Perfusate

It is usual to use Krebs-Henseleit solution (original recipe and variations).

Composition (variant):

Salts

(anhydrous) g/l mmol/l

NaCl 6.9 118.0

KCl 0.35 4.7

CaCl₂ 0.28 2.52

MgSO₄ 0.20 1.64

NaHCO₃ 2.09 24.88

KH₂PO₄ 0.16 1.18

Glucose 1.09 5.55

Na-pyruvate 0.22 2.0

Krebs-Henseleit solution should be made up fresh each day and should then be filtered before use. The pore size of the filter used is usually 0.5 μm for the fine filter and 5 μm for the pre-filter. If no compressed air filter or suction filter is available, a suitably powerful roller pump can be useful. For filtration you require then only a disc filter holder (approx. 50 mm dia.) and suitable filter disc in the appropriate pore sizes (suppliers e.g.: Sartorius, Millipore).

*****Filtration should not be omitted. Even high-grade starting chemicals contain insoluble constituents which lead to micro embolisms in the perfused organ if the solution is not filtered. *****

Aeration

In general Carbogen gas (5% CO₂, 95% O₂ v/v) is used for equilibrating the perfusion solution. This percentage composition should be adhered to as accurately as possible. Otherwise a wrong pH is obtained after equilibrating the perfusate. With constant aeration, Krebs-Henseleit perfusate maintains a pH of 7.4.

Ensure that you use only Carbogen gas of the composition mentioned above. When making up Carbogen or other mixtures yourself you have to ensure that you use only gas of the highest purity. For example, industrial nitrogen contains large amounts of oil.

Carbogen gas is normally provided in high-pressure gas cylinders. Before the gas is introduced into the apparatus its pressure should be reduced to an inlet pressure of 0.5 to 1 bar by means of a finely adjustable pressure reducing valve. If higher pressures are applied the tubing may burst or may slip off from the connection nipples. The pressure regulator used should be provided with a pressure gauge with a suitable scale. The pressure setting must be indicated reliably and be read easily.