• Media composition, pH, osmolality and the volume andfrequency of replenishment.

• Incubation conditions such as temperature, relative humidityand gas composition can be regulated, as can the form andand gas composition can be regulated, as can the form andcomposition of the physical substrate for cell attachment

Dr Saeb Aliwaini

Culture medium

1- Nutrients: Should provide all the essential nutrients, vitamins,cofactors, metabolic substrates, amino acids, inorganic ions andtrace elements needed to support cellular functions and thesynthesis of new cells.

2- Buffers : buffering is required in order to maintain the cellswithin their normal pH range and limit the effects of acidic wastewithin their normal pH range and limit the effects of acidic wasteproducts generated by the cell, most notably CO2 and lactic acid.

3- non-essential ingredients

Phenol Red

However, this is not in itself sufficient to support cell viability formore than a few hours.

Fetal bovine serum (FBS– also known as fetal calf serum) is themost common.

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• Eagle’s minimal essential medium (EMEM) and its derivatives –these include alpha minimal essential medium (αMEM),Dulbecco’s modified Eagle’s medium (DMEM), Glasgowminimal essential medium (GMEM) and Joklik’s minimalessential medium (JMEM).essential medium (JMEM).

• Media designed at the Roswell Park Memorial Institute (RPMI).The most popular of these is probably RPMI 1640, but othersinclude RPMI 1629 and RPMI 1630.

• Basal media designed for use in serum-free formulation, forexample Iscove’s modified Dulbecco’s medium (IMDM), CMRL1060, Ham’s F10 and derivatives such as F12, TC 199 andderivatives, the MCDB series, NCTC series, and Waymouth’s.

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• Basal media designed for use with sera, for example Fischer’s,Leibovitz’s L-15, Trowell’s, and Williams’ Medium E.

• Basal media are composed of:

Balanced salt solutions comprise a mixture of inorganic saltsBalanced salt solutions comprise a mixture of inorganic salts(Na+, K+, Mg2+, Ca2+, Cl–, SO42–, PO43– and HCO3–)designed to:

- Osmotic pressure

- Buffer

- Membrane potential

- Cofactors in enzyme reactions and in cell attachment.

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• Selenium as trace element but not with sera.

• glucose is often added as an energy source for the cells

- Types of balanced salts solutions

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Dulbecco’s phosphate-buffered saline (DPBS)

Hanks’ balanced salt solution (HBSS)

Eagle’s spinner salt solution (ESSS)

Earle’s balanced salt solution (EBSS).

• Most cell lines grow well at pH 7.2 -7.4

• Phenol red is commonly used as an indicator.

• It is red at pH 7.4 and becomes orange at pH 7.0, yellow at pH6.5, lemon yellow below pH 6.5, more pink at pH 7.6, andpurple at pH 7.8.purple at pH 7.8.

• Bicarbonate is frequently used for this purpose, but requiresan atmosphere of 5–10% CO2in order to maintain the pH thatthe medium was designed to have (at least until the cellsthemselves produce significant quantities of CO2 and/or lacticacid).

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• Each of the basal media has a recommended bicarbonateconcentration and CO2 tension to achieve the correct pH andosmolality .

• Carbon dioxide in the gas phase dissolves in the medium,establishes equilibrium with HCO3− ions, and lowers the pH.establishes equilibrium with HCO3− ions, and lowers the pH.Because dissolved CO2, HCO3−, and pH are all interrelated

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• The amount of dissolved CO2 in water is dependent on theamount of atmospheric CO2 and the temperature.

• Increasing CO2 tension in the absence of sodium bicarbonateleaves the medium acidic.

• The addition of sodium bicarbonate in the presence of CO2• The addition of sodium bicarbonate in the presence of CO2will drive the equation to the left to allow for an equilibriumto be achieved and the pH maintained at 7.2-7.4

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Bicarbonate buffer is still used more frequently than any otherbuffer because of its low toxicity, low cost, and nutritional benefitto the culture.

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid )

• HEPES, at 10 to 20 mM, is a much stronger 6.8 to 8.2

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• HEPES has a pKa of 7.3 at 37 ◦C, and thus buffers very effectively

without CO2 in the pH 7.2–7.6 range.

• However, bicarbonate is also essential to cells as a nutrient, so there

must always be sufficient in the medium for this purpose.

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• When preparing a new medium for the first time, add the specifiedamount of bicarbonate and then sufficient 1 N NaOH such that themedium equilibrates to the desired pH after incubation in a Petri dish at37◦C, in the correct CO2 concentration, overnight.

• The inclusion of pyruvate in the medium enables cellsto increase their endogenous production of CO2, makingthem independent of exogenous CO2, as well as HCO3−to increase their endogenous production of CO2, makingthem independent of exogenous CO2, as well as HCO3−

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Leibovitz L15 medium contains a higher concentration of sodium pyruvate(550 mg/L) but lacks NaHCO3 and does not require CO2 in the gas phase.L15 is sometimes recommended for the transportation of tissue samples.

Dr Saeb Aliwaini

Dr Saeb Aliwaini

• An energy source

• culture media contain 4 to 20 mM glucose

• It is a carbon source for glycolysis, generating lactic acid as anend product, oxygen is in relatively short supply.

• Shall we raise the O2 tension, this will generate free radicalspecies that are toxic to the cell, so O2 is usually maintained atatmospheric levels.

• This results in anaerobic conditions and the use of glycolysis forenergy metabolism.

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• However, the citric acid cycle remains active, and it has becomeapparent that amino acids can be utilized as a carbon source byoxidation and entry into the citric acid cycle

• L-glutamine provides nitrogen for NAD, NADPH and serves as a• L-glutamine provides nitrogen for NAD, NADPH and serves as asecondary energy source for metabolism.

• L-glutamine concentrations for mammalian cell culture mediacan vary from 0.68 mM in Medium 199 to 4mM in Dulbecco’sModified Eagles’s Medium..

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• Deamination of the glutamine tends to produce ammonia,which is toxic and can limit cell growth, but the use ofdipeptides, such as glutamyl-alanine or glutamyl-glycine,appears to minimize the production of ammonia and has theadditional advantage of being more stable in the medium.

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A buffer may be incorporated into the medium to stabilize the pH,but in (1) exogenous CO2 may still be required by some cell lines,particularly at low cell concentrations, to prevent the total lossof dissolved CO2 and bicarbonate from the medium

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Dr Saeb Aliwaini

Dr Saeb Aliwaini

Sterile Filtration.

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Dr Saeb Aliwaini

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Primary culture

• Iinitiation of a primary cell culture

• Enzymes Used in Disaggregation

• Trypsin, collagenase, elastase, and hyaluronidase, alone or invarious combinations.

• Trypsin and pronase give the most complete disaggregation butmay damage the cellsmay damage the cells

• Pronase is a mixture of several nonspecific endo- andexoproteases that digest proteins down to single amino acids.

• At least 10 proteases are in the mixture

• Collagenase and Dispase, in contrast, give incompletedisaggregation, but are less harmful

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Trypsin is a pancreatic serine protease(Proteolytic) enzyme with specificityforpeptides bonds. It is the mostcommonly used enzyme for cellharvesting in cell culture.

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Common Features of Disaggregation

• Fat and necrotic tissues are best removed during dissection.

• The tissue should be chopped finely with sharp scalpels to causeminimum damage

• Enzymes used for disaggregation should be removed subsequentlyby gentle centrifugation.

• The concentration of cells in the primary culture shouldbe much higher than that normal

• A rich medium, such as Ham’s F12, is preferable to asimple medium, such as Eagle’s MEM, and if serum isrequired, fetal bovine often gives better survival thandoes calf or horse.

• Embryonic tissue disaggregates more readily

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Mouse Embryo

• mouse embryo fibroblasts

• Full term is about 19 to 21 days

• Optimal around 13 days

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Dr Saeb Aliwaini

Dr Saeb Aliwaini

Dr Saeb Aliwaini

Primary Explantation

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• Attaching explants

• Cover slip, collagen

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Enzymatic Disaggregation

• Cell–cell adhesion in tissues is mediated by a varietyof homotypic interacting glycopeptides (cell adhesionmolecules, or CAMs).

• some of which are calcium dependent (cadherins) and henceare sensitive to chelating agents such as EDTA or EGTA.

• Intercellular matrix and basement membranes contain otherglycoproteins, such as fibronectin and laminin, which areprotease sensitive.

• Embryonic tissue disperses more readily and gives a higheryield of proliferating cells than newborn or adult tissue.

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Warm Trypsin

• Dissociated cells should be collected every half hour

• The warm trypsin technique is useful for the disaggregationof large amounts of tissue in a relatively short time,particularly for chopped whole mouse embryos or chickembryosembryos

• Not for adult

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Trypsinization with Cold Preexposure

• After preparation and washing …

• Add 10 mL/g of tissue of 0.25% trypsin in RPMI1640

• Place the mixture at 4◦C for 6 to 18 h

• Remove and discard the trypsin carefully, leaving the tissuewith only the residual trypsin.with only the residual trypsin.

• Place the tube at 37◦C for 20 to 30 min

• Add warm medium, approximately 1 mL for every100 mg of original tissue, and gently pipettethe mixture up and down until the tissue iscompletely dispersed

• Dilute the cell suspension to 1 × 105 to 1 × 106/mL in growth medium

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• The cold trypsin methodusually gives a higher yieldof viable cells, withimproved survival after 24-h culture and preservesmore different cell typesthan the warm method.than the warm method.

• Cultures from mouseembryos contain moreepithelial cells whenprepared by the coldmethod

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Mechanical Disaggregation

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