Cowles - J25 Nutrition of cells in culture & media preparation Page 1 F9 - Defined media and supplements When tissue culture was first developed cells were grown in natural media such as body fluid extracts. However, as larger amounts of consistent media were needed synthetic media were developed. These were created via a knowledge of body fluids and nutritional biochemistry, then fine tuned for various conditions and cell lines. It must have been a huge job to try to figure out all the materials in body fluids that were required for cell growth! I'm sure glad we don't have to figure that out for ourselves. Serum is still added to most media. However, several more complex media have been developed so serum is not needed. This prevents the introduction of pathogens and simplifies later processing. pH most cells grow best at a pH of 7.4. Phenol red is often used to monitor pH since it noticeably shifts away from red as the pH drifts from 7.4. It is useful to set up a control container in order to notice small color changes. I wonder who came up with phenol red and a pH indicator. It would be interesting to see if they were trying to find an indicator that bracketed optimal cellular pH, or if it was discovered in some other way and only used for cell culture later. CO2 and bicarbonate increased CO2 levels create HCO3 in solution and lower the pH. This can be offset by the addition of bicarbonate. The amount of bicarbonate needed can be determined using a table. However, this amount may need to be varied depending on the buffers present in the media and the preparation. It appears that cells cannot be without atmospheric CO2, even if the proper pH is maintained. If pyruvate is included in the medium the cells produce their own CO2 and it doesn't need to be added. Great idea! the inclusion of pyruvate would drive the Krebs cycle, which would create CO2. That's a really creative way to get around adding CO2. Buffering buffering is needed in open cultures and at high cell concentrations. Bicarbonate is the most common buffer since it is non toxic, cheap, and nutritional to the cells. However, other buffers are also effective. Oxygen Cultured cells get most of their O2 dissolved in the media. This makes them susceptible to free radicals. Because of this a balance has to be created between enough O2 for respiration but not so much that it becomes toxic. Most energy in cultured cells comes from anaerobic respiration. However, the amount of O2 needed varies greatly between different culture types. Cowles - J25 Nutrition of cells in culture & media preparation Page 2 I wonder if O2 carrying pigments such as hemoglobin or hemocyanin could be used to carry oxygen to the cells. You wouldn't need to add new cells to the culture since several pigments are extracellular (ex hemocyanin). Osmolality Most cultures have a wide osmolality tolerance. Starting solutions slightly hypotonic can compensate for evaporation during incubation. Measurement of osmolality can insure that the proper ingredients were added in the correct amounts. Temperature The proper temperature for a culture depends on the animal it came from and the part of the animal. For most animals this is around 37C. Most cells do far better at lower than normal temperature than they do at higher than normal temps. Poikilotherms have an even wider temperature tolerance. Temperature also influence the pH due to changes in CO2 solubility. Viscosity Viscosity is usually due to the serum used and does not influence cell growth. It is important when suspended cells are agitated and can reduce cell damage. Surface tension and foaming Foaming may increase protein denaturation and contamination risk. Defoaming agent can be used to reduce foaming. Balanced salt solutions (BSS) are made up of inorganic salts and sometimes glucose. it is used to dilute amino acids and vitamins to produce complete media and for short culture times. The BSS chosen depends on CO2 tension and whether the cells will be suspended or attached. It's interesting that a change in the amounts of different salts present changes the affinity of cells to be suspended or attach to the container. Complete medium is media that has all the components needed for its specific use and may be simple or complex. All the essential amino acids have to be added. However, several non-essential AA may also be added due to the cells' inability to make them efficiently or some other reason. Maximum number of cultured cells appears to be limited by AA availability. Vitamins are also a key component of media. Most media have the water soluble vitamins, but only a few have fat soluble. Vitamins limit growth rate and survival rather than max cell number like AA. It's interesting that while AA and vitamins are both required for cell growth, they have different effects when in low supply. Does this suggest that vitamins are associated with cell maintenance while AA are needed mainly for the creation of new cells? Cowles - J25 Nutrition of cells in culture & media preparation Page 3 Salts are another important part of complete medium. They modulate osmolality and enable adhesion molecules such as cadherin. Glucose is usually included in media as an energy source. Some of the glucose may also continue through the krebs cycle, although glutamate may be used instead. antibiotics were used in the past to limit contamination. However this is not usually the case now since they have significant drawbacks such as creating resistant bacteria and hide low level contamination. They should be used only if necessary. Serum is the source of growth factors, hormones and adhesion factors. Calf and fetal bovine are the most common serums. Human serum is used but need to be screened for infection first. Protein is a major component of serum, but much of its activity is unknown in vitro. Several proteins are needed for culture, such as albumin and fibronectin. However there are likely others that are needed but haven't been identified yet. growth factors control cell proliferation. Platelet-derived growth factor (PDGF) causes more proliferation that the cells original serum. Other growth factors such as FGF can be released by cells or purchased and added. It amazes me how little we know about the exact growth factors that are needed by each cell type and how different factors effect different cell types. We still have so much to learn! I wonder if some growth factors make some cells grow but slow down the growth of other cells. Various hormones present in serum cause effects varying from changes in differentiation to attachment ability. Serum may also contain trace amounts of minerals and cell proliferation inhibitors (although these may be added inadvertently during preparation). It can be hard to choose the right media type since there are so many. However, you find the media used for similar cell lines in the literature. Simple media is usually sufficient unless complex procedures are being used. Price may also factor in to the media used. Serum can be reserved at suppliers while different batches are tested. The serum should be tested for the number of cells that survive, how fast they grow, and sterility. I hadn't fully considered all the things that have to go into media. Even with many years of accumulated knowledge of what to put into media we still need to add serum to most media. This underscores the complexity in the cellular environment. F10 - Serum free media Unlike traditional media, serum free media don't need the addition of serum to support cells. However, these media are much more complex and more specific; a different combination of components in needed for each cell type or cell line. Cowles - J25 Nutrition of cells in culture & media preparation Page 4 It amazes me that it's possible to culture cells without serum at all. the huge complexity of animal systems would make it very difficult to do so. Some of the problems with serum are high variability, poor shelf life, changes in quality, availibility, contamination, difficulty standardizing, accidental inclusion of growth inhibitors, and high cost. However the cost of individual components isn't appreciably lower than the cost of serum. One of the advantages of serum free media is its cell selectivity. This inhibits the growth of cells that are not of interest. This could be a real pain since a new combination of components has to be developed for each cell type. However, I guess this offers advantages as well. The disadvantages of serum free media include the large number of different recipes that are needed, unintended cell selection based on media used, high purity requirements, slower growth, and availability of serum free media. The essential components of serum are adhesion factors, peptides, essential nutrients, and hormones. Protease inhibitors are also needed without serum after trypsin-mediated subculture. Various hormones are needed to promote growth. Some hormones have been shown to have effects that would not be expected. these may have effects previously unknown or may be broken down into hormones that do have an effect. Growth factors are needed to promote growth and proliferation. There are quite a few, but most have been shown to be fairly unspecific. this is probably because they are usually released as a paracrine factor in vivo and thus have a limited range. they work with each other either additively or synergistically. Finally, something that doesn't have to be tailored for each specific cell type! The inclusion of proteins in the media may increase growth rate but adds uncertainty and possible contamination. Serum free media should be chosen based on if it will be used to eliminate unwanted cell types or whether a specific line will be grown on it. The media may be mixed yourself or purchased. If it is purchased it is ideal if the seller has grown your cell type on it in a testing environment. Another option is synthetic serum. However, this still has some of the drawbacks of true serum such as batch variation. Cell lines can be adapted to growth on serum free media. However, it is important to ensure that the unique cell line characteristics are not lost during the process. The adaption process is best performed over several serial cultures with the concentration of serum being lowered each time. Cowles - J25 Nutrition of cells in culture & media preparation Page 5 there are several different ways to develop serum free media for new cell lines. the easiest way is to start with a basic media and change the concentrations of important factors until they are optimized. It may also be possible to use protein free media, although this has proved to be difficult. While serum free media has many advantages, the increased difficulty it introduces to cell culture has made it a less frequent choice. I'm surprised that it's possible at all, even with lots of time and effort! T3 - Getting started with tissue culture tissue culture labs need to be properly equipped with the necessary equipment, such as a sterile hood. The hood needs to be large enough to hold all the materials you're working with as well as provide ample room for movement. It should be sterile and use a HEPA filter and the surface should be sterilized before use. All work should be performed on a sterile surface such as a petri dish. water that is free of contamination is needed to clean equipment and make culture media. this may be made through distillation, reverse osmosis or other advanced filtering methods. A room need to be set aside as a place to store cultures once they have been prepared. It generally does not need to be climate controlled, although it should stay near room temperature. It may also need full spectrum lights for some types of cultures. there are many different tissue culture mediums. For the best results it is important to choose the one that is best suited to your species of interest and what you're trying to do with your samples. Development of the various formulas has been the result of trial and error by many different individuals. Tissue culture media is made up of several components: 1. inorganic minerals: needs are species dependent. Macrosalts are the minerals that are needed in higher amounts. These are N, K, Mg, Ca, P and S and are usually provided in different salts. Microsalts are minerals that are needed in trace amounts such as cobalt, iron, boron, manganese, and molybdenum. Impurities in the media may provide enough of these. However, it seems safer to me to add them rather than assuming the media is 'properly' contaminated. 2. organic compounds: sugar is an important part of the media since the explants aren't usually making their own through photosynthesis. Vitamins and amino acids are also included in some media. Complex organics are undefined additions such as tomato juice or birch sap. Charcoal may also be used to absorb unwanted organic compounds released by the explants. 3. plant growth regulators (PGRs): have dramatic effects, even at very low concentrations. The most important are the auxins and cytokinins, while other PGRs are hormones or synthetic molecules. Low levels of auxin lead to root development, while high levels cause callus formation. Cytokinins promote cell division and shoot growth. agar is extracted from algae and used to solidify the media and enable explants to be placed on the surface of the media. Agarose is a purified form or agar and is clear rather than slightly opaque. Cowles - J25 Nutrition of cells in culture & media preparation Page 6 There are several different units of concentration used in tissue culture: 1. volume percent (v/v) 2. weight percent (w/v) 3. molarity 4. mg/L 5. ug/L 6. parts per million (ppm) stock solutions can be made up before hand and used to create media later, thus saving time. Solutions of the mineral salts should be made in 100x or 1000x the required concentration. Serial dilution can be used to add tiny amounts of something to the media. The pH of the media needs to be adjusted to between 5.3 and 5.8 for best growth. This should be done just before the agar is added. media can be stored effectively for a month as long as it's sealed and doesn't get wet. in addition to sterile equipment, it is also important to sterilize plant tissues before they are put on the media. Sterilization should be performed in an autoclave at 121 degrees C for and 15psi for at least 15 minutes. Plant tissues should be disinfested prior to explanting, usually with sodium hypochlorite. T4 - nutrition of callus cultures Callus is mostly undifferentiated paranchyma cells. Tissues from various plant parts can be induced to form callus. Many factors are involved in callus formation, such as light, PGRs, temperature, and the genotype. Research is required to determine what combination of factors work for each species. The research must be intense trial and error. It would be worse than animal culture too, since the tissues are slower growing and the generation time is greater. The experiments in this chapter will show that one medium can support several different plant species and some ingredients are more important than others. An important fact is that different species will display different growth characteristics on the same media. This will be shown by growing tobacco stem pith on media specialized for it as well as several other species on the same media. The tobacco explants should form the largest callus, while the callus of the other species will be smaller. Another important concept is that tissues require a specific mix of nutrients and PGRs for max growth. The second experiment grows tobacco on media ranging from nutrient free to complete nutrients. the expected results are that growth should be poor on all the media without the full nutrient complement. growth should be good on the media with all the nutrients, and even better when supplemented with coconut milk. Cowles - J25 Nutrition of cells in culture & media preparation Page 7 Coconut milk must have nutrients that are missed by other media. I wonder what the best way to find out what these are. I suppose they might not work as well if they were isolated from the rest of the components of coconut milk though. Questions 1. T/F: A plant's genotypes influences the media it grows on best. 2. What are the advantages of serum free media? 3. define microsalts and macrosalts. 4. define complete media and list 3 of its components. 5. list and briefly describe 4 of the physiochemical properties of media.
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