safety cabinets

2. General scientific strategies
Descriptive strategies:
The whole system (cell, organism) is observed without influencing it.
Advantage: physiological states are not altered
Disadvantage: it is difficult to elucidate cause-effect relationships
• Manipulative strategies:
Various factors are kept constant, while others are altered on purpose
to observe their role in the whole system.
• Advantage : cause-effect relationships can be monitored or
detected
• Disadvantage : The physiological steady state is altered and
influenced. Results might be artefacts of the measurement
system.

3. In vitro, biochemical systems:
Investigation in a solution (e.g. enzyme reactions): many
parameters can be fixed (temperature, pH, buffer
composition…).Example: enzyme activity assay, EMSA …
In vitro, cell biological systems:
Including cellular structures e.g. in vitro-
transcription/translation with membrane components, in
vitro-fusion of endosomes, nuclear import assays with
isolated nuclei etc.
• Experimental conditions have to be set in a way that
cellular structures are not damaged (e.g. isotonic buffer,
physiological pH….);
• Reaction partners can be influenced widely (e.g. antibodies
can be added…)

4. cell culture systems
• immortalized cell lines: unlimited passages
• primary cells: limited passage number, more
• complicated cell culture (e.g. coated plates…)
• co-culture of different cell types: e.g. keratinocytes +
fibroblasts in a collagen matrix.
xenograft systems:
• cells are injected into immuno-compromized mice
(nude mice, SCID mice); e.g. subcutaneously
• tissue recombination systems (e.g. prostate epithelial
cells with mesenchymal cells injected into the renal
capsule of SCID mice)
Organ cultures: (e.g. skin sheets, brain slices…)
Organ perfusions:
Animal Experiments: Transgene and knock-out animals

5. Cell culture system
1. • cell culture of immortalized cells
2. • cell culture of primary cells (often just a few
3. passages)
4. • culture of polarized cells
5. • co-culture of different cell types
6. • organ culture and organ-like culture
7. • Organ-perfusion

7. Class II Cabinets
• These cabinets are designed to give operator
protection as well as a sterile environment
• The air is directed downwards from the top of the
cabinet to the base, when working in these cabinets it
is important not to pas non-sterile objects over sterile
ones
• Because air is also drawn in from the front of the
cabinet, this area is not sterile
• Most work with Human or Primate cells is done in
Class II containment

10. Aseptic Techniques
1. Controlled environment
2. „Traffic, air flow
3. Sterile media and reagents
4. Avoid aerial contamination of solutions
5. Avoid manual contamination of equipment
6. Avoid repeated opening of bottles
7. 70% ethanol swab
8. UV irradiation before and after
9. Only use disposable equipment once

11. Aseptic Techniques
• Lab coat, Gloves, sterile and autoclaved tips
• Tip does not touch the tube
• Pipette aid motorized with potable battery,
disposable pipette

13. Factors affecting cell behaviour in the
• complex in vivo environment
• The local micro-environment: metabolites,
• local growth factors, ECM, architecture
• Cell-cell interactions
• Circulating proteins, cytokines, hormones

14. • Most adherent cells require attachment to
proliferate
• Change charge of the surface, Poly-L-lysine
• Coating with matrix proteins
• „Collagen, laminin, gelatine, fibronectin

16. Media
• Initial studies used body fluids
• „Plasma, lymph, serum, tissue extracts
• Early basal media
• „Salts, amino acids, sugars, vitamins
• Supplemented with serum
• More defined media
• „Cell specific extremely complex

17. Media
• Bacteria or yeast don‘t need complex media and grow
in minimal media
• mammalian cells are more complex and need media with
vitamins, amino acids, hormones and growth factors
• Serum, mostly fetal calf serum (FCS), is the source of growth
factors (such as FGF, EGF…)
• Common composition
• - DMEM (Dulbecco‘s Modified Essential Medium)
• - 10% FCS
• - 2 mM Glutamine (unstable amino acid, has to be added
• again, if the medium is older than app. 6 weeks)
• - Penicillin (100 u/ml)
• - Streptomycin (100 µg/ml)

18. Media
• Inorganic ions
Osmotic balance –cell volume
• Trace Elements
„Co-factors for biochemical pathways (Zn, Cu)
• Amino Acids
„Protein synthesis
„Glutamine required at high concentrations
• Vitamins
Metabolic co-enzymes for cell replication
• Energy sources
glucose

20. pH
• Physiological pH 7
pH can affect
• „Cell metabolism
• „Growth rate
• „Protein synthesis
• „Availability of nutrients
CO2 acts as a buffering agent in
• combination with sodium bicarbonate in
• the media

26. SOME CONSIDERATIONS
Cultures can be initiated from
• „tissue or organ fragments
• „single cell suspensions
Choices to be made
• „Disaggregation techniques
• „Media
• „Culture conditions
• „Selection procedures

27. • Sensitivity to mechanical dispersal or enzymes; cell-cell
• contact may be required for proliferation
• Dispersed cells in culture are vulnerable
• Most primary cells require satisfactory adherence
• Some cells are not normally adherent in vivo and can be
grown in liquid suspension
• In a mixed primary culture differences in growth rate may
mean a loss of the cell type of interest –selection
techniques
• Some cells are prone to spontaneous transformation
• Limited life span of some cultures

28. Dispersal of tissues
• Mechanical: Mincing, shearing, sieves
• Chemical
• Enzymatic (proteases that affect ECM)
„Trypsin, pronase, collagenase, dispase
• Can be a combination