[Effect of serum and osmotically active substances on metabolism in 262 tissue cultures of pig's corneas--improved preservation of donor tissue for keratoplasty].
BACKGROUND
The storage time of corneal organ cultures in limited in the closed system mainly used in European eye banks. In addition, swelling during storage reduces the quality of the donor material for keratoplasty. Therefore, dextrane was added in concentrations of 2.5%, 5%, 7.5% and 10% to the culture medium, and the energy producing metabolism was investigated.


METHODS
The experiments were carried out with 262 pig corneas. MEM with some supplements was used at 31 degrees C. No serum was added, because glucose consumption, lactate production and ATP levels proved to be the same or better than with serum supplement. After 6 and 12 days, the corneas from organ cultures were extracted with perchloric acid. The levels of glucose and lactate in the stroma and of ATP and ADP in the epithelium were analysed with enzymatic-optical tests.


RESULTS
Dextrane inhibited glycolysis and the production of ATP in corneal organ cultures during twelve days. With 7.5% and 10% dextrane in the medium, lactate levels in the cornea decreased from 6.09 to 4.5 and 4.6 microM/g H2O instead of increasing. At the same time, glucose increased paradoxical from 1.3 to 3.4 and 2.2 microM/g H2O, respectively. With 5% dextrane, glycolysis operated sufficiently producing an increase of lactate levels from 6.0 to 8.8 microM/g H2O and consuming glucose from levels of 1.27 down to 0.57 microM/g H2O. After 12 days with 7.5% and 10.0% dextrane, ATP levels were reduced, from 4.54 to 0.98 and 0.49 microM/g dry weight, and ATP/ADP ratios from 1.9 to 1.1 and 0.7 respectively. With 2.5% dextrane, the ATP was diminished from 4.5 to 2.2 microM/g dry weight. When 5% dextrane were added to the culture medium, the hydration was at optimum by 4.1, and ATP levels were reduced only from 4.5 to 2.6 microM/g dry weight. Moreover, the ATP/ADP ratios were at 2.1 as good as without dextrane.


CONCLUSIONS
From the results it was concluded, that serum free medium may be used, and that permanent dewelling proved to be beneficial for the energy producing metabolism of the cornea in organ culture. Such conditions may improve morphological and metabolic quality of donor material. From previous publications, it was recommended, to use instead of toxic dextran the well tolerated HES (hydroxyethyl starch) and to apply a storage temperature of 21 degrees C, which slowed down glucose consumption without impairing the energy producing metabolism.
Rheinisch-Westfälische Technische Hochschule, Aachen, Augenklinik der Medizinischen Fakultät, Klinikum, Pauwelsstrasse 30, 52057 Aachen.
M
M
Reim
F J
FJ
Pantenburg
C D
CD
Ziegler
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D002951:Citrates / Q000494:pharmacology; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism*; D015715:Corneal Edema / Q000378:metabolism / Q000517:prevention & control*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D046508:Culture Techniques / Q000379:methods*; D003911:Dextrans / Q000378:metabolism / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D009995:Osmosis; D010952:Plasma Substitutes / Q000378:metabolism / Q000494:pharmacology*; D013552:Swine
D002951:Citrates; D016895:Culture Media, Serum-Free; D003911:Dextrans; D010952:Plasma Substitutes; D019344:Lactic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C007999:hydroxycitric acid; D005947:Glucose