unit i media and components for growth

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MEDIA AND COMPONENTS FORMEDIA AND COMPONENTS FOR

GROWTH, CONSTITUENTSGROWTH, CONSTITUENTS

AND THEIR FUNCTIONSAND THEIR FUNCTIONS

Dr. Sesha Srinivas VutukuruDr. Sesha Srinivas Vutukuru,, M.Tech., PhD, PG DEMM.Tech., PhD, PG DEM

Associate Professor Associate Professor

Department of BiotechnologyDepartment of Biotechnology

Sreenidhi Institute of Science and TechnologySreenidhi Institute of Science and Technology

HyderabadHyderabad--501 301501 301

Unit I:



Contents

MediaMedia compositioncomposition

ComponentsComponents for for growthgrowth

ConstituentsConstituents and and their their functionsfunctions



RICH MEDIA ARE REQUIRED TO CULTURE ANIMAL CELLS

Nutrients (solid or liquid) are used to cultivate cells

A nutrient is chemical substance that enters a cell and is used as a

substrate for biosynthesis or energy metabolism

Examples: amino acids

fatty acids

sugars

ions

trace elements

vitamins

cofactors

growth factors



Basal salt solution:

Balanced salt solution (BSS) is composed of

inorganic salts that maintain the physiological

pH and osmotic pressure

Inorganic ions such as Na+,K+,mg2+,ca2+,cl-,so42-

,Po43-

, HCO3-

maintain membrane potential andalso work as cofactors in enzyme reactions



Fibroblast-like 3T3 cells derived from

mouse embryos

Most cells grow well at

pH 7.4

Cell growth varies

relatively little among

different strains.

Fibroblast cells perform

best at pH 7.0 ² 7.7

Epidermal cells could

be maintained at pH 5.5Epithelial-like cell line (Cl-9) derived fromrat liver



Phenol red is commonly used as an indicator Phenol red is commonly used as an indicator

It is red at pH 7.4 and becomes orange at pHIt is red at pH 7.4 and becomes orange at pH7.07.0

Yellow at pH 6.5Yellow at pH 6.5

More pink at pH 7.6 andMore pink at pH 7.6 and

purple at pH 7.8purple at pH 7.8



Basal Salt Solution: 4 types

EBSS: Earle·s Balanced salt solution

DPBS: Dulbecco·s phosphate-buffered saline

HBSS: Hank·s Balanced salt solution

ESSS: Eagle·s spinner salt solution

Eagle·s Basal Medium(1955)

Eagle·s Minimum Essential Medium- MEM (1959)



Basal Medium :Other Constituents

Carbohydrates

Protein supplements

Essential Amino Acids

PhospholipidsVitamins

Hormones

Growth factors

Metals

Antibiotics



Carbohydrates: Source of energy

Sugars such as glucose, maltose, sucrose,mannose, fructose, and galactose

Protein supplements: Fibronectin, albumin,

transferrin, fetuin, -globulin

Albumin is a transport protein - carries vitamins,

lipids and hormones into cellsTransferrin binds and transports iron

Insulin is essential for all media



Essential Amino Acids:Nine amino acids, referred to as the essential amino acids,

cannot be synthesized by adult vertebrate animals and thusmust be obtained from their diet

Animal cells grown in culture also must be supplied with thesenine amino acids, namely, histidine, isoleucine, leucine, lysine,

methionine, phenylalanine, threonine, tryptophan, and valine.(HILLMPTTRV)

In addition, most cultured cells require cysteine, glutamine,and tyrosine

In the intact animal, these three amino acids are synthesizedby specialized cells; for example, liver cells make tyrosine fromphenylalanine, and both liver and kidney cells can makeglutamine



Glutamine serves both

as an energy source andcarbon source in the

synthesis of nucleic

acids

It is the most unstable

amino acid

The concentration of amino acids influences

cell yield, survival and

growth rates



During the lag phase of growth cycle , there is

an enhanced requirement of cysteine,glutamine, isoleucine and serine

Deficiency of Amino Acid:

Amino acid deficiency inhibits cell division ,induces chromosomal damage and increaseslysosomal activity and cell death

Imbalance of Amino acid concentration can alsoproduce karyotype changes



Vitamins: Required as cofactors in metabolism Eg.Niacin, NAD(P), riboflavin, FMN, FAD etc.

Retenoids help cells to adhere to the substrate

Hormones: Glucocorticoid hormones likeHydrocortisone and dexamethasone can either stimulate

cell growth or inhibit cell growth depending on cell type

Phospholipids: Choline is necessary for incorporation into

membrane phospholipids

Phospholipids and cholesterol, and prostaglandins E and F2 are

involved in cell growth along with growth factors



Growth factors: Nerve growth factor (NGF),Epidermal growth factor (EGF), Fibroblast

growth factors (FGF) promote cell growth

Metals: Copper, Zinc, Cobalt, Manganese,

Molybdenum, and Selenium acitivates

enzymes and protect against free radicals,which cause damage to DNA



Antibiotics: should have no toxicity and

preferably should be a broad spectrum type

Antibacterial: Penicillin (100 IU/ml) and

Streptomycin (50µg/ml)

Antifungal: Gentamycin (50 µg/ml) andNystatin (25 µg/ml) are expensive butwidely used



Chemical constituents Eagle's

MEM

Dulbecco 's modification Ham's F12

1. Amino Acids

L-asparagine 126 84 211

L-cystine 24 48 -

L-glutamine 292 584 146

Glycine - 30 7.5

L-histidine HC1.H20 42 42 21

L-isoleucine .52 42 21

L-leucine 52 105 13.1

L-lysine.HCI 73.1 146 36.5

L-methionine 15

30

4.48

L-phenylalanine 33 4.96

L-prolinc 34.5

L-serine

42

10.5

L-threonine 48 95 11.9

L-tryptophan 10 16 2.04



2. Vitamins2. Vitamins Eagle'sEagle'sMEMMEM

Dulbecco 's modificationDulbecco 's modification Ham's F12Ham's F12

BiotinBiotin -- -- 0.00730.0073

DD--CaCa--pantothcnate pantothcnate 11 44 0.480.48

Choline chlorideCholine chloride 11 44 1414

Folic acidFolic acid 11 44 1.31.3

InositolInositol 22 7.27.2 1818

Nicotinamide Nicotinamide 11 44 0.040.04

Pyridoxal.HClPyridoxal.HCl 11 44 0.0620.062

RiboflavinRiboflavin 0.10.1 0.40.4 0.0380.038

Thiaminc.HCIThiaminc.HCI .. -- 1.361.36

Vitamin B12Vitamin B12 -- -- 1.361.36

Pyridoxin.HCIPyridoxin.HCI -- -- 0.0620.062



3. lnorganic Salts Eagle's

MEM

Dulbecco 's

modification

Ham's

F12

CaCI2 (anhydrous) 200 200 -

CaCl,.2H20 - - 44

Fe(NO3)3.9H2O - 0.1 -

KCI 400 400 224

MgCl2.6H20 - - 122

MgS04.7H20 200 200 -

NaCl 6800 6400 7599

NaHCO3 2200 3700 1176

Na2H2PO4.H3O 140 125 -

Na2HPO4.7HjO - - 268

FeSO4.7H2O - - 0.834

ZnS04.7H20 - - O.863

CuS04.5H20 - - 0.00249



ChemicalChemical

constituentsconstituents

Eagle'sEagle's

MEMMEM

Dulbecco'sDulbecco's --

modificationmodification

Ham's F12Ham's F12

4. Other Chemicals4. Other Chemicals

DD--g!ucoseg!ucose 10001000 45004500 18021802

Lipoic acidLipoic acid -- -- 0.210.21

Phenol redPhenol red 1010 15

15

1212Sodium pyruvateSodium pyruvate -- 110110 110110

HypoxanthineHypoxanthine -- -- 4.14.1

Linolcic acidLinolcic acid -- 0.0840.084 --

Putrescine.2HClPutrescine.2HCl -- -- 0.1610.161

ThymidineThymidine -- -- 0.730.73

COCO22 (gas phase)(gas phase) 5%5% 10%10% 5%5%



Other Media

CMRL 1066CMRL 1066

RPMI 1640RPMI 1640

M199M199L15L15

McCoy¶s 5AMcCoy¶s 5A

Fischer Fischer MB 752/1MB 752/1



Co2 and Bicarbonate

Carbondioxide in the gas phase dissolves in the

medium , establishes equilibrium with HCO3- ions ,

and lowers the pH.

The bicarbonate concentration and carbondioxide

tension must be in equilibrium to achieve correct pH

and osmolarity.

H20 +CO2 H2 CO3 H+ + HCO3-

(Carbonic Acid)Bicarbonate ions



Dissolved gasesDissolved gases

to control pH together with NaHCOto control pH together with NaHCO33

HH22O + COO + CO22 HH22COCO33 HH++ + HCO+ HCO33--

HandersonHanderson--Hasselbach eqn predicts pH:Hasselbach eqn predicts pH:

pH = 6.0966+log ([HCOpH = 6.0966+log ([HCO33-- ]/[CO]/[CO22 ])])

5-10% ambient COCO22

dissolves in the medium

decreasing pH

addition of NaHCONaHCO33

increases HCOHCO33-- thusthus

promotes Hpromotes H22C0C033 formationformation

increasing pHincreasing pH



The increased HCO3

- Concentration pushes tothe left until equilibrium is reached at pH 7.4

Media needs buffered to compensate the

evolution of CO2 and production of lactic acidfrom glucose metabolism

Metabolism of the cultured cells can excrete

various types of metabolites in to the mediaincluding the desired product, which can alter the pH of the medium



The most frequently used buffering system in cell culture

media is the bicarbonate³ carbon dioxide system

HEPES(4-(2-hydroxyethyl)-1-piperazine ethane sulfonicacid ) alone can maintain pH values around (7.2 -7.6) in

the absence of exogenous CO2

TES(N-Tris(hydroxymethyl)methyl-2-amino

ethanesulfonic acid) (7.4), DIPSO(7.6), and HEPPSO (7.8)

Buffering system in cell culture media



a) Natural media: Natural sources of nutrients

sufficient for growth and proliferation of animal

cells. eg. serum, plasma, amniotic fluid etc

b) Synthetic media :It is prepared artificially by

adding several nutrients (organic and inorganic),

vitamins, salts, 02 and Co2 gas phases, serumproteins, carbohydrates and cofactors

Media : TypesMedia : Types



PlasmaPlasma is the strawis the straw--colored liquid in which thecolored liquid in which theblood cells are suspended.blood cells are suspended.

Plasma transports materials needed by cellsPlasma transports materials needed by cellsand materials that must be removed from cells:and materials that must be removed from cells:

various ions (Navarious ions (Na++, Ca2, Ca2++, HCO, HCO33

íí, etc., etc.

glucose and traces of other sugarsglucose and traces of other sugars

amino acidsamino acids

other organic acidsother organic acids

cholesterol and other lipidscholesterol and other lipidshormoneshormones

urea and other wastesurea and other wastes



Serum containing media Serum free medium

Serum: is blood plasma without fibrinogen andother clotting factors

Bovine calf, Fetal bovine, Horse, Human serum

Calf serum and Fetal bovine serum are mostly used added to medium by 1-20% v/v

Synthetic media is 2 types



(Serum )



S erum

Serum is one of the very important components of animal cell culture

Eagles minimal essential medium (serum freemedium) when supplemented with 5-20 % serum isgood nutrient medium for culture of most types of cells

It should be supplemented along with the medium

because it contains growth factors which promotecell proliferation, cell attachment and adhesionfactors



The serum provides various plasma proteins,

peptides, carbohydrates, minerals, and someenzymes

Serum is also a source of various amino acids,

hormones (insulin), lipids, vitamins, polyamines and

salts containing ions such as calcium, chloride,

ferrous, ferric, potassium etc.

It acts as buffer



A major role of serum is to supply proteins eg.

fibronectin, which promote attachment of cellsto substrate

It provides several binding proteins eg.albumin, trasferrin which carry other

molecules to cell

Protease inhibitors protect cells , especially

trypsinised cells from proteolysis



SERUM-CONTAINING MEDIUM (EAGLE'S MEDIUM)

Essential amino

acids

The essential amino acids histidine, isoleucine,

leucine, lysine, methionine, phenylalanine,

threonine, tryptophan, and valine plus cysteine,

glutamine, and tyrosine (all at 10 4 to 10 5 M)

Vitamins Choline, folic acid, nicotinamide, pantothenate,pyridoxal, and thiamine (all at 1 mg/L); inositol (2

mg/L); riboflavin (0.1 mg/L)

Salts Na+, K+, Ca2+, Mg2+, Cl , PO43 , HCO3

Glucose 0.9 g/L

Dialyzed serum* 5 to 10 % of total volume



Disadvantages of Serum

Physiological variability ² Major constituents effectscell growth

Shelf-life and consistency ² serum varies from batch tobatch. Best batch will last for one year

Quality control ² changing serum batches requirestesting to ensure as close to previous batch



Specificity ± If more than one cell type is used , each type

may require a different batch of serum(Coculture problem)

Availability ± supply of serum; sometimes high demand

Downstream Processing - contents interfere with what cellsproduce

Contamination- serum is frequently contaminated with virus

Cost ± Cost of a bottle is ten times more than chemicalconstituents



Disadvantages of Serum in the Culture MediumDisadvantages of Serum in the Culture Medium

1. Serum may inhibit growth of some cell types, e.g., epidermalkeratinocytes

2. Serum may contain some cytotoxic or potentially cytotoxicconstituents. For example, fetal calf serum contains the enzymePolyamine oxidase, which converts polyamines like spermidine

and spermine (secreted by fast growing cells) into cytotoxicpolyaminoaldehyde

3. There is a large variation in serum quality from one batch toanother; this requires costly and time consuming testing everytime a new batch has to be used

4. Some growth factors may be inadequate for specific cell typesand may need supplementation

5. It interferes with downstream processing when cell cultures areused for production of biochemicals

6. The supply of serum is always lower than its demand

unit i media and components for growth

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