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Biochemistry, physiology and genetics of mammalian cell culture


In vitro cell culture methods are based mainly on empirical knowledge and less on accurate science. To highlight the complexity of this technology, this lecture is somewhat different to classical cell culture text books. Most of the experimental, methodological aspects of mammalian cell culture will be discussed. However, the focus will be on the biology of the cells to give the audience a better understanding how cells behave in an artificial in vitro “plastic milieu” and how relevant it is compared to an in vivo environment. The lecture is suitable for biology and life science master students, medical students and PhD students.



University of Würzburg, master program. Lecture numbers: 0611019 (Biol. Faculty), 0356510 (Med. Faculty)

Venue: University of Würzburg, Biocenter Hubland.

2-day block lecture WS 2017/2018: 26th and 27th of March 2018, 8:30 am to 5 pm. Lecture room A101, Biocenter. Language: English. Date of written exam: to be agreed upon. 5 ECTS points.


Technical Fakultät, Dept. of  Bioengineering, University of Erlangen, master program

Venue: University of Erlangen, Konrad-Zuse-Str. 3/5, seminar room SR 00.030

4-day block lecture WS 2017/2018: 5th to 8th of March 2018, 8:30 am - 5 pm. Language: German. Date of written exam: to be agreed upon. 5 ECTS points..

 


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. CO 2 /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

 

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