Lecture 14 Cryopreservation 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  Cryopreservation and Storage of Cells Lecture Overview Introduction: Why discuss this topic Main discussion: Cryopreservation for long term storage of mammalian cells Conclusion: Take home message BIOT6012 Mammalian Biotechnology Lecture 14 Slide 2  Cryopreservation and Storage of Cells Introduction Cell Producing a Product e.g. drugs, food products, beverages Valuable Raw Material Need to protect and ensure an indefinite supply of cells Freeze for long term storage - cryopreservation BIOT6012 Mammalian Biotechnology Lecture 14 Slide 3  Cryopreservation and Storage of Cells A cell line might be impossible to replace; at best, replacement would be expensive and time-consuming. It is, therefore, essential to protect this considerable investment by preserving the cell line. Why cryopreserve: - contamination by microorganisms - cross-contamination by other cell lines - misidentification due to careless handling - incubator failure - saving time and materials by not maintaining lines other than those in current use - need for distribution to other users - senescence and the resultant extinction of the cell line BIOT6012 Mammalian Biotechnology Lecture 14 Slide 4  Cryopreservation and Storage of Cells Cryopreservation: a process where cells or whole tissues are preserved by cooling to sub-zero temperatures. Need to minimise intracellular ice crystal formation and reduce damage from high-concentration solutes formed when intracellular water freezes. This is achieved by: - freezing log phase cells with a high viability (>90%) - freezing slowly to allow water to leave the cell - using a cryoprotectant - thawing rapidly BIOT6012 Mammalian Biotechnology Lecture 14 Slide 5  Cryopreservation and Storage of Cells Freezing Medium The cell suspension is frozen in the presence of a cryoprotectant such as glycerol or dimethyl sulfoxide (DMSO) DMSO appears to be the more effective, possibly because it penetrates the cell better than glycerol. Concentrations of between 5% and 15% have been used, but 10% is more usual. For most monolayer cultures the cryoprotectant is removed at the first medium change (as soon as the cells have attached). Many laboratories also increase the serum concentration in freezing medium BIOT6012 Mammalian Biotechnology Lecture 14 Slide 6  Cryopreservation and Storage of Cells Successful Cryopreservation and Subsequent Resuscitation: Cultures should be healthy with a viability of >90% and no signs of microbial contamination. Cultures should be in log phase of growth Freeze cells slowly and thaw quickly to ensure maximum viability. A high concentration of serum/protein (>20%) should be used. Use a cryoprotectant such as dimethyl sulphoxide (DMSO) Cryogenic storage vial BIOT6012 Mammalian Biotechnology Lecture 14 Slide 7  Cryopreservation and Storage of Cells Cooling Rate Most cultured cells survive best if they are cooled at 1°C/ min. - compromise between fast freezing minimizing ice crystal growth and slow cooling encouraging the extracellular migration of water Place the cryovials in a Mr Frosty (Thermo Scientific) or CoolCell (Bioscision) freezing and place at −70°C or −80°C. Provides the critical repeatable -1°C/minute cooling rate required for successful cell cryopreservation and recovery. Store at -196°C for long term stability. BIOT6012 Mammalian Biotechnology Lecture 14 Slide 8  Cryopreservation and Storage of Cells Ultra-low Temperature Storage of Cell Lines: Following controlled freezing in the presence of cryoprotectants, cell lines can be cryopreserved in a suspended state for indefinite periods provided a temperature of less than -135°C is maintained. Such ultra-low temperatures can only be attained by specialised electric freezers or more usually by immersion in liquid nitrogen. BIOT6012 Mammalian Biotechnology Lecture 14 Slide 9  Cryopreservation and Storage of Cells Ultra-low Temperature Storage of Cell Lines: Storage in Liquid Nitrogen is currently the most satisfactory means of long-term storage of cell lines If Liquid Nitrogen storage is not available, the cells may be stored in a conventional freezer. The temperature in this freezer should be as low as possible, preferably between–140°C and –150°C Little deterioration has been found at –196°C, but significant deterioration may occur at –70°C. BIOT6012 Mammalian Biotechnology Lecture 14 Slide 10  Cryopreservation and Storage of Cells Liquid Nitrogen Freezer The investment in the contents of a nitrogen freezer can be considerable and must be protected by a strict monitoring regime and electronic liquid-level alarms. Wide-necked with storage in drawers; piped liquid nitrogen perfused through freezer wall and level controlled automatically by high- and low-level sensors shown at top left (high capacity, even temperature throughout chamber BIOT6012 Mammalian Biotechnology Lecture 14 Slide 11  Cryopreservation and Storage of Cells Inventory Control: All ultra-low temperature storage vessels should include a racking / inventory system designed to organise the contents for ease of location and retrieval. This should be supported by accurate record keeping and inventory control incorporating the following: Each ampoule should be individually labelled, using “wrap around”, resistant labels with identity and date of freezing The location of each ampoule should be recorded ideally on a spreadsheet There should be a control system to ensure that no ampoule can be deposited or withdrawn without updating the records BIOT6012 Mammalian Biotechnology Lecture 14 Slide 12  Cryopreservation and Storage of Cells Cryopreservation Procedure (harvest Freezing Cell Lines cells in the log phase of growth) 1. View cultures using an inverted microscope to assess growth and absence of contamination. 2. Release adherent cells into suspension using trypsin/ EDTA. Suspension cell lines can be used directly. 3. Perform a cell count. Ideally, the cell viability should be in excess of 90%. 4. Centrifuge the culture and decant supernatant. 5. Re-suspend cells at required density in freeze medium. 6. Pipette 1ml aliquots of cells into cyroprotective ampoules that have been labelled. 7. Freeze the cells in a controlled rate freezing apparatus, decreasing the temperature approx. 1°C per minute. 8. Transfer to ultra-low temperature freezer. BIOT6012 Mammalian Biotechnology Lecture 14 Slide 13  Cryopreservation and Storage of Cells Thawing Cells Resuscitation of Frozen Cell Lines 1. Prepare the flasks - label with cell line name, passage number and date. 2. Remove ampoules from storage and place quickly in a 37°C water bath. 3. Wipe ampoule with a tissue soaked in 70% alcohol prior to opening. 4. Pipette the whole content of the ampoule into a sterile tube. 5. Add 5ml pre-warmed medium. 6. Determine the viable cell density. Transfer the appropriate volume of cell suspension to a flask to achieve the required cell seeding density. 7. Incubate at the required temperature and CO2 level. 8. Examine cells microscopically (phase contrast) after 24 hours and subculture as necessary. BIOT6012 Mammalian Biotechnology Lecture 14 Slide 14  Cryopreservation and Storage of Cells Conclusion Cryopreservation: used to ensure a long term store of cells To ensure viability: - freezing log phase cells with a high viability (>90%) - freezing slowly to allow water to leave the cell - using a cryoprotectant - thawing rapidly Long term storage: electric freezers but preferably liquid nitrogen. BIOT6012 Mammalian Biotechnology Lecture 14 Slide 15