Mammalian Biotechnology Lecture 12 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  Growing Mammalian Cells Lecture Overview Introduction: Why discuss this topic Main discussion: Vessels, reagents and procedures for growing mammalian cells Conclusion: Take home message BIOT6012 Mammalian Biotechnology Lecture 12 Slide 2  Growing Mammalian Cells Introduction Mammalian cell culture markedly different to growing microbial cells This lecture will describe specific reagents, growth vessels and methods required BIOT6012 Mammalian Biotechnology Lecture 12 Slide 3  Growing Mammalian Cells Culture Vial Plastic screw cap vials used for cryogenic storage of cultures. Cultures grown to a specific cell density, mixed with a cryoprotectant and placed in vial for long term storage. Tissue Culture Flasks Plastic flasks available in a wide variety of sizes with surface areas ranging from 25 to 175cm2. Roller Bottles Pre-sterilised disposable plastic bottles. Cylindrical screw cap vessels used for anchorage dependent cells. Bottles are set up to revolve slowly which bathes the cells that are attached to the inner surface with medium. Spinner Flasks Plastic or glass bottles with a central magnetic stirrer shaft and side arms for the addition and removal of cells and medium, and gassing with CO2 enriched air. BIOT6012 Mammalian Biotechnology Lecture 12 Slide 4  Growing Mammalian Cells Small Scale Bioreactor Made of glass or stainless steel. Wave Bioreactors Sterile disposable plastic bag placed on a special rocking platform. Rocking motion induces waves in the culture fluid. Waves provide good mixing and oxygen transfer. Large Scale Bioreactor Made of stainless steel. BIOT6012 Mammalian Biotechnology Lecture 12 Slide 5  Growing Mammalian Cells Solutions Used Used to wash/remove excess serum. Phosphate Buffered Saline Ca2+ Mg2+ Free (PBS Must be warmed in the water bath so cells are not shocked by cold liquid. Cell counts Trypan Blue Living cells cannot take up the dye and will appear bright. Dead cells will absorb the dye and appear blue. An enzyme used to detach the cells from a culture dish. Activity neutralized by serum. Trypsin-EDTA Note: too long an exposure will reduce cell viability. Mg2+/Ca2+ ions inhibit the enzyme. EDTA is a chelator which will "mop" up these ions. BIOT6012 Mammalian Biotechnology Lecture 12 Slide 6  Growing Mammalian Cells Cell Culture: Growth Conditions Temperature - 37°C for cells pH - 7.2-7.5 Humidity is required CO2 – 5 to 10% maintained using a CO2 gas cylinder Cells should be cultured in the dark and exposed to room light as little as possible BIOT6012 Mammalian Biotechnology Lecture 12 Slide 7  Growing Mammalian Cells Successful growth of mammalian cells depends on ability to mimic conditions of the body (in vivo) in the lab (in vitro). In Vivo In Vitro Nutrients Blood Use of serum Temperature 37ºC Use of incubator pH 7.2 – 7.5 Bicarbonate – CO2 buffering system Humidity ------------ 95% (pan of water) Tonicity Isotonic Media formulation BIOT6012 Mammalian Biotechnology Lecture 12 Slide 8  Growing Mammalian Cells Growth conditions maintained using a CO2 incubator Maintains CO2 level, humidity and temperature (37oC) to simulate in vivo conditions. The cells are grown in an atmosphere of 5-10% CO2 because the medium used is buffered with sodium bicarbonate and the pH must be strictly maintained. Culture flasks should have loosened caps or filter caps to allow for sufficient gas exchange. BIOT6012 Mammalian Biotechnology Lecture 12 Slide 9  Growing Mammalian Cells The bicarbonate buffer system in the blood Acid-base homeostatic mechanism involving the balance of carbonic acid (H2CO3), bicarbonate ion (HCO−3), and carbon dioxide (CO2) in order to maintain pH in the blood The bicarbonate buffer system in cell culture media Designed to mimic the bicarbonate buffer system in the blood. CO2 dissolves into the culture media and reacts with water to form carbonic acid. As the cells metabolize and produce more CO2, the pH of the medium decreases. Sodium bicarbonate, NaHCO3, dissociates into sodium and bicarbonate ions. Free bicarbonate ions then react with the extra H+ ions to form carbonic acid “shifting the reaction to the left”, stabilising pH BIOT6012 Mammalian Biotechnology Lecture 12 Slide 10  Growing Mammalian Cells Cell Culture: Growth Pattern Lag Phase A period of no growth, immediately after reseeding. Allows the cells to recover from trypsinization, secrete matrix to aid attachment, and spread out on the substrate, enabling them to reenter cell cycle. Log Phase Exponential growth - the cell population doubles over a definable period, known as the doubling time Plateau Phase As the cell population becomes crowded, the cells eventually withdraw from the cell cycle. They then enter the plateau or stationary phase. Cells may be subcultured from plateau, but preferable to subculture log phase cells, as the recovery time (lag period) will be shorter if the cells are harvested from the top end of the log phase. BIOT6012 Mammalian Biotechnology Lecture 12 Slide 11  Growing Mammalian Cells Cell Culture: Subculture Suspension culture: fed by dilution into fresh medium. Adherent cultures: When the cells become semi- confluent, several methods are used to remove the cells from the growing surface so that they can be diluted. Proteolytic enzymes – Trypsin usually in combination with EDTA, causes cells to detach from the growth surface. -fast and reliable -can damage the cell surface -terminated by the addition of serum BIOT6012 Mammalian Biotechnology Lecture 12 Slide 12  Growing Mammalian Cells Cell Culture: Subculture Mechanical – a cell scraper can be used to physically remove the cells from the growth surface. - quick and easy - disruptive to the cells and - may result in significant cell death This method is best when harvesting many different samples of cells for preparing extracts, i.e., when viability is not important. BIOT6012 Mammalian Biotechnology Lecture 12 Slide 13  Growing Mammalian Cells Cell Culture Procedure for Anchorage Dependent Cells View cells using inverted phase microscope Aseptically aspirate media Rinse media with PBS (Mg2+ , Ca2+ free) Add Trypsin-EDTA to cells (source: porcine or recombinant) View cells using the inverted phase microscope Neutralise trypsin with fresh medium containing serum Centrifuge to pellet cells Re-suspend pellet with fresh complete medium Take sample and count cells. Calculate how many cells are needed to add to new plate or flask Seed new flask with the required cell density BIOT6012 Mammalian Biotechnology Lecture 12 Slide 14  Growing Mammalian Cells If using a serum free medium than cannot neutralize trypsin by adding a serum containing medium. Alternative: Use of TrypLE Express instead of trypsin. High purity, recombinant fungal enzyme produced by fermentation. It is a serine protease with trypsin-like activity (i.e. it cleaves at the same two amino acid sites–arginine and lysine–as trypsin. Inactivated by dilution. BIOT6012 Mammalian Biotechnology Lecture 12 Slide 15  Growing Mammalian Cells Figure 1. Effect of Trypsin and TrypLE cell-detaching procedures on cell recovery and cell viability. (A) Number of the cells recovered by each cell-detaching reagent and incubation time indicated. (B) Survival rate of the detached cells B by each reagent. Data are represented as the average and standard deviation of six samples. *p < 0.05. N.E., not examined; n.s., not significant. Based on the information in Figure 1 do you consider TryplE an effective replacement for Trypsin? BIOT6012 Mammalian Biotechnology Lecture 12 Slide 16  Growing Mammalian Cells Observing Cells in Culture Macroscopic observation Check color of media – Healthy growth usually leaves media slightly orange. – Too yellow, bacterial growth. – Too purple, low carbon dioxide Microscopic observation Observe cell morphology under phase microscope – Spread out or rounded? – How confluent? – Microbial contamination? BIOT6012 Mammalian Biotechnology Lecture 12 Slide 17  Growing Mammalian Cells What to do with growing cells. 70- 80% Confluent Not very Confluent Subculture them Feed them – Remove old media and replace with fresh, warm media. If cell density gets too high: Nutrients in media get depleted. Waste products build up in media. In the case of some cells (such as animal cells), cell- to-cell contact can cause cells to stop dividing (“contact inhibition”) BIOT6012 Mammalian Biotechnology Lecture 12 Slide 18  Growing Mammalian Cells Confluency An estimated percentage, so 10% confluency means that 10% of the surface the dish or flask used is covered with cells, 100% means that it is entirely covered. Confluency is not a hard measure, but rather an estimate of the cell density. BIOT6012 Mammalian Biotechnology Lecture 12 Slide 19  Growing Mammalian Cells Conclusion Specific reagents: trypsin, PBS, cell culture medium, trypan blue Specific growth vessels: T-flasks, spinner flasks, wave bags etc. Sub-culture: method dependent on whether cells are adherent or non-adherent Deciding to sub-culture: observe the colour of the growth medium, confluency of the monolayer and cell morphology BIOT6012 Mammalian Biotechnology Lecture 12 Slide 20