Lecture 13: Monitoring Mammalian Cell Growth PDF

Week 1 Tue 10th Sept Lecture Module Introduction Week 2 Mon 16th Sept Lecture 1 Use of mammalian cells Tue 17th Sept Lecture 2 Cell Culture Laboratory Lab layout, Equipment and Materials Week 3 Mon 23rd Sept Lecture 3 Contamination control...

Week 1 Tue 10th Sept Lecture Module Introduction Week 2 Mon 16th Sept Lecture 1 Use of mammalian cells Tue 17th Sept Lecture 2 Cell Culture Laboratory Lab layout, Equipment and Materials Week 3 Mon 23rd Sept Lecture 3 Contamination control Tue 24th Sept Lecture 4 Contamination control Week 4 Mon 30th Sept Lecture 5 Contamination control Tue 01st Oct Lecture 2, 3, 4 and 5 recap and sample assessment questions Week 5 Mon 07th Oct Lecture 6 Nutrient uptake Tue 08th Oct Lecture 7 Nutrient uptake and sample assessment questions Week 6 Mon 14th Oct Lecture 8 Biology of Culture Cells Tue 15th Oct Lecture 9 Cell culture media Week 7 Mon 21st Oct Lecture 10 Cell culture media postponed Tue 22nd Oct Lab 3 data analysis Reading Week Week 8 Mon 04thNov Lecture 10 Cell culture media Tue 05 Nov th Lecture 11 Cell Culture Media Week 9 Mon 11th Nov Lecture 12 Growing mammalian cells Tue 12th Nov Lecture 8, 9, 10 and 11 recap and sample assessment questions Week 10 Mon 18th Nov Lecture 13 Monitoring growth Tue 19th Nov Lecture 14 Cryopreservation of cells and Lecture 12, 13 and 14 recap and sample assessment questions Week 11 Mon 25th Nov Lecture 15 Innate immune response Tue 26th Nov Lecture 16 Adaptive immune response & Bioassays Lecture 15 and 16 recap and sample assessment questions Week 12 Mon 02nd Dec Revision Tue 03rd Dec  Monitoring Mammalian Cell Growth Lecture Overview Introduction: Why discuss this topic Main discussion: Direct and Indirect methods for monitoring growth of mammalian cells Conclusion: Take home message BIOT6012 Mammalian Biotechnology Lecture 13 Slide 2  Monitoring Mammalian Cell Growth Introduction For any bioprocess using cells need to be able to monitor the growth of the cells Methods can be: - direct or indirect - in-line or off-line BIOT6012 Mammalian Biotechnology Lecture 13 Slide 3  Monitoring Mammalian Cell Growth Cell quantification is required for applications that require the use of cells The accurate determination of cell growth is pivotal to monitoring a bioprocess 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 BIOT6012 Mammalian Biotechnology Lecture 13 Slide 4  Monitoring Mammalian Cell Growth Direct Measurements Cell Counts Hemacytometer Concentration of cell suspension determined by placing cells in a haemocytometer and counting using a phase contrast microscope Specialized chamber with etched grid used to count the number of cells in a sample. Use of trypan blue allows differentiation between living and dead cells Count all cells within the four corner areas. BIOT6012 Mammalian Biotechnology Lecture 13 Slide 5  Monitoring Mammalian Cell Growth Direct Measurements Cell Counts Hemacytometer Phase Contrast Microscope A phase contrast microscope with objectives below the specimen. A phase plate will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast BIOT6012 Mammalian Biotechnology Lecture 13 Slide 6  Monitoring Mammalian Cell Growth Direct Measurements Using the haemacytometer What the cells look like Remove the hemacytometer and Living cells - Bright refractile coverslip (carefully), wash and dry “spheres” thoroughly. Dead cells - blue Center coverslip on hemacytometer Barely fill the grid under the coverslip with your cell suspension. Count cells in four squares. BIOT6012 Mammalian Biotechnology Lecture 13 Slide 7  Monitoring Mammalian Cell Growth Direct Measurements Using the haemacytometer Count all the cells in the four corner squares. Separate count of live and dead cells. Include cells on top and left line. Do not count cells touching middle line at bottom and right. Total volume counted – 0.4µl (0.1µl when averaged) Cells/ml = cell count x dilution factor x 104 4 BIOT6012 Mammalian Biotechnology Lecture 13 Slide 8  Monitoring Mammalian Cell Growth Cells/ml Count *dilution * 104 4 % Viability Live cells * 100 Total cells BIOT6012 Mammalian Biotechnology Lecture 13 Slide 9  Monitoring Mammalian Cell Growth A. 150 cells (average) B. 110 cells (average) 125 live 105 live 25 Dead 5 Dead 10µl cells + 90µl Trypan Blue 50µl cells + 50µl Trypan Blue Live cell count/ml? Total cell count/ml? % Viability % Viability C. 10 live 16 live 2 dead 1 dead Count all cells within the four corner areas. 13 live 17 live 4 dead 2 dead 20µl cells + 80µl Trypan Blue Live cell count/ml? % Viability BIOT6012 Mammalian Biotechnology Lecture 13 Slide 10  Monitoring Mammalian Cell Growth Direct Measurements Countess Automated Cell Counter Front View Automated Side View cell counter that performs cell count and viability Slide Port Power measurements using trypan blue USB Port Button stain A single, sample measurement within Touchscreen a minute provides the following data: LiveFocus and dead cell Focus Knob Lock concentration/mL Total cell concentration/mL Viability (% live cells to total cells) Mean diameter Cell images Graphical data representation BIOT6012 Mammalian Biotechnology Lecture 13 Slide 11  Monitoring Mammalian Cell Growth Cell Density Calculation Calculate the volume of stock cell culture that must be taken and the volume of diluting growth medium that must be added to accomplish a final concentration in the new flask of 1 x 104 cells/ml in a final volume of 10ml. Stock cell culture = 1 x 106 cells/ml Required concentration in new flask = 1 x 104 cells/ml in a final volume of 10ml. BIOT6012 Mammalian Biotechnology Lecture 13 Slide 12  Monitoring Mammalian Cell Growth Direct Measurements Electronic cell counter Cell suspension is forced through a small hole. Electrodes on either side of the hole measures electrical resistance. Electrical resistance increases each time a cell passes through the hole – recorded as a signal by the electronic counter. Advantage – speed of analysis makes it suitable for counting a large number of samples Disadvantage – does not distinguish between live and dead cells - cell aggregates can result in underestimated counts BIOT6012 Mammalian Biotechnology Lecture 13 Slide 13  Monitoring Mammalian Cell Growth Indirect Measurements Indirect methods of estimating cell growth involve the chemical analysis of a culture component or a measure of metabolic activity. - nucleic acid or protein (estimate of biomass) - glucose depletion (estimate of cellular activity) Main substrates Waste Products Glucose Lactate Amino Acids Ammonia Alanine BIOT6012 Mammalian Biotechnology Lecture 13 Slide 14  Monitoring Mammalian Cell Growth Indirect Measurements Glucose is the main substrates of mammalian cells. - utilised by the cells in the highest amount of all available substrates. Cells recruit most of their energy from glucose. Limitation results in: - cessation of cell growth - cell death - loss of productivity Control of glucose concentration is crucial for any bioprocess using mammalian cells. - photometric enzyme-based assays (off-line) - glucose sensors (in-line) BIOT6012 Mammalian Biotechnology Lecture 13 Slide 15  Monitoring Mammalian Cell Growth Indirect Measurements Photometric enzyme-based assays as per BIOT6012 Lab 3 E.g. Glucose oxidase assay can be used to measure glucose concentration. - colourimetric assay that utilises two enzymes. Glucose oxidase D-glucose + H2O + O2 D-gluconic acid + H2O2 Peroxidase 2H2O2 + phenol + 4-aminophenazone red quinone + 4H2O Coloured complex measured with spectrophotometer BIOT6012 Mammalian Biotechnology Lecture 13 Slide 16  Monitoring Mammalian Cell Growth YSI Glucose Analyser (automated enzyme analysis) Measure important nutrients and metabolites including glucose, lactate, glutamine, glutamate, ammonium, potassium, ethanol, methanol and more. BIOT6012 Mammalian Biotechnology Lecture 13 Slide 17  Monitoring Mammalian Cell Growth Remove a sample from No removal the growth of a sample vessel from the growth vessel Off-line and in-line monitoring of growth Haemocytometer Sample Automated removed counters Off-line from Electronic analysis culture counters vessel for Glucose analysis analysis BIOT6012 Mammalian Biotechnology Lecture 13 Slide 18  Monitoring Mammalian Cell Growth Cell Counting Off-line In-line Example: Aber Biomass Probes Remove Bioreactor - sterilisable probe that can be sample inserted via a port into the vessel. from culture vessel - works in complex industrial media - suitable for suspension and attached cells Manual counting - measures viable cells using capacitance (Haemocytometer) - insensitive to media, cell debris and gas Automated counting (Countess bubbles or other) BIOT6012 Mammalian Biotechnology Lecture 13 Slide 19  Monitoring Mammalian Cell Growth Conclusion The accurate determination of cell growth is pivotal to monitoring a bioprocess DIRECT methods of measuring microbial growth Counting cells – can be manual or automated. Can determine % viability INDIRECT methods of measuring microbial growth Not counting cells directly metabolic activity e.g. glucose measurement Direct and indirect methods can be in-line or off-line Measuring device Sample removed e.g. sensor inserted from the growth into growth medium vessel and measured BIOT6012 Mammalian Biotechnology Lecture 13 Slide 20