Mammalian Cell Culture Media Lecture Notes 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  Mammalian Cell Culture Media Lecture Overview Introduction: Why discuss this topic Main discussion: Mammalian cell culture media sterilisation and quality control Conclusion: Take home message BIOT6012 Mammalian Biotechnology Lecture 11 Slide 2  Mammalian Cell Culture Media Introduction Following preparation of cell culture media, can sterilise using membrane filtration Not necessarily required if media prepared using aseptic reagents in an aseptic environment Need to perform quality control checks on media prior to use i.e. sterility checks and growth promotion BIOT6012 Mammalian Biotechnology Lecture 11 Slide 3  Mammalian Cell Culture Media Media sterilisation Cell culture media must be free of harmful contaminants and adventitious biological agents: - bacteria - fungi - viruses - mycoplasma Quickly colonise and flourish in the rich environment provided by cell culture media. For cell culture media sterility rely on: - Laminar flow (biological safety cabinets / cleanrooms) - Suitable work surfaces (easily cleaned) - Trained personnel - Reagents and media (QC tested) - Aseptic Technique - Apparatus and equipment (regular maintenance) BIOT6012 Mammalian Biotechnology Lecture 11 Slide 4  Mammalian Cell Culture Media Media sterilisation Media components: - highest quality and purity - proper storage to prevent deterioration - certified for use Media containers - must be sterile - typically sterile disposable plastic flasks and containers Autoclaving typically not an option, as it may destroy critical cell culture media components and biomolecules needed for normal cell growth. Filtration - method of choice Sterile filter the media - use 0.2µm-rated (or smaller) sterilising grade membrane filters. BIOT6012 Mammalian Biotechnology Lecture 11 Slide 5  Mammalian Cell Culture Media Media sterilisation – membrane filtration https://synderfiltration.com/2014/wp-content/uploads/2014/05/Cross-Flow-Ultrafiltration.jpg Separation by size using an applied pressure Particles larger than the pore size of the filter are retained. Particles smaller than the pore size of the filter pass through the filter along with the liquid. BIOT6012 Mammalian Biotechnology Lecture 11 Slide 6  Mammalian Cell Culture Media Media sterilisation – membrane filtration Materials of construction for a sterilizing filter must be proven inert - neither adding nor removing components (other than those larger than the pore size). Cell growth studies should confirm that sterile filtration has no adverse effects on growth promoting properties of the media or protein expression. BIOT6012 Mammalian Biotechnology Lecture 11 Slide 7  Mammalian Cell Culture Media Cell growth assessing the impact of sterile filtration on the growth promoting properties of the medium Figure. Filtration of RPMI media containing 10% FBS using Nalgene Rapid-Flow aPES filters BIOT6012 Mammalian Biotechnology Lecture 11 Slide 8  Mammalian Cell Culture Media Quality Control Sterile filter: When filtration is complete the filter should be tested to ensure that the membrane is still intact: - if not, media may be compromised Test can check if the filter has a tear Medium: A medium that is prepared in the laboratory needs to be tested before use Tests: growth promotion and sterility tests If the medium is purchased readymade then it should be possible to rely on the quality control carried out by the supplier BIOT6012 Mammalian Biotechnology Lecture 11 Slide 9  Mammalian Cell Culture Media Quality Control Growth Promotion All of these tests should be performed on the new batch of medium with your regular medium as a control. Plating efficiency: the number of cells that grow into colonies per 100 cells inoculated. That is, it is the proportion of cells that attach and grow to the number of cells originally plated, expressed as a percentage. PE(%)=(Colonies Counted / Cells Inoculated) x 100 Growth curve: growth in old and new lots of media and compare growth curves Preservation of cell culture characteristics: compare using old and new lots of media BIOT6012 Mammalian Biotechnology Lecture 11 Slide 10  Mammalian Cell Culture Media Quality Control Sterility Test Standard microbiological tests for bacteria and yeast - incubating aliquots of media into several different broths e.g. Trypticase Soy broth or nutrient broth (bacteria) Sabouraud Dextrose broth (yeast) - incubate at required temperature for required time - observe for growth BIOT6012 Mammalian Biotechnology Lecture 11 Slide 11  Mammalian Cell Culture Media Sterility Test Testing for sterility of samples from sterile filtration or from supernatant medium of culture (TSB = trypticase soya broth used to isolate aerobic and facultative aerobic organisms; TGB = thioglycollate broth used to isolate anaerobic and microaerophilic organisms; SAB = Sabourard’s broth use for isolation of fungi). BIOT6012 Mammalian Biotechnology Lecture 11 Slide 12  Mammalian Cell Culture Media Sterility Test of Cell Culture Media TSB Media SDB Media No Inoculate No Inoculate inoculation with 0.1ml inoculation with 0.1ml S. E. coli 1mk BHK media cerevisiae 1mk BHK media Negative Positive Test Negative Positive Test Control Control Control Control Incubate at 37 C Incubate at 22 C for two days for five days Contaminated Evidence of contamination: - cloudiness If contamination is observed batch is discarded. All media should be visually checked prior to use. BIOT6012 Mammalian Biotechnology Lecture 11 Slide 13  Mammalian Cell Culture Media Sterility Test as per lab 4: Aseptic Technique and Making Medium TSB Media – Sterility Test Results SDB Media – Sterility Test Results -ve control +ve control BHK -ve control +ve control BHK Media Media Test Test Sterility test results acceptable? Explain your answer. BIOT6012 Mammalian Biotechnology Lecture 11 Slide 14  Mammalian Cell Culture Media Media sterilisation To prepare the final medium, components (defined and non defined) are dissolved in purified water before sterilisation. Generation of water of suitable purity is key for the growth of mammalian cells. Potable water (i.e. drinking water) must be subject to further in-house purification prior to its use. Prepared medium sterilised by filtration and tested for sterility Preparation details should be recorded (including details of the raw materials used) and an expiry date assigned to each batch. BIOT6012 Mammalian Biotechnology Lecture 11 Slide 15  Mammalian Cell Culture Media Media sterilisation Potable water In-house purification Purified Water for Injection water (WFI) Distinguished on the basis of the range and quantity of allowable contaminants with WFI being the most pure. BIOT6012 Mammalian Biotechnology Lecture 11 Slide 16  Mammalian Cell Culture Media Water Purification Incoming Potable Water Organic material, chlorine, disinfectants solids Anion Depth Filtration Organic Trap Activated Charcoal Exchange ions Water for Distillation / Cation Filtration Reverse Osmosis Exchange Injection microorganisms contaminants Purified Water Red text indicates contaminants eliminated at each stage of the purification process BIOT6012 Mammalian Biotechnology Lecture 11 Slide 17  Mammalian Cell Culture Media Media sterilisation Parameter Water for Injection Purified Water USP EP USP EP Electrical