introduction to balanced salt solution and its modifications
TRANSCRIPT
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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