Introduction to Balanced Salt Solution & Simple growth medium &

its modifications

Presented By:Devendra Kumar

M.Sc. (Biotechnology)Enrolment No. 1386/14

Balanced Salt Solution

BSS provides an environment that maintains the structural and physiological integrity of cells in vitro condition

BSS most commonly includes – Na, K, Ca, Mg, Cl and elements which required by the cells in vitro condition during culture

BSS are used for washing tissues and cells and are usually combined with other agents to treat the tissues and cells.

BSS provide water and inorganic ions to maintaining a physiological pH and osmotic pressure 

Most commonly BSS are :

Earle’s Balanced Salt Solution (EBSS)

Hank’s Balanced Salt Solution (HBSS)

(Dulbecco's) Phosphate Buffer Saline (DPBS)

Ringer’s Balanced Salt Solution (RBSS)

Phosphate Buffered Saline (PBS)

Balanced Salt Solution

How BSS work

Na and K regulate tonicity and permeability

Ca and Mg maintain the integrity of cell membranes and internal structures

phosphate (HPO4-H3PO4) and/or bicarbonate (HCO3) control the hydrogen ion concentration through their buffering effect

BSS also include glucose, which provides a readily available energy source for cells

Media used for tissue (cell) culture

Cell culture medium are complex mixture of salts

Medium mainly includes carbohydrates, vitamins, amino acids, growth factors, hormones and trace elements

Sodium bicarbonate, phosphate and HEPES for pH and osmolality

Phenol red, a pH indicator is also added for colorimetrically monitor changes in pH

Media for Tissue culture

2 types of media used for culture of animal cells and tissues

Natural media

Synthesized media

Natural media Natural sources of nutrient sufficient for

growth and proliferation of animal cell and tissues

Used to promote cell growth

Divided in 3 categories Coagulant Biological fluids Tissue extracts

Coagulants Plasma clots are used as coagulants

Commercially available in the form of liquid plasma kept in silicon ampoules or lyophilized plasma

Also prepared in laboratories taking out blood from male fowl and adding heparin or anticoagulant

Biological fluid Serum is basically used as biological

fluid

Serum is important component

& source of various amino acid, hormones, lipids, vitamins,

polyamines

Also contain growth factors which promotes cell proliferation, cell attachment & adhesion factors

Salts containing ions such as Calcium, ferrous, ferric, potassium etc.

Serum is obtained from human blood, placental, cord blood, horse blood and calf blood

Other biological fluids are coconut water, amniotic fluid, pleural fluid, aqueous humour and culture filtrate etc.

Biological fluid

Tissue extracts

Embryo extracts-

extracts from tissues such as

embryo,

liver,

spleen,

leukocytes,

tumour,

bone marrow etc.

Synthetic media

Prepared artificially by adding several organic and inorganic nutrients such as

Vitamins

Salts

Serum

Proteins

Carbohydrates

Cofactors etc.

Synthetic media are prepared for different types for cell and tissue culturing

Basically divided in following types:

Serum containing media

Serum free media

Protein free media

Chemically defined media

Synthetic media

Serum containing medium

MEM (Minimum Essential Media):

it contains >20 types of substances which can be divided in 4 subgroups

CO2 with glucose

Amino acid:

Arginine, Cysteine, Isoleucine, Lucien,

Lysine, Methionine, phenilanine, Thionine,

Tryptophan, Histidine, Tyrosine and Valine

Vitamins:

choline, chloride, folic acid, inositol, nicotinamide, thiamine, riboflavin, pyridoxine

Inorganic salts:

NaCl, KCl, CaCl, MgSO4, NaHCO3, NaH2PO4

Eagle’s Minimum Essential Media (EMEM):First widely used medium formulated by Harry Eagle contains EBSS, nonessential amino acids, sodium pyruvate and higher level of serum

Serum containing medium

Serum containing medium

Dulbecco’s Modified Eagle’s Medium (DMEM):

It has roughly twice the conc. of Amino acids & 4times vitamins as EMEM, as well as ferric nitrate, sodium pyruvate & some supplementary amino acids

Hybri-Care Medium:Modification of DMEM supplemented with insulin, oxalacetic acid and HEPES

used for propagation of hybridomas and other fastidious cell lines

Serum containing medium

Serum containing medium

Iscove’s Modified Dulbecco’s Medium(IMDM):Formulated for growth of lymphocytes and Hybridomas

It has additional amino acids, vitamins and inorganic salts compared to DMEM.

It also contain HEPES and selenium as a buffer solution

McCoy’s 5A and RPMI-1640: Used to grow Novikoff hepatoma cells and supports the growth of primary cultures

RPMI-1640 is a modification of McCoy’s 5A and developed for long-term culture of peripheral blood lymphocytes

RPMI-1640 also supports growth of variety of cells in suspension as well as monolayer culture

DMEM/F12 Medium:Mixture of DMEM and Ham’s F-12 in the 1:1

Extremely rich and complex medium supports the growth of a broad range of cell type in both serum and serum free formulation

Ham’s Nutrients Mixtures:Developed to supports the clonal out growth of Chinese Hamster Ovary (CHO) cells

EMEM modified with including Ham’s F-12 medium

Leibovitz’s L-15 Medium:Formulated for use without CO2 incubation as is found in teaching laboratories or when collecting biopsy samples

NaHCO3/ CO2 buffering system is replaced by a combination of phosphate buffers, free-base amino acids, higher level of sodium pyruvate and galactose

Serum free media (SFM) Used to grow specific type cell

perform a specific application in the absence of serum

Advantages:Easier purification and further processing

More consistent performance

Increase growth or productivity

Better control over physiological responses

Enhance the detection of cellular mediator

Protein free media (PFM) Protein free growth medium is a medium

which does not contain any hormone or growth factor

Medium is optimized for cultivation Chinese hamster ovary (CHO) cells specially

Absence of protein in the medium eliminates any transmission risk of blood bone diseases and hence is safer

Chemically defined media

Medium in which chemical nature of all the ingredients and their amounts are known

Rat tail collagen:It is used as either a thin layer on tissue culture surface to enhance the cell attachment, proliferation and promote expression of cell morphology and function

Suitable for variety of cells- hepatocytes, fibroblast and epithelial cells

Chemically defined media

Media should contains inorganic salts, and other nutrients capable of sustaining cells in culture such as

Amino acids

Fatty acids

Sugars

Ions

Trace elements

Vitamins

Cofactors

Glucose for energy source

Phenol red for pH indicator of medium

Culture media modifications

Essential requirement for tissue culture are special incubators to maintain the level of O2, CO2,

Gaseous modifications

Temperature &pHTempIn most of mammalian cell culture, the temperature is maintained at 37.C in the incubators as the body of Homo Sapiens

pHThe suitable pH for most cells is 7.2-7.4The main substance causing pH changes is CO2 produced in cell metabolism process. CO2 can combine H2O2 to produce carbonic acid and thus reduce the pH value of the medium. Synthesized medium employs NaHCO3-CO2 buffer system to solve this problem.

Amino acids

Amino acid is the raw material for the cell to synthesize protein.

All the cells need twelve essential amino acids: arginine, cystine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, histidine, tyrosine and valine, which are L-amino acids.

glutamine is another component playing important role in the cell metabolism process. The nitrogen contained in glutamine is not only the source of purine and pyrimidine of nucleic acid,

Monosaccharides

Cultured cells use aerobic glycolysis and anaerobic glycolysis of hexose as main energy source.

hexose is used for the synthesis of some amino acid, fat and nucleic acid.

Cell absorptive capacity varies among different monosaccharides, with the highest for glucose and the lowest for galactose.

Vitamins & trace elements Vitamins mainly act as coenzymes or prosthetic

groups in cell metabolism processes.

Biotin, folate, nicotinamide, pantothenic acid, pyridoxine, riboflavin, thiamine and cynocobalamine are common vitamins in culture medium.

Inorganic ion and trace element also required for cell culture

cell growth needs some trace elements, such as molybdenum, vanadium, iron, zinc and selenium, copper, manganese.

Hormones & Somatomedin

Cells grown in vivo are always regulated by somatomedin and hormones.

Some hormones have promoting growth effects on different cell types. For instance, insulin can promote the use of glucose and amino acids in the cell.

Some hormones are cell-type specific, as hydrocortisone that can promote the growth of epidermal cells and prolactin that induces the proliferation of mammary epithelial cell.

Osmotic pressure

Cells need an isotonic environment and human plasma osmotic pressure is about 290 mOsm/kg, which is thought to be ideal osmotic pressure to culture human cells.

Mouse plasma osmotic pressure is about 320 mOsm/kg.

Osmotic pressure of 260-320 mOsm/kg fits for most mammalian cells.

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