Cell Culture and Contamination Control

Weekly Schedule

• Week 1: Lecture module introduction
• Week 2: Lecture 1: Mammalian cell use; Lecture 2: Cell culture lab layout, equipment, and materials
• Week 3: Lecture 3: Contamination control; Lecture 4: Contamination control
• Week 4: Lecture 5: Contamination control; Lecture recap of lectures 2, 3, 4, and 5, plus sample assessment questions
• Week 5: Lecture 6: Nutrient uptake; Lecture 7: Nutrient uptake and sample assessment questions
• Week 6: Lecture 8: Biology of culture cells; Lecture 9: Cell culture media
• Week 7: Lecture 10: Cell culture media (postponed); Lab 3: Data analysis; Reading week
• Week 8: Lecture 10: Cell culture media; Lecture 11: Cell culture media
• Week 9: Lecture 12: Growing mammalian cells; Lecture recap of lectures 8, 9, 10, and 11, plus sample assessment questions
• Week 10: Lecture 13: Monitoring of growth; Lecture 14: Cryopreservation of cells; Lecture recap of lectures 12, 13, and 14, plus sample assessment questions
• Week 11: Lecture 15: Innate immune response; Lecture 16: Adaptive immune response and bioassays; Lecture recap of lectures 15 and 16, plus sample assessment questions
• Week 12: Revision

Lecture 13, Slide 2

• Lecture Overview
  • Introduction: Why discuss this topic?
  • Main discussion: Direct and indirect methods for monitoring growth of mammalian cell growth
  • Conclusion: Take home message

Lecture 13, Slide 3

• Introduction
  • For any bioprocess using cells: Monitor cell growth
  • Methods can be:
    • Directr or Indirect
    • In-line or off-line

Lecture 13, Slide 4

• DIRECT methods of measuring mammalian growth
  • Counting cells
    • Microscopic counts (manual, automated)
    • Electronic counters
• INDIRECT methods of monitoring mammalian growth
  • Not counting cells directly
    • Metabolic activity (e.g., glucose measurement)

Lecture 13, Slide 5

• Direct Measurements
  • Cell Counts Hemacytometer
    • Concentration of cell suspension determination
    • Placing cells in a hemacytometer
    • Counting using a phase contrast microscope
    • Specialized chamber with etched grid to count cells
    • Use of trypan blue for differentiation between living and dead cells

Lecture 13, Slide 6

• Direct Measurements
  • Cell Counts Hemacytometer
    • Phase contrast microscope
      • Objectives below specimen
      • Phase plate exploiting refractive index differences in different areas of cells and surrounding areas to create contrast

Lecture 13, Slide 7

• Using the haematocytometer
  • Remove and clean the hemacytometer and coverslip
  • Center the coverslip on the hemacytometer
  • Fill the grid with the cell suspension
  • Count cells in four squares
  • Living cells: "Bright refractile spheres"
  • Dead cells: blue

Lecture 13, Slide 8

• Using the hemacytometer
  • Count all cells in four corner squares
  • Separate live and dead cell counts
  • Include cells on top and left lines, excluding those touching middle lines at bottom and right
  • Total volume counted: 0.4µl (0.1µl when averaged)
  • Cells/ml = cell count x dilution factor x 10⁴ / 4

Lecture 13, Slide 9

• Calculations
  • Cells/ml calculation (count x dilution x 10⁴ / 4)
  • % Viability calculation (live cells x 100 / total cells)

Lecture 13, Slide 10

• Calculations
  • Sample cell count calculations
    • Viability determination
  • Additional examples providing data for calculation practice

Lecture 13, Slide 11

• Automated Cell Counter
  • Countess Automated Cell Counter Description and function
  • Measurements: Live/dead cell concentration, total cell concentration, viability, mean diameter, and images and graphical data output

Lecture 13, Slide 12

• Cell Density Calculation
  • Calculate stock cell culture volume
  • Diluting growth medium volume needed
  • Achieve 10⁴ cells/ml in a 10ml final volume

Lecture 13, Slide 13

• Electronic Cell Counter
  • Cell suspension forced through a small hole
  • Electrodes on either side of the hole that measure electrical resistance
  • Electrical resistance increases with each passing cell, which is registered by the electronic counter
  • Advantages - speed of analysis for large numbers of samples
  • Disadvantages - does not distinguish between living and dead cells, cell aggregates can lead to underestimated cell counts

Lecture 13, Slide 14

• Indirect Measurements
  • Methods involving chemical analysis of a culture component or measuring metabolic activity
  • Examples:
    • Nucleic acid/protein (biomass estimate)
    • Glucose depletion (cellular activity estimate)
  • Main substrates - Glucose, Amino Acids
  • Waste Products - Lactate, Ammonia, Alanine

Lecture 13, Slide 15

• Indirect Measurements (Glucose)
  • Glucose as the primary substrate for mammalian cells
  • High utilization rate compared to other substrates
  • Glucose limitation results in cessation of cell growth, cell death, and productivity loss
  • Control of glucose concentration crucial for bioprocesses using mammalian cells
  • Photometric enzyme-based assays (off-line)
  • Glucose sensors (in-line)

Lecture 13, Slide 16

• Indirect Measurements (Glucose Assay)

  • Glucose oxidase assay
  • Colourimetric assay using two enzymes (glucose oxidase and peroxidase)
  • D-glucose + H₂O + O₂ → D-gluconic acid + H₂O₂
  • 2H₂O₂ + phenol + 4-aminophenazone → red quinone + 4H₂O

Lecture 13, Slide 17

• YSI Glucose Analyzer
  • Automated enzyme analysis measuring important nutrients and metabolites (e.g., glucose, lactate, glutamine, glutamate, ammonia, potassium, ethanol, methanol, and more)

Lecture 13, Slide 18

• Off-line and in-line monitoring
  • Differentiating off-line vs in-line methods of analysis (i.e., removal vs. no removal of sample from growth vessel)

Lecture 13, Slide 19

• Off-line and in-line monitoring (example systems)
  • Off-line: Manual counting (e.g., haemocytometer), automated counting (e.g., Countess or other)
  • In-line: Bioreactor-attached probes (e.g., Aber Biomass Probes) that measure viable cells using capacitance, measuring media and cell debris, or gas bubbles

Lecture 13, Slide 20

• Conclusion
  • Accurate cell growth determination is key for bioprocesses
  • Direct methods: Cell counting (manual, automated); viability determination
  • Indirect methods: Measuring metabolic activity (e.g., glucose measurement)
  • Monitoring methods: In-line or off-line
  • Measuring devices: Sensors inserted into growth medium

Test your knowledge on the principles of cell culture, including techniques, contamination control, and nutrient uptake. This quiz covers material from multiple lectures over a few weeks. Perfect for students engaged in biological sciences or related fields.