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Lecture 4a culture equipment
- 1. Fig. 3.2 Laminar flow cabinet (class II)
- 4. Fig. 3.6 Tissue culture flasks (T-flasks)
- 5. Fig. 3.7 Multi-well plates
- 6. Fig. 3.10 Spinner bottle with suspended paddle
- 7. Fig. 3.11a Roller bottles of various sizes
- 8. Bottles in a roller system Fig. 3.11b
- 9. Fig. 10.17 A confluent layer of BHK cells covers two microcarriers
- 10. Cell yield on microcarriers with varying charge
- 13. Fig. 10.18 Comparison of a unit system and a multiple system
- 14. Cell encapsulation Fig. 10.19
- 15. Typical c ulture vessels suitable for cell growth Culture vessel Number of culture wells/ unit Max. culture volume (ml) Vessel size Growth surface (cm 2 ) Material Multiple well plate 96 0.37 10.8 x 6.4 mm (D x diam.) 0.32 plastic " " 24 3.4 17.6 x 15.5 mm " 1.88 plastic " " 12 6.9 17.6 x 22.1 mm " 3.8 plastic " " 6 16.8 17.6 x 34.6 mm " 9.4 plastic Medical flat bottle 10 125 ml 22 glass " " " 15 250 ml 30 glass Roux bottle 50 500 ml 200 glass T-flask, 25 5.0 50 ml 25 plastic " , 75 15-30 250 ml 75 plastic " , 150 75 600 ml 150 plastic " , 175 50-100 750 ml 175 plastic
- 16. Typical c ulture vessels suitable for cell growth continued Culture vessel 100-200 1250 ml 490 plastic Culture vessel 100-250 2200 ml 850 plastic Culture vessel 100-500 4900 ml 1750 plastic Culture vessel 100 250 ml glass Culture vessel 250 500 ml glass Culture vessel Number of culture wells/ unit Max. culture volume (ml) Vessel size Growth surface (cm 2 ) Material
- 17. Fig. 3.12a Inverted microscope
- 18. Fig. 3.12b Standard microscope
- 20. Double chamber CO 2 incubator Fig. 3.3
- 21. Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 34.
Editor's Notes
- Recirculates air through HEPA filters (High efficiency particulate air) Filter – series of glass fibres – particles in the air will be removed. remove 0.3 microns sterile environment – and air circulates – protects the operator Allows sterile manipulation and also protects the environment and operator Useful for Level 2 containment (Laboratory)
- Level 1. Examples: Bacillus subtilis , canine hepatitis , Escherichia coli , varicella ( chicken pox ), as well as some cell cultures and non-infectious bacteria. Level 2: or are difficult to contract via aerosol in a lab setting, such as hepatitis A , B , and C , influenza A , Lyme disease , salmonella , mumps , measles , HIV [3] , scrapie . Level 2 – primate cells Level 1 non-primate cells Level 3 – generally they are disease causing viruses (antharax, west nile virus, yellow fever, small pox) Sealed Cabinet and air locked storages.
- Level 3 : Includes various bacteria and viruses that can cause severe to fatal disease in humans, but for which vaccines or other treatment exist, such as anthrax , West Nile virus , Venezuelan equine encephalitis , Eastern Equine Encephalitis , SARS , smallpox , tuberculosis , typhus , Rift Valley fever , Rocky Mountain spotted fever , yellow fever . All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. Level 4: such as Bolivian and Argentine hemorrhagic fevers , dengue fever , Marburg virus , Ebola virus , hantaviruses , Lassa fever , Crimean-Congo hemorrhagic fever , and other various hemorrhagic diseases. When dealing with biological hazards at this level the use of a Hazmat suit and a self-contained oxygen supply is mandatory. The entrance and exit of a Level Four biolab will contain multiple showers, a vacuum room, an ultraviolet light room, designed to destroy all traces of the biohazard.
- Most labs use disposable flasks Generally sulfonated plastic – so that cells can grow in the inner surface containers are gamma radiated and sterile inside. loosen caps to allow gas exchanege Some manufacturers have covers that have holes that has a plastic inside to cover (no leaks) to allow gas exchange T-25, T-75, T-120 = numbers are square centimetres – surface area for cells to grow
- Could use a lot of replicate cultures Come in various sizes
- Autoclave sterilization Glass, screw cap on top that holds a column that contains a magnetic stir bar and teflon paddle stirs media Other arms can be used for sampling or probes depending on the size of bottle Max = 10 L Limitation is for 10L – power of stirring – efficiency of stirring is compromised. Stirred tank bioreactor (STB / STR) over 10 L – require a motor for stirring
- T 25 – surface area of 25 square cm, original were made out of glass 3 standard sizes (490, 850, 1750) Square centimetre required for anchorage dependent growth Bought as sterile units and are only meant for single use
- 5-60 rev /h Inner surface is constantly in contact with media ^ Simple lab setup, there are also machines that can hold 30 000 roller bottles
- BHA cells growing on the microcarriers (150 microns in diameter) about 250 cells on the surface of microcarrier Increase surface area = Suspend microcarriers in the stirred tank Each microcarrier needs to have clonal population on it Unit scale up: increase the volume in each flask – value: labour intensity is less i.e. Initial 1 L roller flask to an upgrade of 10 L bioreactor Multiple scale up: increase the number of rollers flasks – value: labour intensifies i.e. initial 4 roller flasks to an upgrade of 20 or more roller flasks
- Exchange capacity – miliequivalents/g – measure of the charge on the microcarrier Productivity = cell growth Very narrow window of charge where cells have high growth potential.
- Matrix in cytodex is dextran – polymer of glucose Cells only grow on the surface of the microcarrier Cytodex 1 is the most widely used Cytodex 3 has a good advantage – collagen – primary cells may require an attachment surface
- - Cytopore – dextra nmesh – larger pores – cells buries inside
- - Sodium alginate – liquid at room temperature mix with cells – drop a droplet in CaCl2 solution and bead hardens – cells therefore grows within the bead
- -Inverted microscope – stage is fairly wide – can fit a t flask on a stage and can be maginified through the microscope - Inverted – light source in on top
- - Stage is fairly narrow and cannot fit a t flask
- Bicarb-CO 2 system operates in blood in vivo. Balance between CO 2 from atmosphere and bicarbonate in the media. pKa of 6.3 is adequate, but not ideal. Buffering system +/- pKa value Relatively cheap system Concentration of CO 2 is important: if the concentration is too low, the pH increases. If the concentration is too high you get a drop in pH. Concentration of the CO 2 in the incubator ranges from 5 – 10%, supplied by a CO 2 cylinder, depends on the concentration of bicarbo in the media. Consequences of cells growing at a higher temperature is greater than growing cells in a lower than optimal temperature
- Magnetic stirring
- -Two CO 2 tanks attached in tandem. % of CO 2 is detected by an infrared gas analyzer to maintain an accuracy of +/- 0.1 %. - CO2 is filtered in, keeping CO2 sterile -Air filtered in to ensure an even temperature throughout the incubator chamber. - Temperature is controlled by a water jacket maintaining a temperature +/- 0.2 C. May also use heating elements. -Must keep incubator humid to prevent excessive evaporation from the media. A low concentration of disinfectant is kept in the water to prevent microbial growth.