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: 06110190 (Biol. Faculty), 03565100
(Med. Faculty)
Venue: University
of Würzburg, Biocenter Hubland.
2-day block lecture, 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. 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
