Role of serum and supplements in culture medium k.s
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The document discusses the role of serum in cell culture media, highlighting its importance as a source of nutrients, growth factors, and proteins that aid in cell growth and function. It notes the advantages of serum, such as promoting cell attachment and acting as a buffering agent, while also addressing its drawbacks including variability in composition, potential contamination, and the impact on experimental consistency. Additionally, it covers alternative media supplements like amino acid hydrolysates and conditioned medium, emphasizing the need for careful consideration of osmolality and shelf life when adding these supplements.
![Embryo Extract
• Embryo extract is a crude homogenate of 10-day-old chick embryo that is
clarified by centrifugation.
• The crude extract was fractionated by Coon and Cahn [1966] to give
fractions of either high or low molecular weight.
• The low-molecular-weight fraction promoted cell proliferation, whereas the
high-molecular-weight fraction promoted pigment and cartilage cell
differentiation.](https://image.slidesharecdn.com/roleofserumandsupplementsinculturemediumk-210523112237/85/Role-of-serum-and-supplements-in-culture-medium-k-s-24-320.jpg)
![Conditioned Medium
• Puck and Marcus [1955] found that
the survival of lowdensity cultures
could be improved by growing the
cells in the presence of feeder
layers.
• In that instance the effect was due
to soluble factors as the clones and
feeder layer were kept separate.](https://image.slidesharecdn.com/roleofserumandsupplementsinculturemediumk-210523112237/85/Role-of-serum-and-supplements-in-culture-medium-k-s-26-320.jpg)
![• Hauschka & Konigsberg [1966] showed that the conditioning of culture
medium that was necessary for the growth and differentiation of myoblasts
was due to collagen released by the feeder cells.
• Using feeder layers and conditioning the medium with embryonic fibroblasts
or other cell lines remains a valuable method of culturing difficult cells.
• However, conditioning medium adds undefined components and should be
eliminated if possible after the active constituents are determined.](https://image.slidesharecdn.com/roleofserumandsupplementsinculturemediumk-210523112237/85/Role-of-serum-and-supplements-in-culture-medium-k-s-27-320.jpg)
Role of serum and supplements in culture medium k.s
- 1. KAILASH M.Sc BIOTECHNOLOGY 2nd SEMESTER SUBMITTED TO: Dr. S.K. GAHLAWAT DEPTT. OF BIOTECHNOLOGY CDLU (SIRSA)
- 2. Serum in media • Serum is a complex mix of albumins, growth factors and growth inhibitors. • Serum is one of the most important components of cell culture media and serves as a source for amino acids, proteins, vitamins (particularly fat-soluble vitamins such as A, D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace elements.
- 3. • Serum from fetal and calf bovine sources are commonly used to support the growth of cells in culture. • Fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells. • Calf serum is used in contact-inhibition studies because of its lower growth- promoting properties. • Normal growth media often contain 2-10% of serum.
- 4. Supplementation of media with serum serves the following functions : • Serum provides the basic nutrients (both in the solution as well as bound to the proteins) for cells. • Serum provides several growth factors and hormones involved in growth promotion and specialized cell function. • It provides several binding proteins like albumin, transferrin, which can carry other molecules into the cell. For example: albumin carries lipids, vitamins, hormones, etc. into cells.
- 5. • It also supplies proteins, like fibronectin, which promote the attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they begin to divide. • It provides protease inhibitors which protect cells from proteolysis. • It also provides minerals, like Na+, K+, Zn2+, Fe2+, etc. • It increases the viscosity of the medium and thus, protects cells from mechanical damages during agitation of suspension cultures.
- 6. • It also acts a buffer. • Due to the presence of both growth factors and inhibitors, the role of serum in cell culture is very complex. • Unfortunately, in addition to serving various functions, the use of serum in tissue culture applications has several drawbacks .
- 7. proteins • Although proteins are a major component of serum, the functions of many proteins in vitro remain obscure; • it may be that relatively few proteins are required other than as carriers for minerals, fatty acids, and hormones. • Those proteins for which requirements have been found are albumin which may be important as a carrier of lipids, minerals, and globulins which promotes cell attachment. • Protein also increases the viscosity of the medium, reducing shear stress during pipetting and stirring, and may add to the medium’s buffering capacity.
- 8. Hormones • Insulin promotes the uptake of glucose and amino acids. • Growth hormone may be present in serum—particularly fetal serum—and, in conjunction with the somatomedins (IGFs), may have a mitogenic effect. (mitogen: a mitogen is a peptide or small protein that induces a cell to begin cell division: mitosis.) • Hydrocortisone is also present in serum—particularly fetal bovine serum—in varying amounts, and it can promote cell attachment and cell proliferation but under certain conditions (e.g., at high cell density) may be cytostatic and can induce cell differentiation
- 9. Nutrients and Metabolites • Serum may also contain amino acids, glucose, oxo (keto) acids, nucleosides, and a number of other nutrients and intermediary metabolites. • These may be important in simple media but less so in complex media, particularly those with higher amino acid concentrations and other defined supplements.
- 10. Lipids • Linoleic acid, oleic acid, ethanolamine, and phosphoethanolamine are present in serum in small amounts, usually bound to proteins such as albumin.
- 11. Minerals • Serum replacement experiments have also suggested that trace elements and iron, copper, and zinc may be bound to serum protein, probably albumin.
- 12. Inhibitors • Serum may contain substances that inhibit cell proliferation. • Some of these may be artifacts of preparation (e.g., bacterial toxins from contamination before filtration, or antibodies that cross-react with surface epitopes on the cultured cells) • but others may be physiological negative growth regulators, such as TGF-β. Heat inactivation removes complement from the serum and reduces the cytotoxic action of immunoglobulins without damaging polypeptide growth factors, but it may also remove some more labile constituents and is not always as satisfactory as untreated serum.
- 16. Advantages of serum in media • Serum contains various growth factors and hormones which stimulates cell growth and functions. • Helps in the attachment of cells. • Acts as a spreading factor.
- 17. • Acts as a buffering agent which helps in maintaining the pH of the culture media. • Functions as a binding protein. • Minimizes mechanical damages or damages caused by viscosity.
- 18. Disadvantages of serum in media • Lack of uniformity in the composition of serum • Testing needs to be done to maintain the quality of each batch before using • May contain some of the growth inhibiting factors • Presence of serum in media may interfere with the purification and isolation of cell culture products.
- 19. • Serum Can Be a Source of Contamination • Another potential issue with serum is viral or bacterial contamination. Again, lot-to-lot variability is in play here, and heat inactivation may not wholly neutralize microbial pathogens. • Adding contaminated serum to your culture will affect the health and growth of your cells and render them unusable for experiments.
- 20. • Serum Is Highly Variable • For starters, serum is an animal-derived natural product, and each new lot of material will vary. • While serum does contain items important for the health of your cells, it also contains things like immune complement proteins that can harm your cells. • To get around this, serum is usually heated to inactivate harmful proteins. Of course, heat will also denature beneficial proteins too, which can increase the variability between media preparations.
- 21. • While this might not be a big issue for some types of research, variability in factors critical for cell growth and survival can confound your results if you are running, for example, a cell-based assay measuring the effect of a growth factor or drug on a given cellular function. • In this case, one of the biggest reasons for going serum-free is to increase the standardization of culture conditions for more consistent results. • This type of standardization is even more critical in regulated laboratories and using a defined medium allows greater control over your cell culture and your experiments.
- 22. Media supplements • In addition to serum, tissue extracts and digests have traditionally been used as supplements to tissue culture media. • These components, supplements, help sustain proliferation and maintain normal cell metabolism.
- 23. Amino Acid Hydrolysates • Many such supplements are derived from microbiological media. • Bactopeptone, tryptose, and lactalbumin hydrolysate (BD Biosciences) are proteolytic digests of beef heart or lactalbumin and contain mainly amino acids and smallpeptides. • Bactopeptone and tryptose may also contain nucleosides and other heat- stable tissue constituents, such as fatty acids and carbohydrates. • Sterility is easily achieved as they are autoclavable.
- 24. Embryo Extract • Embryo extract is a crude homogenate of 10-day-old chick embryo that is clarified by centrifugation. • The crude extract was fractionated by Coon and Cahn [1966] to give fractions of either high or low molecular weight. • The low-molecular-weight fraction promoted cell proliferation, whereas the high-molecular-weight fraction promoted pigment and cartilage cell differentiation.
- 25. • Although Coon and Cahn did not fully characterize these fractions, more recent evidence suggests that the low-molecular-weight fraction may contain peptide growth factors and the highmolecular- weight fraction proteoglycans and other matrix constituents. • Embryo extract was originally used as a component of plasma clots to promote cell migration from the explant and has been retained in some organ culture techniques. • It should always be frozen and thawed at least twice to ensure that there is no carryover of live cells from the embryo.
- 26. Conditioned Medium • Puck and Marcus [1955] found that the survival of lowdensity cultures could be improved by growing the cells in the presence of feeder layers. • In that instance the effect was due to soluble factors as the clones and feeder layer were kept separate.
- 27. • Hauschka & Konigsberg [1966] showed that the conditioning of culture medium that was necessary for the growth and differentiation of myoblasts was due to collagen released by the feeder cells. • Using feeder layers and conditioning the medium with embryonic fibroblasts or other cell lines remains a valuable method of culturing difficult cells. • However, conditioning medium adds undefined components and should be eliminated if possible after the active constituents are determined.
- 28. Precautions to take while adding suppliments: • since the addition of supplement can change the osmolality of the complete growth media which can negatively affect the growth of cells, it is always best to recheck the osmolality after supplements are added. • For most of the cell lines, optimal osmolality should be between 260 mOSM/kg and 320 mOSM/kg. • The shelf life of the growth media changes after the addition of supplements. Complete media containing protein supplement tend to degrade faster than basal media alone.
- 29. Reference: • Culture of Animal Cells A Manual of Basic Technique and Specialized Applications by R. Ian Freshney. • www.labome.com