Cell Culture and Contamination Control

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Questions and Answers

What is the primary purpose of a haemocytometer?

To culture mammalian cells

To measure glucose levels in cell cultures

To analyze the refractive index of liquids

To determine the concentration of cell suspensions (correct)

Which method helps differentiate between living and dead cells while using a haemocytometer?

Cell motility assessment

Phase contrast imaging

Trypan blue staining (correct)

Fluorescent staining

When counting cells in a haemocytometer, which cells should you include in your count?

Only the cells that are fully within any square

All cells touching the left and top edges (correct)

Only the cells in the center squares

All cells touching the middle line

What type of microscope is typically used with a haemocytometer?

Phase contrast microscope Signup and view all the answers

What characteristic indicates a living cell when viewed under a phase contrast microscope?

Bright refractile 'spheres' Signup and view all the answers

What is a primary advantage of using speed of analysis in counting cell samples?

It allows for counting a large number of samples quickly. Signup and view all the answers

Which of the following is a limitation of indirect measurements in estimating cell growth?

They do not measure glucose concentration directly. Signup and view all the answers

What happens to mammalian cells when there is a limitation of glucose?

Cessation of cell growth. Signup and view all the answers

What is the main substrate utilized by mammalian cells for energy?

Glucose Signup and view all the answers

What type of assay is a glucose oxidase assay considered?

Photometric enzyme-based assay. Signup and view all the answers

How is the cell concentration in cells/ml calculated?

Cells/ml = cell count x dilution factor x 10^4 Signup and view all the answers

What is the formula to calculate percentage viability?

% Viability = (Live cells / Total cells) x 100 Signup and view all the answers

If 150 cells are counted, with 125 live and 25 dead, what is the % viability?

83.33% Signup and view all the answers

Which dilution factor is used for the cell count of 10 live and 2 dead cells?

Dilution factor is 4 Signup and view all the answers

What is the total cell count/ml for a scenario with 13 live and 4 dead cells counted?

17 cells/ml Signup and view all the answers

What is essential for any bioprocess using cells?

Monitoring the growth of the cells Signup and view all the answers

Which method is NOT classified as a direct method of measuring mammalian growth?

Metabolic activity analysis Signup and view all the answers

What are the two primary classifications of methods for monitoring cell growth?

Direct and indirect methods Signup and view all the answers

Which of the following is a direct method for monitoring mammalian cell growth?

Cell counting Signup and view all the answers

What are 'indirect methods' of monitoring mammalian cell growth aimed at?

Evaluating metabolic activity Signup and view all the answers

Which statement about cell quantification is correct?

It is pivotal for successful monitoring of a bioprocess. Signup and view all the answers

What type of methods can be used for monitoring growth of mammalian cells?

Direct and indirect methods Signup and view all the answers

Which of the following options is NOT typically used as a method for monitoring mammalian cell growth?

Migration assays Signup and view all the answers

What method is employed by the Countess Automated Cell Counter to assess cell viability?

Trypan blue stain Signup and view all the answers

How is cell density calculated using the stock cell culture concentration and desired final concentration?

By calculating the dilution factor needed Signup and view all the answers

What is the final desired concentration of cells in the new flask?

1 x 10^4 cells/ml Signup and view all the answers

What does the electronic cell counter measure to count cells?

Electrical resistance Signup and view all the answers

What additional information does the Countess Automated Cell Counter provide besides cell count?

Mean cell diameter Signup and view all the answers

What is the main purpose of using a diluting growth medium in cell culture?

To reduce cell density Signup and view all the answers

Within what time frame can the Countess Automated Cell Counter provide cell measurement results?

1 minute Signup and view all the answers

Which of the following is NOT a feature of the Countess Automated Cell Counter?

Nutrient composition analysis Signup and view all the answers

What is the main reaction catalyzed by glucose oxidase?

D-glucose + H2O + O2 --> D-gluconic acid + H2O2 Signup and view all the answers

What component is measured with a spectrophotometer in the reaction involving peroxidase?

Red quinone Signup and view all the answers

Which of the following nutrients can the YSI Glucose Analyser measure?

Glutamine Signup and view all the answers

What is a key difference between off-line and in-line monitoring of cell growth?

Off-line monitoring can be done without removing samples. Signup and view all the answers

What type of equipment can be used for cell counting in a laboratory setting?

Haemocytometer Signup and view all the answers

What type of probe can be used for in-line cell growth monitoring in a bioreactor?

Aber Biomass Probe Signup and view all the answers

Which of the following is a characteristic of automated enzyme analysis?

It can analyze a variety of metabolites. Signup and view all the answers

What is the role of peroxidase in the biochemical reaction mentioned?

To convert hydrogen peroxide to water Signup and view all the answers

Study Notes

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

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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.