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, i t is sometimes necessary to store the tissue between removal from the organ ism and freezing. However, when excised tissue is stored in buffer, the ele mental concentrations in the cell may change, In the present study, it was attempted to develop a storage buffer that would retain the cellular elemen tal concentrations close to their in situ values. To start, the NaCl compon ent 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 soluti on, part of the K-gluconate was replaced by an equivalent amount of NaCl. I ncubation of excised rat liver (4 degrees C, 4 h) in 85% high K+ solution r esulted in retention of cellular Na, K, Ca, S and Mg concentration most clo sely to the it? 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 Kf solution resulted in optimal retention of cellula r Na, It, Ca, P, S and Mg, while Cl was retained best in a 70% high K+ solu tion. Based on these results, an optimally modified Krebs-Ringer solution f or the liver would consist of 119 mM K+, 26 mM Na+ and 45 mM Cl-. An optima lly 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, It 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 th e rest of the incubation, whereas in the submandibular gland tissue it incr eased again after 4 h, The increase in cell volume (oedema) was less in tis sue stored in the modified solutions, than in the 100% high K+ solutions. I ncubation in high Naf buffers (4 degrees C, 6 h) resulted in a progressive increase in the percentage of cells showing trypan blue uptake. A similar i ncrease in trypan blue uptake was seen in the modified solution, but this i ncrease levelled off after 4 h. After cholinergic stimulation in high Na+ s olution (25 degrees C, 1 min), the expected decrease in cellular Cl concent ration was seen in submandibular gland cells that had previously been prese rved (4 degrees C, 4 h) in the modified solution, but not in those that had been preserved in the 100% high K+ solution.