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Basics of Cell Culture

Basics of Cell Culture. Part 1: Basic Growth Requirements. Overview. Growing bacterial cells Growing mammalian cells Things in common Unique things. Using Cultured Cells. Cells can be grown as individuals Bacteria and yeast Animal cells Plant cells. Cell Growth Profile.

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Basics of Cell Culture

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  1. Basics of Cell Culture Part 1: Basic Growth Requirements

  2. Overview • Growing bacterial cells • Growing mammalian cells • Things in common • Unique things

  3. Using Cultured Cells • Cells can be grown as individuals • Bacteria and yeast • Animal cells • Plant cells

  4. Cell Growth Profile

  5. Growth Curve Data

  6. Primary vs. Secondary Metabolites • Growing cells focus on “primary metabolites” • Required for living and making macromolecules • Only kind in eukaryotic • Cells after log growth phase make secondary metabolites • Often useful to us • Micro-organisms mainly

  7. Cell Culture Requirements • For all cells • Nutrients • Dissolved oxygen • Waste removal (or dilution) • Appropriate pH and temperature • For some cells • Surface for attachment • Growth stimulating signals

  8. Nutritional Requirements • Basics are common to all cells • Energy source (carbon) • Nitrogen source • Salts (Na+, K+, Ca++) • Details vary with specific cells • Bacteria more self-sufficient • Mammalian cells more sensitive, helpless

  9. Carbon source Molasses Malted grain Cellulose Plant oils and fats Wood pulp waste Nitrogen source Corn steep liquor Yeast extracts Peptones Soybean meal Bacterial Media

  10. Other Ingredients • Water • Trace minerals • Inhibitors (if needed) • Dissolved oxygen • Antifoam agents

  11. Oxygen • Must be dissolved, but water has low capacity • Continually supplied from above • Continually supplied from below

  12. Growth Conditions • Proper temperature • Proper pH • Low levels of waste metabolites • Growth conditions are maintained by a fermentor

  13. pH • Cell growth affected by pH • Biphasic curve (rate vs. pH) • Low at low and high • Best in middle • pH is altered by cell growth • Metabolic wastes include acids, ammonia • Control by buffering, medium replacement or adding alkaline or acid

  14. Temperature • Cell growth affected by temperature • Biphasic curve (rate vs. temperature) • Low at low and high temperature • Best in middle (range defined by cell type) • Cell growth produces heat • Regulate temperature of environment • Incubator • Cooling/warming coil • Water jacket

  15. Jacketed Tank (concept) • Resembles a thermos bottle • Container surrounded by hollow space • Difference: inlet and outlet for heat exchange solution

  16. Large Scale Fermentor

  17. Other Tank Types • Rely on air for mixing • Bubble tank • Sparged air acts to mix • As air rises, mixing occurs • Air-lift tank • Divided tank • Shown at left • Both tend to have foam

  18. Culture Types • Batch • Closed • Open • Advantages • Continuous • Advantages

  19. Mammalian Medium • More complicated recipes • Cells less capable than microbes • Need more “processed” ingredients • May need lipids and amino acids • May need vitamins and hormones • May require serum supplementation • Animal serum provides many “factors” • Growth factors • Attachment factors

  20. Simple Mammalian Medium AMINO ACIDS: L-Arginine-HCl 0.398 L-Cystine 0.200 L-Alanyl-Glutamine 4.03 Glycine 0.399 L-Histidine HCl-H2O 0.20 L-Isoleucine 0.802 L-Leucine 0.802 L-Lysine-HCl 0.798 L-Methionine 0.201 L-Phenylalanine 0.400 L-Serine 0.400 L-Threonine 0.078 L-Tryptophan 0.078 L-Tyrosine 0.398 L-Valine 0.803 + 10% Fetal Bovine Serum INORGANIC SALTS: Calcium Chloride 1.80 Ferric Nitrate 0.000248 Potassium Chloride 5.30 Magnesium 0.813 Sodium Chloride 110.34 Sodium Bicarbonate 44.10 Sodium Phosphate 0.906 OTHER: D-Glucose 5.55 Phenol red 0.0346 Sodium Pyruvate 1.00 VITAMINS: D-Ca pantothenate 0.0083 Choline Chloride 0.0285 Folic Acid 0.00906 i-Inositol 0.04 Niacinamide 0.0328

  21. Serum Supplements • Provide important nutrients • Add growth factors • Recreate “animal” environment • Potential source of infectious agents • Expensive • Lot-to-lot variation

  22. Attachment Surface • Mammalian cells • Most “normal” cells require • Exceptions are blood cells • Glass or treated plastic • Surface charge groups • Proteins in serum help with attachment • Bacteria and yeast grow in suspension • Commonly used insect cells go either way

  23. Surface Growing vs. Suspension Tank withsuspendedcells at aconcentrationof 106/ml 64 plates with 106 cells/plate

  24. Cheating for Suspension (I) Micro-carrier bead allows cells to grow on surface in suspension

  25. Cheating for Suspension (II) Cells can be grown inside hollow fibers bathed in medium

  26. Cell Density • Cells grow best in specific density range • Range is wider for single cell organisms • Narrower for dissociated cells (tissue or organ) • Mammalian cells secrete growth factors • “Conditioned medium” stimulates growth • Density too low and these signals too low • Use gradual increase in culture volume • Small initial volume, large final volume • Density of cells ~ same throughout process

  27. Cell Culture Train

  28. Review • Cells can be grown in vitro • Cells have requirements for growth • Require nutrients, etc. • Require dissolved oxygen • Require “space” • Industrial cell culture uses specialized equipment • Small scale to large scale • Specifics vary with cell needs

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