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PTC - C8 (Basic Cell Culture Medium) Cheat Sheet (DRAFT) by

Brief summary of Chapter 8 (Basic Cell Culture Medium) of Plant and Tissue Culture Subject

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Media

1. Natural Media
a) Coagulant, such as plasma clots
-Blood before coagul­ation; Plasma can also be prepared in the laboratory by taking out blood and adding heparin to prevent blood coagul­ation.
-natural sources of nutrient sufficient for growth and prolif­eration of animal cells and tissues
b) Biological fluids such as serum
-Blood after coagul­ation; serum: com­po­nents of animal cell culture which is the source of various nutrients.
   
-serum: Contains growth factors which promotes cell prolif­era­tion, cell attachment and adhesion factors
   
-other forms of biological fluids used are coconut wa­ter, amniotic fluid, pleural fluid, insect haemolymph serum, culture filtrate, aqueous humour, from eyes etc.
 
c) Tissue extracts for example Embryo extracts
-Extracts from tissues such as embryo, liver, spleen, leukoc­ytes, tumour, bone marrow etc. are also used for culture of animal cells.
2. Synthetic Media
a) Serum containing media
-prepared artifi­cially
b) Serum-free media
-Recipe: or­ganic and inorganic nutrients, vitamins, salts, serum proteins, carboh­ydr­ates, co-fac­tors, etc.

Base Media

-many different types of base medium available
-Addit­ional supple­ments are added to this bottle
-Some cell types prefer one type of medium to another, be sure to know all this inform­ation ahead of time.

Types of Base Medium

Basic Consti­tuents of Media

Consti­tuents
Function
1. Inorganic Salts
-retain the osmotic balance of the cells
 
-regulate membrane potential by provision of sodium, potassium and calcium ions
 
-required in the cell matrix for cell attachment and as enzyme cofactors
2. Carboh­ydrates (glucose)
-main source of energy
major sugars used are glucose and galactose, however, some media contain maltose or fructose
-media containing the higher concen­tration of sugars are able to support the growth of a wider range of cell types
3. Amino Acids
-building blocks of proteins
 
-‘Esse­ntial’ amino acids must be added to culture media as cells are not able to synthesize these themselves
 
-The concen­tration of amino acids in the culture medium will determine the maximum cell density that can be achieved
 
-once depleted the cells will no longer be able to prolif­erate
 
-once depleted the cells will no longer be able to prolif­erate
 
-Commonly the necessary amino acids include cysteine and tyrosine, but some non-es­sential amino acids may be needed.
 
-Adding supple­ments of non-es­sential amino acids to media both stimulates growth and prolongs the viability of the cells in culture.
4. Vitamins
-precu­rsors for numerous co-factors
 
-Many vitamins, especially B group vitamins, are necessary for cell growth and prolif­eration and for some lines the presence of B12 is essential.
 
-Many vitamins, especially B group vitamins, are necessary for cell growth and prolif­eration and for some lines the presence of B12 is essential.
5. Fatty Acids and Lipids
-important in serum free media since they are normally present in serum
 
-E.g. choles­terol and steroids essential for specia­lised cells.
6. Proteins and Peptides
-parti­cularly important in serum free media
 
-most common proteins and peptides include albumin, transf­errin, fibron­ectin and fetuin and are used to replace those normally present through the addition of serum to the medium
7. Buffering Systems - pH
-Close control of pH is essential for optimum culture conditions
 
-Most cells require pH conditions in the range 7.2-7.4
 
-Phenol red indicator to monitor pH (pH 6.5 = yellow, pH 7.0 = orange, pH 7.4 = red, pH 7.8 = purple)
 
-Could monitor contam­ination
8. Trace Elements
-E.g. zinc, copper, selenium and tricar­boxylic acid interm­ediates
 
-Selenium is a detoxifier and helps remove oxygen free radicals
9. Antibi­otics and Anti-m­ycotics (anti-­fungal)
-Unless good sterile conditions can be maintained (e.g., using laminar flow hoods) it is ne­cessary to incorp­orate antibi­otics and antimy­cotics into the media.
 
-To prevent contam­ination in culturing
 
-Specific antibi­otics - penici­l­l­in/­str­ept­omycin solutions
 
-Broad spectrum antibi­otics - kanamycin or amphot­ericin B
 
-should not be toxic to the cells in culture
 
-should de­pend on the type of contam­ination
 
-Some antibi­otics are used for selecting transf­ected animal cells, such as geneticin
 
->T­ran­sfected cell acquire resistant selectable marker and able to survive on medium containing geneticin
 
->N­on-­tra­nsf­ected cell could not survive
10. Serum
-super­natant of clotted blood
 
-Many undefined growth factors are contained, including many proteins and metal ions.

Types of Serum

  

Regulation of pH

Regulation of pH is usually achieved by one of two buffering systems
1. “natural” buffering system
-Gaseous CO2 balances with the CO3/HCO3 content of the culture medium
-Bicar­bonate buffer requires 5-10 % CO2 (supplied in a CO2 incubator) to maintain pH 6.9-7.4
-Bicar­bon­ate/CO2 is low cost, non-toxic and also provides other chemical benefits to the cells.
2. chemical buffering using a zwitterion called HEPES (4-(2-­hyd­rox­yet­hyl­)-1­-pi­per­azi­nee­tha­nes­ulfonic acid)
-HEPES has superior buffering capacity in the pH range 7.2-7.4
-but is relatively expensive and can be toxic to some cell types at higher concen­tra­tions (above ~100nM­olar).
-HEPES buffered cultures do not require a controlled gaseous atmosp­here.

Use of the phenol red indicator in media

Pros and Cons of Serum in Medium

Advantage
Disadv­antage
1. Serum binds and neutra­lizes toxins
1. It is not chemically defined, its compos­ition varies a lot
2. Serum contains a complete set of essential growth factors, hormones, attachment and spreading factors, binding and transport proteins
2. It is sometimes source of contam­ination by viruses, mycopl­asma, prions, etc.
3. Serum contains the protease inhibitors
3. Sera could contain inhibitors or toxins
4. Serum increases the buffering capacity
4. Some ingred­ients could cause unwanted reactions
5. Serum provides trace elements

Serum-free media (SFM)

-A defined SFM is one in which a group of components of known purity, present at a known concen­tra­tion, are formulated together to optimize perfor­mance for a given cell type
 
The two main approaches generally followed in designing a serum-free medium are:
1. Reduced serum
-conce­ntr­ation of serum in the basal medium is progre­ssively reduced whilst other compon­ents, e.g. growth factors and hormones, are added to identify the factors capable of restoring growth to the level obtained in the presence of serum
2. Basal medium
-add components (singly or in combin­ations) to a basal medium in a stepwise manner until a medium is progre­ssively ‘built up, to give a similar or equivalent cell growth to the serum-­sup­ple­mented medium

Pros and Cons of Serum Free Media

Advantage
Disadv­antage
1. Avoids qualit­ative and quanti­tative fluctu­ations of nutrients
1. Multip­licity of media-Each cell type appears to require a different recipe
2. Growth conditions are defined and controlled
2. Select­ivi­ty-Some media may select a sublineage that is not typical of the whole population
3. Small chances of contam­ination
3. Reagent purity-The removal of serum also requires that the degree of purity of reagents and water and the degree of cleanl­iness of all apparatus be extremely high, as the removal of serum also removes the protec­tive, detoxi­fying action that some serum proteins may have
4. More consistent perfor­mance
4. Cell prolif­era­tio­n-G­rowth is often slower in serum-free media, and fewer genera­tions are achieved with finite cell lines
5. Allows the possib­ility of studying the effects of one component present in serum, while elimin­ating the rest
6. Avoid ethical issues