Retaining ionic concentrations during in vitro storage of tissue for microanalytical studies

When human or animal tissue is to be investigated by X-ray microanalysis, it is sometimes necessary to store the tissue between removal from the organism and freezing. However, when excised tissue is stored in buffer, the elemental concentrations in the cell may change. In the present study, it was attempted to develop a storage buffer that would retain the cellular elemental concentrations close to their in situ values. To start, the NaCl component in Krebs-Ringer buffer was exchanged for K-gluconate and KCl for NaCl. This buffer was called a '100% high K+ solution'. Starting from this solution, part of the K-gluconate was replaced by an equivalent amount of NaCl. Incubation of excised rat liver (4 degrees C, 4 h) in 85% high K+ solution resulted in retention of cellular Na, K, Ca, S and Mg concentration most closely to the in situ state, whereas cellular Cl was retained best when the tissue was incubated in 75% high K+ solution. For rat submandibular gland, incubation in 80% high K+ solution resulted in optimal retention of cellular Na, K, Ca, P, S and Mg, while Cl was retained best in a 70% high K+ solution. Based on these results, an optimally modified Krebs-Ringer solution for the liver would consist of 119 mM K+, 26 mM Na+ and 45 mM Cl-. An optimally modified Krebs-Ringer bicarbonate solution for the submandibular gland would be composed of 96 mM K+, 53 mM Na+ and 46 mM Cl-. After incubation in the modified solutions (at 4 degrees C), cellular Na, Mg, S, Cl, K and Ca in both tissues were maintained close to the in situ state throughout a 6-h incubation. The cellular P concentration was reduced after incubation for 1 h; thereafter, in the liver cells it remained at this lower level for the rest of the incubation, whereas in the submandibular gland tissue it increased again after 4 h. The increase in cell volume (oedema) was less in tissue stored in the modified solutions, than in the 100% high K+ solutions. Incubation in high Na+ buffers (4 degrees C, 6 h) resulted in a progressive increase in the percentage of cells showing trypan blue uptake. A similar increase in trypan blue uptake was seen in the modified solution, but this increase levelled off after 4 h. After cholinergic stimulation in high Na+ solution (25 degrees C, 1 min), the expected decrease in cellular Cl concentration was seen in submandibular gland cells that had previously been preserved (4 degrees C, 4 h) in the modified solution, but not in those that had been preserved in the 100% high K+ solution.