Lecture 9: Mammalian Cell Culture Media 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 Tue 22nd Oct Lab 3 data analysis Reading Week Week 8 Mon 04thNov Lecture 11 Cell Culture Media Tue 05 Nov th Lecture 8, 9, 10 and 11 recap and sample assessment questions Week 9 Mon 11th Nov Lecture 12 Growing mammalian cells Tue 12th Nov Lecture 13 Monitoring growth Week 10 Mon 18th Nov Lecture 14 Cryopreservation of cells Tue 19th Nov 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: Components of mammalian cell culture media Conclusion: Take home message BIOT6012 Mammalian Biotechnology Lecture 9 Slide 2  Mammalian Cell Culture Media Introduction Mammalian cell culture is the removal of animal cells and subsequent propagation and cultivation in vitro in an artificial environment that is suitable for growth Create the conditions of the body (in vivo) in an artificial in vitro environment In the body, the blood carries oxygen and nutrients to cells. Cell culture media is designed to replicate the role of the blood in the body i.e. provision of nutrients BIOT6012 Mammalian Biotechnology Lecture 9 Slide 3  Mammalian Cell Culture Media Animal cells and microbial cells – functionally similar but structurally distinct Therefore, ingredients in media vary considerably from bacteria and yeast to mammalian cells BIOT6012 Mammalian Biotechnology Lecture 9 Slide 4  Mammalian Cell Culture Media Why is mammalian growth medium so complex? A mammal is made up of hundreds of different cell types, each specialized to perform one or a few functions. Different cell types synthesize the many different molecules needed - are secreted into the extracellular fluid and are circulated by the blood throughout the body. The mammalian cell culture medium is an attempt to recreate this extracellular fluid. BIOT6012 Mammalian Biotechnology Lecture 9 Slide 5  Mammalian Cell Culture Media Criteria for a successful culture medium for animal cells Nutrients Basic nutritional conditions, amino acids, Isotonic monosaccharide, Human plasma osmotic vitamins, inorganic ions, pressure is about trace elements 290mOsm/kg, ideal to culture human cells Minimum Hormones Requirements In vivo cells are regulated by hormones so must be added to in vitro cell pH culture medium 7.2-7.4, maintained using CO2 and sodium Temperature bicarbonate 37°C, body temperature BIOT6012 Mammalian Biotechnology Lecture 9 Slide 6  Mammalian Cell Culture Media Basic Constituents of Animal Cell Culture Media Inorganic salts Carbohydrates Amino Acids Vitamins Fatty acids and lipids Proteins and peptides Trace elements Serum or replacements are used to supplement the above basic components BIOT6012 Mammalian Biotechnology Lecture 9 Slide 7  Mammalian Cell Culture Media What is serum and where does it come from? Blood – four components White blood cells Red blood cells Platelets Plasma Clotting components removed from plasma = serum (blood fraction remaining after coagulation of blood followed be centrifugation to remove blood cells. Fetal bovine serum - most widely used serum-supplement for the in vitro cell culture - low level of antibodies - contain high levels of growth factors BIOT6012 Mammalian Biotechnology Lecture 9 Slide 8  Mammalian Cell Culture Media Artificial Media Categories: Serum containing media: Fetal Bovine serum most common Serum-free media: addition of purified proteins from serum Animal-free: components are derived from non-animal sources Low protein media: where minimal quantities of proteins are employed BIOT6012 Mammalian Biotechnology Lecture 9 Slide 9  Mammalian Cell Culture Media Nutritional Requirements – Mammalian Cell Growth Serum containing mammalian growth media has two parts 1. Defined - Culture medium in 2. Non-defined - Culture which all the constituents are medium that contain a mixture known of ingredients in unknown proportions. i.e. serum Defined + non-defined = complete animal cell culture medium BIOT6012 Mammalian Biotechnology Lecture 9 Slide 10  Mammalian Cell Culture Media Nutritional Requirements – Mammalian Cell Growth 1. Defined (synthetic) Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarbonate Trace elements - iron, zinc, selenium Sugars - glucose is the most common Vitamins Amino acids BIOT6012 Mammalian Biotechnology Lecture 9 Slide 11  Mammalian Cell Culture Media Nutritional Requirements – Mammalian Cell Growth The Essential and Nonessential Amino Acids for Mammalian Cell Systems Essential Amino Acid Nonessential Amino Acid Glutamine* Alanine * Glutamine provides nitrogen for NAD, Isoleucine Arginine NADPH and nucleotides Leucine Asparagine and serves as a secondary energy Lysine Aspartic acid source for metabolism. Phenylalanine Glutamic Acid Glutamine is an unstable amino acid Threonine Glycine that, with time, Tryptophan Proline converts to a form that cannot be used by Valine Serine cells, and should be Methionine Tyrosine added to media just before use Histidine BIOT6012 Mammalian Biotechnology Lecture 9 Slide 12  Mammalian Cell Culture Media Nutritional Requirements – Mammalian Cell Growth Defined Components: Amino Acids Obligatory ingredients as amino acids are the building blocks of proteins. Essential amino acids must be included in the culture media as cells cannot synthesize these by themselves. Nonessential amino acids may also be added to the medium to replace those that have been depleted during growth. ROLE: required for the proliferation of cells and their concentration determines the maximum achievable cell density Carbohydrates Most of the media contain glucose and galactose. ROLE: major source of energy BIOT6012 Mammalian Biotechnology Lecture 9 Slide 13  Mammalian Cell Culture Media Nutritional Requirements – Mammalian Cell Growth Defined Components: Proteins and peptides The most commonly used proteins and peptides are albumin, transferrin, and fibronectin and are particularly important in serum-free media. Fatty acids and lipids Again, they are particularly important in serum-free media as they are generally present in serum. Vitamins Serum is a major source of vitamins. ROLE: essential for growth and proliferation of cells. Inorganic salt ROLE: retain osmotic balance of the cells BIOT6012 Mammalian Biotechnology Lecture 9 Slide 14  Mammalian Cell Culture Media Nutritional Requirements – Mammalian Cell Growth Defined Components: Trace elements Normally found in serum so must be supplemented in serum-free medium. ROLE: micronutrients are essential for many biological processes, e.g. the maintenance of the functionality of enzymes. BIOT6012 Mammalian Biotechnology Lecture 9 Slide 15  Mammalian Cell Culture Media Nutritional Requirements – Mammalian Cell Growth Defined Components: Phenol Red – pH indicator No nutritive value so why is it added? BIOT6012 Mammalian Biotechnology Lecture 9 Slide 16  Mammalian Cell Culture Media Characteristics of Bacterial Contamination of Mammalian Cell Cultures A B Both flask A and B were inoculated with BHK cells and examined after 24 hours incubation at 37°C, 5% Co2. Which flask is contaminated and why? BIOT6012 Mammalian Biotechnology Lecture 9 Slide 17  Mammalian Cell Culture Media Nutritional Requirements – Mammalian Cell Growth Defined Components: Antibiotics Antibiotics were originally introduced into culture media to reduce the frequency of contamination. However, the use of biological safety cabinets, coupled with strict aseptic technique, makes antibiotics unnecessary. Therefore, it is strongly recommended that routine culture be performed in the absence of antibiotics Disadvantages of antibiotics??? encourage the development of antibiotic-resistant organisms hide the presence of low-level, cryptic contaminants e.g. mycoplasma encourage poor aseptic technique Therefore, it is strongly recommended that routine culture be performed in the absence of antibiotics BIOT6012 Mammalian Biotechnology Lecture 9 Slide 18  Mammalian Cell Culture Media Conclusion To successfully grow mammalian cells in vitro need to supply the right mix of nutrients Basic components: amino acids, monosaccahrides, vitamins, ionorganic ions, trace elements Additional requirements e.g. hormones, growth factors, attachment factors provided by serum or serum replacements Phenol red, no nutritive value, added as an indicator of pH (based on colour) Not recommended to routinely culture mammalian cells in medium containing antibiotics BIOT6012 Mammalian Biotechnology Lecture 9 Slide 19