic stem cells, induced pluripotent stem cells, pluripotent stem cells, genetic instability in vitro)
Hypoxia cell culture (e.g. physiology and genetics of hypoxia)
Stable and transient genetic transfectants (e.g. plasmids, siRNA/shRNA, lipofection, electroporation, viral transduction)
Cell fusion (e.g. hybridomas)
8. Cell types: normal and tumor cells
Cell types and in vivo tissue complexity (e.g. in vitro cell differentiation, mesenchymal and bone marrow stem cells)
Characteristics of senescent and permanent cell cultures (e.g. crisis, spontaneous transformation of rodent cells, genetics of aging)
Cell transformation and immortalization (e.g. tumor viruses, TERT)
Physiology and genetics of mutagenes
Biosafety classification of cell lines (e.g. S1, S2, S3)
9. In vitro complexity of growth factor actions
Essentials of cell growth, cell activation and in vitro complexity
Genetics of normal and aberrant cell signaling (e.g. mutations in tumor cells)
Cytokines/growth factors/interleukins (e.g. definitions, examples, family members, in vitro doses)
Cellular heterogeneity of action and production of cytokines (e.g. network actions, hematopoietic stem cell niche, wound healing)
Cell death (apoptosis, prim./second. necrosis, oncosis)
10. Applications, prediction and cell line identity of in vitro cell models
Applications of in vitro cell cultures (e.g. basic research, disease models, diagnosis, toxicity prediction, production of biologicals)
In vitro vs in vivo: signaling pathway stability (e.g. environment and alteration of cell physiology)
In vitro vs in vivo prediction (e.g. problem selection of cell lines and culture format, example drug development and in vivo toxicity prediction)
Problem cell line identity: mix-ups and classifications
11. Cell culture and laboratory equipment (optional)
Overview cell culture facility and equipment
Safety aspects: biohazard levels 1 to 3
Guidelines for safe laboratory practice and risk assessment
Safety aspects protection, disinfection, waste disposal, cleaning
Sterile workplaces: laminar flow and biosafety cabinets (subclassifications, HEPA filters)
Incubators, centrifuges, water baths, microscopes
12. Essentials of cell analytical technologies (optional)
Cell morphology
Cell viability/death analysis
Proliferation
Migration/invasion/angiogenesis assays
Karyotyping, chromosome analysis, in situ hybridization
Gene array technology; correlation mRNA/protein expression
Immunocytochemistry, FACS protein analysis
Intermediate filaments analysis
Single cell analysis: microscopy and flow cytometry
Special aspect: multidimensional effector functions and normalization methods
13. Special cell culture lecture: Cancer therapeutics – targets and drug development (optional)
Cancer therapeutic targets and drug development: from hits to leads to clinical compounds
Drug development low-molecular weight compounds
Drug development monoclonal antibodies
More drug strategies: immunotherapy, DNA vaccination, cytokines, immune cell transfer
14. Special cell culture lecture: Recombinant cell lines (optional)
Cell lines generation: isolation of cells from tissues
Generation of recombinant cell lines: nucleic acid transfer techniques
Generation of recombinant cell lines: lethal and proliferation inhibitory genes
Reporter molecules: GFP, artifical cell surface receptors
Cytotoxicity: definition and selection of marker genes for recombinant cells
