Basics of Cell Culture

Part 1:

Basic Growth Requirements

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Overview

• Growing bacterial cells

• Growing mammalian cells

• Things in common

• Unique things

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Using Cultured Cells

• Cells can be grown as individuals– Bacteria and yeast– Animal cells– Plant cells

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Cell Growth Profile

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Tuesday (last year)

00.20.40.60.8

11.21.41.61.8

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Series1

Series2

Series3

Growth Curve Data

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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

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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

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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

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Bacterial Media

• Carbon source– Molasses– Malted grain– Cellulose– Plant oils and fats– Wood pulp waste

• Nitrogen source– Corn steep liquor– Yeast extracts– Peptones – Soybean meal

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Other Ingredients

• Water

• Trace minerals

• Inhibitors (if needed)

• Dissolved oxygen

• Antifoam agents

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Oxygen

• Must be dissolved, but water has low capacity

• Continually supplied from above

• Continually supplied from below

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Growth Conditions

• Proper temperature• Proper pH• Low levels of waste

metabolites• Growth conditions are

maintained by a fermentor

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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

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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

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Jacketed Tank (concept)

• Resembles a thermos bottle

• Container surrounded by hollow space

• Difference: inlet and outlet for heat exchange solution

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Large Scale Fermentor

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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

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Culture Types

• Batch– Closed– Open

• Advantages

• Continuous • Advantages

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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

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Simple Mammalian Medium

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

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

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Serum Supplements

• Provide important nutrients

• Add growth factors

• Recreate “animal” environment

• Potential source of infectious agents

• Expensive

• Lot-to-lot variation

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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

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Surface Growing vs. Suspension

Tank withsuspendedcells at aconcentrationof 106/ml

64 plates with 106 cells/plate

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Cheating for Suspension (I)

Micro-carrier bead allows cells to grow on surface in suspension

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Cheating for Suspension (II)

Cells can be grown inside hollow fibers bathed in medium

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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

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Cell Culture Train

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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