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 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

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 INORGANIC SALTS: Calcium Chloride 1.80 Ferric Nitrate Potassium Chloride 5.30 Magnesium Sodium Chloride Sodium Bicarbonate Sodium Phosphate OTHER: D-Glucose 5.55 Phenol red Sodium Pyruvate 1.00 VITAMINS: D-Ca pantothenate Choline Chloride Folic Acid i-Inositol 0.04 Niacinamide AMINO ACIDS: L-Arginine-HCl L-Cystine L-Alanyl-Glutamine 4.03 Glycine L-Histidine HCl-H2O 0.20 L-Isoleucine L-Leucine L-Lysine-HCl L-Methionine L-Phenylalanine L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine % Fetal Bovine Serum

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 with suspended cells at a concentration of 10 6 /ml 64 plates with 10 6 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