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.

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…)

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

Cell culture system

1. • cell culture of immortalized cells2. • cell culture of primary cells (often just a few 3. passages)4. • culture of polarized cells5. • co-culture of different cell types6. • organ culture and organ-like culture 7. • Organ-perfusion

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

Aseptic Techniques

1. Controlled environment2. Traffic, air flow�3. Sterile media and reagents4. Avoid aerial contamination of solutions5. Avoid manual contamination of equipment 6. Avoid repeated opening of bottles7. 70% ethanol swab8. UV irradiation before and after9. Only use disposable equipment once

Aseptic Techniques

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

pipette

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

• Most adherent cells require attachment to proliferate

• Change charge of the surface, Poly-L-lysine• Coating with matrix proteins• Collagen, laminin, gelatine, fibronectin�

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�

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)

Media

• Inorganic ionsOsmotic balance –cell volume• Trace Elements Co-factors for biochemical pathways (Zn, Cu)�• Amino Acids Protein synthesis� Glutamine required at high concentrations�• VitaminsMetabolic co-enzymes for cell replication• Energy sourcesglucose

pH

• Physiological pH 7pH 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

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�

• 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

Dispersal of tissues

• Mechanical: Mincing, shearing, sieves• Chemical• Enzymatic (proteases that affect ECM) Trypsin, pronase, collagenase, dispase�• Can be a combination