2. INTRODUCTION
• Need: to maintain tissue viability outside the
animal body to mimic the internal environment of
ions and nutrients
• Generally termed as Ringer solution
3. • Despite over 100 years of their use in in vitro experiments
most of the standard salt physiological solutions are still poor
substitutes for blood.
4. PROBLEMS ENCOUNTERED
WITH BLOOD
• Coagulation has to be prevented.
• Blood is easy to oxygenate, but attempting to do this often
results in problematic bubbling because of the surface active
properties of proteins in the blood.
• Contact of blood with glass and other foreign surfaces results
in the release of pharmacologically active compounds, e.g.
amines (5HT) and kinins as well as the loss of platelets and
other blood cells
• Blood is a very good nutrient for bacteria.
5. VANE CASCADE
• EXTRACORPOREAL source of blood
• blood taken from a large artery used to flow over and thereby
maintain isolated tissue which when used is returned via a
suitable vein.
• This technique is the basis of the superfusion cascade assay
system
7. ROLE OF EACH INGREDIENT
• Na+ - major extracellular cations - maintains the electrolyte level in the
tissue - makes the solution isotonic by maintaining the osmolarity
• K+ - major intracellular cation - important role in nerve conduction,
muscle contraction,. - maintains heart rhythm
• CaCl2 - controls excitabilty of muscle, nerves, and glands. maintains
cellular integretity, permeabilty
• MgCl2- 2nd most important intracellular cation reduces spontanoeus
activity, role in neurtransmission
• HCO3- , NaHPO4 – buffer(pH7.4)
• glucose: major nutrient(fatty acid : cardiac muscle)
8. VARIANTS
• Krebs - nerve muscle preparation - may be used for
any tissue
• Tyrode - exp with non inervated muscle - esp
mammalian smooth muscle.
• Frog Ringer - amphibian tissue.
• Ringer Locke - heart muscle preparation.
• DeJolan, McEwen :mammalian isolated
oragan(nerve associates) and avian skeletal muscle.
9. METHODS FOR DELIVERING
PHYSIOLOGICAL SOLUTION TO
TISSUE
• Perfusion (directly connected to the vascular system)
• Tissue bath (sample immersed in fluid)
• Superfusion (solution running over the tissue
surface)
10. • However, oxygen solubility in water is low and thus carries
little oxygen. Temperature is adjustable for homeotherms
(warm blooded), room temp for poikilotherms (cold blooded).
• Normally the bathing temperature is kept somewhat below
body temperature so as to reduce metabolic needs and so
preserve tissue.
11. PRECAUTIONS
• CaCl2 should be added at the last
• pH to be maintained at 7.3-7.4
• Distilled water or double distilled water or deionised water is
to be used
• Carbogen or oxygen is used for aeration
• error < 1%
12. STORAGE
• Fresh solution is always preferred
• Max storage time: 24hrs in refrigerator
• Risk of microbial growth : due to prescence of glucose
• If storage > 24hrs is required : Ca2+ and glucose is added only
at the time of experiment
