Lecture 4a culture equipment


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Industrial Microbiology Dr. Butler 2011

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  • 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.
  • Lecture 4a culture equipment

    1. 1. Fig. 3.2 Laminar flow cabinet (class II)
    2. 2. Biosafety Levels <ul><li>Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard Precautions against the biohazardous materials in question are minimal, most likely involving gloves and some sort of facial protection. </li></ul><ul><li>Biosafety Level 2 moderate potential hazard to personnel and the environment. Includes various bacteria and viruses that cause only mild disease to humans, </li></ul>
    3. 3. Biosafety levels contd. <ul><li>Biosafety Level 3 is applicable to agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. The laboratory has special engineering and design features. </li></ul><ul><li>Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, agents which cause severe to fatal disease in humans for which vaccines or other treatments are not available. Multiple airlocks are employed and are electronically secured to prevent both doors opening at the same time. </li></ul>
    4. 4. Fig. 3.6 Tissue culture flasks (T-flasks)
    5. 5. Fig. 3.7 Multi-well plates
    6. 6. Fig. 3.10 Spinner bottle with suspended paddle
    7. 7. Fig. 3.11a Roller bottles of various sizes
    8. 8. Bottles in a roller system Fig. 3.11b
    9. 9. Fig. 10.17 A confluent layer of BHK cells covers two microcarriers
    10. 10. Cell yield on microcarriers with varying charge
    11. 11. Non-porous microcarriers <ul><ul><li>Cytodex </li></ul></ul><ul><ul><ul><li>Cells grow adherent to the surface of the bead </li></ul></ul></ul><ul><ul><ul><li>Used for adherent cells </li></ul></ul></ul><ul><ul><ul><li>Allows for the growth of adherent cells in scaled up processes </li></ul></ul></ul><ul><ul><li>3 varieties: 1, 2, and 3 </li></ul></ul><ul><ul><ul><ul><li>1 = positively charged throughout the bead </li></ul></ul></ul></ul><ul><ul><ul><ul><li>2 = positively charged on the surface of the bead </li></ul></ul></ul></ul><ul><ul><ul><ul><li>3 = collagen coated to promote attachment </li></ul></ul></ul></ul><ul><ul><ul><ul><li> 1 & 3 Most commonly used Cytodex species </li></ul></ul></ul></ul>
    12. 12. Porous microcarriers <ul><ul><li>Cytopore </li></ul></ul><ul><ul><ul><li>Dextran mesh increases surface area </li></ul></ul></ul><ul><ul><ul><li>Cells can lodge themselves in the dextran mesh </li></ul></ul></ul><ul><ul><ul><ul><li>Protection from shear forces </li></ul></ul></ul></ul><ul><ul><ul><li>Designed for CHO cells </li></ul></ul></ul><ul><ul><ul><li>Positive charge throughout the bead </li></ul></ul></ul><ul><ul><li>2 varieties: 1 and 2 </li></ul></ul><ul><ul><ul><ul><li>1 = 1.1 mEq charge </li></ul></ul></ul></ul><ul><ul><ul><ul><li>2 = 1.8 mEq charge </li></ul></ul></ul></ul>Amersham Biosciences
    13. 13. Fig. 10.18 Comparison of a unit system and a multiple system
    14. 14. Cell encapsulation Fig. 10.19
    15. 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 &quot; &quot; 24 3.4 17.6 x 15.5 mm &quot; 1.88 plastic &quot; &quot; 12 6.9 17.6 x 22.1 mm &quot; 3.8 plastic &quot; &quot; 6 16.8 17.6 x 34.6 mm &quot; 9.4 plastic Medical flat bottle 10 125 ml 22 glass &quot; &quot; &quot; 15 250 ml 30 glass Roux bottle 50 500 ml 200 glass T-flask, 25 5.0 50 ml 25 plastic &quot; , 75 15-30 250 ml 75 plastic &quot; , 150 75 600 ml 150 plastic &quot; , 175 50-100 750 ml 175 plastic
    16. 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. 17. Fig. 3.12a Inverted microscope
    18. 18. Fig. 3.12b Standard microscope
    19. 19. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media <ul><li>Culture Conditions (Incubator) </li></ul><ul><li>cells grow best at 37 o C and pH 7.4 </li></ul><ul><li>grow slower at slightly < 37 o C, > 37 o C (39-40 o C) may destroy cells </li></ul><ul><li>pH of liquid cultures maintained by bicarbonate-CO 2 buffer system </li></ul><ul><li>CO 2 + H 2 O  HCO 3 - + H + , pKa = 6.3 </li></ul><ul><li>pH further dependent on enriched CO 2 atmosphere in the incubator </li></ul>
    20. 20. Double chamber CO 2 incubator Fig. 3.3
    21. 21. Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 34.
    22. 22. <ul><li>“ Good” Buffers </li></ul><ul><li>- Maintaining pH of a cell culture </li></ul><ul><ul><li>HEPES (pKa = 7.4) </li></ul></ul><ul><ul><li>MOPS </li></ul></ul><ul><ul><li>Tris </li></ul></ul><ul><li>Each has a specific pKa value </li></ul><ul><li>HEPES expensive </li></ul><ul><li>Allow pH to drift downwards due to lactic acid accumulation, HEPES will buffer the system and maintain it +/- of 7.4. </li></ul>
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